Funded Assay Projects
A Screen For Drugs That Promote Oligodendrocyte Formati*
APPEL, BRUCE
VANDERBILT UNIVERSITY
1 R03 MH076346-01
1
DESCRIPTION (provided by applicant): The goal of this project is to develop zebrafish as a model system to investigate regeneration of oligodendrocytes, the myelinating cell type of the vertebrate central nervous system, from resident populations of neural stem and progenitor cells. The ability to promote formation of new oligodendrocytes might aid therapies to restore nervous system function following disease or injury. Zebrafish offer numerous advantages for studying neural regeneration. Zebrafish develop entirely outside the mother and embryos and young fish are optically clear, providing the opportunity for direct and extended observations of neural cells. To facilitate direct observation of oligodendrocytes, we have produced transgenic reporter gene lines that express fluorescent proteins in oligodendrocytes at all stages of their differentiation. Because they readily take up chemical compounds, our transgenic zebrafish can be used to screen for drugs that promote oligodendrocyte replacement. Thus, this work seeks to utilize the Zebrafish Chemical Screening Center to search for drugs that promote formation of oligodendrocytes. Neural diseases and injuries have devastating impacts on human health. This work will provide a foundation for identifying and investigating genes and drugs that promote neural regeneration and recovery. -
Screening of DNA-PKcs Autophosphorylation Blockers via *
CHEN, BENJAMIN P
UNIVERSITY OF TEXAS SW MED CTR/DALLAS
1 R03 MH076515-01
1
DESCRIPTION (provided by applicant): Radiation treatment is one of the most important cancer-therapeutic approaches. Non-toxic radiosensitizers, used in conjunction with radiation therapy, would enhance the effectiveness of the radiotherapy and, at the same time, reduce the total radiation dose to prevent unwanted side effects. DNA double-strand breaks (DSBs) are the major DNA lesion induced by ionizing radiation (IR) and the dominant cause of radiation sensitivity. It is well established that lack of DSB repair can be lethal in mammalian cells. DNA-dependent protein kinase (DNA-PK) is the key component of the DNA nonhomologous end-joining (NHEJ) pathway that is the predominant pathway for DSB repair in mammalian cells. We have shown that inhibition of DNA-PK kinase activity and/or abrogation of radiation-induced autophosphorylation of DNA-PKcs (the catalytic subunit of DNA-PK) results in severe radiation sensitivity. As the autophosphorylation of DNA-PKcs is a requisite step for NHEJ mediated DSBs repair, molecules that inhibit autophosphorylation without affecting the protein levels of DNA-PK or its enzymatic activity could selectively radiosensitize cells without affecting other cellular processes; whereas molecules that inhibit the kinase activity of DNA-PK also commonly inhibit other PI-3K like protein kinases (ATM and ATR) and cause significant cellular toxicity. In this proposal, we plan to develop both lysate-based and cell-based high throughput screening (HTS) assay to identify chemicals or natural compounds that would either block the autophosphorylation of DNA-PKcs or inhibit the kinase activity of DNA-PKcs. With regard to the approaches, -
Chemical Genetic Screening of Oncogene RAS-Based Inhibi*
CHENG, XIAODONG
UNIVERSITY OF TEXAS
MEDICAL BR GALVESTON
1 R03 MH076387-01
1
DESCRIPTION (provided by applicant): Pancreatic cancer is one of the most aggressive and devastating human disease. Almost all patients diagnosed with the disease die from the malignancy with a medium survival of less than six months. Clinically, the disease often evades detection during its early stages due to the lack of specific symptoms and limitation in diagnostic methods. Adding to this problem, pancreatic cancer is resistance to most forms of treatments such as chemotherapy, radiation, and combination therapy. One single most important cause for such a dismal clinic prognosis of pancreatic cancer is the lack of fundamental understanding of basic biology of human pancreas both under physiological and pathophysiological conditions. A recent study by the National Cancer Institute Pancreatic Cancer Progress Review Group concluded that pancreatic cancer is disproportionately underrepresented in both clinical and basic research compared with other cancer sites. Although many of the genetic changes associated with pancreatic cancer development have been revealed, significant gaps exist in our understanding of how these genetic alterations lead to the initiation, development, and maintenance of pancreatic cancer. This information is essential for develop new and effective diagnostics and treatments for pancreatic cancer. One major roadblock for pancreatic cancer research is the lack of genetically tractable human cancer cell model systems to investigate the origins and progression of pancreatic cancer. To overcome this barrier, we have recently developed a genetically defined human pancreatic cancer cell model using genetic components known to be frequently altered in pancreatic cancer. We will use our genetically defined human pancreatic cancer model to screen chemical libraries for the purpose of discovering of chemical compounds that preferentially kill the cancer cells but not normal human pancreatic cells. These studies may lead to the identification of potential novel and effective treatments for pancreatic cancer. In addition, the identification of such mechanism-based chemical inhibitors will also aid the elucidation of down-stream signaling pathways of these well-defined genetic alterations that are important for the initiation and development of this destructive disease. -
FP Assay for Isolation of Hsp90 Inhibitors
CHIOSIS, GABRIELA
SLOAN-KETTERING INSTITUTE FOR CANCER RES
1 R03 MH076499-01
1
DESCRIPTION (provided by applicant): The long-term goal of this initiative is to identify novel Hsp90 inhibitors with use in cancer therapy and in the treatment of neurodegenerative diseases. The goal of the current effort is to translate our Hsp90 fluorescence polarization assay to MLSCN centers. This assay probes the competitive binding of red-shifted cy3B-labeled geldanamycin to Hsp90 from cell lysates. Funding for the development of the assay has been provided in part by R03 NS050838-01; Chiosis, G (PI). Hsp90 is a chaperone with important roles in maintaining transformation and in elevating the survival and growth potential of cancer cells. Recent evidence suggests additional applications of Hsp90 inhibitors in neurodegenerative diseases, nerve injuries, inflammation and infection. Several natural products that inactivate Hsp90 function have anti-tumor effects in in vitro and in vivo models of cancer. However, due to the role of Hsp90 in normal cellular homeostasis, it remained unclear whether Hsp90 inhibitors will be sufficiently specific for use as therapeutic agents. Early clinical results with 17AAG, the first Hsp90 inhibitor to enter clinical trials, suggest that these fears may be unfounded. These studies confirm that Hsp90 is a promising target for novel cancer therapeutics and pave the road for the introduction of Hsp90 inhibitors in the clinic. The potential of Hsp90 inhibitors as therapeutics in other diseases has been less explored due to limitations with the current inhibitors (i.e. 17AAG is not crossing the blood-brain barrier, BBB). Currently there is thus, increasing interest in developing novel inhibitors of this protein. We have conducted pioneering research in the area and designed the first synthetic class of Hsp90 inhibitors, the purine-scaffold class (PU-class). We have also designed and developed several assays that probe biochemical and cellular inhibition of Hsp90. One such assay is proposed here for use in MLSCN. Discovery of novel Hsp90 inhibitors based on diverse chemical skeletons is necessary to fully harvest the therapeutic potential of the chaperone, and we propose here that such diversity may be obtained by screening large libraries of compounds against this target. -
Isolation of Inhibitors of Her-Kinase Expression
CHIOSIS, GABRIELA
SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH
1 R03 MH076408-01
1
DESCRIPTION (provided by applicant): Human, cancers frequently express high levels of transmembrane tyrosine kinases of the Her family. Overexpression of at least one of these, the Her2 receptor, has been associated with a more aggressive behavior. Several therapeutic strategies targeting the receptor are now in various stages of clinical development. Most of the known agents block the activation or inhibit the activity of Her2; however, a more significant therapeutic outcome may result from degrading this oncoprotein. We propose here a cell-based assay that may be used to conduct high-throughput screens (HTS) to isolate Her2 kinase expression inhibitors. The compounds identified from these efforts will represent important leads that will serve as the starting point to obtain drugs that modulate Her2 expression. These drugs could, in principle, be used to treat patients with advanced cancers driven by Her2-overexpression/oversignalling. Given the importance of Her-kinases in human disease, the time is ripe to design and perform a large-scale screening regimen to identify Her-kinase expression modulators. To our knowledge, this has not been accomplished, and we believe that we are uniquely positioned to initiate this goal. -
Multiplexed HTS of Serine and Cysteine Proteases
DIAMOND, SCOTT L
UNIVERSITY OF PENNSYLVANIA
1 R03 MH076406-01
1
DESCRIPTION (provided by applicant): A multiplexed microarray HTS campaign is proposed for a series of proteins relevant to blood coagulation, complement function, neutrophil platelet crosstalk, and lysosomal processes. Protocols are defined where the compound library will be printed in replicate sets on microarrays. Each set of microarrays (3072 compounds/microarray and 33 microarrays per 100,000 compound screen) will be activated with an individual enzyme and a matched fluorogenic substrate. A total of 9 proteases have been fully profiled on microarrays for substrate specificity using 722-member fluorogenic substrate libraries (Ala-P3-P2-K/R libraries). The proposed targets for multiplexed microarray HTS are: Serine proteases (Coagulation) Human factors IXa, Xla, Xa; Serine proteases (Complement) Human C1s, C1r, Factor D; and Human cathepsins (Lysosomal) Cathepsins B, S, G. The use of multiplexed assay on microarrays allows the generation of critical data for the chemical protease interactome. -
Assay for Formylpeptide Receptor Family Ligands
EDWARDS, BRUCE
UNIVERSITY OF NEW MEXICO ALBUQUERQUE
1 R03 MH076381-01
1
DESCRIPTION (provided by applicant): We have developed a high-throughput methodology for flow cytometry, HyperCyt(r), that enables automated, quantitative, high-content analysis of 40 or more samples per minute. A long-term goal is to adapt this methodology to high content screening of diverse molecular interactions in cells and cell-free multiplex beadbased assays. A more immediate goal is to screen small molecule compound libraries for structures that block binding of defined fluorescent ligands to receptors expressed on cells. We have established and characterized a homogeneous no-wash HyperCyt(r) assay for sensitive detection of compounds that block ligand binding to the human formylpeptide receptor (FPR) and the FPR family member, FPR-like 1 (FPRL1). Since ligand binding is measured directly, the assays detect active compounds independently of potential complexities in cell physiological response patterns. We propose a minor, easily implemented technical modification, color-coding of FPRL1-expressing cells, to allow integration of FPR and FPRL1 assays into a duplex assay capable of simultaneously measuring compound interactions with both receptors in a single sample. Our FPR screening experience supports a high probability that screening of the NIH Small Molecule Repository will identify novel FPR and FPRL1 ligands amenable to optimization as high affinity biological probes. We expect that such compounds will prove to be important tools with which to advance the understanding and therapy of inflammation-associated diseases. -
AlphaScreen Technology for High Throughput Screening of*
FU, HAIAN
EMORY UNIVERSITY
1 R03 MH076385-01
1
DESCRIPTION (provided by applicant): Through phosphorylation-dependent binding to target proteins, 14-3-3 proteins regulate a wide range of physiological processes, including cell proliferation, cell cycle progression, suppression of apoptotic program, and neuronal functions. In order to generate research tools for study the functions of 14-3-3 in various biological systems and potentially to develop 14-3-3-targeted therapeutic strategies, we have developed an AlphaScreen technology based high throughput assay for isolation of small molecules that can disrupt the 14-3-3/client protein interactions. We have optimized and miniaturized the assay for adaptation in a 384-well plate format for robotic operations. The goal of this proposal is to utilize this assay in the Molecular Libraries Screening Centers Network for isolation of small molecule compounds that can interfere with the interaction of 14-3-3 with its client proteins. -
Yeast Lifespan Chemical Screening
GOLDFARB, DAVID S
UNIVERSITY OF ROCHESTER
1 R03 MH076395-01
1
DESCRIPTION (provided by applicant): The goal of this proposal is to exploit a novel high throughput assay capable of screening hundreds of thousands of compounds for affects on replicative lifespan in the yeast Saccharomyces cerevisiae. Key aspects of aging are conserved between yeast and humans, including signaling, regulatory, and effector pathways. Yeast has been a valuable model system for the discovery and elucidation of conserved aging genes such as SIR2 and their modes of regulation, but only the surface of the field has been scratched. The screening of chemical libraries with this assay should result in the identification of compounds that affect the activities of known and as yet undiscovered cellular targets with roles in aging and lifespan. The “death of daughters” (DEAD) replicative lifespan assay provides cell-based phenotypic screen that will allow us to address an entirely new order of questions about the molecular mechanism and control of aging and lifespan. This high throughput assay will allow us to investigate the genomics of lifespan, screen chemical libraries for compounds that affect lifespan, and study the genetics of lifespan among natural populations. Preliminary studies screening a library of ~2500 compounds indicates that the DEAD assay can be performed in 384 well plates without constant shaking. When the starting DEAD cell culture is prepared and handled properly, the optical density read-out is readily controlled and highly reproducible. The aim of this proposal is to facilitate DEAD assay screening of chemical libraries in the NIH Small Molecule Repository at Molecular Libraries Screening Centers. Promising compounds can be readily assessed using other lifespan assays in yeast, worms, and fruit flies. The assay results can be easily formatted in raw and processed formats to maximize their sharing and utility to others. -
In Vitro High Throughput Screening Assay for MKP-3
LAZO, JOHN S
UNIVERSITY OF PITTSBURGH
1 R03 MH076390-01
1
DESCRIPTION (provided by applicant): Mitogen activated protein kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that has a central role regulating intracellular signal transduction by controlling the phosphorylation status of mitogen activated protein kinases. There is considerable evidence indicating MKP-3 could be a potential molecular target for several human diseases. Nonetheless, no potent or selective small molecule inhibitors of this protein phosphatase have been identified. This proposal will provide sufficient reagents to permit a high throughput screening of 100,000 compounds -
Chemical Complementation Assay for MKP-3
LAZO, JOHN S
UNIVERSITY OF PITTSBURGH
1 R03 MH076330-01
1
DESCRIPTION (provided by applicant): Mitogen activated protein kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that has a central role regulating intracellular signal transduction by controlling the phosphorylation status of mitogen activated protein kinases. There is considerable evidence indicating MKP-3 could be a potential molecular target for several human diseases. Nonetheless, no potent or selective small molecule inhibitors of this protein phosphatase have been identified. Cell-based chemical complementation assays provide a novel platform technology to screen for cell-active inhibitors of this protein phosphatase. This proposal will provide sufficient reagents, including cells and DMA transfection vectors, to permit the high throughput screening of 100,000 compounds. -
In Vitro High Throughput Screening Assay for MKP-1
LAZO, JOHN S
UNIVERSITY OF PITTSBURGH
1 R03 MH076391-01
1
DESCRIPTION (provided by applicant): Mitogen activated protein kinase phospahtase-1 (MKP-1) is a dual specificity phosphatase that has a central role regulating intracellular signal transduction. MKP-1 is a potential molecular target for several human diseases. No potent or selective small molecule inhibitors of this protein phosphatase are known. This proposal will provide sufficient reagents to permit a high throughput screening of 100,000 compounds. -
Chemical Complementation Assay for MKP-1
LAZO, JOHN S
UNIVERSITY OF PITTSBURGH
1 R03 MH076407-01
1
DESCRIPTION (provided by applicant): Mitogen activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that has a central role regulating intracellular signal transduction by controlling the phosphorylation status of mitogen activated protein kinases. There is considerable evidence indicating MKP-1 could be a potential molecular target for several human diseases. Nonetheless, no potent or selective small molecule inhibitors of this protein phosphatase have been identified. Cell-based chemical complementation assays provide a novel platform technology to screen for cell-active inhibitors of this protein phosphatase. This proposal will provide sufficient reagents, including cells and DMA transfection vectors, to permit the high throughput screening of 100,000 compounds. -
Cellular Assay for the Presentation of the Adhesion Mol*
MAYER, THOMAS
COLUMBIA UNIVERSITY HEALTH SCIENCES
1 R03 MH076343-01
1
DESCRIPTION (provided by applicant): Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. The more common inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and psoriasis. Although each disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and immobilizing lymphocytes, allowing infiltration of the tissue to produce a full-blown inflammatory response. A critical event in this process is cell surface expression of VCAM1, which is essential for recruitment of activated lymphocytes. The goal of the proposed project is to use a cell based assay to screen for compounds which block activation of the endothelial cell. Primary human cells (HUVEC) will be stimulated by cytokine and the activation determined by observing expression of the adhesion molecule, VCAM1. A cell surface VCAM assay for automated image analysis by the InCell Analyzer 3000 imaging system was successfully implemented to stage III (Analysis/result acquisition). The assay can generate data with high confidence and a Z’ factor of 0.80. We have also shown that the assay is a powerful tool to screen for bioactive compounds. -
Cellular Assay for TNF Alpha Induced NF Kappa B Translo*
MAYER, THOMAS
COLUMBIA UNIVERSITY
HEALTH SCIENCES
1 R03 MH076344-01
1
DESCRIPTION (provided by applicant): Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. The more common inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and psoriasis. Although each disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and immobilizing lymphocytes, allowing infiltration of the tissue to produce a full-blown inflammatory response. An early event in this pathway is translocation of the transcription factor, NFKB into the nucleus. The goal of the proposed project is to use a cell based assay to screen for compounds which block activation of the endothelial cell. Primary human cells (HUVEC) will be stimulated by different cytokines and the activation determined by observing translocation of the transcription factor NFKB into the nucleus. An NFKB translocation assay for automated image analysis by the InCell Analyzer 3000 imaging system was successfully implemented to stage III (Analysis/result acquisition). The assay can generate data with high confidence and a Z’ factor of 0.79. We have also shown that the assay is highly versatile to screen for bioactive compounds. -
Cellular Assay for TNF Alpha Induced E-Selectin Express*
MAYER, THOMAS
COLUMBIA UNIVERSITY HEALTH SCIENCES
1 R03 MH076509-01
1
DESCRIPTION (provided by applicant): Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. The more common inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and psoriasis. Although each disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and immobilizing lymphocytes, allowing infiltration of the tissue to produce a full-blown inflammatory response. A critical event in this process is cell surface expression of E-selectin, which is essential for lymphocyte adherence. The goal of the proposed project is to use a cell based assay to screen for compounds which block activation of the endothelial cell. Primary human cells (HUVEC) will be stimulated by cytokine and the activation monitored by cell surface expression of the adhesion molecule E-selectin. A cell surface E-selectin assay for automated image analysis by the InCell Analyzer 3000 imaging system was successfully implemented to stage III (Analysis/result acquisition). The assay can generate data with high confidence and a Z’ factor of 0.77. We have also shown that the assay a powerful tool to screen for bioactive compounds. -
Development of the HTS Assay for E. Coli RNA Polymerase
MUSTAEV, ARKADY
PUBLIC HEALTH
RESEARCH INSTITUTE
1 R03 MH076325-01
1
DESCRIPTION (provided by applicant): RNA polymerase (RNAP) is the principal enzyme of gene expression and the target for genetic regulation. It has been a proven target for antimicrobial therapy, and its mutations are central to the problem of multi-drug resistant (MDR) TB. The long-term objective of this research is the development of interventions in gene expression using small compounds that specifically interfere with the RNAP’s catalytic function or its interaction with cellular regulatory factors. For search of RNAP inhibitors we have designed the assay for automated high throughput screening (HTS) of large libraries of chemical compounds. The proposed assay utilizes nucleotide substrate analogs carrying a fluorescent group, which is activated upon release in the polymerization reaction. The assay has been optimized in respect to concentration of the components, reaction conditions and adapted for HTS. The feasibility of the assay has been demonstrated by retrieving of RNAP inhibitors from the collection of 16,000 compounds in HTS setup. The follow-up assays have been developed to detect false positives identified in the primary assay. Experiments are proposed to further increase the assay performance by miniaturization and introduction of the additional follow-up assay to screen out the compounds that target template DNA rather than RNAP. The methodology would be useful for identification of new inhibitors of RNAP suitable for anti-microbial therapy particularly in overcoming the MDR problem. It will also open new approaches to discovery of drugs targeted to specific genes and regulatory pathways. In addition, new inhibitors of RNAP would serve as a potent tool for understanding of the fine mechanisms of RNA synthesis and its regulation. -
Measurement of GPCR-mediated thallium flux through GIRK*
NISWENDER, COLLEEN M
VANDERBILT UNIVERSITY
1 R03 MH076398-01
1
DESCRIPTION (provided by applicant): Pharmacological agents targeting G-protein coupled receptors and ion channels represent promising clinical agents spanning a multitude of human diseases. We have developed a screening strategy for Gi/o-linked receptors by measuring thallium flux through G protein regulated Inwardly Rectifying Potassium (K+) (GIRK) channels. Using this technique in HEK cells stably expressing GIRK 1 and 2 channels, we have generated preliminary data using muscarinic and adrenergic agonists and antagonists and have observed dose dependent regulation of these channels by endogenous M2/M4 receptors and ?2C adrenergic receptor selective agents. We have adapted this assay to a 384 well plate format and have performed initial Z’ experiments that indicate that the assay is amenable to HTS. We propose to conduct a high throughput screen for novel agonists at the ?2C receptor; in the course of these studies, it is anticipated that we will identify novel agonists of GIRK 1/2 channels as well. Very few specific pharmacological tools exist targeting the ?2C receptor and due to its proposed role in psychiatric disorders and normal cardiac function it is anticipated that agonists targeting this receptor would provide critical tools to test ?2C receptor function in vivo. GIRK1/2 channels are known to be involved in neuronal excitability and it has been proposed that agonists for this receptor may provide new therapeutic avenues for epilepsy and pain. Lay summary: G-protein coupled receptors (GPCRs) and ion channels represent accessible therapeutic targets in human disease. We have developed a new assay, amenable to high throughput strategies, which can be used to screen large chemical libraries for compounds acting within the signal transduction pathway from receptor to ion channel. We propose to use this assay to identify compounds interacting with the ?2C adrenergic receptor as well as agents directly modulating the potassium channel employed in our assay system. Identified compounds will provide new pharmacological tools to test the role of the ?2C adrenergic receptor in psychiatric and cardiovascular disorders and the role of GIRK 1/2 channels in the modulation of neuronal excitability. -
Unraveling the Molecular Mechanisms of Phagocytosis
PAUMET, FABIENNE
COLUMBIA UNIVERSITY HEALTH SCIENCES
1 R03 MH076502-01
1
DESCRIPTION (provided by applicant): Phagocytosis is a special form of endocytosis in which large particles such as microorganisms and dead cells are ingested via large endocytic vesicles called phagosomes. In mammals, three classes of white blood cells act as professional phagocytes: macrophages, neutrophils and dendritic cells. These cells defend us against infection by ingesting invading pathogens. Macrophages have also an important role in scavenging senescent cells and cells that have died by apoptosis. In quantitative terms, the scavenging dead cells are by far the most important: our macrophages phagocytose more than 1011 senescent red blood cells in each of us every day, for example. Although multiple cellular elements are known to be involved in phagocytosis, the regulation of this pathway is still obscure. For example, it is unclear which membranes are recruited for the formation of the phagosome and under which conditions each compartment is mobilized. It is also unclear which proteins are involved in the process (in particular the fusion proteins). In this application, we seek to address these questions. By using a cell assay based on fluorescent latex beads, we will investigate 1- which compounds are influencing the internalization of different size particles and 2- which regulatory elements are involved. Understanding the molecular mechanism of phagosome formation and maturation is fundamental for the discovery of new drug targets and the development of new treatments for a wide variety of infectious diseases. The leading re-emerging infectious disease is tuberculosis caused by mycobacterium tuberculosis (and to much lesser extent mycobacterium bovis). M. tuberculosis is one of several pathogens that exploit host cell phagocytic machinery to gain access to the cell interior and to escape immune surveillance. -
Aggregation and Clearance of Mutant Huntingtin
ROTHMAN, JAMES E
COLUMBIA UNIVERSITY HEALTH SCIENCES
1 R03 MH076348-01
1
DESCRIPTION (provided by applicant): Accumulation and aggregation of mutant proteins are a common link across a wide array of neurodegenerative disorders. Recently, an exciting theme has emerged: if mutant protein accumulation is eliminated, symptomatic progression not only halts but also leads to recovery from disease. The first indication that neurodegenerative diseases are reversible comes from an inducible mouse model of the polyglutamine disorder Huntington’s disease (HD). In the presence of expanded polyglutamine huntingtin, mice recapitulated HD-like symptoms. When mutant gene expression was abolished, not only did the aggregates disappear, the symptoms regressed. These findings signal that neurodegenerative diseases need no longer be considered a death sentence. Unfortunately, however, the pathway underlying clearance of these mutant proteins are not yet clear. We believe that screening a well-designed cell-based assay with a chemical compound library will allow us to not only further clarify which degradative pathway is important, but may also reveal new means by which these pathways can be activated. We have therefore designed a functional cell-based assay that monitors not only aggregation of mutant huntingtin protein, but also its clearance. To do so, we created a stable cell line that conditionally expresses the N-terminus of huntingtin protein with polyQ proteins of different polyQ lengths fused to variants of GFP. These cell lines permit high throughput confocal microscopy to examine the state of the expressed mutant protein in live cells. We hypothesize that by eliminating the accumulated protein we will bring about recovery of the neurodegenerative process, as shown in several animal models of the disease. -
Regulation of Neurotransmitter Transporter Recycling
ROTHMAN, JAMES E
COLUMBIA UNIVERSITY HEALTH SCIENCES
1 R03 MH076384-01
1
DESCRIPTION (provided by applicant): Clearance of neurotransmitter from the synaptic cleft, by high affinity transport proteins (neurotransporters), is an important stage in the regulation of neuronal signalling. A major mechanism by which neurotransporters are regulated occurs at the level of subcellular localization. Internalization of neurotransporters away from the cell surface, into an intracellular pool reduces the number of available neurotransporter molecules at the synaptic cleft. Changes that affect the regulation of neurotransporters are important in the progression of a number of neurodegenerative diseases. Here we describe an assay that can be developed into a high throughput screen to identify small molecules (drugs) that affect neurotransporter trafficking. The assay measures changes in the subcellular localization of fluorescently tagged neurotransporters in response to stimuli such as protein kinase C (PKC) signaling. Screening chemical compounds against this assay has the potential to identify small molecule effectors that can be used as tools to pharmacologically manipulation of neurotransmitter clearance. This will aid research into pathological conditions caused by alterations of neurotransporter regulation. In addition, identification of such molecules may yield compounds that can be developed into new therapeutic drugs, or provide information about potential therapeutic targets. -
MLSCN HTS Assays R03 – S1P1
SANNA, M GERMANA
SCRIPPS RESEARCH INSTITUTE
1 R03 MH076534-01
1
DESCRIPTION (provided by applicant): Higher throughput scientific approaches can provide a foundation of enabling data that facilitates rapid and efficient progress in biomedical science. The discovery of chemical probes and proof-of-concept (POC) molecules provides enabling tools that remove a potential limiting factor in the progress of biomedical science and this can be achieved on occasion from high-throughput screening deliverables alone. The key hypothesis surrounding the development of the MLSCN proposal is that high throughput chemical approaches, when integrated with state-of-the art post-genome sequence, cell, molecular and in vivo biology, provides a rapid and facile mechanism for enhancing the progress of biomedical science. With the following proposal we intend to address the following aims: Aim #1: Establish a 96 well assay capable of being implemented in a 384-well format by the MLSCN Aim #2: Transfer the assay conditions and reagents to MLSCN for small molecule screening Aim #3: Provide selectivity screens for count-screening MLSCN hits RATIONALE Screening the NIH library to find POC molecules that act as agonists and antagonists for LPA1 receptor to study receptor function in vitro and the implication in the growth of neuronal cells and ovarian tumor migration. The goals of small molecule screening have been to provide useful chemical tools for the study of biological processes at molecular, cellular and in vivo levels. The success of the present process can be defined as the selection or identification of one or more selective, reasonably high affinity ligands for the proposed target. MATERIAL AND METHODS: Screening will be performed in a 96 or 384 well fluorescence imaging plate reader (FLIPR) format which allow throughput of >10,000 compound per day, screening throughput of 50-100,000 compounds per day can be achieved in homogenous assay formats. -
Human 5HT 1E Serotonin Receptor Drug Development
TEITLER, MILT
ALBANY MEDICAL COLLEGE OF UNION UNIV
1 R03 MH076345-01
1
DESCRIPTION (provided by applicant): The 5HT1E serotonin receptor is one of fourteen serotonin receptors expressed in humans. It is especially highly expressed in human cortical brain tissue, indicating modulation of this receptor by drugs may produce robust effects on cognition and/or mood. There has been surprisingly little information on the inherent function of this receptor, presumably due to the lack of selective drugs to stimulate or antagonize the 5HT1E receptor. In order to advance the field of 5HT1E receptor pharmacology we propose to develop a high throughput screen (NTS) for selective agonists and antagonists for the human 5HT1E receptor. The specific aims of this proposal are to demonstrate that the recombinant cell line possesses the properties that will allow the application of HTS using SPA (Scintillation Proximity Assays) to the development of novel, specific agonists and antagonists for this understudied human brain serotonin receptor. Selective agonists and antagonists for the 5HT1E receptor should, at the least, provide information on the function of this highly expressed receptor, and may lead to novel therapeutics for brain dysfunction. -
Identification of O-Glc NAc Transferase Inhibitors by H*
WALKER, SUZANNE
HARVARD UNIVERSITY
(MEDICAL SCHOOL)
1 R03 MH076518-01
1
DESCRIPTION (provided by applicant): The goal of this project is to implement a developed donor displacement assay for O-GlcNAc transferase (OGT) in high throughput format using the MLSCN. OGT is the sole enzyme that mediates the attachment of O-GlcNAc groups to serine and threonine residues in the nucleus and cytoplasm of eukaryotic cells, and no specific inhibitors are known. This type of glycosylation is involved in signal transduction and plays a key role in many essential cellular processes. High O-GlcNAc levels have been correlated to insulin resistance and type 2 diabetes. In addition to elucidating the role of O-GlcNAc in cellular processes, inhibitors of OGT might be useful in studying and perhaps treating type 2 diabetes. -
High Throughput Screen for Mycobacterium Tuberculosis P*
WHITE, E. LUCILE
SOUTHERN RESEARCH INSTITUTE
1 R03 MH076412-01
1
DESCRIPTION (provided by applicant): It is estimated that one third of the world’s population is infected with tuberculosis (TB), with about 8 million new cases annually. Multi-drug resistant TB, defined as forms resistant to two or more of the front line anti-TB agents, has become an increasing health problem, especially in HIV infected individuals. These forms of TB are more often fatal and more difficult to treat. Consequently, development of molecular research tools or drugs targeting novel pathways has become increasingly important. The biosynthetic pathway of pantothenate is such a target. Pantothenate is an essential precursor in mycobacteria for the synthesis of co-enzyme A and acyl carrier protein which are involved in fatty acid synthesis. Pantothenate synthetase, encoded by the panC gene, catalyzes the formation of pantothenate in bacteria. M. tuberculosis pantothenate synthetase has been characterized enzymatically and a high resolution crystal structure has also been solved. More recently several compounds have been identified which inhibit this enzyme. Unfortunately, none of these compounds affect the growth of Mycobacterium tuberculosis in vitro. To address the need for molecular research tools to study the pantothenate pathway in M. tuberculosis the specific aims of this proposal are: 1) To transfer the existing HTS assay for pantothenate synthetase to the Molecular Libraries Screening Center Network. . 2) To follow up hits from the HTS assay in a series of confirmatory assays available through the TAACF that would evaluate their in vitro and in vivo efficacy against M. tuberculosis. 3) To study the effect of these novel inhibitors on the pantothenate pathway in M. smegmatis. 4) To co-crystallize these compounds with pantothenate synthetase to study their mode of binding -
HTS Assay for Inhibitors of BAP1, a BRCA1 Associated De*
WILKINSON, KEITH D
EMORY UNIVERSITY
1 R03 MH076382-01
1
DESCRIPTION (provided by applicant): Individuals who carry mutations in the breast cancer susceptibility gene, BRCA1, are predisposed to early onset breast and ovarian cancer, some of the most common malignancies in Western societies. Such mutations in BRCA1 account for almost all families with inherited breast and ovarian cancer and for approximately half of families with breast cancer only. The detection of loss-of-heterozygosity (LOH) affecting the wild-type BRCA1 allele in tumors from BRCA1 carriers implies that BRCA1 is a tumor suppressor. The involvement of BRCA1 in breast cancer is complex; to date, more than 100 unique, naturally occurring BRCA1 germline mutations have been identified. The study of BRCA1 has important implications for breast cancer research and attempts to elucidate its biochemical function have included identifying its protein partners, such as BAP1. BAP1 is a member of the UCH family of deubiquitinating enzymes (DUB). These are proteases that reverse the conjugation of ubiquitin to proteins targeted for degradation by the proteasome or relocalization in response to ubiquitination. The conjugation of ubiquitin has been shown to be important in control of many regulatory pathways including; cell cycle regulation, chromatin structure, DNA repair and genome stability, transcription, viral pathogenesis, immune response, and protein quality control. Deubiquitinating enzymes are likely to be useful drug targets in at least some pathological conditions. With the exception of neuronal UCH-L1, no DUB has been the target of a systematic screen. We have developed the means to produce significant amounts of a generic DUB substrate at affordable costs and have chosen to focus our efforts first on a DUB associated with the BRCA1 tumor suppressor. We have begun to optimize the screening conditions and have shown that the assay is adaptable to the 384 well format. We will mount a conventional high throughput drug screen of available NIH compound libraries to identify inhibitors and activators of DUB action. This screen will result in useful molecular probes of BAP1 function and help to clarify the role of BAP1 in BRCA1 mediated events. -
HTS for Inhibitors of Schistosoma Mansoni Peroxiredoxins
WILLIAMS, DAVID L
ILLINOIS STATE UNIVERSITY
1 R03 MH076449-01
1
DESCRIPTION (provided by applicant): Schistosomiasis is an important, debilitating disease affecting ~250 million people in more than 70 countries. The annual mortality of this disease is estimated to be ~280,000 in sub-Saharan Africa, while 20 million individuals suffer from extreme disability. In the coming years the Bill and Melinda Gates Foundation and endemic country programs will treat tens of millions of people with the single anti-schistosomiasis drug in widespread use, praziquantel. There is already clinical and laboratory evidence for the existence of praziquantel resistance parasites and widespread use is expected to generate strong selective pressure for drug resistance. Clearly, there is an urgent need for new anti-schistosome drugs. Studies on schistosome redox balance mechanisms indicate a distinct and compressed pathway in the parasite compared to its human host. Schistosoma mansoni peroxiredoxins (Prx) are important parasite antioxidant proteins that play a crucial role in redox balance mechanisms. Our data strongly suggest the possible use of Prx as novel drug targets. First, the proteins are essential for the parasite survival. Second, the proteins exhibits sufficient biochemical and structural differences from host Prx proteins. Third, the protein(s) is amenable for study at the molecular level and can be produced in bacteria in large quantities, in soluble and active form. Moreover, Prx activity can be screened in high throughput, inexpensive, and sensitive assays. Since no parasite-specific inhibitors of Prx are currently available, our overall goal is the identification of inhibitors of Schistosoma mansoni Prx by conducting a high throughput screen of the Small Molecule Repository of the Molecular Libraries Screening Centers Network (MLSCN). This will be the first step in the development of novel antischistosome chemotherapies, which are essential for continued public health measures. -
Small-Molecule Modulators of a Family of Voltage-Depend*
ZHOU, MING
COLUMBIA UNIVERSITY HEALTH SCIENCES
1 R03 MH076402-01
1
DESCRIPTION (provided by applicant): Voltage-dependent potassium channels (Kv) are integral membrane proteins that, in response to membrane voltage changes, catalyze potassium ions to diffuse across the cell membrane. Kv channels regulate membrane excitability and are essential to many physiological processes, such as the rhythmic beating of heart, the communication between neurons, and the secretion of hormones. In a cell, Kv channels are always assembled with other proteins, and channel function is regulated by cellular environment through these associated proteins. The beta subunit (Kvbeta), a potential aldo-keto reductase (AKR), attaches to the intracellular side of the Kv1 family channels. They are ubiquitous in the brain and abundantly expressed in heart, and have been implicated in control of cell excitability and in sensing cellular redox state. The long-term goal of this research is to understand the role of Kvbeta in cell biology. We have recently identified two Kvbeta substrates and for the first time demonstrated that Kvbeta is a functional AKR. We also found that the substrates, when perfused to the intracellular side of the channel, modulate channel functions only when Kvbeta is co-expressed. These exciting new results led us to hypothesize that Kv1 channel functions are coupled to Kvbeta enzymatic activities. In this proposal we focus on screening for Kvbeta substrates and we propose the following two specific aims: Specific Aim 1. To identify small-molecule substrates of Kvbeta by a high throughput screen (the primary assay). We will use recombinant purified Kvbeta protein to screen for substrates in an enzymatic assay that follows the consumption of NADPH. Specific Aim 2. To test in an electrophysiological assay the positive hits from the screen (the secondary assay). We will test positive hits from Aim 1 using an electrophysiological assay that monitors channel functional change. Small-molecule substrates of Kvbeta will be used as a tool in our combined approaches of biochemistry, Xray crystallography, and electrophysiology to dissect molecular details of the coupling mechanism. The substrates will also provide a reasonable starting point for developing a new class K channel modulators. -
Discovery of novel allosteric modulators of the M1 muscarinic receptor
CONN, P JEFFREY, PHD
VANDERBILT UNIVERSITY
1 X01 MH077606-01
2
DESCRIPTION (provided by applicant): Evidence suggests that the antipsychotic effects of cholinergic agents may be mediated by the M1 or M4 subtype of muscarinic receptor. Previous compounds developed to selectively activate the M1 receptor have failed in clinical development due to a lack of true specificity for M1 and adverse effects associated with activation of other mAChR subtypes. Furthermore, the lack of highly selective compounds has made it impossible to definitively determine whether the behavioral and clinical effects of these compounds are mediated by M1 and M4 is also a viable candidate for mediating the antipsychotic effects. Previous attempts to develop agonists and antagonists that are highly selective for M1 or other specific mAChR subtypes have failed because of the high conservation of the ACh binding site and difficulty in developing compounds that are truly specific. Novel compounds have now been discovered that act at an allosteric site on M1 receptor rather than the orthosteric ACh-binding site to induce a robust activation of the receptor and provide high receptor subtype specificity. We have been highly successful in the use of high throughput screening technologies for discovery of novel allosteric ligands at multiple other GPCR subtypes. We have now developed a highly sensitive assay for the M1 muscarinic receptor that is suitable for high throughput screening of small molecule libraries for discovery of novel agonists and antagonists of this important GPCR. We propose a series of studies in which an M1 expressing cell line will be used by the MLSCN screening network to identify novel small molecules that act as agonists or antagonists at the M1 muscarinic receptor. Both agonists and antagonists can be identified in a single screen. We will then perform rigorous secondary assays to identify compounds that act at sites other than the orthosteric ACh binding site. Finally, we will use database mining and medicinal chemistry approaches to optimize selected compounds for use as laboratory reagents. Lay summary: The M1 muscarinic receptor is thought to be an important therapeutic target in schizophrenia. We have developed an assay system for high throughput screening to identify compounds with high selectivity for the M1 receptor subtype that act at an allosteric site on the receptor, thus providing increased specificity for this single receptor subtype. It is anticipated that these compounds will provide important tools for the study of muscarinic receptor function in the CNS. -
In Vivo Angiogenesis Assay for HTS
DOAN, THANH N
ZYGOGEN, LLC
1 X01 MH077629-01
2
DESCRIPTION (provided by applicant): The purpose of this proposal is to provide an in vivo zebrafish angiogenesis assay to be used by MLSCN. Identification of angiogenic compounds via use of this assay will potentially provide drugs to combat cancer and cardiovascular diseases. The integration of the zebrafish angiogenesis assay will expedite and reduce the attrition of the drug discovery process by providing a novel quantitative, in vivo tool to screen compounds in a high throughput manner. -
Chemical Genetics Screens to Identify Modulators of Post-Golgi Transport
HARSAY, EDINA
UNIVERSITY OF KANSAS LAWRENCE
1 X01 MH077628-01
2
DESCRIPTION (provided by applicant): Membrane vesicle and tubule-mediated intracellular transport of proteins and lipids is an essential process in all eukaryotic cells. The regulation of membrane transport pathways is required for the control of cell growth and division as well as for maintaining normal cell function and homeostasis of non-dividing cells. The long-term goal of the Harsay laboratory is to define the molecular mechanisms by which cargo is transported from the Golgi to the cell surface, so that improved therapeutic strategies to treat diseases involving transport dysfunction can be developed. Many of the components of the transport machinery have been described for several transport steps and shown to be highly conserved among eukaryotes. However, the complexity and branching of the late, post-Golgi, exocytic pathway has made it difficult to identify proteins that function at late transport steps, and the molecular machinery required for exocytic cargo sorting and exit from the Golgi is still largely unknown. The objective of this application is to identify components of the post-Golgi transport machinery by using a chemical genetic approach. The strategy involves a mutant yeast strain having a block in one branch of the post-Golgi exocytic pathway. By using this strain in high-throughput, cell-based phenotypic screens of diverse compound libraries, it is expected that compounds that target specifically regulators or structural components of the post-Golgi transport machinery will be discovered. Identifying compound targets and target interactors, as well as using biologically active small molecules as tools in the molecular dissection of membrane and protein traffic steps, will result in a better understanding of late secretory transport mechanisms and of the diseases that involve perturbations of these mechanisms. Such diseases include diabetes, neurological and immune disorders, and cancer. Furthermore, many intracellular pathogens exploit host cell transport mechanisms for their survival and proliferation. Therefore, the knowledge and tools gained from this work will allow better understanding of the etiology and pathophysiology of many human diseases, as well as ultimately suggest strategies for therapeutic interventions to prevent and treat such diseases. -
Ligands for premortem diagnosis and treatment of Alzheimer’s disease
KURET, JEFF A, PHD
OHIO STATE UNIVERSITY
1 X01 MH077621-01
2
DESCRIPTION (provided by applicant): Alzheimer’s disease is a dementing illness of the elderly defined by the appearance of characteristic lesions in the brain. It differs from other dementias in the protein composition of the lesions and by their hierarchical emergence in distinct cortical and subcortical regions. For these reasons, their spatial and temporal distributions facilitate differential diagnosis and staging of disease. Neurofibrillary lesions have special utility in this regard. They are composed of tau, a microtubule-associated protein that normally functions to promote tubulin assembly, microtubule stability, and cytoskeletal integrity. Tau that accumulates in neurofibrillary lesions differs from microtubule-associated protein in its state of aggregation. Moreover, fibrillization is accompanied by conformational changes in tau protomers that can be sensed by selective monoclonal antibodies and by small molecules such as thioflavin dyes. These data suggest that tau-bearing lesions present novel binding sites that can be detected with small molecule probes, and potentially exploited to develop ligands capable of selectively detecting their presence. Such agents could have practical usage as contrast agents capable of staging disease pre-mortem, whereas ligands capable of antagonizing aggregation could potentially emerge as therapeutic agents. The present proposal has one Specific Aim to test this hypothesis. Amyloidgenic conformations of tau protein will be subjected to high throughput screening in the Molecular Libraries Screening Centers Network. Active compounds will be subjected to secondary screens capable of quantifying binding affinity and selectivity for tau relative to other aggregating proteins. The ability of selected ligands to inhibit tau aggregation also will be assessed. Successful completion of this project will yield pharmacological agents with the potential to facilitate dissection of the aggregation pathway in biological systems, to clarify the role of filament formation in disease, and to drive early stage diagnosis of Alzheimer’s disease. -
A Screen for Novel Antimicrobials
LEWIS, KIM
NORTHEASTERN UNIVERSITY
1 X01 MH077622-01
2
DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a novel method for antimicrobial drug discovery, based on a targeted search for prodrugs. The potential advantage of prodrugs is considerable – being converted inside a microbial cell into a non-specific active molecule, such compounds will have the capability to “sterilize” an infection, something that currently available antibiotics lack. There is a considerable unmet need for any type of novel antibiotics, due to the rise of multidrug resistant pathogens, and the threat of engineered multidrug resistant bioweapons. The unmet need for compounds that can act against slow-growing or dormant pathogens, and persisting biofilms, is even greater. The rationale of the method is to screen for compounds against strains differentially expressing an essential enzyme potentially capable of converting a prodrug into an antimicrobial. A strain overproducing a converting enzyme will be more susceptible to a prodrug. A combination of genomics with HTP screening makes this straightforward method realistic. Genomics provides us with a list of good candidate enzymes that can potentially convert prodrugs into drugs, and a rational screening design will enable efficient identification and validation of hits. We validated this approach with E. coli overexpressing NfnB, the converting enzyme for metronidazole. The overexpression strain showed >100 fold greater sensitivity as compared to the wild type control (Preliminary Studies). Importantly, the population was completely sterilized, the first observation of this kind with an antibiotic. Lead compounds resulting from this screen will sterilize an infection, and since the converting enzyme is an essential protein, the probability of resistant mutations will be low, limited to rare modifications of the enzyme that specifically exclude the prodrug without affecting the natural substrate. We expect several outcomes from this project, both short- and long-term: (1) The proposed screen will provide proof-of-principle for a targeted search for prodrugs. (2) Validated hits we identify will become leads for antimicrobial drug development. (3) The prodrug screen will be applied to yeast in search of antifungal compounds. (4) A direct prodrug screen for anticancer agents using mammalian cells can be developed as well, based on the same general design that we will use in this project. Specific Aims: 1. A compound library will be screened to identify prodrug hits. This will be achieved in two steps: a. The original library of 500,000 compounds will be screened for direct activity against E. coli. This will be performed in a standard HTP format, detecting inhibition of growth activity by changes in OD. This will produce an active sub-library of ~10,000 compounds. b. Prodrug candidates will be identified from the sub-library by a screen against 50 individual E. coli strains each overexpressing a gene coding for a particular essential enzyme. 2. Validated prodrug candidates will be identified in a secondary screen. Hits will be tested against strains with a decreased expression of enzyme of interest. The activity of a hit compound in such a cell will be decreased, confirming its prodrug nature. -
High Throughput Screening of Select Orphan Nuclear Receptors
LI, XIAOLING, PHD
ORPHAGEN PHARMACEUTICALS
1 X01 MH077624-01
2
DESCRIPTION (provided by applicant): Nuclear receptors are excellent targets for drug design. Orphagen has developed a 384-well assay for three orphan nuclear receptors that have not yet been characterized at a pharmacological level. Basic biological studies suggest that these targets have therapeutic potential in treatment of obesity, prostate cancer, metabolic disease (including type 2 diabetes), and sleep disorders. X-ray crystallography shows that the three receptors have small molecule binding pockets typical of the nuclear receptor family. To explore their potential as novel drug targets, however, small molecule ligands must be synthesized that are bioavailable and pharmacologically active in animal models of disease. A critical first step is the identification and confirmation of hits in high throughput screening (HTS) of diverse small molecule libraries. We propose screening of the three receptors in parallel in a cell-based assay for receptor-mediated gene transcriptional activity. Because the shapes of the small molecule binding pockets for each of the three receptors are substantially different, spurious hits (1-2% of most libraries) are easily identified since they affect all three receptors in the same way, whereas candidate hits will regulate one of the three receptors selectively. These candidate hits are confirmed in dose responsive transcriptional assays. In follow-on studies supported by current SBIR grant funding to Orphagen from the NIH, a secondary cell-free assay that measures compound binding to each of the purified receptors will be developed to substantiate ligand interaction. The characterized receptor assays, along with proven agonist or antagonist ligands, will provide a solid foundation for new drug discovery. Relevance to Public Health: Unexplored drug targets, such as the orphan nuclear receptors, are sources of potential new drug classes for entirely new approaches to chronic disease. Interest in these targets by the pharmaceutical industry is low since they are at the very beginning of the drug discovery pipeline. Therapeutic areas that may be addressed by the studies described here are: type 2 diabetes, obesity, insomnia and other sleep disorders, and prostate cancer. The proposed studies, if successful, will provide workable and proven drug discovery technology for these targets where none exists today. -
Conducting a HTS against a Multi-protein DNA Replication System
MCHENRY, CHARLES S, PHD
UNIVERSITY OF COLORADO DENVER/HSC AURORA
1 X01 MH077636-01
2
DESCRIPTION (provided by applicant): High throughput screens for the replication elongation systems of important pathogens have been developed initially by the McHenry Laboratory in collaboration with Replidyne, a biotechnology company founded by Charles McHenry. Garry Dallmann was also employed at Replidyne as Director of Lead Discovery for a period of 2.5 years. This collaboration enabled significant successes in assay development and finding a plethora of promising hits and early leads, but little has emerged that adds broadly to the scientific community’s knowledge of DNA replication, general principles of drug design against DNA replication targets, or reagents useful for ‘chemical genetics’ approaches to study of DNA replication and coupled processes. This is largely due to a process characterized by weeding out compounds not suited, with other than minimal modification, for human use. This process was perhaps necessary for meeting Replidyne’s short-term objectives, but as a result, many valuable compounds were passed over with no substantial characterization, depriving the scientific community at large of knowledge of potential drug target sites or reagents useful as research tools. Due to Replidyne policies pertaining to the protection of ‘intellectual property,’ these compounds remain unavailable to the McHenry laboratory and researchers outside of the company. In spite of significant success with the replication program, all compounds have been set aside by the company, and Replidyne’s resources are being devoted to late stage in-licensed assets (disclosed on the company’s web site). To position the McHenry laboratory to serve broader scientific and human health interests, Dr. McHenry and Dr. Dallmann have ended their relationships with Replidyne in all capacities (i.e. as an employee, officer or director or consultant). Because the resources of Replidyne are not available to the general scientific community, some efforts within this application duplicate activities that have already taken place within the company. This is necessary though, to reach a place where the technology can be both extended and applied in a manner that advances our communal scientific knowledge. -
Screening of compounds that modulate insulin promoter activity in TRM-6 cells
MERCOLA, MARK
BURNHAM INSTITUTE
1 X01 MH077630-01
2
DESCRIPTION (provided by applicant): The assay being proposed here will screen for small molecule compounds that modulate insulin promoter activity. The assay is based upon a human pancreatic endocrine cell line, TRM-6, that was derived from fetal islets. To develop these cells as the basis for a high throughput assay, they were engineered to express an insulin promoter-fluorescent reporter protein transgene using a lentiviral vector. In addition, the cell line has been engineered to express a panel of transcription factors that together stimulate insulin gene activity; thus, the cells express substantial levels of endogenous insulin mRNA but less than produced by a healthy pancreatic a-cell. Thus, although broadly designed to detect compounds that affect Insulin gene expression, either positively or negatively, the screen is specifically engineered to increase the likelihood of detecting compounds that complement the activities of the known transcription factor inputs controlling insulin production. The secondary assay used to verify hits will be evaluation of the biological effect on the endogenous insulin gene. Preliminary Studies indicate that the insulin promoter-fluorescent reporter transgene mimics the activity of the endogenous insulin gene. Optimization of the assay characteristics, including the cell seeding density and tamoxifen concentration yielded a z’ of 0.6. A small scale screen of 8,000 compounds from the ChemBridge DiverSet library identified multiple compounds that induced and repressed the insulin promoter-GFP transgene. A primary confirmatory assay yielded a true positive rate of approximately 50% of those scored initially as hits. Using the secondary assay of quantitative insulin RT-PCR found that these confirmed hits had significant effects on endogenous insulin promoter activity. Discovering compounds that modulate the insulin promoter has the potential to identify signaling pathways that are involved in the establishment and maintenance of mature a-cells. -
Screening of compounds that modulate insulin promoter activity in MIN-6 cells
MERCOLA, MARK
BURNHAM INSTITUTE
1 X01 MH077631-01
2
DESCRIPTION (provided by applicant): We propose a screen for small molecule compounds that modulate insulin promoter activity. The assay is based upon a mouse insulinoma, MIN6, that normally expresses insulin mRNA. This cell was engineered to stably contain two cassettes that use a fluorescent reporter protein to monitor insulin promoter activity and a housekeeping gene activity. The secondary assay to be used to confirm the hits will be to verify modulation of endogenous insulin mRNA expression relative to control mRNAs. Like all cultured insulin-producing cells, MIN6 cells produce substantially less insulin mRNA and protein than do normal a-cells in the intact pancreas. Thus, the hypothesis is that it should be feasible to identify compounds that both stimulate as well as suppress insulin production. The small molecule modulators of insulin mRNA synthesis should be useful tools to probe the regulatory pathways that control insulin secretion. Knowledge of the pathways and means to modulate them are expected to lead to a knowledge base that will be applied to the treatment of type I and II diabetes. Preliminary studies indicate that the Insulin promoter-eGFP reporter transgene mimics the activity of the endogenous insulin gene. Assay parameters have been optimized, consolidated into a standard operating procedure and used to perform a pilot screen of 8,000 compound subset of the ChemBridge DiverSet collection. Hits that increased and decreased insulin gene expression were identified and confirmed. These hits were used to calculate a z’ value of 0.74 for increase and 0.43 for decrease in eGFP. -
Pharmacological inhibitors of tissue-nonspecific alkaline phosphatase (TNAP)
MILLAN, JOSE LUIS, PHD
BURNHAM INSTITUTE
1 X01 MH077602-01
2
DESCRIPTION (provided by applicant): The mechanisms that regulate tissue calcification are of major importance, as they ensure that calcification of the skeleton proceeds normally while mineralization is prevented elsewhere in the body. Alterations in these regulatory mechanisms, either due to genetic defects or as a result of aging, lead to disease, such as osteoarthritis and arterial calcification. Inorganic pyrophosphate (PPi) is a potent inhibitor of calcification and three molecules have been identified as central regulators of mineralization via their ability to control the pool of extracellular PPi, i.e., PPi is generated outside of the cells by the enzymatic action of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) and it is also transported from the inside to the outside of the cells by the ankylosis protein (ANK). In turn, extracellular PPi is degraded by the enzymatic action of tissue-nonspecific alkaline phosphatase (TNAP). Genetic experiments in mice have revealed that the functional deletion of the NPP1 or the ANK gene lead to very similar disease states that include osteoarthritis, fusion of the ligaments of the spine and arterial calcification. The simultaneous inactivation of the TNAP gene in NPP1-deficient and ANKdeficient mice leads to normalization of extracellular PPi levels, and correction of calcification abnormalities in these mice. The central hypothesis underlying this research proposal is that the pharmacological ablation of TNAP function will lead to an increase in the concentrations of extracellular PPi that will result in amelioration/prevention of arterial calcification. Since arterial calcification is a condition associated with the development of atherosclerosis, we will test this hypothesis using the NPP1-deficient model of osteoarthritis and arterial calcification described above, but also the Apolipoprotein E-deficient mouse model that mimics the development of atherosclerotic plaques as seen in humans. As a first step toward targeting TNAP therapeutically, we have optimized a microtiter plate enzymatic assay using pnitrophenylphosphate as substrate and measuring liberated p-nitrophenol as product, confirming suitable assay performance for the high-throughput environment. We have screened libraries containing 53,080 compounds and succeeded in identifying small molecule, drug-like lead compounds that can be further modified to obtain highly specific TNAP inhibitors for in vivo use. Initial hits from this screen were counter-screened using very similar assay condition but using PPi as substrate and using the Biomol reagent to detect liberated phosphate as product. Subsequent tests will also be run using both assay designs to select those compounds that inhibit TNAP but not other related human phosphatases or NPP1. We propose to use both of these already optimized assays for screening the NIH’s chemical library. We will use a combination of computer modeling, docking experiments and chemical synthesis to further modify the HTS hits to design novel, improved inhibitors of TNAP activity for use as in vivo therapeutics and to test these novel TNAP inhibitors in vivo for their ability to ameliorate/prevent arterial calcification in our animal models of arterial calcification and atherosclerosis. -
HTS for HePTP Inhibitors-A Leukemia Target
MUSTELIN, TOMAS M
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 X01 MH077603-01
2
DESCRIPTION (provided by applicant): HePTP is a tyrosine phosphatase expressed in hematopoietic cells and regulates the MAP kinases Erk and p38. HePTP has been found to be overexpressed in leukemic cells and in myelodysplastic syndrome, a preleukemic disorder. This Molecular Libraries Screening Centers Network (MLSCN) Resource Access Award application seeks to have our screen-ready colorimetric 96-well assay for the hematopoietic tyrosine phosphatase (HePTP) used for high-throughput screening (HTS) to identify hits that will be further developed into potent and specific inhibitors of HePTP. These inhibitors will primarily be used for basic research into the mechanisms of signal transduction and MAP kinase regulation, but may also become useful for treatment of hematopoietic malignancies (e.g. acute myeloid leukemia) where HePTP has been reported to be overexpressed. -
HTS for LYP Inhibitors-An Autoimmunity Target
MUSTELIN, TOMAS M
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 X01 MH077604-01
2
DESCRIPTION (provided by applicant): The goal of the present application is to submit our screen-ready, 96-well, colorimetric phosphatase assay and the needed 16.7 mg of high-quality LYP protein for HTS by the MLSCN. We would prefer the MLSCN screening facility at The Burnham Institute, because we have carried out HTS here before, but we believe that any MLSCN facility could carry out this very simple screen. We anticipate a hit rate of 0.02-0.1 % and are prepared to analyze, counter screen, and further optimize these hits into specific and selective inhibitors of LYP. These inhibitors will be used for studies of LYP in the regulation of Src and Syk kinases, T cell activation, and will be used to explore whether LYP inhibitors will be useful for treatment of autoimmune disease associated with the LYP*W620 allele. Our work on LYP is supported by grant R01 AI53585, which is active until the end of 2007. This grant does not include funds for drug development, but if the HTS by the MLSCN is successful, we will focus the competitive renewal of R01 AI53585 on this task. -
Discovery of novel allosteric agonists of the M4 muscarinic receptor
NISWENDER, COLLEEN M, PHD
VANDERBILT UNIVERSITY
1 X01 MH077607-01
2
DESCRIPTION (provided by applicant): Evidence suggests that the antipsychotic effects of cholinergic agents may be mediated by the M1 or M4 subtype of muscarinic receptor. The lack of highly selective compounds, however, has made it impossible to definitively determine whether the behavioral and clinical effects of muscarinic agonists are mediated by M1 or M4. Previous attempts to develop agonists and antagonists that are highly selective for specific mAChR subtypes have failed because of the high conservation of the ACh binding site and difficulty in developing compounds that are truly specific. Novel compounds have now been discovered that act at an allosteric site on the M1 receptor rather than the orthosteric ACh-binding site to induce a robust activation of the receptor and provide high receptor subtype specificity [3, 4]. We have been highly successful in the use of high throughput screening technologies for discovery of novel allosteric ligands at multiple other GPCR subtypes. We have now developed a highly sensitive assay for the M4 muscarinic receptor that is suitable for high throughput screening of small molecule libraries for discovery of novel and specific allosteric agonists of this important GPCR. We propose a series of studies in which an M4 expressing cell line will be used by the MLSCN screening network to identify novel agonists. We will then perform rigorous secondary assays to identify compounds that act at sites other than the orthosteric ACh binding site. Finally, we will use database mining and medicinal chemistry approaches to optimize selected compounds for use as laboratory reagents. Lay summary: The M4 muscarinic receptor is postulated to be an important therapeutic target in schizophrenia. We have developed an assay system for high throughput screening to identify compounds with high selectivity for the M4 receptor subtype that act at an allosteric site on the receptor, thus providing increased specificity for this single receptor subtype. It is anticipated that these compounds will provide important tools for the study of muscarinic receptor function in the CNS. -
HTS assays for inhibitors of HIV RNase H
PARNIAK, MICHAEL A, PHD
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
1 X01 MH077605-01
2
DESCRIPTION (provided by applicant): The rapid development of HIV-1 resistance to antiretroviral agents is a major clinical problem. This troubling phenomenon has been observed with each class of current clinically used anti-HIV agents. An increasingly serious clinical problem is the emergence of multidrug resistant variants of HIV-1 that show varying levels of resistance to many of the experimental drugs in the preclinical pipeline, since these analogs are directed at the same viral target as those agents to which the virus has developed resistance. There is therefore an urgent need to identify and validate new viral targets for drug discovery. HIV-1 reverse transcriptase associated ribonuclease H (RT-RNH) is one such target. Very few inhibitors of RT-RNH have been described, and those identified to date lack the drug like characteristics needed for therapeutic utility. One of the problems with RT-RNH as a target for drug discovery has been the lack of a suitable high-throughput screening (HTS) assay. We have developed a fluorescence resonance energy transfer assay readily applicable to 96-well and 384-well microplate formats with robotic manipulation to enable high-throughput screening for inhibitors of HIV-1 RT-RNH. The substrate is an 18 nucleotide 3′-fluorescein labeled RNA annealed to a complementary 18 nucleotide 5′-DABCYL modified DNA. The intact duplex has an extremely low background fluorescent signal and provides up to 50-fold fluorescent signal enhancement following hydrolysis. The size and sequence of the duplex are such that RT-RNH cuts the RNA strand four nucleotides from the 3′-end. The labeled tetraribonucleotide readily dissociates from the complementary DNA at ambient temperature with immediate generation of a fluorescent signal. The assay is rapid, inexpensive and robust, providing Z’ factors of 0.8 and coefficients of variation of about 5%. The assay requires only two addition steps with no washing and is thus suitable for robotic operation. Several chemical libraries totaling more than 106,000 compounds were screened with this assay in approximately one month. Hit rates average between 0.1 – 1%. We hope that by using our HTS assay in the MLSCN suitable drug like leads may be identified and optimized. -
Colorimetric assay for HTS discovery of chemical inhibitors of EphA4 receptor antagonists
Pasquale, Elena
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 X01 MH077609-01
2
DESCRIPTION (provided by applicant): Damaged neuronal connections in the adult central nervous system (CNS) do not regenerate. This is due at least in part to the physical barrier formed by glial cells that respond by proliferating and forming a scar and to the presence of inhibitory molecules in the CNS environment. Recent work has shown that the EphA4 receptor tyrosine kinase plays a critical role in the inhibition of axon regeneration that occurs after spinal cord injury. Remarkably, axons in EphA4 knockout mice can regenerate past the site of injury and re-establish severed connections resulting in functional recovery. Other evidence suggests that EphA4 plays an inhibitory role in axonal and dendritic growth in other regions of the central nervous system as well. Furthermore, EphA4 has been implicated in the maintenance of platelet aggregation during thrombus formation and in prostate cancer cell growth. Thus, inhibiting EphA4 function is a very promising new approach with high potential for a number of therapeutic applications. However, EphA4 has not yet been exploited as a target for small molecules. The signaling activity of EphA4 is stimulated by binding several membrane-anchored ligands, called ephrins. As we have recently shown, peptides that antagonize ephrin binding block the physiological activity of the receptor. Furthermore, a pilot high throughput screen performed in the Chemical Library Screening Facility of our Institute on 10,000 compounds has demonstrated the feasibility of screening for small molecules that inhibit ligand binding to EphA4. In this application, we propose to perform a full scale high throughput screen for small molecule EphA4 antagonists and to characterize the potency and selectivity of the active compounds identified. We propose to use an assay protocol similar to the one that was previously successful in our pilot screen. In this assay, we will measure the ability of small molecules to inhibit the binding of an ephrin-A5 alkaline phosphatase fusion protein to the EphA4 extracellular domain immobilized through a carboxy-terminal hexahistidine tag. An advantage of reagents that target the extracellular domain of EphA4 is that they can be highly selective, unlike most tyrosine kinase domain inhibitors, and that they can act without having to penetrate inside the cell. -
High-Through Screen for a Therapeutic Agent for eIF2B-Related Disorders
PAVITT, GRAHAM
Manchester
1 X01 MH077608-01
2
DESCRIPTION (provided by applicant): ‘eIF2B-related disorders’ are genetically inherited diseases caused by mutations in the general translation initiation factor eIF2B. Although eIF2B functions in all cells, affected individuals have tissue-specific disease. The most serious disease is a dysfunction in brain myelin. Progressive demyelination causes severe disability. The impairment leads to an increasingly poor quality of life and early death for affected individuals. ‘Classical’ onset is in children aged 2-5, but severe forms of disease present from as early as birth and milder forms do not present until early adulthood. Milder forms are also associated with germ cell defects. There is currently no therapy or cure available. Experimental work suggests that disease results from a moderate impairment of eIF2B function in many if not all cell types. As control of eIF2B function is important for cellular stress responses, reduced eIF2B function correlates with the observed stress-induced (fever, trauma) deterioration/ degeneration. In theory a chemical intervention that can boost the remaining function of eIF2B should be able to prevent or slow further decline in affected individuals. Alternatively, a compound that is not directly beneficial to patients may also be a useful research tool to probe eIF2B structure and function. As eIF2B is a universally conserved protein among eukaryotic organisms, we have developed a cellular assay system using yeast cells, Saccharomyces cerevisiae. Assays applicable to high throughput screening have been optimized. The first assay is a simple growth assay, where absorbance increase is measured. eIF2B mutations cause cells to grow at a slower rate than normal cells. Compounds that rescue this defect are sort. This assay was validated using a small library of 2000 compounds. The second assay is a reporter gene assay using a luminescent substrate. GCN4 expression is inversely correlated with eIF2B activity in cells. Consequently cells bearing eIF2B mutations have aberrantly high GCN4 expression. Compounds that can correct (lower) this are sort. A program of post-screening analysis of mutations in a variety of human, mammal and yeast model systems is outlined. Post-screening studies involve expertise within collaborating laboratories. Lay Summary-relevance for Human Health: Leukodystrophies are degenerative brain disorders that affect children of all racial groups, including Native Americans. Affected individuals have increasingly poor life quality and die prematurely. Recent work has discovered that one common cause of leukodystrophy is genetically inherited mutations of a protein called eIF2B. eIF2B is known to be an important protein involved in normal growth and development. A reduction in eIF2B function rather than a loss of its function is responsible for the disease. There is currently no therapy or cure available for eIF2B related leukodystrophies. We have developed cell based assays that we wish to employ to screen a large library of compounds, to determine if any may be useful therapeutic agents or research tools for this disease. The aim is to identify a chemical that may specifically boost the remaining activity of eIF2B. If successful in cellular models of the disease this may ultimately result in the slowing or halting of disease progression. -
Identification of Molecular Probes That Reverse MRP-Mediated Drug Resistance
PIAZZA, GARY A
SOUTHERN RESEARCH INSTITUTE
1 X01 MH077620-01
2
DESCRIPTION (provided by applicant): Drug resistance, whether intrinsic or acquired, is a major clinical obstacle that contributes to the marginal efficacy of cancer chemotherapy. Multi-drug resistance (MDR) is a phenomenon by which tumor cells display or develop resistance to a number of structurally and functionally distinct anticancer drugs. A significant factor that contributes to MDR is the overexpression of certain ATP-dependent transporter proteins in tumor cell membranes that cause the efflux of cytotoxic drugs; thereby reducing their intracellular concentration and limiting their effectiveness to inhibit tumor cell proliferation and induce apoptosis. The most well characterized transport proteins responsible for MDR are the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). Inhibition of membrane transporters is an attractive therapeutic strategy to enhance chemotherapy efficacy with minimal additional toxicity. P-gp has been well studied for the past two decades and a number of inhibitors have been evaluated in clinical trials. However, none have received FDA approval due mostly to excessive toxicity when combined with chemotherapy, which is believed to be caused by the protection of normal cells and interference with drug metabolism and elimination. By comparison, less is known regarding the binding and transport properties of MRP and few inhibitors have been identified. Nonetheless, recent studies suggest that it is feasible to reverse MDR by inhibiting MRP without causing cytotoxicity. Given the potential to enhance chemotherapy with minimal additional toxicity and lack of available inhibitors, there is an urgent need to screen a large chemical library of structurally diverse compounds to identify novel MRP inhibitors using a simple cell-based model of drug resistance. The purpose of this proposal is to access the HTS resources of the Molecular Libraries Screening Center Network (MLSCN) with the goal of identifying molecular probes that can reverse MRP-mediated drug resistance. We propose a fluorescence-based cytotoxicity assay involving a MRP over-expressing human small cell lung tumor line, H69/AR. We have devised an innovative screening strategy to identify noncytotoxic, selective and potent MRP inhibitors. The availability of novel MRP inhibitors is anticipated to provide valuable tools to the scientific community that will encourage further study of MRP as a potential drug target. In addition, the resulting structure-activity analysis may help to define key topological features of inhibitors to allow for the development of a pharmacophore model. Our future plans are to identify specific chemical classes of inhibitors that have potential safety advantages, which we intend to evaluate efficacy using in vitro and in vivo models. In summary, this project is anticipated to lead to the discovery of potent and selective MRP inhibitors that can reverse resistance to chemotherapy with minimal additional toxicity. -
MLSCN Assay for Ligands of GRP30 and Classical Estrogen Receptors
PROSSNITZ, ERIC R, PHD
UNIVERSITY OF NEW MEXICO ALBUQUERQUE
1 X01 MH077627-01
2
DESCRIPTION (provided by applicant): Estrogen (17a-estradiol, E2) is a critical hormone in the human body, regulating a multitude of functionally dissimilar processes in numerous tissues. Estrogen represents one of a family of steroid hormones, which also includes progesterone, testosterone, cortisol/glucocorticoids and aldosterone/mineralocorticoids that control many aspects of mammalian physiology. Steroid hormones are synthesized in numerous tissues throughout the body, including the ovaries, testes and adrenal glands. The effects of all hormones, including estrogen, are mediated by specific receptors that recognize and bind the hormone transmitting this information to downstream effectors. We have recently characterized a novel 7-transmembrane G protein-coupled estrogen-binding receptor GPR30 that appears to function along side classical estrogen receptors to effect cellular responses to estrogen. It is therefore essential to develop families of ligands that can distinguish between this receptor and the classical nuclear estrogen receptor. Such probes will be critical to determine the physiological roles of GPR30 as well as potentially develop receptor type-specific drugs. The goal of this application is to perform HTS of compounds that will displace a fluorescent estrogen derivative from binding to this newly discovered G protein-coupled estrogen receptor, GPR30, as well as to the classical estrogen receptors, ERa and ERa. -
MLSCN Assay for Inhibitors of Arrestin-GPR Interactions
PROSSNITZ, ERIC R, PHD
UNIVERSITY OF NEW MEXICO ALBUQUERQUE
1 X01 MH077637-01
2
DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. They are also the target of more than 50% of drugs on the market today. Although they are best known for their interactions with G proteins, another protein named arrestin is recognized as being as potentially an important mediator of GPCR actions as the G proteins themselves. Arrestins bind with high selectivity to the ligand-bound, phosphorylated form of GPCRs, where they mediate receptor trafficking and the scaffolding of signaling molecules, such as MAP kinases. We have developed an in vitro method to reconstitute the GPCRarrestin interaction using only a phosphorylated peptide representing the carboxy terminus of the receptor. This bead-based method is ideally suited to high throughput screening for the purpose of identifying small molecules that disrupt the arrestin-GPCR interaction. Such molecules will serve as important probes and reagents for the further dissection of the functions of arrestins in vivo. They may also serve as the basis for the development of therapeutic agents that modulate the activity of GPCRs. -
Chemical Inhibitors of Anti-Apoptotic Protein Bfl-1
REED, JOHN C
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 X01 MH077632-01
2
DESCRIPTION (provided by applicant): Apoptosis is governed in part by Bcl-2-family proteins. The human genome contains six genes that encode anti-apoptotic members of the Bcl-2 family. Each of these proteins can be inhibited by endogenous proteins that contain a conserved peptidyl domain called BH3. The binding of fluorochrome-conjugated BH3 peptides to anti-apoptotic Bcl-2-family proteins thus provides the basis for construction of Fluorescence Polarization Assays (FPA), suitable for high throughput screening (HTS). Among the anti-apoptotic Bcl-2-family members is Bfl-1 (also known as A1 in mice), a NF-kB-inducible member of the Bcl-2 family. Unlike the other anti-apoptotic members of the Bcl-2 family that have all been successfully ablated in mice, the mouse ortholog of Bfl-1 consists of a cluster of four replicated genes (i.e., four copies of the gene, termed A1a, A1b, A1c, and A1d). Thus, chemical inhibitors are needed as research tools for neutralizing Bfl-1 in human and mouse cells. We propose to identify and optimize chemical inhibitors of Bfl-1. To this end, we have devised procedures for producing multi-milligram quantities of purified recombinant Bfl-1 protein and we have devised a fluorescence polarization assay (FPA), using a Bfl-1-binding synthetic peptide conjugated with FITC. A preliminary screen has been performed of ~10,000 compounds, demonstrating the suitability of this homogeneous assay for the high-throughput environment. -
Chemical inhibitors of antigen receptor-induced NF-?Beta activation
REED, JOHN C, MD
BURNHAM INSTITUTE
1 X01 MH077633-01
2
DESCRIPTION (provided by applicant): Many cellular pathways leading to activation of NF-?B-family transcription factors have been identified, participating in host-defense, immunity, inflammation, and cancer. Recently, a unique pathway activated by antigen receptors on T- and B-lymphocytes has been revealed, involving a cascade of participating proteins that includes CARMA1 (Blimp), Bcl-10, paracaspase (MALT1), TRAF6, and Ubc13. This pathway is initiated by Protein Kinase C-theta, which induces phosphorylation of components of this signaling pathway. Treatment of cells with the combination of phorbol ester PMA and calcium-ionophore ionomycin triggers this pathway, resulting in NF-?B activation. We have devised a stably transfected reporter cell line that contains a luciferase gene driven by a NF-?B responsive promoter. In pilot studies, we have used this cell-based assay to screen collections of compounds, confirming suitable assay performance for the high throughput environment. Hits from this screen are counter-screened using the same reporter cell line stimulated with alternative NF-?B activators, including TNFa, thus identifying pathway-specific chemical inhibitors. We propose to use this cell-based HTS assay for screening the NIH’s chemical library. The compounds identified using this assay will be useful research tools for analysis of the physiological roles of this NF-?B activation pathway. -
Zebrafish Lipid Metabolism Assay
RUBINSTEIN, AMY L
ZYGOGEN, LLC
1 X01 MH077634-01
2
DESCRIPTION (provided by applicant): The purpose of this proposal is to submit an assay to screen compounds as part of the Molecular Libraries Screening Centers Network. The assay described in this proposal provides an in vivo, quantitative measure of lipid absorption and processing in the digestive system of a vertebrate organism, the zebrafish. The zebrafish processes lipids through the digestive system in a manner similar to mammals. Because zebrafish larvae are essentially transparent, such processing can be readily observed in the whole organism, with the aid of fluorescent lipid substrates, which are swallowed by the zebrafish larvae and transported from the intestine to the liver and gall bladder. Cardiovascular disease is the number one cause of death in the United States today. High levels of cholesterol and triglycerides have been identified as major contributing factors to heart disease. Thus, an assay that can identify compounds with lipid lowering properties would be of substantial benefit to human health. -
Fluroscence polarization assay for PLK1 inhibitors
SHARLOW, ELIZABETH R., PHD
UNIVERSITY OF PITTSBURGH
1 X01 MH077611-01
2
DESCRIPTION (provided by applicant): Polo-like kinase 1 (PLK1) is a serine-threonine protein kinase that functions as a key regulator of mitosis. Numerous studies indicate an involvement of PLK1 in human cancer. In addition, there is increasing evidence implicating PLK1 in neurogenerative diseases such as Alzheimer’s (disease) and diseases of viral origin, including simian immunodeficiency virus (SIV) and human cytomegaloviral and papillomaviral infection. Research designed to study the functional role of PLK1 in these various disease states is hindered by the existence of few known specific inhibitors of PLK1 activity. The goal of this grant application is to transition a low throughput PLK1 Immobilized Metal Antibody-free Polarization (IMAP)-based fluorescence polarization (FP) assay to a high throughput capacity assay. The PLK1 IMAP FP assay would be used to screen large compound libraries for novel PLK1 inhibitors. Once identified and characterized these inhibitors can then be used as research tools to study the functional role of PLK1 in human disease. -
MLSCN Assay for Allosteric Ligands for the VLA-4 Integrin
Sklar, Larry A, PHD
UNIVERSITY OF NEW MEXICO ALBUQUERQUE
1 X01 MH077638-01
2
DESCRIPTION (provided by applicant): Leukocyte integrins play key roles in vascular cell adhesion in host defense, inflammation, hemostasis, and metastasis. Our data suggest that the conformation of VLA-4 (“very late antigen”), the alpha4beta1 heteordimer, and its molecular extension (moving the ligand binding site away from the cell membrane) are associated with the affinity of VLA-4 for its ligand VCAM-1 (“vascular cell adhesion molecule”). An increase in affinity is accomplished through a decrease in molecular dissociation rate or increased residence time of the ligand. In turn, the extended conformation contributes to cell adhesion avidity (particularly the efficiency of cell adhesion). VLA-4 can be induced to change conformation and to extend by divalent cations and inside-out signal transduction (e.g. the pathway from a chemokine receptor to VLA-4) in conjunction with ligand binding. We have further suggested that the ability of VLA-4 to extend under the shear forces of cell adhesion promote extension of the molecule, increasing affinity and strength of cell-cell adhesion, and leading to intracellular signaling which further impacts the molecular conformation. At the current time, molecular probes for integrin function and conformation make use of both isosteric and allosteric regulators. The isosteric small molecules are targeted to the binding site of the native ligand. We have already developed a fluorescent peptide analogue for the ligand binding site of VLA-4. This high affinity ligand has been used with flow cytometry to measure VLA-4 affinity, its conformational state through fluorescence resonance energy transfer, and the time courses associated with their regulation. While allosteric regulators have been developed for other integrins, there are as yet no allosteric regulators for VLA-4. An allosteric regulator would not block the ligand binding site. Rather, distinct sets of allosteric molecules have the potential either to block the conformational and affinity changes of the integrin or to initiate them. Thus, the allosteric agonist is expected to cause integrin extension, increase affinity and increase avidity. It might promote intracellular signaling assocated with adhesion under shear. The antagonist of the allosteric site is expected to block extension, block affinity change, and block avidity change. It might also inhibit shear induced intracellular signaling. We have in hand several cell lines and physiological models with appropriate levels of integrin expression for high throughput screening for discovery of small molecule allosteric regulators by flow cytometry. These models provide the appropriate signaling and conformational characteristics to test all of the activities of the new small molecules that will be discovered. We have established a generic, homogeneous no-wash flow cytometric assay involving fluorescent peptides and stable cell lines expressing the desired receptor. We have validated that this assay can be performed in a high throughput model with VLA-4 and our fluorescent ligand with appropriate Z’. -
ATP Hydrolysis-dependent disassembly of the 26S proteosome as an HTS assay for fl
SKOWYRA, DOROTA, PHD
ST. LOUIS UNIVERSITY
1 X01 MH077613-01
2
DESCRIPTION (provided by applicant): We propose to develop an HTS assay at the University of New Mexico, Albuquerque (Larry Sklar, PI), based on a new regulatory phenomenon recently discovered by my research group, the controlled disassembly of the 26S proteasome in the presence of endogenous proteasome-interacting proteins (Babbitt et al. 2005, Cell: 121, 553-565). Ubiquitin-mediated protein degradation by the proteasome has only recently been recognized as critical for cell growth and proliferation. Already, perturbations of this system have been implicated in multiple aspects of cancer pathogenesis, marking the proteasome as an attractive therapeutical target. The project I propose here stems from my long-term goal to understand the molecular mechanisms by which the proteasome recruits substrates and facilitates their destruction. My research group addresses this goal via biochemical dissection of protein degradation in vitro, using purified substrates and components of the SCF ubiquitin ligase pathway of yeast S. cerevisiae, which we reconstructed in vitro and characterized (Skowyra et al. 1997, Cell: 91, 209-219; Skowyra et al. 1999, Science: 284, 662-665; Kamura et al. 1999, Science: 284, 657-661; Deffenbaugh et al. 2003, Cell: 114, 611-622; Babbitt et al. 2005, Cell: 121, 553-565). This pathway is conserved and controls the degradation of major G1 cell cycle regulatory proteins and signaling molecules in all organisms, be they yeast or human. The information obtained with yeast is therefore directly relevant and, frequently, key to understanding SCF-mediated proteolysis in human cells. In our studies, we seek to uncover features of the proteasome that could serve as targets for pharmacological regulation of its activity at the steps of substrate recognition and processing for degradation, but not degradation itself. This knowledge will likely prove of considerable significance for the development of novel strategies for targeting the proteasome in cancer and other diseases linked to abnormal protein degradation. In collaboration with Larry Sklar, we have developed several flow cytometry-based assays for monitoring the rate-limiting, regulatory steps in the SCF-proteasome pathway (Deffenbaugh et al. 2003, Cell: 114, 611- 622; Babbitt et al. 2005, Cell: 121, 553-565; and data unpublished). The assay proposed here will allow us to identify, in a single screen, a collection of small molecules that could interfere with the catalytic cycle of the proteasome via a variety of mechanisms. Subsequent analysis of these molecules in the multiple in vitro assays available in my laboratory will allow us to identify the precise mechanisms by which the compounds function. As I explain below, such molecules are likely to become an invaluable research and therapeutical tool. -
Inhibitors of the NS3 Proteinase of West Nile and Dengue Viruses
STRONGIN, ALEX Y, DSC, PHD
BURNHAM INSTITUTE
1 X01 MH077601-01
2
DESCRIPTION (provided by applicant): The purpose of this application is to describe our low risk/high reward, timely and sharply-focused structure-based drug design effort against West Nile virus and Dengue hemorrhagic fever. The target of our drug design effort is the essential processing NS2b-NS3 serine proteinase of flaviviruses, a single proteinase encoded by the viral genome. The presence of the functionally active, mature NS3 proteinase is absolutely essential for the virus life cycle, virus propagation and disease progression. The immediacy and intensity of our effort is directly responsive to the US government’s decision that flaviviral infections caused by these viruses are a potential weapon of a bioterrorist. These viruses are categorized by NIAID as Category A-C priority pathogens. There are also hundreds of millions of cases of flaviviridae infections worldwide and, in addition, thousands of cases of West Nile virus in the US. Currently, there are no effective countermeasures against flaviviral infections. Despite the needs of Biodefense and a growing number of naturally-infected patients, there is currently no specific treatment or vaccine to cure or prevent West Nile and Dengue infections. Obviously, there is an urgent need for a potent and safe antiflavivirus therapy. The Burnham Institute team that will implement any research program resulting from this proposal will be drawn from the laboratories of the Infectious and Inflammatory Disease Center, and the Center for Proteolytic Pathways of the Burnham Institute (La Jolla, CA). The team will be lead by Dr. Alex Strongin, PI. Currently, Dr. Strongin is guiding several NIH-funded projects on proteinases including the NIAID-funded proposals “Structure-based Drug Design for Smallpox Therapy” and “Develop effective inhibitors of anthrax lethal factor”, and his overarching stewardship will insure program-to-program cost effectiveness and scientific value. THE SPECIFIC AIMS OF OUR PROJECT ARE: 1. Identify drug-like small molecule inhibitors of the NS3 West Nile virus protease by high throughput screening of chemical libraries. 2. Identify drug-like small molecule inhibitors of the NS3 Dengue virus protease by high throughput screening of chemical libraries. Future plans include optimization the structure of the novel, low-molecular weight, synthetic inhibitors of the West Nile and Dengue virus NS3 proteinase and to validate the selectivity and potency of the selected drug leads in additional in vitro tests and assays. In addition, we will determine, at the atomic resolution level, the structure of the NS3 protease bound to lead antagonists. The foundation for this drug development project is the expertise and experience of the Burnham team in the discovery of novel drugs for infectious and immune diseases and the recent exciting discoveries directly related to this particular project. Vast amount of structure-activity data, which will flow from this project, will facilitate creation of therapeutically important, anti-viral, drugs. -
Development of a HTS Assay for Inhibitors of Bacterial DnaK
STURGESS, MICHAEL ALAN
CHAPERONE TECHNOLOGIES, INC.
1 X01 MH077612-01
2
DESCRIPTION (provided by applicant): Antibiotic resistance is a major concern that has influenced both the clinical uses of established antimicrobials and the development of new agents. In clinical settings, gram-negative bacteria were a major problem in the 1970′s, whereas the past decades have seen a climb in the number of incidents with multidrug resistant gram-positive strains. Currently, the rapid emergence of resistant strains involves both grampositive and gram-negative pathogens. It is not an overstatement to claim that one of the most serious, and urgent, topics in clinical and vetinary health is the timely development of antibacterial compounds that kill bacteria in a manner completely different from those utilized by the currently marketed antimicrobial compounds. The goal of this program application is to advance the knowledge and utilization of DnaK inhibitors as the next generation of antibacterial agents. Our previous efforts in this field have demonstrated the validity of DnaK inhibitors as antimicrobial agents, albeit in the realm of peptide-based inhibitors. By advancing this effort, and the anticipated small molecule DnaK inhibitors, into the academic realm it is expected that a more thorough and precise understanding of the role of microbial chaperone systems in the procession of pathogenic infections will result, thereby allowing a greater understanding of the role of environmental adaptations in the establishment of such disease states. Through optimization and validation of the preliminary screening format a viable HTS protocol will be developed that may be applied to a number of diverse homologs of E.coli DnaK, leading to a family of HTS formats for the identification and optimization of organism-selective antimicrobial agents. It is the goal of Chaperone Technologies to develop these agents as effective antimicrobial treatments for clinically significant drug-resistant infections such as E.coli and K.pneumoniae drug-resistant urinary-tract infections, MRSA infections and VRE/GRE infections. The occurrence of such clinical infections is rapidly increasing and will represent a major challenge in the coming years. It is forecast that the current generation of antibiotics will not be sufficiently active to adequately control these cases, necessitating the discovery and development of fundamentally novel antimicrobial agents. -
Allosteric Modulators of D1 Receptors
WATTS, VAL J, PHD
PURDUE UNIVERSITY WEST LAFAYETTE
1 X01 MH077619-01
2
DESCRIPTION (provided by applicant): The proposal will use high throughput screening (HTS) of chemical libraries to identify allosteric modulators of D1 dopamine receptors. Allosteric modulators hold great promise as therapeutics because they provide activity-dependent spatial, temporal, and receptor specificity. Specific Aim 1 shall provide initial validation tests and reagents to the appropriate MLSC to establish a cell-based assay as a tool to identify compounds acting as positive allosteric modulators of D1 dopamine (D1) receptors. In the proposed cell model, D1-stimulated increases in cyclic AMP lead to activation of the cyclic AMP response element (CRE) which then leads to increase luciferase expression (Luc) that can be measured in a high throughput setting. We have previously used this model to screen successfully a small (2000 compounds) chemical library supplied by NCI. We propose to validate and establish the assay performance minimums required for HTS using equipment within one of the MLSCN’s HTS Centers. Specific Aim 2 will use the validated D1-CRE-Luc cell model to perform a large scale HTS screen to identify allosteric modulators of D1 dopamine receptors. Compounds identified here will be subjected to a follow up HTS in the presence of a non-selective activator of adenylate cyclase, forskolin. This high throughput follow-up screen will be used to minimize rapidly false positives that have actions inconsistent with those of a D1 dopamine receptor allosteric modulator. Specific Aim 3 will validate potential lead compounds as allosteric modulators of D1 dopamine receptors using a HTS cyclic AMP accumulation assay as a more direct readout of D1 receptor function. These experiments will include concentration-response curves in the absence and presence of dopamine to examine the potency and the activity of each allosteric modulator. Specific Aim 4 seeks to identify potential structural classes of compounds by mining chemical libraries and performing chemical synthesis. Each series of structurally-related compounds will then be subjected to the pharmacological studies described in Specific Aim 3 to identify the structure-activity relationships. All compounds from these screens will be directly deposited into PubChem to promote information sharing. Relevance to Public Health: Dopamine receptors are critical neuroreceptors that are involved motor function, memory, mood, and drug addiction. The D1 dopamine receptor has been identified as a key target associated with Parkinson’s disease and schizophrenia. We propose to identify new chemical probes that can be used to safely and effectively modulate D1 dopamine receptors to improve human health. -
Identifying novel Hsp90 Inhibitors by disrupting its cochaperone interaction usin
Yi, Fang, PHD
YALE UNIVERSITY
1 X01 MH077625-01
2
DESCRIPTION (provided by applicant): Specific Aims: To identify small molecules that inhibit Hsp90 activity by inhibiting its interaction with the co-chaperone HOP. Inhibition of this interaction will prevent Hsp90-dependent folding, thus lead to the degradation of oncogenic signaling proteins. Aim 1: Test the MLSCN compound libraries to identify compounds that inhibit the Hsp90-TPR2A interaction, using an AlphaScreen based high throughput screening assay. Aim 2: Validate compounds identified in the screen described in Aim 1 by testing their efficacy in inhibiting the Hsp90-TPR2A interaction, using independent in vitro and in vivo assays. Aim 3: Test the effectiveness of the compounds validated in Aim 2 in inhibiting cancer cell growth. -
Identification of lkappaBalpha Stabilizers in a Human Lymphoma Cell Line Using A
AULD, DOUGLAS S
NIH/NHGRI
1 X01 MH078957-01
3
DESCRIPTION (provided by applicant): The goal of this project is to identify chemical probes that inhibit “upstream” points in activation of the NF-kappaB (NFkB) pathway using cell lines from a type of lymphoma that is largely incurable by conventional chemotherapy. Many cell types normally activate the NFkB signaling pathway temporarily in vital functions such as immunity, but certain cancers display constitutive NF-kB activation and can resist therapy. While it is clear that the later stages of NF-kB activation are common to both normal and malignant cells, the upstream mechanisms of constitutive NF-kB activation in cancer are largely unknown. In the classical pathway of NF-kB activation, the midpoint is rapid degradation of the critical regulatory protein IkappaBalpha (IkBa) after phosphorylation by an activated IkappaB kinase (IKK) complex. We have found that cell lines of the ABC-DLBCL type of human lymphoma display high constitutive IKK activity, on which they depend for survival. Conventional cell-based assays of NF-kappaB activation measure its endpoint, and present several problems for HTS. We have addressed these with an innovative cell-based assay in ABC-DLBCL lines that interrogates the midpoint of the NF-kB pathway. In stably-engineered reporter lines, green light-emitting beetle luciferase is fused to IkBa and increases upon inhibition of IkBa degradation, while unfused red luciferase provides for normalizing the signal to cell number and check cell viability. Development and testing of this assay has confirmed its precision, robust response to known small-molecule inhibitors of IKK activity or IkBa degradation, and suitability for miniaturization and HTS. Screening to date of 11,707 compounds has yielded a small number of actives, some of which are structurally similar to known IKK inhibitors. Further screening will increase the chance that compounds can be found that inhibit upstream causes of constitutive NFkB activation in these lines, or identify novel inhibitors of IKK activity or IkBa degradation, with the potential for mechanistic insights and therapy development. Large-scale validation of this assay will promote its use in other applications, such as cell lines from other types of cancer with constitutive NFkB activity. The chemical probes yielded by the project should be useful tools in providing a better understanding of this process and perhaps leading to therapies that would specifically inhibit constitutive NFkB activation in cancers without causing side effects such as immunosuppression. -
High-Throughput Screen for Antagonists of the Plasmodial Surface Anion Channel (P
DESAI, SANJAY A
NAT’L INST OF ALLERGY/INFEC DISE
1 X01 MH078943-01
3
DESCRIPTION (provided by applicant): We propose a high-throughput screen for antagonists of a novel ion channel on P. falciparum-infected red blood cells (RBCs). This channel, the plasmodial surface anion channel (PSAC), is highly conserved in all studied human, rodent, and primate malaria models. Because PSAC dramatically increases RBC permeability to amino acids, purines, vitamins, and precursors for phospholipid biosynthesis (all required for in vitro parasite growth), it likely serves an essential role in intraerythrocytic parasite nutrient acquisition. Our high-throughput screen aims to identify high affinity, specific PSAC antagonists that kill parasites by interfering with nutrient uptake. PSAC has already been recognized as an important antimalarial drug target by the Medicines for Malaria Venture, a nonprofit organization created to discover, develop and deliver new antimalarial drugs through effective public-private partnerships. With MMV support, the Principal Investigator has initiated collaborations with academic and pharmaceutical screening facilities. Our previous high throughput screens, carried out in 384-well and in 3,456-well formats, identified high affinity antagonists that have provided insights into PSAC structure and biological role. Some antagonists identified may be starting points for collaborative drug discovery programs. Lay language statements: The deadly malaria parasite, Plasmodium falciparum, lives and grows within human blood cells. Because it has a high metabolism, it must acquire nutrients from serum. It uses an unusual ion channel known as the Plasmodial Surface Anion Channel (PSAC), induced on the blood cell membrane by the parasite, to facilitate nutrient uptake. We propose to find inhibitors of PSAC by screening large collections of drug-like molecules. Identified inhibitors may be developed into desperately needed antimalarial drugs. -
ADAMTS/MMP Drug Development
FIELDS, GREGG B
FLORIDA ATLANTIC UNIVERSITY
1 X01 MH078948-01
3
DESCRIPTION (provided by applicant): The natural breakdown of collagen is critical to physiological processes such as embryogenesis and bone remodeling. On the other hand, the destruction of collagen’s triple-helical structure can also give rise to a variety of pathologies, including tumor cell spreading (metastasis), arthritis, glomerulonephritis, periodontal disease, and tissue ulcerations. Our laboratory has developed a target-based approach for profiling of extracellular matrix (ECM) degrading proteinases. More specifically, collagen-model conformationally constrained fluorescence resonance energy transfer (FRET) substrates have been utilized to quantify activities for collagenolytic matrix metalloproteinases (MMPs) and aggrecan-degrading members of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family. These FRET substrate assays are fully compatible with multi-well formats. The specific aims for the present proposal are to (a) transfer the FRET substrate assays to the MLSCN for screening of collagenolytic MMP and aggrecandegrading ADAMTS family members that have been implicated in osteoarthritis (MMP-13 and ADAMTS-4) using the NIH Small Molecule Repository, and (b) verify and counter-screen inhibitor “hits” for MMP-13 and ADAMTS-4. Because the collagen-model FRET substrates have distinct conformational features that interact with the proteases’ exosites, non-active site binding inhibitors can be identified that bind to MMPs and ADAMTSs. Exosites are secondary substrate binding sites, and have been shown to represent unique opportunities for the design of selective inhibitors. Our laboratory is uniquely positioned to utilize these HTS assays, based on our expertise with FRET triple-helical substrates for collagenolytic MMPs and FRET collagen-model substrates for aggrecanases. Initial clinical trials with MMP inhibitors were disappointing, with one of the problems being a lack of selectivity. In the case of aggrecanases, few inhibitors have been described. Selective inhibitors for MMP-13 and ADAMTS-4 would allow for a more definitive evaluation of these proteases in osteoarthritis, as well as representing a potential next generation in metalloproteinase therapeutics. -
Small Molecule Inhibition of Staphylococcus Aureus Virulence
GRESHAM, HATTIE D
UNIVERSITY OF NEW MEXICO ALBUQUERQUE
1 X01 MH078952-01
3
DESCRIPTION (provided by applicant): Quorum sensing is a cell-to-cell communication system that permits members of a bacterial population to coordinate their behavior dependent on cell density. The mediators of this communication system are small, diffusible pheromones or autoinducers that are secreted by the bacteria and that accumulate extracellularly. At the appropriate concentration threshold that reflects a sufficient number or quorum of bacteria, the autoinducers signal gene expression programs that direct the coordinated action of the population. The list of bacterial pathogens that use this method of communication to regulate virulence is expanding and now includes some of the most common bacterial pathogens of humans including the medically important pathogen Staphylococcus aureus. Because antibiotic resistance is an emerging problem in this pathogen and vaccines are of limited efficacy, quorum sensing is becoming a therapeutic target for treatment of this infection. Attacking virulence by these strategies, termed “quorum quenching,” has proven successful in two animal models of S. aureus infection. We recently published data demonstrating that phagocyte-derived reactive oxidants inactivate the peptide thiolactone autoinducer of S. aureus and that this is important for host defense against this infection (Rothfork et al, PNAS 101:13867, 2004). Our data demonstrate that targeting virulence by chemical inactivation of the quorum sensing pheromone represents a viable treatment option. We hypothesize that small molecule inhibitors of the peptide autoinducing pheromone (AIP) can abrogate virulence dependent gene expression. To test this hypothesis, we are applying to the Molecular Libraries Screening Centers Network (MLSCN) to pursue the following specific aims: Specific Aim #1: To screen libraries of small molecules in a high throughput fluorescence-based screening assay to identify compounds capable of suppressing pheromone-dependent activation of the promoter for a global regulator of Staphylococcus aureus virulence, RNAIII. Specific Aim #2: To confirm that the compounds that inhibit RNAIII promoter activation also inhibit expression of the virulence genes that are regulated by RNAIII. -
Novel sEH Inhibitors for the Therapeutic Treatment of Hypertension and Inflammat
HAMMOCK, BRUCE D
UNIVERSITY OF CALIFORNIA DAVIS
1 X01 MH078954-01
3
DESCRIPTION (provided by applicant): Hypertension and vascular inflammation are associated with cardiovascular diseases (CVD), the primary cause of death in our society. Because a large proportion of patients are not responding to current therapies, the next generation of drugs will not only need to reduce BP but also treat vascular and renal inflammation as well as reduce smooth muscle cell proliferation which in turn should also reduce hypertension related organ damage. Using inhibitors developed previously in our laboratory, we showed that the inhibition of soluble epoxide hydrolase (sEH) has therapeutic application in the treatment of hypertension and several inflammatory diseases. While these inhibitors are scientifically useful, the low solubility and relatively fast metabolism of our first generation inhibitors makes them less than therapeutically efficient, underlying the need for novel inhibitor structures. Toward such a goal, we recently developed a new fluorescent assay for sEH that is adequate for high throughput screening. Therefore, we are proposing to use this assay to screen the MLNSC library of compounds to identify new chemical leads for sEH inhibition, and thus for the therapeutic treatment of hypertension and inflammation. -
Targeting a Protein Interaction Site on Smad Transcription Factors
HOFFMANN, MICHAEL
UNIVERSITY OF WISCONSIN
1 X01 MH078945-01
3
DESCRIPTION (provided by applicant): A high throughput screening assay will be provided to screen the MLSCN library for compounds that disrupt a key protein-protein interaction site on the Smad transcription factors that mediate cellular responses to transforming growth factor beta (TGF-a). There are currently no small molecule ligands that target Smad proteins. Inhibition of Smad function will be an important research tool in elucidating the multiple biological functions of TGF-a. Inhibition of TGF-a signaling is also a validated target in several advanced cancers because of its role in facilitating cancer cell migration, proliferation, metastasis to bone and/or evasion of the immune response. It is also a validated target in all forms of fibrotic disease including idiopathic pulmonary fibrosis and diabetic nephropathy. Current strategies for inhibiting TGF-a signaling focus on disruption of the ligand-receptor interaction with neutralizing antibodies or inhibition of the TGF-a receptors using small molecule inhibitors of the kinase activity. This proposal targets the transcriptionally active Smad complexes to inhibit signaling. The working hypothesis is that disruption of only one binding site on Smad will block binding of some but not all Smad binding partners (over two dozen different transcription factors, coactivators and co-repressors), thereby interfering with only a subset of the gene expression responses mediated by Smad complexes. One advantage of this strategy over inhibiting ligand binding or receptor kinase activity is that it might provide selective inhibition of some but not all TGF-a responses. The assay is a homogeneous time resolved fluorescence resonance transfer assay that measures peptide binding to a specific protein binding site in the hydrophobic corridor of Smad2 and Smad3. The binding site is present only in Smad2 and Smad3 and not in Smads 1, 4, 5, 6, 7, or 8, therefore, ligands to this site should have preferential effects on Smad2 and Smad3 functions. Furthermore, missense mutations in the binding site indicate that the ligands for the site, the FoxH1 SIM, the SARA rigid coil and the nuclear porin FG repeat, contact overlapping but not identical amino acids, suggesting that a small molecule ligand might preferentially block binding of one of these proteins to Smad2 or Smad3. The assay has a Z’ factor of 0.7- 0.8 in a 384-well plate format and has been used to screen 16,000 compounds at the Keck-UWCCC Small Molecule Screening Facility. This preliminary screen led to the identification of two compounds that in secondary assays also inhibit TGF-a stimulation of reporter gene expression in HepG2 cells. If the aims of this application are achieved, new compounds targeted to the Smad protein hydrophobic corridor would become critical research reagents to demonstrate which normal or pathological responses to TGF-a depend on this Smad protein binding site. Given the need for new therapeutic approaches in diseases where TGF-a signaling is an important target, such as glioma and idiopathic pulmonary fibrosis, active compounds identified by the proposed assay would stimulate additional pharmaceutical development and clinical trials. -
Screen for Coactivator Binding Inhibitors to Block Estrogen Action
KATZENELLENBOGEN, JOHN A
UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN
1 X01 MH078953-01
3
DESCRIPTION (provided by applicant): The overall aim of this research is to identify novel compounds that can act as effective coactivator binding inhibitors (CBIs) to block gene activation through the estrogen receptors, ERa and ERa, by directly inhibiting their interaction with important coregulator proteins. The ERs well validated targets for women’s health and breast cancer prevention and treatment. The CBIs are not targeted at the traditional site on the ERs, namely, the ligand binding pocket; rather, their target is a hydrophobic groove through which the ERs interact with the coactivator proteins of the p160 family, the steroid receptor coactivators SRC1 and SRC3, critical mediators of estrogen signaling and the regulation of gene transcription by the ERs. By blocking estrogen signaling through ERa and ERa by direct competition with their binding to SRC1 and SRC3 in the hydrophobic groove rather than indirectly by competitive ligand-based antagonism within the ligand binding pocket, we hypothesize that we will be able to achieve a more complete blockade of estrogen action and possibly overcome the development of resistance to endocrine therapy that typically occurs in the treatment of breast cancer both with antiestrogens and aromatase inhibitors. In the past, we have used structure-based design to guide the preparation of CBIs, and we have developed fluorescence-based assays to measure CBI activity. While we have found CBIs with Ki values in the 5-20 fM range, we have not succeeded in finding CBIs having nanomolar potencies. We now propose to work with members of the Molecular Libraries Screening Centers Network (MLSCN) to refine a time-resolved fluorescence resonance energy transfer (TR-FRET) assay and use it to screen large compound libraries in a much broader search for hits of novel structure. After confirming these hits in mechanistically appropriate counter screens and alternative assays of CBI activity and selectivity, we would plan to optimize them by medicinal chemistry approaches, to obtain receptor-specific CBIs that have nanomolar affinities, so that in future studies they can be used in cell-based and animal assay systems at concentrations and doses that will not be toxic. This cooperative assay development/molecular library screening work will also provide a basis for expanding the search for CBIs that are specific for other nuclear hormone receptors or those that block ER interaction with other proteins that might be mediating the development of endocrine resistance in breast cancer therapy. -
High Throughput Screening for Protein Misfolding Disease Therapeutics
KELLY, JEFFERY W
SCRIPPS RESEARCH INSTITUTE
1 X01 MH078940-01
3
DESCRIPTION (provided by applicant): The most important objective of the proposed screen is to identify folding modulators, small molecules that enable misfolding prone proteins to be folded in the cell and to be trafficked to their destination, avoiding loss-of-function misfolding diseases. Numerous genetic diseases are caused by misfolding, or the failure of a mutated protein to assume its native active conformation. The distinct advantage of molecules that enhance the folding capacity of the endoplasmic reticulum and/or cytoplasm within the cell is that one or a few molecules could be used to treat multiple loss-of-function diseases. Such an approach is significant because although many of these diseases affect a small number of patients, in aggregate, there are, hundreds of thousands of people suffering of loss-of-function misfolding disease. We employ Gaucher disease, a disorder due to loss-of-function of the enzyme b-glucocerebrosidase (GC), as a representative loss-of-function misfolding disease, and employ patient-derived cells for the discovery of small molecule folding modulators – molecules that enhance the ability of the endoplasmic reticulum to fold, and enable trafficking of GC to the lysosome. Specifically, we utilize the L444P GC patient-derived cell line, which affords active GC when grown at 30 xC, but not when grown at 37 xC, even in the presence of chemical chaperones (molecules that bind to GC active site, and stabilize several variant GC in the ER, enabling them to be trafficked to the lysosome). We employ L444P GC fibroblasts for a high throughput cell-based screen to seek compounds that restore the compromised cellular GC activity at 37 xC associated with Gaucher disease. Putative folding modulator hits will be confirmed by their ability to fold and traffic the mutant chloride channel whose absence is associated with the development of cystic fibrosis. In parallel, a second high throughput screen will be performed using a Gaucher disease cell line amenable to GC specific chemical chaperoning. The N370S GC cell line will be used to select pharmacologically appealing compounds for ameliorating Gaucher disease affecting the central nervous system, which represents a currently unmet medical need. -
Screening for Inhibitors of the Mevalonate Pathway in Streptococcus Pneumoniae
LEYH, THOMAS S.
YESHIVA UNIVERSITY
1 X01 MH078936-01
3
DESCRIPTION (provided by applicant): Streptococcus pneumonia (SP) takes the lives of nearly 4000 people daily, the majority of whom are children below the age of five. The organism’s ability to evolve resistance mechanisms has produced strains capable of tolerating our “last line of defense” antibiotics. This laboratory recently discovered that diphosphomevalonate (DPM), an intermediate in the mevalonate pathway, is a potent allosteric inhibitor of the SP mevalonate kinase (MK), and that it does not inhibit the human isozyme. The mevalonate pathway is essential for survival of the organism in mouse lung. DPM and the allosteric site offer a lead compound and target that provide an opportunity to develop a new class of antibiotics that could help eradicate this disease. Our preliminary data demonstrate that compounds based on these principles are capable of killing infectious SP in rich media. We are hoping to expand our repertoire of lead compounds by working together with the Molecular Libraries Screening Centers Network (MLSCN) to screen roughly 150,000 potential inhibitors against each of the three SP enzymes that comprise the mevalonate pathway. These enzymes are members of the GHMP kinase protein superfamily, whose biomedical relevance extends to both orphan diseases and cataract formation. We have determined the structures two of the three enzymes in the pathway, and have an excellent structural model for the third. We have just received word that that our recently submitted RO1 on the mevalonate pathway project (which will fund a protein functionalist, a high resolution NMR spectroscopist, an X-ray crystallographer and a synthetic chemist) will be funded for five years beginning April 2006; hence, we are well positioned to take advantage of the opportunities provided by the MLSCN screens. -
Rapid Mix and Measure HTS Assays for Anti-NCp7 Drug Discovery
MCPIKE, MARK P
ORTHOSYSTEMS, INC.
1 X01 MH078941-01
3
DESCRIPTION (provided by applicant): This project is aimed at exploiting a novel in vitro HTS assay to discover new anti-AIDS agents. The target is a specific NCp7-RNA interaction that is responsible for selecting full-length viral RNAs into budding virus particles. We have developed an indicator molecule that generates an optical signal upon binding their cognate protein target. These sensors operate free in solution which makes them very amenable for competition based “mix and measure” HTS assays for discovering small molecule inhibitors. The assay has been validated with the NCI diversity set of ~2000 compounds, where we identified 20 promising leads. These screens were conducted in-house using a Biomek(tm) 2000 liquid handler and SpectraMAX(tm) fluorescent plate reader. Our aim in collaborating with the MLSCN, is to scale-up and further optimize the assay to identify and develop lead compounds that demonstrate high affinity and specificity toward the HIV-1 NCp7 protein for therapeutic applications. -
Chemical Tools to Investigate the Protein Folding/Degradation Machinery
PELLECCHIA, MAURIZIO
BURNHAM INSTITUTE
1 X01 MH078942-01
3
DESCRIPTION (provided by applicant): The misregulation of protein folding quality control and protein degradation often results in a variety of deleterious consequences on cellular function that range from the accumulation of non-native protein species or protein aggregates leading to neurological disorders, to the inhibition of apoptosis in cancer cells. Several components of the proteinfolding/degradation machinery have been identified and characterized. A large body of evidence suggests that the regulation of the protein folding/degradation machinery represents a viable target for the development of novel potential therapies against several human malignancies. However, further research in this area is hampered by the lack of effective pharmacological tools. To this end, we propose an innovative approach aimed at the identification of small molecule probes capable of modulating the activity of the chaperone Hsp70 by targeting its substrate binding-domain. Such probe-ligands would open the way to a plethora of experiments with human cancer cells to dissect the multitude of activities that have been attributed to the chaperone in the onset and progression of cancer and ultimately may provide a framework onto which to develop potentially novel anti-cancer therapies. Two other possible targets are also discussed that are involved in the early stages of protein folding and degradation. -
Screening Chemicals to Suppress MT1-MMP Synthesis in Cancer
STRONGIN, ALEX
BURNHAM INSTITUTE
1 X01 MH078949-01
3
DESCRIPTION (provided by applicant): The purpose of this application is to describe our drug design effort to target the synthesis of MT1-MMP in cancer cells. MT1-MMP is a key player in cancer progression and metastasis. Insufficient knowledge of how MMPs including MT1-MMP work was the main reason for the failure of clinical trials in cancer. We have now identified previously uncharacterized, but highly important, additional functions of MT1-MMP. We discovered that MT1-MMP protects malignant cells against the host immunity. We also discovered that MT1- MMP is trafficked to centrosomes where this active protease cleaves integral centrosomal proteins and these events cause mitotic spindle aberrations and aneuploidy. Aneuploidy is a genetic marker of malignancy. In sum, these novel mechanisms allow neoplasms to survive immune attack, to invade the tissues, and to metastasize. We hypothesize that it is absolutely mandatory to suppress the protein synthesis of cellular MT1-MMP, as opposed to merely inhibiting its catalytic activity, in order to prevent malignant progression. This hypothesis warrants us to search for chemicals capable of suppressing the synthesis of MT1-MMP. Consistent with our hypothesis, we have already demonstrated that suppressing synthesis of MT1-MMP by siRNA significantly reduces the tumor growth of human fibrosarcoma HT1080 cells. Our specific aims are: (1) To identify low molecular weight chemicals capable of efficiently suppressing the MT1-MMP transcription. (2) To identify and to discard toxic compounds in an additional cell-based assay. We developed the cytoblotting assay employing HT1080 cells stably transfected with the luciferase reporter plasmid as the main tool for the screening of chemicals. This assay can be easily adapted to fit 384-well or 1536-well plates. This assay (Z’-factor = 0.8-0.9) is readily adaptable to automation. The coefficient of variation does not exceed 5%. Reproducibility between plates in day-to-day experiments also does not exceed 5%. The main technical parameters of the primary assay are as follows: assay volume, 0.1 ml; amount of cells per well, 10,000 in 96-well flat bottom plates; temperature, 37oC; assay time, 10 min. The reconstituted substrate stock is stable at -20′C for up to two weeks. The HT1080 cells stably transfected with the reporter plasmid and the detailed experimental protocol will be provided to the screening center. To discard toxic compounds, we will analyze viability of the cells co-incubated with the selected hits (50 fM) in an additional MTT-based assay. We are convinced that our efforts will result in the identification of efficient drug-like suppressors of MT1-MMP synthesis. These compounds can then be used alone or in a combination with existing cancer therapies. -
“Activators of Prostate Cell Differentiation”
THOMPSON, TODD A
UNIVERSITY OF NEW MEXICO ALBUQUERQUE
1 X01 MH078937-01
3
DESCRIPTION (provided by applicant): Prostate cancer is the most common malignancy to develop among men in the United States. The factors contributing to the high incidence of prostate cancer among US men are unknown. Differentiation is a vital physiologic process for all glandular organs. The incomplete progression of prostate cells to fully differentiate may play a critical role in prostate cancer development. Therefore, strategies to prevent and treat prostatic diseases by targeting prostate differentiation could be instrumental in reducing the suffering produced by these ailments. However, prostate glandular differentiation is a poorly understood process. This is due in large part to a lack of reagents for understanding pathways critical in glandular differentiation. Here, we propose to perform a high throughput screen for small molecules that induce a differentiated phenotype in prostate cells using the LNCaP human prostate cell line. We have established that side-scatter, a simple but highly reliable measure of intracellular granularity using flow cytometry, is a robust primary method for evaluating prostate cell differentiation. As a secondary assay, we have established that changes in mitochondrial mass in prostate cells also accurately reflect the induction of prostate cell differentiation. The HyperCyt system, developed by Dr. Bruce Edwards, Dr. Larry Sklar, and their team at UNM, is proposed as a high throughput flow cytometric procedure to perform the primary and secondary assays for identifying small molecules that induce prostate cell differentiation. High throuphput flow cytometry using the HyperCyt system is available for this proposal through the New Mexico Molecular Libraries Screening Center. The long-term goal of this research is to determine pathways (i.e., molecular targets) that are critical in prostate cell terminal differentiation, which will be essential in developing differentiation-based strategies for the prevention and treatment of prostatic diseases, such as prostate cancer. Relevance: Novel approaches to prostate cancer prevention and treatment are desperately needed. Differentiation is a process subverted in prostate cancer progression that may serve as a useful target for new measures of prostate cancer therapeutics. An innovative method is proposed to effectively screen for small molecules that induce prostate cancer cell differentiation. -
Homogeneous Flourescence Intensity High Throughput Assay for Cdc25B Phosphatase I
TIERNO, MARNI BRISSON
UNIVERSITY OF PITTSBURGH
1 X01 MH078959-01
3
DESCRIPTION (provided by applicant): Cdc25s are protein tyrosine phosphatases that control cell cycle progression. Of the three human isoforms that exist (Cdc25A, B, and C), Cdc25A and Cdc25B have been found to be oncongenic and over-expressed in many cancer cell lines. Cdc25B has been a target in multiple drug discovery endeavors, leading to several weak inhibitors of phosphatase activity. The majority of these compounds, however, are quinones, which inhibit Cdc25B catalytic activity through irreversible oxidation of the catalytic cysteine rather than specific binding at the catalytic domain. We propose to identify potent, selective, non-oxidizing inhibitors of Cdc25B. Initially, we used the catalytic domain of Cdc25B and a homogeneous fluorescence intensity assay to screen the MLSCN starter set of 3,316 compounds at 10 fM in a 384-well high throughput screen. The assay was robust with a Z’ factor of > 0.5. In our preliminary studies, we identified a unique, non-quinone pharmacophore with an IC50 of 8.12 q 0.06. These results suggest that an evaluation of the NIH small molecule repository will identify a significant number of new lead compounds as inhibitors of Cdc25B. -
Inhibitors of Caspase-1 and Caspase-7
WELLS, JAMES A
UNIVERSITY OF CALIFORNIA SAN FRANCISCO
1 X01 MH078950-01
3
DESCRIPTION (provided by applicant): Our primary objective is to discover novel small molecules that selectively inhibit caspase-1 and caspase-7 using a biochemically based high throughput screening assay. We intend to identify reversible inhibitors with better drug-like properties than the currently available set of aspartyl-containing peptidomimetics that covalently bind the active site. We will be especially vigilant to identify allosteric inhibitors to a novel site identified in our laboratory. -
High Throughput Screen for Inhibitors of the HIV Rev-RRE RNA Interaction
WILLIAMSON, JAMES R
SCRIPPS RESEARCH INSTITUTE
1 X01 MH078935-01
3
DESCRIPTION (provided by applicant): This proposal outlines a plan to carry out a high throughput screen (HTS) for inhibitors that block formation of the HIV Rev-Rev Response Element (RRE) RNA complex, at one of the MLSCN Centers. Rev is an RNA binding protein that is critical in regulating the shift from early to late patterns of viral gene expression, and it is specifically implicated in facilitating nuclear transport of viral mRNAs into the cytoplasm. The RRE is an RNA secondary structure element in the HIV mRNA that is the binding site for the Rev protein. This represents a novel target for inhibition of HIV for which there are no therapeutics. A validated HTS for Rev-RRE inhibitors has been developed using a FRET assay to monitor binding of a synthetic peptide and a synthetic RNA that have been labeled with fluorescent groups. Statistical analysis of control plates that validate the assay are presented, as well as the results from a pilot scale screen of ~24,000 compounds. A plan for follow-up screens for the primary hits from the MLSCN screen is presented. In addition, cell-based assays for inhibition of the Rev-RRE interaction are described. Finally, the use of Rev-RRE inhibitors to further our biochemical, biophysical, and cellular understanding of Rev function is outlined. -
High Throughput Screening for Inhibitors of the Polo Box Domain of Human Polo-Lik
YAFFE, MICHAEL
MASSACHUSETTS INST OF TECHNOLO
1 X01 MH078944-01
3
DESCRIPTION (provided by applicant): The long term goal of this project is to identify lead compounds for anti-cancer drug development that specifically target Polo-like kinase 1 (Plk1). Plk1 is a key regulator of cell division and is highly expressed in many forms of human cancer. We have developed a high throughput fluorescence polarization-based assay to screen for small molecule inhibitors of the Polo box domain (PBD) of Plk1. We previously identified the PBD as a phosphopeptide binding domain and showed that this phosphopeptide binding is essential for Plk1 localization and substrate targeting. The assay utilizes a fluorescently labeled phosphopeptide that exhibits fluorescence polarization (FP) when bound to the PBD. Small molecules that compete with the phosphopeptide for PBD binding will reduce the FP signal. We implemented this assay in a small scalescreen of 3362 compounds. Hit compounds were confirmed using a biochemical assay for inhibition of PBD binding to phospho-protein ligands from cell lysate, and a cell-based assay, which identified one compound that inhibited the proliferation of mammalian cells in culture. However, the chemical properties and aqueous solubility of this compound are not drug-like, and we are therefore proposing to apply our assay to the MLSCN compound collection to identify additional PBD inhibitors with more desirable properties. Hit compounds identified in this primary screen will be confirmed by the secondary assays that we have developed. We will also pursue crystal structures of PBD-inhibitor complexes to facilitate structure-guided optimization of hit compounds. Relevance: Polo-like kinase 1 (Plk1) is an anti-cancer drug target that is essential for the proliferation of tumor cells. Using a rapid high throughput screen of a large collection of small molecule compounds, the proposed study aims to identify specific inhibitors of Plk1, and to conduct follow up studies to help develop these inhibitors into actual anti-cancer drugs. -
HTS for Identification of Glucocerebrosidase Activators and Inhibitors as Pharmac
ZHENG, WEI
NIH/ NHGRI
1 X01 MH078932-01
3
DESCRIPTION (provided by applicant): The goal of this project is to screen the compound library to identify small molecule chaperones using a developed beta-glucocerebrosidase assay. Gaucher disease is a genetic disorder resulting from the deficiency of the lysosomal enzyme beta-glucocerebrosidase. Preliminary studies suggest that some mutations in the glucocerebrosidase gene result in the improper protein folding and trafficking of the enzyme. It has been proposed that small molecule inhibitors can function as chemical chaperones which might correct the targeting or folding of the mutant enzyme and thus restore its function. We developed a fluorogenic enzyme assay with emission readout at 600 nm for high throughput screening in 1536-well plate format to identify both inhibitors and activators of glucocerebrosidase which are potential pharmacological chaperones for the mutated enzyme. -
Drug Discovery for Bone Marrow Failure Diseases
DE FIGUEIREDO, PAUL
TEXAS A&M UNIVERSITY SYSTEM
1 X01 MH079865-01
4
DESCRIPTION (provided by applicant): Shwachman-Diamond Syndrome (SDS) is a constitutional bone marrow failure syndrome that also affects the pancreas and skeletal system of patients. The goals of this project are to elucidate the function of SBDS, the gene which, when mutated, results in SDS, and to identify small molecule pharmaceutics that may be useful for treating the disease. We will use a high throughput approach to screen for small molecules that suppress the phenotype of a S. cerevisiae model of SDS. Our hypothesis is that this screen will identify classes of compounds that can provide insight into the genetic pathways affected in SDS, and to the function of SBDS. Additionally, we hypothesize that some of these compounds will improve bone marrow function in SDS patients and will therefore be therapeutically useful. -
Modulators of STAT transcription factors for the targeted therapy of cancer
FRANK, DAVID A
DANA-FARBER CANCER INSTITUTE
1 X01 MH079826-01
4
DESCRIPTION (provided by applicant): Modern cancer therapy is hampered by inadequate efficacy and excessive toxicity. This reflects the fact that chemotherapy is based on the use of non-specific cytotoxic agents that kill cells indiscriminately. To develop the next generation of targeted cancer therapies, it is important to identify specific pathways that are activated inappropriately in cancer cells, and whose inhibition in normal cells causes little effect. Increasing evidence has pointed to a family of transcription factors called STATs that fulfill these criteria. In particular, there is evidence that three family members, STAT1, STAT3, and STAT5, function inappropriately in a number of human cancers including cancers of the lung, breast, prostate, ovary, and pancreas, among others. Furthermore, there is evidence that STATs can be modulated therapeutically to decrease cellular proliferation and enhance apoptosis in cancer. The challenge then, is to identify small molecules that can target these pathways specifically. To achieve this, we have developed cell-based assays to identify modulators of each of these pathways. These assays are based on the expression of a luciferase reporter gene that is activated by each of these transcription factors. We have developed these assays to be robust and reproducible, and we have tested them on a number of small chemical libraries in 384 well format. Thus far, this has led to the identification of several modulators of STAT1 and STAT3 that have beneficial effects in cancer models. Given the success of these systems, we now propose to collaborate with the Molecular Libraries Screening Centers Network to screen libraries orders of magnitude greater in size to identify active compounds. The goal will be to use these molecules as tools to understand the role of STAT signaling in cancer pathogenesis and to rapidly evaluate them for use in clinical trials for patients with cancer. Furthermore, since controlled regulation of STATs may be applicable to the therapy of infectious, autoimmune, ischemic, and neurodegenerative diseases, STAT modulators identified from this approach may have widespread medical usefulness. -
HTRF screen for identifying SRC-1 selective agonists of PPARgamma
GRIFFIN, PATRICK R.
SCRIPPS RESEARCH INSTITUTE
1 X01 MH079861-01
4
DESCRIPTION (provided by applicant): PPAR?, a ligand-dependent transcription factor and member of the nuclear receptor superfamily, is the molecular target of the glitazones. These compounds have been shown to improve muscle insulin resistance, a symptom or cause of type II diabetes, and are referred to as insulin sensitizers. The glitazones are widely prescribed in the type 2 diabetes population. However, these drugs have limited utility in use for mild insulin resistance or in patients with history of cardiovascular disease (CVD) due to specific receptor mediated side affects associated with the glitazones, such as weight gain, fluid retention, and plasma volume expansion. In addition, recent data indicates that TZD based PPAR? agonists have carcinogenic potential in rodents which has prompted the FDA to require the completion of two-year carcinogenicity studies in rodents prior to the use of any new PPAR? modulator in phase II studies. The main focus of this MLSCN proposal is to identify coactivator-selective modulators of PPAR?, and to use these modulators as chemical tools to generate desired functional response. Ligand interaction with PPAR? dictates dissociation of corepressors and association of coactivators thereby regulating specific target gene transcription. Our goal is to develop relationships of the structure of PPAR? ligand modulators with their coactivator recruitment selectivity (SAR) and obtain PPAR? agonists with specific coactivator preference. We have developed a validated HTRF assay for ligand dependent recruitment of the coactivator to PPAR? for a large scale HTS for identifying coactivator selective agonists of the receptor. The results obtained from this proposal will provide a molecular insight into how coactivator recruitment modulates PPAR? activation and shed light on the role of specific coactivators in the pharmacological behavior of PPAR? modulators. -
High Throughput Screening for Cocaine Antagonists
GU, HOWARD H
OHIO STATE UNIVERSITY
1 X01 MH079819-01
4
DESCRIPTION (provided by applicant): Cocaine abuse and addiction continue to be a problem around the world. Currently, there are no effective pharmacological treatments for cocaine addiction. Neurotransmitters are chemical messengers released by nerve cells to communicate with other cells. Neurotransmitter transporters reuptake and recycle the released transmitters and thus play a crucial role in regulating neurotransmissions. Cocaine binds and inhibits the transporters for neurotransmitters dopamine, serotonin and norepinephrine. Ample evidence suggests that cocaine inhibition of dopamine transporter (DAT) primarily mediates cocaine’s rewarding and addictive effects. Compounds that antagonize cocaine actions have been long sought for their great potential as effective drugs to treat cocaine addiction. There have been significant efforts in the search for cocaine antagonists. Several classes of compounds have been found to antagonize cocaine binding to DAT, but they are DAT inhibitors themselves and they do not antagonize cocaine’s action of inhibiting DAT function. We propose a different screening procedure, which is to screen directly for compounds that antagonize cocaine inhibition of DAT function by high throughput screening. In follow up studies, we will characterize the hit compounds and improve their ability to counter cocaine actions in cultured cells and in animals. If successful, this project may lead to the development of the first specific pharmacological treatment for cocaine addiction. -
EphB4-Ephrin-B2 Antagonists for Angiogenesis and Carcinogenesis Inhibition
KUHN, PETER
SCRIPPS RESEARCH INSTITUTE
1 X01 MH079857-01
4
DESCRIPTION (provided by applicant): Eph receptor tyrosine kinases and their ephrin ligands are linked to angiogenesis and tumorigenesis. Recent evidence shows that agonizing EphB4 signaling or antagonizing ephrin-B2 signaling leads to an inhibition of angiogenesis in vivo. Since many tumor cell types overexpress EphB4, modulation of the EphB4-ephrin-B2 interaction would slow tumor growth by restricting the blood supply. We have expressed and purified the human EphB4 ligand-binding domain (LBD) as well as its cognate ligand, ephrin-B2 (extracellular domain). We have also determined the X-ray co-crystal structure of this complex, as well as the complex structure of EphB4 with an EphB4-specific nanomolar affinity peptide which was identified by phage display (see Appendix). Taken together, these structures provide a unique opportunity to understand the specificity and affinity determinants unique only to this Eph receptor. In addition, we have generated EphB4 mutants with altered affinity for both ephrin-B2 and the antagonistic peptide, providing valuable information about specific EphB4 residues critical for receptor-ligand specificity and affinity. While protein and peptide ligands are useful as biochemical tools, small molecule compounds provide a more tractable path towards developing therapeutics that will modulate the EphB4-ephrin-B2 interaction. Using the EphB4-LBD/peptide and EphB4-ephrin-B2 structures as starting models, we have initiated virtual ligand screening experiments to identify compounds capable of modulating the EphB4-ephrinB2 protein-protein interaction. Positive hits have been identified, and commercially available compounds are currently being screened against the EphB4-LBD to determine relative affinities using a fluorescence polarization (FP) assay that is currently performed in a 384-well plate format. To complement our virtual ligand screening experiments, we propose to perform a high-throughput screen to identify EphB4 receptor antagonists. HTS hits will be further characterized by co-crystal structure determination and cell based functional assays (e.g. measuring EphB4 kinase activity in cells expressing EphB4). Improvement of hit compounds would be facilitated by iterative cycles of structure-based compound modification followed by binding assay characterization and co-crystal structure determination. Identification of compounds that bind the EphB4 extracellular ligand binding domain and either agonize the EphB4 signaling cascade, or antagonize reverse signaling through ephrin-B2, will provide a more selective approach to targeting EphB4 compared to identification of inhibitors of their kinase domains. Those compounds have the potential to become valuable antitumorigenic therapeutics. -
Development of Screening Assays for NF-KB-Rel Inhibitors
LIOU, HSIOU-CHI
IMMUNETARGET, INC.
1 X01 MH079841-01
4
DESCRIPTION (provided by applicant): The main stay of immunosuppressive therapy for the past 50 years has been corticosteroids. Steroids are extremely effective in dampening the signs and symptoms of inflammation and the resultant immunopathology in almost all inflammatory disorders. Unfortunately, corticosteroids cause severe systemic side effects that impact almost all organ systems, and which preclude their chronic administration. Therefore, there is a great need for novel immunosuppressive therapeutics that are both safe and efficacious. There have been multiple attempts to develop safer steroids based on the mechanism of action. It is now known that much of the clinical benefit of steroids is due to reduction in pro-inflammatory cytokines, much of it due to reduction in NF-kB/Rel activity, a transcription factor very important in immune activation. Accordingly, one of the new approaches to developing therapeutics mimicking the benefit of corticosteroids is by inhibiting NF-kB/Rel. Twelve years of proof-of-concept research by Dr. Liou (ImmuneTarget Founder) and her team using the c-Rel knockout mouse model have shown that deleting c-Rel ameliorates asthma and a wide spectrum of autoimmune diseases including multiple sclerosis and type I diabetes. The c-Rel knockout mice also support long-term survival of allografts. Most remarkable feature is that deletion of c-Rel in mice does not elicit systemic toxicity or any other readily recognizable abnormality. We hypothesize that NF-kB/Rel is a desirable therapeutic target for the development of safer immunosuppressive medicines that will benefit many diseases including transplantation, inflammation, and autoimmune diseases. Although transcription factors are perceived as non-drugable targets, recent advance in structural biology has suggested the existence of “hot-spots” in many of these targets that are amenable for intervention. As a proof-of-principle for identifying NF-kB/Rel inhibitors, a novel screening strategy not used routinely in the pharmaceutical industry is described here. We have successfully developed a high-throughput screening assay and identified 19 hits (IB series) confirmed by a secondary validation assay. The purpose of this application is to expand the compound classes by screening the comprehensive compound library at the Molecular Libraries Screening Centers Network. ImmuneTarget, Inc intends to further develop NF-kB/Rel based anti-inflammatory therapeutics through lead optimization of the active compounds identified in the project. Aim 1: Procedure for primary screening assay and secondary confirmation assay Aim 2: Biochemical assays to determine in vitro efficacy and selectivity of active compounds Aim 3: Assays and procedure to assess cellular efficacy of active compounds -
High Throughput Screens for Epigenetic Modulators
MARTINEZ, ELISABETH D
UNIVERSITY OF TEXAS SW MED CTR/DALLAS
1 X01 MH079860-01
4
DESCRIPTION (provided by applicant): Over the last several years, a few compounds have been identified that inhibit the methylation or de-acetylation pathways mediated by DNA methyltransferases and histone deacetylases. These compounds have immediate application in the treatment of cancers as they reactivate aberrantly silenced tumor suppressor genes. There is clear need to identify additional small molecules that interact with these enzyme families as well as new targets involved in the epigenetic control of gene expression. Our overall goal is to expand the available repertoire of small molecules that modulate gene expression and to evaluate their basic mechanism of action. Toward this goal, we have recently developed and validated a unique mammalian cell-based system to screen large chemical libraries in 1536-well format. Unlike methods used to date, our image-based assay has the advantage of measuring the exact biological event it seeks to target: the reversal of transcriptional repression in mammalian cells. The assay has been screened in 96-well format against ~3000 NCI compounds that resulted in eight confirmed actives, one of which revealed HDAC inhibitor properties in secondary assays. In collaboration with the NIH Chemical Genomics Center (NCGC), we have miniaturized the assay into 1536-well format and validated it by screening the LOPAC collection in a concentration-titration series, a process developed at the NCGC termed quantitative HTS (qHTS). In an ongoing collaboration with the NCGC, we seek to continue the qHTS of this assay against the Small Molecule Repository to identify novel small molecules and characterize their activity in secondary functional assays. -
Screen for Compounds that Promote Cardiomyocyte Cell Cycle Entry
MERCOLA, MARK
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 X01 MH079847-01
4
DESCRIPTION (provided by applicant): We propose a screen for small molecule compounds that promote cardiomyocyte cell cycle entry. Normally, cardiomyocytes cease cell cycle shortly after birth and show no or negligible replication during childhood through adulthood even in the face of heart muscle damage. Stimulation of cell cycle reentry might be used to expand the number of immature cardiomyocytes derived from fetal, neonatal or stem cell sources for research or clinical application. Thus, small molecules that promote cardiomyocyte cell cycle would be useful reagents and as probes of the cellular constituents that block entry into cell cycle. The assay is based on rat neonatal ventricular cardiomyocytes, which normally cease dividing 2-3 days after birth. Cells will be isolated from the rat neonatal hearts and used to screen for compounds that stimulate entry into cell cycle as visualized by immunostaining with anti-Ki67, which marks S, G2, and M phase cells. The secondary, counter assay used to verify specific activity will be to score entry into S phase by increased incorporation of propidium iodide as measured by flow cytometry. Follow up experiments are described that will evaluate: 1) an effect on the cell cycle regulatory machinery, 2) specificity for cardiomyocytes, 3) whether treatment reversibly or irreversibly blocks the ability of the cardiomyocytes to continue terminal differentiation into mature cardiomyocytes, 4) when (or whether) prolonged replication of the cells leads to cellular senescence, and 5) function of treated cardiomyocytes upon transplantation and function of the probes on transplanted cardiomyocytes. -
Identification of Compounds that Modulate Human Deubiquitinase Function
NICHOLSON, BENJAMIN
PROGENRA, INC.
1 X01 MH079852-01
4
DESCRIPTION (provided by applicant): In the USA, cancer is the second leading cause of death, with one in every four people dying from this disease. Recently it has been shown that the metabolic enzyme fatty acid synthase plays a role in the development of prostate cancer and that the fatty acid synthase levels are decreased when the isopeptidase USP2a is inhibited. It is proposed to look for novel inhibitors of USP2a thus enabling specific reduction in fatty acid synthase levels, selective killing of prostate cancer cells, and ultimately, effective treatment of prostate cancer. Progenra proposes to transfer its assay for USP2a to the NIH for high throughput screening at MLSCN. It is hoped that compounds will be identified that will give rise to anticancer therapeutics. -
Molecules that Enhance CREB Activity
NIRENBERG, MARSHALL WARREN
U.S. NATIONAL HEART LUNG & BLOOD INS
1 X01 MH079867-01
4
DESCRIPTION (provided by applicant): CREB is a transcription factor whose activation enhances some types of long-term memory. The goal of this project is to screen a large library of compounds in a high throughput format to find compounds that enhance CREB activation mediated by forskolin activation of adenylyl cyclase, but have no effect on CREB in the absence of forskolin. Compounds found are candidate compounds that may stimulate the establishment of certain types of long-term memory. CHO-K1 cells stably transfected with a vector that contains a CRE-a-lactamase reporter gene will be used. A low concentration of forskolin partially activates adenylyl cyclase, catalyzing cAMP synthesis, which activates cAMP-dependant protein kinase, which catalyzes phosphorylation and activation of CREB, which binds to the CRE nucleotide sequence in DNA, thereby inducing the transcription of the a-lactamase reporter gene. a-Lactamase catalyzes the cleavage of a cephalosporin derivative, CCF4, that contains coumarin and fluorescein moieties separated by a a-lactam ring. Excitation of the coumarin donor at 409 nm leads to fluorescence energy resonance transfer and emission of green light by the fluorescein acceptor. a-Lactamase catalyzed hydrolysis of the substrate separates the donor and acceptor. The donor coumarin moiety then emits blue fluorescence when excited while the fluorescein acceptor is quenched. Positive compounds found are candidate drugs that can be studied further to determine whether they enhance long-term memory in mice and man. -
Cell-Based Screening for Anti-Angiogenic Agents
QU, ZHICAN
SOUTHERN RESEARCH INSTITUTE
1 X01 MH079851-01
4
DESCRIPTION (provided by applicant): Angiogenesis is a process of new blood vessel formation. Imbalance in this process contributes to numerous malignant, inflammatory, and immune disorders. In adult, blood vessels remain stable and endothelial cells are not usually activated. During angiogenic process, endothelial cells become activated, leading to new blood vessel formation. Targeting endothelial cell activation associated with pathological conditions is an approved strategy of therapeutic treatments for cancer and blindness. We propose a high throughput screening (HTS) with a cell-based assay using human endothelial cells and normal human fibroblasts for a broad search of small molecular inhibitors with selective biological activity in activated endothelial cells. Proof-of-concept experiments in our preliminary study have successfully demonstrated the proposed approach. First, the endothelial cell-based assay for robotic screening has been optimized and validated with reliability and reproducibility during normal HTS running conditions. Second, a control compound, targeting VEGF receptor and other kinases, has shown differential inhibition activity in endothelial cells in comparison to fibroblasts, demonstrating that a secondary screening against fibroblasts is able to assess selectivity of the HTS primary hits. Small molecular probes that selectively interact with biological targets of activated endothelial cells are important research tools for understanding the mechanisms of angiogenesis. With the MLSCN mission, we will share the data generated from this proposed high throughout screening project to facilitate the development of anti-angiogenic therapeutics with the long-term goal of benefiting public health. -
High Throughput Screening Assay for Hsp70 Inhibitors
REED, JOHN C
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 X01 MH079863-01
4
DESCRIPTION (provided by applicant): Hsp70 consists of two domains, an N-terminal ATP-binding domain, and a C-terminal peptide-binding domain, which function in a coupled manner to facilitate folding of protein substrates through cycles of ATP binding, ATP hydrolysis, and ADP/ATP exchange. The 3-dimensional structure has been determined for the full-length Hsc70 protein and for the ATP-binding domain of Hsp70, providing a foundation for drug discovery. Here we describe generation and validation of a Fluorescence Polarization Assay (FPA) for use in high throughput screening (HTS) to identify chemical inhibitors of Hsp70 that compete for binding at the ATP site. We propose to use this screen to identify chemical antagonists of Hsp70 and Hsc70 within the NIH chemical library. Various downstream counter-screens and secondary assays will be employed to further characterize the selectivity of the hits, setting the stage for subsequent compound structure activity relation (SAR) studies and laying a foundation for chemical optimization. -
MLSCN Screen of the Amyloid precursor protein 5′untranslated region
ROGERS, JACK T
MASSACHUSETTS GENERAL HOSPITAL
1 X01 MH079854-01
4
DESCRIPTION (provided by applicant): The 5′untranslated region of the mRNA for the Alzheimer’s Amyloid Precursor Protein (APP 5′UTR) is a key translational regulatory element that sets the amount of APP production in any given cell. IL-1 enhances the interaction between Iron-regulatory Proteins (IRPs) and APP 5′UTR RNA secondary structure via an Iron-responsive Element (IRE). This regulation suggested the APP 5′UTR is an excellent drug target. We established APP 5′UTR constructs and stable cell lines available for screening small molecules from the MLSCN library of 500,000 small molecules. Our screening strategy to identify APP 5′UTR drug hits was conducted for our FDA and LDDN library screens, and now to be adapted for this NIMH solicitation. As prior validation, we screened an FDA mini-libary for APP 5′UTR directed inhibitors of a luciferase reporter gene and identified 17 leads, including paroxetine and metal chelators. These FDA leads limited APP 5′UTR driven translation using transiently transfected neuroblastoma cells. Secondary western blot-based assays showed that paroxetine (but not other SSRIs such as fluoxetine) and dimercaptopropanol and desferrioxamine (not other metal chelators) selectively reduced APP holoprotein expression (and amyloid secretion). As proof that the APP 5′UTR screen could generate drug selectivity, these two hits did not change APLP-1 and APLP-2 expression in SH-SY5Y cells (Payton et al., 2003). Paroxetine reduced the amyloid burden in a transgenic mouse model for AD (TgCRND8 mice) (Tucker et al., (2005/2006). Our laboratory has constructed parallel stable cell lines that express luciferase reporter genes translationally driven by the 5′UTRs of other target proteins relavant to neurodegenerative diseases, including alpha synuclein (Parkinson’s disease) and Prion protein (PrP, mad cow disease). These reagents will ensure target selectivity for the APP 5′UTR but can also be used for performing further primary screens of the 500,000 NIH compounds. In the pipeline these hits may well identify future Prion and alpha-synuclein 5′UTR specific inhibitors relevant to expresion of PrP (Jccob Creutzfeldt-Jacob disease) and alpha synuclein (PD). -
High throughput screens for targets of the proprotein convertases Furin, PC5, SKI-1 and PCSK9 using cell-based assays
Seidah, Nabil G.
Clinical Research Institute of Montreal
1 X01 MH080376-01
4
DESCRIPTION (provided by applicant): There is ample evidence that the protease Furin plays a key role in cancer/metastasis, dyslipidemias, and viral infections3. Inhibitors of this enzyme will find numerous applications in the treatment of these and other pathologies. Our general goals involve the detailed definition of the physiological roles of secretory proteolytic enzymes that are selective for pairs of basic amino acids, and are involved in the processing of various precursors implicated in health and disease. The targeted enzyme Furin is involved in proliferative, cardiovascular, viral infections, and neurodegenerative disorders. The main hypothesis of this group is “Furin is central to many physiological processes and dysregulation of this enzyme results in pathology”. Thus, we are applying to the NIH for high throughput drug screening to obtain specific inhibitors of Furin for applications in cancer/metastasis, dyslipidemias, and viral infections. -
Promiscuous and Specific Inhibitors of B-lactamase
SHOICHET, BRIAN K.
UNIVERSITY OF CALIFORNIA SAN FRANCISCO
1 X01 MH079825-01
4
DESCRIPTION (provided by applicant): High-throughput screening (HTS) is the dominant technique in early phase drug discovery and has had an important impact on human health. Two key problems in HTS are the occurrence of false-positive, promiscuous hits, and the choice of molecules to screen. One of the most common mechanisms underlying promiscuous inhibition is compound aggregation. In the first aim, we investigate how many of the DPISMR screening collection form promiscuous aggregates. The second aim tests the ability of virtual screening to predict the specific inhibitors found in the HTS campaign, directly comparing the two techniques. The screen for promiscuous and specific inhibitors can be accomplished at the same time, making the second aim “free”, requiring only a docking calculation (to be done at UCSF). The specific aims are: 1. To determine the number of DPISMR Library compounds that act as promiscuous inhibitors. We have recently developed a high throughput assay to identify molecules that form promiscuous aggregates, using enzyme inhibition. Compounds that inhibit a-lactamase in the absence but not the presence of detergent are likely aggregators. This assay was developed on 1030 drug-like compounds, here we extend it to the 70,000 compounds in the current NIH molecular library collection. The most important question is how many of these inhibit through promiscuous aggregation? Answering this question will flag promiscuous inhibitors in this widely-used screening collection, which alone will have considerable impact. It will also establish a robust assay for use by other HTS groups on other collections. 2. To compare docking hit rates with HTS hit rates. The HTS assay affords us an unprecedented opportunity to compare high throughput with virtual screening, an issue that has received much recent attention. We will prospectively dock the full DPISMR set against the x-ray structure of a-lactamase and compare the docking hits to those subsequently found by HTS in the presence of detergent, which should return specific inhibitors. We will ask: which hits does docking capture, what are the false positives and the false negatives? We can also investigate classes of compounds that the HTS misses owing to a restricted screening deck. We will dock 650,000 commercially available “lead-like” compounds, testing several tens of docking hits. Can docking “fill-in” regions of chemical space necessarily overlooked by any given screening deck? Of interest to both techniques, all hits will be followed up with x-ray structures. -
HTS for BCI-2 Family Multiplex
SKLAR, LARRY A.
UNIVERSITY OF NEW MEXICO ALBUQUERQUE
1 X01 MH079850-01
4
DESCRIPTION (provided by applicant): Apoptosis is governed in part by Bcl-2 family proteins. The human genome contains six genes that encode anti-apoptotic Bcl-2 family members. Each of these proteins can be bound to endogenous proteins that contain a conserved peptidyl domain called the Bcl-2 homology region 3 (BH3). Pro-apoptotic family members include both multidomain proteins, including Bak, and “BH3-only” proteins, including Bim. As proof of concept, proapoptotic BH3 peptides that dock at this site in Bcl-2 and Bcl-XL also increased apoptosis of leukemia and lymphoma cells in culture and in SCID mice (Holinger et al., 1999; Wang et al., 2000; and Walensky et al., 2004). The binding of fluorochromeconjugated BH3 peptides to Bcl-2 family proteins thus provides the basis for construction of fluorescence assays, suitable for high throughput screening (HTS). The Burnham team has described two fluorescent peptides, F-Bim and F-Bak that bind to six and four members of the Bcl-2 family, respectively. The Burnham team has already devised procedures for producing multi-milligram quantities of purified recombinant proteins and devised a generic fluorescence polarization assay (FPA), using F-Bim (Zhai et al., 2006). A preliminary screen has been performed of ~10,000 compounds with Bfl-1 and F-Bim, demonstrating the suitability of this homogeneous assay for the high-throughput environment. The UNM team has taken advantage of the reagents prepared by the Burnham team and developed a multiplex analysis by HyperCyt high throughput flow cytometry. The results from assays performed one at a time are shown in the table. We have shown that all six members of the family can be screened simultaneously. Potential advantages of such an approach include single step analysis of specificity and selectivity, small reaction volumes and the discrimination of free and bound assemblies that is conservative with respect to reagent usage as compared to fluorescence polarization assays. The comparison of such an important assay across different platforms is likely to have significant impact on the Molecular Library Screening Center Network. This project proposes a high throughput flow cytometry screen for inhibitors of the Bcl-2 family of six proteins with the peptides BIM and BAK. The multiplex capability of flow cytometry will allow all six proteins to be evaluated simultaneously against each peptide. The screen is proposed to be carried out as a collaboration between two molecular libraries screening centers in San Diego and New Mexico. -
Synthetic Lethal Screen for Compounds to Kill Cancer Cells with p53 Mutation
SUN, YI
University of Michigan-Ann Arbor
1 X01 MH079844-01
4
DESCRIPTION (provided by applicant): Tumor suppressor gene p53 plays a key role in suppression of tumorigenesis and is most frequently inactivated during human carcinogenesis. Our long range goal is to discover and develop a novel class of small molecule drugs that selectively kill cancer cells harboring a p53 mutant. The objective of this application is to gain the access to the MLSCN (Molecular Libraries Screening Centers Network) HTS (High Throughput Screening) and chemical optimization resources to identify and characterize small molecules that induce the synthetic lethality to p53 mutation in lung cancer cells. Our central hypothesis is based upon the synthetic lethal concept. Synthetic lethality is a situation where a cancer mutation (e.g., p53 mutation) and a drug that inhibits a targeted protein, together cause the death of cancer cells. A p53 mutation itself is non-lethal, nor a drug, but put them together, the cancer cells die. Thus identified synthetic lethal compound should preferentially kill the p53 mutant cancer cells with a minimal toxic effect on normal cells. Specific aims are 1) to identify small molecules that selectively kill human cancer cells with a mutant p53 via synthetic lethal mechanism through the screening of 100,000 compounds in the Small Molecule Repository through MLSCN with SAR (Structure Activity Relationship) follow-up and 2) to validate the hits, using both in vitro and in vivo biological assays in a variety of lung cancer cells with p53 mutations as well as in normal counterparts. The screening assay has been developed in our laboratory in a 96-well cell based format and has been miniaturized into 1536-well plate format in a collaboration with Dr. Wei Zheng at the NIH Chemical Genomics Center (NCGC). The proposed screening will be conducted at the NIH Chemical Genomics Center using H1299-p53A138V cells. The p53-null H1299 lung cancer cells were transfected with a temperature sensitive p53 mutant (p53-A138V). The transfected cells adopt a mutant p53 conformation when grown at 39 ‘C and a wild type conformation at 32 ‘C. The primary screening will be conducted in the 1536-well format using H1299-p53A138V cells grown in both 39 xC and 32 xC with seven drug concentrations to generate an IC50 curve. The compounds that kill the mutant p53 cells with an efficacy 5 to 10-fold higher than the wild type p53 cells will be counter-screened with H1299-Neo control cells grown at both 39 ‘C and 32 ‘C to filter out false positive associated with temperature shifting. Identified hits (with an IC50 of 5 to 10-fold lower in H1299-p53A138V cells, but less than 2-fold difference in H1299-Neo cells at 39 ‘C vs. 32 ‘C) will be optimized with SAR study in collaboration with Dr. Wei Zheng. The optimized hits will then be further validated in Dr. Yi Sun’s laboratory using various biological assays. Upon successful completion of this proposed study, we expect to identify and validate a novel class of small molecular compounds that selectively target mutant p53-containing cancer cells via synthetic lethal mechanism. Given the fact that p53 is mutated in more than 50% of human cancers and cancer cells harboring a p53 mutation are often more resistant to current cancer therapies due to the lack of p53 induced apoptosis, the application of synthetic lethal screening strategy to identify chemical compounds that target mutant p53-containing human cancer cells is of significant importance in mechanism-driven anti-cancer drug discovery & in identification of p53 synthetic lethal targets. -
Ligand Discovery for Thyroid-Stimulating Hormone Receptor (TSHR) by Virtual and H
GERSHENGORN, MARVIN C
U.S. NATIONAL INST DIABETES/DIGST/KIDNEY
1 X01 MH080680-01
5
DESCRIPTION (provided by applicant): Ligand discovery for thyroid-stimulating hormone receptor (TSHR) by virtual and high- throughput screening Seven transmembrane-spanning receptors (7TMRs) are a large family of related cell surface proteins that are potential targets for therapeutic intervention in human disease. Thyroid- stimulating hormone receptor is a member of the glycoprotein hormone receptor subfamily of 7TMRs. This project seeks to identify ligands for TSHR through high-throughput screening (HTS) of the NCGC library. The HTS will assess both agonist and antagonist activity in a single plate by initially adding the library compound, measuring activity and then adding native agonist TSH and again measuring activity. The cell-based assay utilizes the ACT:one cell line stably- expressing the TSHR and has been evaluated successfully at the NCGC. Ligands for TSHR identified in the HTS may have therapeutic potential. Agonists could replace costly recombinant glycoprotein hormone TSH in screening for thyroid cancer while antagonists might be effective for treatment of hyperthyroidism resulting from autoimmune disease or TSHR-activating mutations. Possible lead compounds identified by HTS will be chemically modified where feasible to explore structure-activity relationships with the objective of enhancing efficacy and/or potency. While ligand discovery for 7TMRs has mostly relied on HTS, more rational approaches that could streamline identification of high affinity ligands would be beneficial. Because a crystal structure is available for only bovine rhodopsin in the 7TMR family, structural information for 7TMRs is limited to homology modeling and refinements based on structure function relationships or mutational analysis. This application proposes to use several computer- based virtual screening strategies to predict which compounds from the NCGC chemical library will bind to TSHR. Virtual screening for TSHR will rely on a combination of receptor-based and ligand-based methodologies using a binding pocket within the transmembrane region that has been identified and characterized using an agonist with low micromolar potency. The virtual screening results will be compared with results from HTS of the same library with the TSHR target. The HTS results will enable evaluation of the effectiveness of virtual screening approaches and may suggest refinements to virtual screening methodologies that will lead to better agreement with HTS. These studies may identify new compounds with potential for therapeutic modulation of TSHR activity and will provide unique opportunities to evaluate and optimize virtual screening methodologies that may be broadly applicable to the discovery of new ligands for 7TMR targets. Ligand discovery for thyroid-stimulating hormone receptor (TSHR) by virtual and high- throughput screening. This project aims to identify small drug-like molecules that will activate (agonists) or inhibit (antagonists) the activity of the thyroid-stimulating hormone receptor (TSHR). Thyroid cancer is the most common endocrine cancer and a small TSHR agonist could replace the use of costly recombinant TSH in diagnostic tests for thyroid cancer. TSHR antagonists identified in this study may lead to clinically useful therapies for hyperthyroidism mediated either by autoantibodies (Graves’ disease) or by activating mutations in TSHR that in total affect over 3 million Americans. -
Identification of Target-Specific
Antimicrobials
HASELBECK, ROBERT JOHN
RX3 PHARMACEUTICALS, INC.
1 R03 MH079846-01A1
5
DESCRIPTION (provided by applicant): The emergence of bacterial strains resistant to most of the current antibacterial agents has a significant negative impact on public health. To combat bacterial resistance, new classes of antibacterial agents are needed that have expanded spectrum that includes the resistant strains. Particularly attractive are new classes of antibacterial drugs that inhibit novel bacterial targets. The potential benefits of these drugs are increased antibacterial potencies, expanded spectrum and significantly reduced frequencies for the emergence of new resistant strains. Rx3 Pharmaceuticals has developed a highly effective and experimentally facile method for MOA detection of antimicrobial hit compounds against the biowarfare pathogen B. anthracis. We have shown that we can apply this assay to hit compounds derived from high-throughput screens to determine their target specificity. Knowledge of the MOA of a hit compound enables SAR and SBDD efforts to improve potency and drug like properties. Quick identification of such lead-potential antimicrobial compounds within the MLSCN library will greatly accelerate the overall development of new antimicrobial drugs. Moreover, cross-correlation of these findings to information derived from other screens, such as mammalian cell toxicity, could greatly facilitate the prioritization of those compounds with the greatest potential to become the next clinically useful antibiotic against B. anthracis and other important bacterial pathogens. The goal of this work is identify novel leads for antibacterial drugs to address the rising threat of antibiotic- resistant infections and the potential threat of biowarfare bacterial pathogens. To accomplish this, we propose to screen the MLSCN compound collection for antibacterial compounds and then identify those compounds that act through a novel mechanism. -
A Screen for Small Molecule Compounds that Inhibit Bacterial Toxins
HASLAM, DAVID B.
Washington University
1 R03 MH081267-01
5
DESCRIPTION (provided by applicant): Shiga toxin (Stx), cholera toxin (Ctx) and ricin are important mediators of human disease and are potential agents of biowarfare. These toxins follow a complex intracellular pathway in order to kill susceptible cells. After binding to cell surface molecules, these toxins are internalized and trafficked in retrograde fashion through the Golgi to the endoplasmic reticulum (ER). From the ER lumen, the toxins must gain access to the cytoplasm, where they carry out their cytotoxic activities. This recently discovered pathway likely represents a recycling and cellular quality control mechanism that has as yet been poorly characterized. The retrograde transport pathway, exploited by toxins of widely different origin, is an attractive target for therapeutic intervention. However, the mechanisms of retrograde traffic and the host molecules involved are largely unknown. We are employing a small molecule screen for inhibitors of Stx susceptibility as a tool to dissect the components of toxin transport pathways. We have developed several assays to examine the effects of inhibitory compounds on retrograde and forward (secretory) transport. The goal of this proposal is to identify several more compounds that, among them, inhibit toxins at multiple sites in their transport from plasma membrane to ER lumen. Our assay is based on luciferase activity as a reporter for toxin resistance. Cells are transduced with an adenoviral construct expressing luciferase that has been modified to be degraded rapidly by the proteasome. Exposure to Stx, which inhibits protein synthesis, results in markedly diminished luciferase content. Compounds that inhibit Stx transport or activity result in a rescue of the luciferase activity and are readily detected in a luminometer. A counterscreen, consisting of incubation with cycloheximide rather than Stx, was incorporated to exclude false-positive hits. The utility of the screen and counterscreen were validated at the ICCB facility, where we found z and z’ scores to be > 0.6 and demonstrated the ability to exclude compounds with nonspecific effects on luciferase activity. Based on our previous round of screening we anticipate identifying at least 50 inhibitory compounds from a screen of 100,000 molecules. We will follow up on these compounds by determining their potency, specificity, toxicity, reversibility, and a preliminary assessment of their effect on toxin trafficking in vitro. Based on the results of this analysis, the compounds will be prioritized for their inclusion into studies that will identify the site of toxin inhibition and the intracellular targets of inhibitory compounds. Investigations proposed here will identify potent and specific inhibitors of toxin trafficking. These compounds will be used as probes to identify trafficking pathways and host molecules involved in various stages of toxin transport. In future experiments, some of these compounds will be optimized for potential use as therapeutic agents. Shiga toxin, cholera toxin and ricin are important agents of human disease and are recognized as potential agents of biowarfare. These and other toxins follow a complex intracellular pathway in order to kill susceptible cells. This transport pathway is an attractive target for therapeutic intervention. We are employing a small molecule screen for inhibitors of shiga toxin action. Compounds with activity against shiga toxin will be examined for their ability to inhibit ricin and cholera toxin. The compounds will then be characterized with respect to their potency, toxicity, reversibility, and mechanism of action. The resulting compounds will be valuable tools to dissect toxin and vesicle trafficking pathways. In the final aim, the host targets of inhibitory molecules will be identified. Over the long term, compounds that inhibit toxin trafficking and display minimal toxicity in vitro will be explored as potential therapeutic agents. -
Modulation of Alternative Splicing by HTS Identified Compounds
KOLE, RYSZARD
UNIVERSITY OF NORTH CAROLINA CHAPEL HILL
1 X01 MH080684-01
5
DESCRIPTION (provided by applicant): The long-term goal of this project is to identify compounds that modulate splicing of target genes. This is important because 70% of all human genes are alternatively spliced. Moreover, incorrect splicing is frequently associated with diseases ranging from inherited disorders to cancer. The focus of this application is restoration of correct splicing of aberrantly splice pre-mRNAs, which carry thalassemic splicing mutations in human a-globin gene. The aims of this project are: 1) To screen ~70, 000 compounds using HeLa EGFP-654 cell line as a test system. These cells stably express a modified, alternatively spliced enhanced green fluorescent protein (EGFP) gene, which is interrupted by a human a-globin intron 2 with a thalassemic splicing mutation (EGFP- 654). The cells produce EGFP only if correct splicing of the EGFP-654 pre-mRNA is restored. 2) To verify by several independent methods that the positive hits identified in Aim 1 are indeed modifying splicing of the target RNA. These experiments will identify clinically interesting compounds and also will shed some light on the mechanism and sequence specificity of the effects. In future experiments, the most effective hits will be tested in vivo in transgenic mouse models, which express EGFP-654 transgene, and in thalassemic knock-in mice, which express IVS2-654 a-globin gene in their bone marrow. These mouse models are available and currently in use in the PI laboratory. The progression from initial screen to validated drug-like compounds and tests in clinically relevant animal models is an important strength of this application. Equally importantly, the methodology developed under this proposal will be applicable to other alternatively spliced and disease associated genes. By screening close to 100,000 compounds at the state of the art NIH MLSCN facility we anticipate to identify small, drug-like molecules that control alternative splicing, an important cellular process, the defects of which are associated with disease. Because most of human genes are alternatively spliced, developed methods will be applicable to a large number of gene targets. This project will focus on compounds that may be useful as therapeutics for an orphan disease, thalassemia. -
Screening C. Albicans Biofilms for Lethal Potentiators of Miconazole
LEWIS, KIM
NORTHEASTERN UNIVERSITY
1 X01 MH080686-01
5
DESCRIPTION (provided by applicant): The long-term goal of this project is to develop an effective therapy against relapsing vaginosis caused by Candida albicans. The disease occurs in 8-9% of all women, and is recalcitrant to currently available therapies. Formation of highly drug tolerant biofilms by C. albicans apparently contributes to the recalcitrance of the disease. The goal of this screening project is to identify compounds that will be subsequently developed into leads for the development of a therapeutic capable of sterilizing a yeast infection. We have developed a screen for potentiators of miconazole, a standard therapy against vaginosis. A pilot screen of 5,000 compounds produced a compound, AC9 that completely eradicates both planktonic and biofilm populations of C. albicans in combination with miconazole. Neither AC9, nor miconazole alone have any activity against the biofilm cells. This preliminary finding provides proof-of-principle for the proposed approach. AC9 acts at a rather high concentration, and the aim of this screening proposal is to use a large library to identify highly active potentiators of miconazole. Once hits are identified, they will be verified by retesting under the same conditions. Confirmed hit compounds will then be tested for direct activity. Those that do not show direct activity will be tested in a secondary confirmatory assay for their ability to kill C. albicans in the presence of miconazole. Compounds that pass this validation test will become early leads. The leads will then be tested for cytotoxicity. Subsequent lead development, including medicinal chemistry optimization, will ultimately lead to an antifungal therapeutic capable of sterilizing an infection. C. albicans is the major human fungal pathogen that forms essentially untreatable biofilm infections. This includes relapsing fungal vaginosis which afflicts 8% of all women and is recalcitrant to current therapies. This project will develop lead compounds that will form the basis for an effective therapeutic against vaginosis. -
A HTS Assay for Identifying Anti-Viral Drugs Against Arbovirus Infections
LI, QIANJUN
SOUTHERN RESEARCH INSTITUTE
1 R03 MH081271-01
5
DESCRIPTION (provided by applicant): In this proposal, we propose to transfer our HTS-ready, florescence based caspases-3/7 assay to identify novel anti-viral drugs against arboviruses infection based on the different host apoptotic responses in BTV infection model system. Arthropod borne viruses (arboviruses) are important human and/or animal pathogens that cause acute virus infections with severe diseases and/or death. Several recent human and/or animal epidemics are caused by arboviruses, including Dengue virus (DNV) in Asia, West Nile virus (WNV) in North America and Bluetongue virus (BTV) in Europe. Arboviruses are unique because they are transmitted to their vertebrate hosts by arthropod vectors, therefore, they must be capable of replicating in two very divergent host taxa–vertebrates and insects. Although arboviruses replicate efficiently in both vertebrate and insect cells, the respective host cellular responses are quite different. For example, BTV infection induces a rapid apoptotic response in vertebrate cells, whereas such apoptotic response is unapparent in insect cells, despite the productive virus replication in both host cells. While apoptosis has been positively linked to arbovirus diseases in infected animals/human, insects show no detectable signs of any diseases. Little attention has been paid to the significance of such different host cellular responses and the possibility of adapting such strategy for drug discovery to the protection of vertebrate hosts. We hypothesize that compounds preventing BTV-induced apoptosis in vertebrate cells could act either via inhibiting apoptosis or via interfering viral life-cycle. A secondary assay will allow us to separate anti-viral hits from the apoptosis inhibitors. These anti-viral compounds could presumably also protect host cells from host apoptotic response induced by other arboviruses, including DNV and WNV. Our long term goal is to develop new prevention and control measures for arbovirus diseases in human. The objectives of this application will be achieved by carrying out the following Specific Aim: To screening designated compound library using the developed efficient HTS assay in the BTV infection model in vertebrate cells. Based on our preliminary data showing that BTV-induced apoptosis in vertebrate cells via intrinsic apoptotic pathway, an efficient HTS assay have been designed, developed, optimized and validated using this model system. We propose to transfer this assay to the designated screening center to reproduce, miniaturize and automate the assay. A secondary assay using apoptosis inducers including Staurosporine will also be implemented to confirm hits and exclude false positives including apoptosis inhibitors. The possible mechanism of these hits will also be examined to prioritize these hits for further investigations against DNV and WNV infection. Arthropod borne viruses (arboviruses) are important human and/or animal pathogens that cause acute virus infections with severe diseases and/or death, several arboviruses cause recent human or animal epidemics, including Dengue virus in Asia, West Nile virus in North America and Bluetongue virus in Europe. In this proposal, we proposed to transfer our HTS-ready, florescence based caspases- 3/-7 assay to identify novel targets against arboviruses infection based on the different host apoptotic responses in arbovirus infection to protect hosts from arboviruses infections, including Dengue virus and Bluetongue virus. Our long term goal is to develop new prevention and control measures for arbovirus diseases in human and animals. -
Small Molecule Inhibitors of Cilia
MARSHALL, WALLACE
UNIVERSITY OF CALIFORNIA SAN FRANCISCO
1 R03 MH081217-01
5
DESCRIPTION (provided by applicant): We have developed a simple and reliable assay to identify small molecule inhibitors of cilia assembly and function. Cilia are sensory and motile organelles that play critical roles in development and disease, including polycystic kidney disease, hydrocephalus, syndromic obesity, chronic sinusitis, bronchiectasis, and retinal degeneration. Small molecule modulators would provide critical tools for studying dynamic events in cilia, and might even point the way to development of pharmacological approaches to ciliary diseases, for which no drugs are currently available. Our assay takes advantage of the model organism Chlamydomonas, which uses cilia to swim to the bottom of an assay well. Because the cells are green, this causes a dark green pellet to self-organize in the well. Mutations or pharmacological treatments which block ciliary motility, or which cause cells to have no cilia, prevent this organization. We have developed image-analysis software that can distinguish these two results in a 96-well format. We have verified the performance of this assay and found it has a Z- value of 0.7 with low plate-to-plate variability and high day-to-day reproducibility. The assay can tolerate DMSO up to 1% and is completely compatible with high throughput robotic methods. We anticipate that a screen using this assay may provide a critical step forward in understanding the mechanisms and treatment of ciliary diseases. Cilia are cellular antennas which project from the surface of most cells in the human body, and sense the surrounding environment. These sensory cilia play critical roles in kidney function, and defects in cilia lead to a devastating disease called polycystic kidney disease. Cilia also play roles in fat metabolism and retina function, and cilia defects can result in genetic obesity and retinal degeneration. Cilia also act like oars to move fluid around cells, and defects in cilia movement can lead to a number of disease symptoms including chronic lung inflammation due to poor mucus clearance, and to hydrocephalus (water on the brain). Currently there are no drugs available to treat diseases, and little is known about how cilia assemble or function. We have developed a new assay for identifying chemical compounds that can affect cilia. These will not only provide important tools for basic research on how cilia work, they would also serve as lead compounds for drug development efforts aimed at treating the large number of ciliary diseases (ciliopathies) that are now known. -
HTS to Identify Small Molecule Inhibitors of Phospho-Specific Protein-Protein Int
NATARAJAN, AMARNATH
UNIVERSITY OF TEXAS MEDICAL BR GALVESTON
1 R03 MH081227-01
5
DESCRIPTION (provided by applicant): Protein-protein interactions form the backbone of nearly all the signaling networks in the cells. BRCA1 is an 1863 amino acid protein that has a phosphoprotein / peptide binding site (hot spot) in its carboxy terminus domains (BRCT). BACH1 and CtIP are two proteins that bind to the BRCT hot spot and these interactions are key for a variety of cellular events such as checkpoint regulation, transcription etc. BRCT domains are riddled with cancer causing mutations and M1775R is one which prevents BACH1/CtIP binding to BRCT. Based on preclinical and retrospective clinical studies there is an emerging theory that suggests that BRCT mutations sensitize cancer cells to DNA damage based chemotherapeutics. We hypothesize that reversible inhibitors that target the hot spot on BRCT and functionally mimic the M1775R mutation will sensitize cells to DNA damage based chemotherapeutics. To test this hypothesis we developed a fluorescence polarization (FP) assay and optimized it to a 384-well format which is ideal to carry out high throughput screening. Here we would like to submit this assay to screen the compounds in the small molecule repository at the MLSCN and identify inhibitors that can be used as probes to dissect the BRCA1 signaling network. We also have developed a second FP assay that probes the protein-protein interaction between ZAP70 – N-cbl. This assay can be used as to counter screen the hits from the BRCA1 FP assay and identify target specific inhibitors. A cell based reporter based dual luciferase transcription assay that is currently available in our laboratory can be used as a secondary assay to test for mechanism specific inhibition. Using this combination of assays we propose to work with the MLSCN to identify and subsequently optimize small molecule inhibitors that can be used as chemical probes to interrogate the transient protein-protein interactions involving BRCA1. BRCA1 is and 1863 amino acid protein that has a binding site for phosphorylated proteins. The BRCA1 mediated protein-protein interactions is part of a complex signaling network. This application describes a set of biological assays that can be used to identify small molecule inhibitors of BRCA1. These inhibitors are valuable chemical probes that can be used to better understand tumorigenesis in patients with BRCA1 mutations. -
Chemical Genetic Screens for Mitochondrial Division and Fusion Inhibitors
NUNNARI, JODI M
UNIVERSITY OF CALIFORNIA DAVIS
1 R03 MH081279-01
5
DESCRIPTION (provided by applicant): We propose to conduct a high through put screen in the Molecular Libraries Screening Centers Network identical to the one that, on a smaller screening scale, has already successfully identified mitochondrial fission and fusion inhibitors. Specifically, we will employ straightforward growth-based assays in S. cerevisiae strains that monitor mitochondrial fission and fusion activity to identify additional small molecule inhibitors. Given our success, we are confident that many novel compounds that will be found. The medical importance of events regulated by mitochondrial membrane dynamics, such as apoptosis, indicates that the chemical genetic approach may also lead to the identification and development of novel therapeutic agents for stroke, myocardial infarction, neurodegenerative diseases, and cancer. We believe that the novel therapeutic potential of these compounds, our expertise in finding their targets, and our unique ability to exploit them to more fully understand mechanism, justifies our request to expand our screen under this funding mechanism. We propose to conduct a high through put screen in the Molecular Libraries Screening Centers Network identical to the one that, on a smaller screening scale, has already successfully identified mitochondrial fission and fusion inhibitors. Specifically, we will employ straightforward growth-based assays in S. cerevisiae strains that monitor mitochondrial fission and fusion activity to identify additional small molecule inhibitors. Given our success, we are confident that many novel compounds will be found. The medical importance of events regulated by mitochondrial membrane dynamics, such as apoptosis, indicates that the chemical genetic approach may also lead to the identification and development of novel therapeutic agents for stroke, myocardial infarction, neurodegenerative diseases, and cancer. We believe that the novel therapeutic potential of these compounds, our expertise in finding their targets, and our unique ability to exploit them to more fully understand mechanism, justifies our request to expand our screen under this funding mechanism. -
Screening for Small Molecule Inhibitors of Eukaryotic Translation Initiation
PELLETIER, JERRY
MC GILL UNIVERSITY
1 R03 MH081216-01
5
DESCRIPTION (provided by applicant): Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. – insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, this process is deregulated in many human cancers. Over-expression of certain translation factors can lead to malignant transformation and many of the components of the translational apparatus are over-expressed in human cancers. Several tumor suppressor genes directly influence the translation process and recently, chemoresistance in vivo has been linked to deregulated translation initiation. In a transformed setting, where translation can be inhibited by a small molecule modulator (e.g. rapamycin), decreased translation rates are associated with reversal of chemoresistance, possibly by inhibition of (a) pro-survival or resetting of (a) pro-apoptotic program(s). These results validate translation initiation, and in particular eIF4F, a heterotrimeric complex involved in the ribosome/mRNA recruitment phase, as a potential chemotherapeutic target. The Specific Aims of the current application are to implement a High Throughput Screen (HTS) at the MLSCN in order to identify small molecules that alter eIF4F complex integrity, by disrupting the association between two of its subunits, eIF4E and eIF4G. Following the initial identification of compounds that show activity in the HTS assay, false-positives will be identified and eliminated using a counterscreen designed to identify non-specific effects of compounds on the TR-FRET assay or on protein-protein interaction (for example, denaturants or chemically reactive compounds). The activity of compounds that are not eliminated in the counterscreen will then be confirmed in a secondary assay that monitors the interaction between eIF4E and eIF4G, but utilizes a different readout. Follow-up studies with optimized compounds will be performed to characterize their biological properties in vitro and in vivo. Should any of the optimized hits show activity in vivo, relative cytotoxicities will be determined in lymphomas of defined genotypes generated from a mechanism-based mouse cancer model. -
High Throughput Screening-Based Identification of Measles Virus Probes
PLEMPER, RICHARD K.
EMORY UNIVERSITY
1 R03 MH080836-01
5
DESCRIPTION (provided by applicant): Measles virus (MV), a member of the paramyxovirus family, remains one of the ten most lethal human pathogens despite the existence of a live-attenuated vaccine. The virus is endemic in Africa, Asia and parts of Europe, and cases are reported annually in the US. No therapy is available for management of severe cases of measles or rapid control of local outbreaks. It is therefore the long-term objective of this project to develop novel inhibitors of MV. Towards this goal multiple assays have been established in pilot studies and a research plan with three specific aims will be implemented. The first specific aim provides a robust assay to the MLSCN for hit identification through high throughput screening (HTS). A cell-based automated assay has been established that employs a fluorescent MV reporter stably expressing green fluorescent protein as additional transcription unit. Assay evaluation has revealed favorable z’-values of 0.8 and a pilot screen of a 34,000 compound in- house library at Emory has yielded amongst others a promising new inhibitor class, providing proof-of-concept for the approach. The second specific aim backs up the automated hit discovery with three independent manual assays that quantify suppression of virus-induced cytopathicity and reduction of virus yields in the presence of compound, and determine inherent compound cytotoxicity. Collectively, these assays allow hit confirmation, calculation of active concentrations, and assessment of compound selectivity. The third specific aim determines the target specificity of potent hits towards MV in comparison with other members of the paramyxovirus family, and subjects selected candidates to an initial characterization of the mechanism of antiviral activity. Candidate probes with high biologic activity towards MV will be subjected to testing against closely related canine distemper virus and distantly related human parainfluenza virus type 2. In individual assays, the effect of selected compounds on receptor binding, membrane fusion, and viral gene expression and genome replication will then be examined. Combined, this screening exercise will enable us to identify the most promising small molecule probes that merit future biochemical characterization of docking modes, further in vitro optimization through generation of quantitative structure-activity relationships and pharmacophore extraction, and in vivo toxicity and efficacy testing using the cotton rat small animal model for MV infection. Collectively, paramyxoviruses are responsible for significant morbidity and mortality worldwide. Measles virus (MV) alone, a member of this virus family, accounts for approximately 500,000 deaths annually, placing it among the most lethal human pathogens despite the existence of a vaccine. This is partially due to the exceptionally high infectivity of MV and the fact that no therapy is available for management of severe cases of measles or rapid control of local outbreaks. The high-throughput screening-based identification of novel biological probes that interfere with MV replication and spread, and the further development of applicable drugs based on these probes is therefore the ultimate objective of this project. -
Chemical Antagonists of IAP-Family Anti-Apoptotic Proteins
REED, JOHN C
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 R03 MH081277-01
5
DESCRIPTION (provided by applicant): Apoptosis plays essential roles in many aspects of normal development and physiology, becoming dysregulated in myriad diseases characterized by insufficient or excessive cell death. Caspases are the executioners of apoptosis. These intracellular proteases are suppressed by Inhibitor of Apoptosis Proteins (IAPs), a family of evolutionarily conserved anti-apoptotic proteins. Proteins released from mitochondria (SMAC and HtrA2) can competitively displace IAPs from the Caspases, thus helping to drive apoptosis. It has been shown that only a few residues at the N-terminus of activated SMAC protein (4mer) are sufficient to affect the release of IAPs from Caspases. Thus, it is plausible to identify chemical compounds that mimic the effect of SMAC in antagonizing IAPs by causing them to release Caspases. Non-peptidyl chemical inhibitors would have advantages over SMAC peptides, in terms of cell permeability, stability, and in vivo pharmacology. To this end we have developed a binding assay based upon fluorescence polarization, using a short peptide representing residues from the N-terminus of activated SMAC with an attached fluorochrome. This fluorescence polarization assay (FPA) forms the basis for a high-throughput competitive displacement assay that we have optimized for chemical library screening. We propose to screen the NIH compound library using this FPA and thus identify chemical compounds that compete with SMAC peptide for binding to IAPs. Then, using 3 types of secondary assays we have already devised, the hits will be independently confirmed. Structure Activity Relations (SAR) studies of analogs will be performed for a prototypical member of the IAP-family, XIAP. Finally, to define the selectivity of the compounds, SAR studies will be performed using assays configured for additional members of the IAP family (cIAP1, cIAP2, ML-IAP, ILP2). Altogether, these efforts will result in validated chemical probes for studying the biology of IAPs in a variety of cellular and organismal contexts. Cell death is a normal facet of physiology. The average human produces and in parallel eradicates 50-70 billion cells in his or her body, with most of this cell death occurring via a process known as “apoptosis.” Defects in the normal regulation of apoptosis are at the core of many diseases, including cancer where insufficient cell death permits abnormal cell accumulation, and degenerative diseases where excessive cell death leads to tissue loss and organ dysfunction. Apoptosis is accomplished by intracellular proteases, called Caspases. Like all proteolytic systems, the Caspases are under fine control by networks of proteins that either promoter their activation or suppress their activity. The chief endogenous antagonists of Caspases are IAPs (Inhibitor of Apoptosis Proteins), an evolutionarily conserved family of proteins that bind to and inhibit the activity of Caspases or that induce Caspase degradation by ubiquitin-dependent mechanisms. Our objective is to identify chemical compounds that bind sites on IAPs, competitively displacing Caspases. The resulting compounds will be useful as research tools for understanding the biology of IAPs and for ascertaining their roles in diseases such as cancer, where IAP over-expression is commonly observed. -
Screening for Chemicals that Potentiate TRAIL-Induced Apoptosis of Cancer Cells
ROZANOV, DMITRI
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 R03 MH081232-01
5
DESCRIPTION (provided by applicant): The purpose of this R03 application is to describe our proposed drug design effort designed to potentiate TRAIL-induced apoptosis in cancer cells. Apoptosis plays an essential role in the regulation of cell number in many physiological and pathological settings. Apoptosis mediates therapy-induced cytotoxicity in chemotherapy, ?-irradiation and immunotherapy. Many tumors, however, have proved to be drug- and treatment-resistant. TRAIL is a highly promising cancer therapeutic because it can induce apoptosis in a broad spectrum of cancer cell types but not in normal cells. Unfortunately, certain tumor cell lines have already acquired resistance to TRAIL. There are chemotherapeutic agents that augment TRAIL-mediated apoptosis when co-administered with TRAIL. These existing combined therapies, however, have demonstrated unacceptable side-effects when therapy affects both tumor and normal cells. We believe that the most realistic and practical means to employ TRAIL-induced apoptosis in cancer treatment is to identify chemical leads that specifically potentiate TRAIL-mediated anti-tumor cytotoxicity. We fully expect to identify these leads by using HTS techniques to screen comprehensive libraries of chemicals. Hypothesis/Aims. Because it is known that TRAIL induces apoptosis in a broad spectrum of cancer cells but not in normal cells and that available drugs used in combined therapy targeting the apoptotic pathways have serious deleterious side-effects, we hypothesize: (1) that it is of the utmost importance to discover and identify chemicals that can be developed into drugs that specifically augment TRAIL-mediated anti-tumor toxicity, and (2) that the use of HTS techniques within the MLSCN is the most efficient manner to accomplish this task. Our approach is based on the sequential treatment of human fibrosarcoma HT1080 cells with chemical library compounds followed by treatment with the trimeric, yeast-derived, TRAIL-LZ construct with a known low hepatotoxicity. The initial hits that augment TRAIL-induced apoptosis will be validated in an additional assay to select chemicals that are non-toxic to normal human hepatocytes. Our Specific Aims are: (1) To identify low molecular weight chemicals that can efficiently enhance TRAIL-mediated apoptosis in human fibrosarcoma HT1080 cells. (2) To identify, among selected hits, compounds that are non-toxic to normal cells in an additional cell-based assay. (3) To determine which of the hits specifically sensitize tumor cells to TRAIL (and other apoptosis inducing TNF family cytokines) without impacting the sensitivity of cells to other types of apoptosis pathways. The resulting compounds will be specific sensitizers of the “extrinsic” pathway and consequently will serve as valuable research tools for improving our understanding of the mechanisms of TRAIL resistance. In addition, these compounds can provide a starting point for the development of a novel class of less toxic and more powerful anticancer drugs. One liter of Pichia conditioned medium provides us with 5 mg of purified TRAIL-LZ. This amount is sufficient for the analysis of 500,000 individual chemicals. Future Plans include: (1)To identify the putative targets and to determine the efficiency and selectivity of the apoptosis activation mechanism exerted by initial hits in cell-based assays; (2) Optimize the structure of the selected hits to improve their properties such as potency and ADMET (absorption, distribution, metabolism, excretion, and toxicity) attributes; (3) Confirm the selectivity and efficiency of the derivatized compounds to potentiate TRAIL-mediated apoptosis employing multiple cancer cell types; (4) Confirm the efficiency and safety of the derivatized hits in vivo in tumor xenograft models. Assays. We propose to use the cell-based assays employing HT1080 cells (primary screen) and human hepatocytes (secondary screen). The cells will be incubated with chemicals followed by TRAIL-LZ treatment. This assay with a Z’-factor equal to 0.6-0.9 is readily adaptable to automation to fit 384-well or 1536-well plates. We are confident that reproducibility between plates and day-to-day experiments will be greater than 95% and the coefficient of variation (CV) will not exceed 5%. To select compounds non-toxic to normal cells, we will evaluate the toxicity of the identified hits in human primary hepatocytes. The normal human primary hepatocytes, TRAIL-LZ, and the detailed experimental protocol will be provided to the screening center. The main technical parameters of the primary assay are as follows: assay, CellTiter-Glo Luminescent Cell Viability Assay (Promega); assay volume, 0.1 ml; number of cells/well, 50,000; plates, 96-well transparent, flat bottom; temperature, ambient; cost, unless a bulk purchase is arranged, the $2,159.0 costs of the commercially available CellTiter-Glo Cell Viability Assay will be sufficient for the analysis of 10,000 compounds in a 96-well plate format. The cost of all other assay reagents is minimal. Stability of the cells and the substrate: DMSO up to a 1% concentration does not affect the viability of HT1080 target cells. The HT1080 cells, human primary hepatocytes, TRAIL-LZ, and the detailed experimental protocol will be provided to the screening center. -
A Cell Based HTS Approach for the Discovery of New Inhibitors of the H5N1 Virus
SEVERSON, WILLIAM E
SOUTHERN RESEARCH INSTITUTE
1 R03 MH081270-01
5
DESCRIPTION (provided by applicant): Currently, there is no commercially available vaccine to protect humans against the highly pathogenic avian influenza H5N1 virus that is spreading across Asia, Europe, and Africa. Since humans have no immunity against any H5 viruses, the Centers for Disease Control and Prevention estimates that the economic impact of the next influenza pandemic could cost the United States billions of dollars, devastate the world economy and potentially kill one billion people worldwide. Thus, there is a critical need for antiviral drugs to supplement vaccine development and existing chemotherapeutics. A high throughput screening (HTS) approach provides an opportunity to screen large compound libraries. We have developed and validated a 384-well cell-based assay that measures CPE induced in Madin Darby canine kidney (MDCK) cells by influenza virus infection, using a luminescent-based detection system for signal endpoint. This molecular screen will provide the scientific community with an assay that allows for the rapid identification of potential inhibitors of influenza virus by evaluating large compound libraries in vitro. The proposed studies aim to gain access to the Molecular Libraries Screening Center Network (MLSCN) high throughput screening resources to facilitate the discovery of new molecular probes for the inhibition of avian influenza strain H5N1 virus. To achieve the objective of this proposal, the specific aim is to assist the MLSCN in transferring the validated HTS assay to the appropriate screening center and provide technical support for follow up confirmatory assays on single dose ‘hits’ from the primary assay in a HTS dose response format. -
High Throughput Screening for Toll-Like Receptors
TOBIAS, PETER S
SCRIPPS RESEARCH INSTITUTE
1 R03 MH081265-01
5
DESCRIPTION (provided by applicant): The specific aim of this proposal is to use a high throughput screening assay that we have developed to find inhibitors of TLR4 signaling. There are a number of diseases whose etiology involves chronic or acute, but sterile, inflammation. Examples are atherosclerosis and kidney transplantation. In some of these diseases, notably the two just mentioned, inflammation initiated by Toll-like receptor (TLR) signaling is clearly involved. However, there are no inhibitors of TLR signaling that might serve as models for small molecule inhibitors of the activity of TLRs and which might be useful, or serve as starting points for development of therapeutic compounds. We have developed an assay that exhibits a positive signal when TLR4 and its intracellular signaling adapter MyD88 associate. The signal develops when two fragments of a-lactamase, expressed as chimeric proteins with TLR4 and MyD88, reconstitute a-lactamase activity when the two chimeric proteins associate. Thus the assay measures a-lactamase activity indicating TLR4 – MyD88 association. Inhibition of the a-lactamase activity signals detection of an inhibitor of TLR4 – MyD88 association. Following high throughput screening with this assay we will eliminate false positive hits by assaying for a-lactamase inhibitors. We will also assay positive hits for their ability to inhibit TLR4-MyD88 association by co-immunoprecipitation assays. Finally we will assess for effects of the positive hit on association of MyD88 with other TLRs. There are a number of human diseases, such as atherosclerosis and kidney disease, which involve inflammation generated by the activity of cell surface proteins called Toll-like receptors (TLRs). There are no good candidates for drugs that would block the activity of TLRs that could be useful in treating these diseases. This project will utilize an assay that we have recently developed to search for such compounds. -
HTS to Identify Specific Small Molecule Inhibitors and Activators of Ras and Ras-
WANDINGER-NESS, ANGELA
UNIVERSITY OF NEW MEXICO ALBUQUERQUE
1 R03 MH081231-01
5
DESCRIPTION (provided by applicant): Ras and Ras-related, small molecular weight GTPases function in the regulation of signaling and cell growth control and collectively serve to control cell proliferation, differentiation and apoptosis. There are over 150 known human proteins belonging to the Ras superfamily of GTPases. When mutant or hyperactivated, Ras family members contribute to oncogenesis and hereditary disorders affecting vision, immune and neurologic function. Strategies for inhibiting Ras to date have relied on altering membrane recruitment with drugs affecting prenylation. Inhibition of prenylation enzymes lacks specificity and is problematic because the cellular prenylation machinery is required for the proper function of many Ras superfamily members. The demonstrated efficacy of targeting drugs to the nucleotide binding pocket of specific kinases offers a paradigm that may be applied to the GTPases. The PI and co-I have established a fluorescent GTP-binding assay for GST-GTPase chimeras that can be monitored by flow cytometry. In addition, through outreach, the new Mexico Molecular Libraries Screening Center has established the ability to screen multiple GTPases simultaneously with multiplex analysis on a HyperCyt??high throughput flow cytometer. Advantages of the multiplex approach include single step analysis of drug specificity and selectivity, small reaction volumes and the discrimination of free and bound fluorescence that is conservative with respect to reagent usage when compared to polarization assays. The group has in hand a collection of small Ras-related GTPases as GST fusion proteins that include members of the Rab, Rho, and Ras families. It is the aim of the present application to identify lead compounds that interfere with fluorescent GTP-binding to individual Ras superfamily members. Subsequent functional assays will be used to identify the compounds as direct and allosteric inhibitors or activators of GTPase function. The identification of small molecule nucleotide binding modulators of small GTPases has not yet been undertaken and is expected to have important utility in the future treatment of cancer and neurologic diseases where GTPase function is specifically altered. -
A High Throughput Screen for Inhibitors of a Novel Necrotic Cell Death Pathway
YUAN, JUNYING
HARVARD UNIVERSITY (MEDICAL SCHOOL)
1 R03 MH081226-01
5
DESCRIPTION (provided by applicant): The objective of this proposal is to isolate additional necrostatins for understanding the molecular mechanism of necroptosis. Apoptotic pathways have been studied extensively during the past decade. However, it has become increasingly clear that apoptosis is not the only cellular suicide mechanism. For example, in a subset of cell types, inhibition of caspases when cells are stimulated by FasL or TNFa lead to inhibition of apoptosis but cells die with necrotic morphology through a cellular process termed necroptosis. Necroptosis has been shown to be a promising target for the treatment of stroke with an extended time window. Necrostatins are small molecules that specifically inhibits necroptosis but not apoptosis. This application is to carry out a mechanistic study of necroptosis by utilizing unique chemical resources at NIH to generate small molecule affinity reagents for target identification, and to identify new necrostatins in order to meet the highly challenging goal of developing an anti-stroke drug. This project is to identify small molecule inhibitors of a novel cell death pathway, termed necroptosis. Necroptosis has been shown to be a promising target for the treatment of stroke as it represents a type of delayed cell death in stroke and offers an extended time window for therapy. -
Quantitative High Throughput Screening for Anthrax Toxin Inhibitors
BUGGE, THOMAS H
U.S. NAT’L INST/DENTAL/CRANIOFACIAL RES
1 X01 MH082337-01
6
DESCRIPTION (provided by applicant): The overall objective of this proposal is to perform a quantitative high throughput screen to identify novel inhibitors of anthrax toxin intoxication. This objective will be pursued through the following two Specific Aims: Specific aim 1: Perform automated quantitative high-throughput screening of chemical libraries to identify novel inhibitors of anthrax toxin intoxication. Specific aim 2: Determine the mechanism by which the novel anthrax toxin inhibitors prevent anthrax toxin intoxication. The aims will be achieved by using an established assay for non-invasive optical imaging of the cellular uptake of anthrax toxin to perform a quantitative screen of a large library of chemical compounds. Compounds with inhibitory activity will be subjected to a secondary heterologous confirmatory screen. The mode of inhibition of verified compounds and compounds with related chemical structure will be determined thereafter using established biochemical and cell biological techniques. The proposed research is important to human health because it will: 1) Provide novel candidate agents for post-exposure treatment of anthrax. 2) Generate important new knowledge of the pathogenesis of anthrax toxin intoxication. The proposed research will increase our understanding of why people that have been accidentally or deliberately infected with anthrax and related disease-causing bacteria get sick. Furthermore, the research will identify a number of chemical compounds that may be useful for treatment of people infected with anthrax. -
The Role of Wnts in Stem Cell
COOMBS, GARY
University of Utah
1 R03 MH082387-01
6
DESCRIPTION (provided by applicant): The connection between Wnt/a-catenin or “canonical” Wnt signaling and cancer is well appreciated. Some cancers, such as colon cancer, have mutations in components of the signaling pathway (i.e. adenomatous polyposis coli protein, “APC”, axin, and a-catenin). Others have upregulated signaling due to methylation of promoters driving expression of secreted signaling inhibitors such as sFRP or DKK1. Still others have upregulated expression of Wnts or their receptors. In breast cancer and some leukemias, cancer stem cell maintenance has been shown to depend on Wnt signaling. Both a-catenin dependent and independent signaling pathways are implicated in stem cell maintenance. It is unclear in most cases exactly how Wnt signaling is upregulated in cancer stem cells. However, in the more overtly Wnt dependent cancers, it is clear that intervention at multiple points in the signaling pathway(s) is required for effective treatment of a broad range of cancers. We have developed and validated a cell based screen for inhibitors of Wnt signaling which can identify inhibitors throughout the entire pathway including secretion from Wnt expressing cells. We have also characterized a series of secondary assays which can place the function of each inhibitor within one of six defined portions or compartments of the signaling pathway. Through high throughput screening, we hope to identify inhibitors in each compartment and characterize them in a broad range of cancer cell based assays. We propose to identify inhibitors of Wnt signaling effectors throughout the signaling cascade, from Wnt secretion to transcription of target genes, and identify their targets and epistatic locations. Much accumulated evidence implicates both a-catenin dependent and independent Wnt signaling in overtly Wnt dependent cancers and cancer stem cell self renewal. With these compounds we propose to identify targets with the highest therapeutic indices and efficacy for treatment of Wnt dependent cancers. -
Discovering Calpain Inhibitors for Neurological Diseases
GLASS, JONATHAN DAVID
Emory University
1 R03 DA024890-01
6
DESCRIPTION (provided by applicant): Calpain inhibitors have enormous potential as therapeutic agents for neurological diseases. Calpains are calcium-activated cysteine proteases that are major contributors to the process of axonal and neuronal degeneration. Degeneration of axons leads to the disconnection of neurons from their motor or sensory targets, leading to weakness, numbness, pain, and cognitive impairment. Axonal degeneration underlies neurological dysfunction in stroke, head and spinal cord injury, peripheral neuropathies, and multiple sclerosis. We have had success in preventing axonal degeneration and neurological dysfunction in a model of peripheral neuropathy. We propose to use the Molecular Libraries Screening Center at Emory University for high throughput screening of a large library of small molecules (up to 100,000) for calpain inhibitor activity. We have developed a secondary assay for independent analysis of “hits”, and plan for follow-up developmental chemistry for optimizine drug design. Thus, the resources of the MLSCN will allow us to identify new calpain inhibitor compounds that may provide important new treatment for patients with a variety of neurological diseases. The degeneration of axons is a pathological feature common to many neurological diseases that causes neurological disability due to disconnection of neurons from their targets. Preventing axonal degeneration is thus protective, and we are proposing to discover small molecules that can be used for this purpose. The target of our small molecule screen is the calcium-activated protease calpain. Inhibitors of calpain will be developed as novel treatments of neurological diseases. -
An HTS Assay Based on Neuronal Human Cells to Identify Small Molecule Activators
GRIMALDI, MAURIZIO
Southern Research Institute
1 R03 MH082367-01
6
DESCRIPTION (provided by applicant): Effective treatment for neurodegenerative diseases, such as Alzheimer’s, is still lacking. Only, marginal symptomatologic treatment is available to data. Alzheimer’s association and other Health agencies, including NIH, have estimated that the economic cost of Alzheimer’s disease patients management in the sole USA amounts to billions. Also the psychological and material burden on the patients’ families accounts for additional and consistent damage done by this devastating disease. Recent statistics have calculated that just slowing down the disease by a few years will have an enormous impact on the economics and social consequences of this tremendous disease. Although, considerable efforts and resources have been deployed in the research for the causes and for an effective treatment, a cure for this disease is far from being discovered. New ideas and approaches are needed, indeed. In this context our proposal is completely novel and can open a new field to advance both knowledge and possibilities to approach these conditions. There are several indications that NF-kB system plays a role in neuronal resilience and in the changes induced by cellular learning such as long term potentiation and depression. Several reports have shown that knocking out NF-kB activity in the brain causes sensitization to toxic stimuli, such as a-amyloid, and excitatory aminoacids and to trauma. In addition, activation of NF-kB is a known anti-apoptosis mechanism. Based on these premises, we believe there is room to investigate the possibility that NF-kB up regulation could be of value in mitigating both neurodegenerative phenomenon and learning and memory failure, a unique possibility. We have developed an original neuronal cell-based assay that will assess NF-kB up-regulation using a luciferase reporter. We propose to use this assay to perform a high throughput screen of small molecules libraries available to us. We will screen these library to identify small molecules able to increase NF-kB expression (usually 2-5% of the total compounds, will turn out as hits). We plan to follow up the screening with in vitro validation of the selected compounds on a number of parameters which will include NF-kB expression, function, in vitro neurotoxicity paradigms to verify that the identified compounds have, in fact, a neuroprotective and/or a neurotrophic effect. We believe that as a result of the proposed studies we will have identified 1) novel small molecules that can be used as research tools to study the effects of NF-kB on signaling pathways; 2) possible drug leads that can serve as a launch pad for a larger effort aiming to develop and test active and safe compounds to help in the battle against neurodegenerative disorders. Treatment options for Alzheimer’s and other neurodegenerative disorders are fairly limited. They are usually symptomatic rather than curative, and in general their effectiveness is poor. Statistics from the Alzheimer’s association and from the NIH have assessed that the cost for the management of this patients rises to billions. An important part of the socially debilitating effect of these disorders is the consequences, both economic and psychological, on the families. This is due to both the expensive long term care and to the witnessing of the effect that these devastating disorders have on their loved one. It has been calculated that if the progression of Alzheimer disease could be delayed even by a few years the repercussions both at economic and psychological level will be enormous. Recent evidence have pointed out that NF-kB signaling system is involved in the resilience of neurons and in their ability to survive disparate insults and in neuronal molecular correlates of learning and memory. NF-kB molecular ablation has shown that neurons from these animals are more sensitive to insults including trauma, a-amyloid toxicity and excitotoxicity. Also a direct neuroprotective effect from both non apoptotic and apoptotic models of neurodegeneration has been attributed to direct or indirect activation of NF-kB signaling. In addition, NF-kB signaling has been involved in the establishment of long term potentiation and long term depression, two models of learning and memory that take place at cellular level. Therefore, NF-kB up regulation in neurons could be useful in attacking at the same time neuronal degeneration and deterioration of neuronal functions associated with loss of learning and memory as seen in Alzheimer’s disease. To pursue this grand scope, we have developed an original neuronal cell-based assay that will assess NF-kB up-regulation using a lucipherase reporter. We propose to use this assay to perform a high throughput screen of small molecules libraries available to us. We will screen this library to identify small molecules able to increase NF-kB expression (usually 2-5% of the total compounds, will turn out as hits). We will follow up the screening with in vitro testing of the selected compounds on a number of parameters which will include NF-kB expression, function, in vitro neurotoxicity paradigms to verify that the identified compounds have, in fact, a neuroprotective and/or a neurotrophic effect. We believe that as a result of the proposed studies we will have identified 1) novel small molecules that can be used as research tools to study the effects of NF-kB on signaling pathways; 2) possible drug leads that can serve as a launch pad for a larger effort aiming to develop and test active and safe compounds to help in the battle against neurodegenerative disorders. -
High Throughput and Virtual Screening for Human 12-LO, 15-LO-1, and 15-LO-2 Inhib
HOLMAN, THEODORE R
UNIVERSITY OF CALIFORNIA SANTA CRUZ
1 R03 MH081283-01A1
6
DESCRIPTION (provided by applicant): High-throughput screening (HTS) is a major method in early drug discovery and has yet to be fully applied to all human lipoxygenase (hLO) isozymes, critical enzymes in the progression of a number of human diseases. We have currently discovered over 20 novel inhibitors against human lipoxygenase primarily utilizing manual screening but have recently developed a high-throughput assay to increase the rate of discovery. We would now like to apply this HTS technology to the approximately 73,000 compounds at the NIH DPISMR to identify specific inhibitors to each of our three LO isozymes (12- hLO, 15-hLO-1 and 15-hLO-2). The specific inhibitors we discover will then be screened against specific cell lines to determine their in-vivo activity against prostate cancer cells and neuronal cells (as ischemia models). In parallel with the HTS, and at no extra cost to the NIH, we will dock the full HTS library against our three lipoxygenase homology models (12-hLO, 15-hLO-1 and 15-hLO-2) and compare the docking hits to those found by the HTS assay. This comparison will allow us to both improve our in silico methods and help us structurally interpret the HTS data we will obtain. Currently, we have shown both the accuracy and the reliability of this screen against a 47 compound flavonoid library and the NCI 3104 compound Mech./Div./Nat.Prod. library. In addition, Anton Simeonov, at NIH Chemical Genomics Center (NCGC), has successfully screened the LOPAC library with the Kalypsys robot and shown known LO inhibitors can be accurately found. Finally, modeling predictions with Prof. Matt Jacobson have already produced inhibitors from earlier screens. All of this data suggests that these aims are not only feasible but highly likely to be successful. Lipoxygenases (LO) are widely distributed throughout the plant and animal kingdoms and play a central role in the biology of these organisms. In plants they are involved in germination and senescence. In human tissue, there are three major human lipoxygenases, 5-, 12-, and 15-LO, whose primary enzymatic difference lies in their positional specificity on arachidonic acid (AA). The products of lipoxygenase are the precursors of hormones, such as leukotrienes and lipoxins, which have been implicated as critical signaling molecules in a variety of inflammatory diseases and cancers. There is currently strong evidence that if scientists can discover potent inhibitors against each specific lipoxygenase isozyme, these may become effective cellular probes and therapeutics against LO. This grant submission proposes to do exactly this for platlet 12-LO, reticulocyte 15-LO-1 and epidermal 15- LO-2. -
HTS for Modulators of Beta-Amyloid Catabolism by Insulin-Degrading Enzyme
LEISSRING, MALCOLM A
The Scripps Research Institute
1 R03 DA024888-01
6
DESCRIPTION (provided by applicant): Alzheimer’s disease (AD) is characterized by abnormal accumulation of the amyloid ?-protein (A?) in brain regions subserving memory and cognition. In recent years, proteases that degrade A??have been identified as potent and rate-limiting regulators of cerebral A??levels and amyloidogenesis in vivo. Significantly, orally bioavailable compounds that activate A?-degrading proteases have already been identified that are effective in reducing AD-type pathology in animal models and are currently entering clinical trials. Converging lines of evidence strongly implicate insulin-degrading enzyme (IDE) as a particularly important A?-degrading protease. Nonetheless, there is a surprising lack of pharmacological tools targeting IDE, or indeed any member of the unusual zinc-metalloprotease superfamily to which it belongs. Importantly, accumulating evidence shows how IDE activity might be augmented pharmacologically by any of several mechanisms, including the displacement of endogenous inhibitors or modulation of its expression and/or secretion into the extracellular space. Moreover, new crystal structures of IDE show that this protease possesses unorthodox structural features that can be targeted to directly activate the protease as much as 4000 percent. The purpose of this proposal is to utilize our well-characterized fluorescence polarization-based A?-degradation assay (Leissring et al., JBC 2003, Appendix) to search for chemical modulators of IDE within the Small Molecule Library of the MLSCN. We propose a series of secondary assays to confirm discovered hits, to establish their specificity for IDE, and to identify cell-penetrant compounds. Probes suitable for use in cultured cells or in vivo will be used in downstream experiments to resolve several outstanding questions about the role of IDE in AD pathogenesis that can only be addressed via a chemical biology approach. In addition, chemical activators of IDE with suitable properties could serve as pharmacophores for the development of novel AD therapies. -
High Throughput Imaging Assay for Beta-Catenin
MCDONOUGH, PATRICK M
Vala Sciences, Inc.
1 R03 MH082378-01
6
DESCRIPTION (provided by applicant): Cancer is one of the most tragic afflictions in modern society, and often results from alterations in the mitotic process. In normal cells, a-catenin is found predominantly associated with the plasma membrane. However, in tumor cells, a-catenin redistributes to the nucleus where it interacts with transcription factors to stimulate the expression of proteins that promote mitosis. The pathway is also important in CNS-related disorders ranging including manic depression, and Alzheimer’s. In response to PAR-06-545 “Solicitation of Assays for High Throughput Screening (HTS) in the Molecular Libraries Screening Centers Network (R03)” we are submitting an assay to quantify the cellular distribution of a-catenin. The assay involves exposing HeLa cells to test compounds that are likely to alter a-catenin distribution; the cells are then immunostained for endogenous a- catenin and photographed using high content robotic microscopy workstations. Specialized image analysis software is used to quantify cellular a-catenin localization. In preliminary experiments, an inhibitor of glycogen synthase kinase 3-a (GSK-3), an enzyme typically regulated by the Wnt-signal transduction pathway, induced nuclear localization of a-catenin and this was quantified by the assay with a Z’ score of 0.70. The proposed assay will help identify chemicals that are likely to regulate GSK-3 or other proteins that control a-catenin localization. Discovery of such chemicals will contribute to our understanding of the regulation of a-catenin localization and potentially help lead to development of novel therapeutics, depression, and dementia. -
HTS Screen of TB RmlC & RmlD dTDP-Rhamnose Formation Enzymes
MCNEIL, MICHAEL R
Colorado State University
1 R03 DA024889-01
6
DESCRIPTION (provided by applicant): New drugs are needed against tuberculosis (TB) for three major reasons. The first reason is because the rate of cure with the present drugs is very slow. Secondly, increasing co-infection of HIV and Mycobacterium tuberculosis is occurring and treatment with present drugs results in harmful HIV/TB drug interactions. Thirdly, is the increasing prevalence of M. tuberculosis resistant to the present drugs. In a TB drug development program, we are targeting the formation of the cell wall of M. tuberculosis, a proven drug target. dTDP-rhamnose is a required biosynthetic precursor for TB cell wall formation. dTDP-rhamnose is not found in humans. Two of the enzymes which act sequentially for its formation are dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC) and dTDP-6-deoxy-L- lyxo-4-hexulose reductase (RmlD). These enzymes have been shown to be essential for the growth of M. smegmatis and M. tuberculosis. The crystal structures of both have been obtained for proteins from non-TB bacterial sources and RmlC has been determined for the M. tuberculosis protein. Moreover TB RmlC has been obtained with the substrate mimic, dTDP-rhamnose, bound in the active site. Both RmlC and RmlD have been over-expressed in active form and a microtiter plate based assay based on the conversion of dTDP-6-deoxy-D-xylo-4-hexulose to dTDP-rhamnose with the concomitant oxidation of NADPH to NADP has been developed. The enzymes are balanced so that an inhibitor of either enzyme will be detected. The assay has been shown to be robust and reproducible. Herein we request that this assay be used to screen for inhibitors of RmlC and/or RmlD by the MLSCN and the resulting hits be optimized in concert with our lab and with our X-ray crystallographer and medicinal chemist collaborators. The purpose of this project is ultimately to develop new drugs against tuberculosis. These drugs are badly needed because of resistant strains of tuberculosis, because the treatment time needs to be decreased, and because co-infection of TB and HIV-AIDS is difficult to treat. The immediate purpose of the project is to find compounds that have the potential to be developed into new drugs because they inhibit enzymes required for the formation of the cell wall of the tuberculosis bacterium. -
Activators of the Pyrophosphatase Activity of Alkaline Phosphatase
MILLAN, JOSE LUIS
Burnham Institute for Medical Research
1 R03 MH082385-01
6
DESCRIPTION (provided by applicant): We have recently reported that the rickets and osteomalacia characteristic in tissue-nonspecific alkaline phosphatase (TNAP)-deficient mice (Akp2-/- mice) results from highly increased levels of inorganic pyrophosphate (PP), a calcification inhibitor PPi and a natural substrate of TNAP, and from the concomitant increase in the expression of skeletal osteopontin (OPN), another calcification inhibitor. These studies suggested the possibility of manipulating the PP/OPN axis as a means of affecting calcification. We recently tested this axis by surmising that transgenic mice over-expressing TNAP might be able to achieve tissular expression of TNAP sufficiently high to be able to lower circulating PPi and OPN concentrations to enhance bone mineral density (BMD) in these animals. Transgenic mice were generated by expressing human TNAP cDNA under control of the Apolipoprotein E promoter, which drives expression of TNAP primarily in the post- natal liver. We examined the expression levels of TNAP in tissues from mice carrying one copy or two copies of the ApoE-Tnap transgene and also from [Akp2-/-; ApoE-Tnap] mice, and examined the ability of their primary osteoblasts to calcify in culture. MicroCT analysis was used to measure BMD in long bones, vertebrae and calvaria. TNAP expression in ApoE-Tnap mice was major in the liver and kidney as expected, with lower but yet detectable levels in bone, brain and lung. Serum AP concentrations were 10 to 50-fold higher than age- matched sibling control wild-type (WT) mice. As predicted, serum levels of PPi and OPN were reduced in the transgenic animals. Furthermore, fCT analysis of femur, vertebrae and calvaria revealed higher BMD in cancellous bone of ApoE-Tnap+ and ApoE-Tnap+/+ mice compared to WT mice. Thus, we have shown that increases in tissular and circulating levels of TNAP lead to higher BMD by reducing the effective levels of the calcification inhibitors PPi and OPN. These data provide a mechanistic interpretation for the correlation between AP and BMD that has been observed in humans and mice. Furthermore, these studies suggest the possibility that administration of recombinant TNAP itself, or of pharmacological activators of TNAP’s pyrophosphatase activity, may serve as therapeutics drugs for the treatment of osteoporosis. Thus, this proposal aims at developing a sensitive assay for the discovery of TNAP activators that may serve as lead compounds for the development of drug-like molecules suitable for in vivo administration. We will use this assay to screen the small molecule repository (MLSMR) for activators of TNAP. The specific aims are to: I) Identify small molecule compounds in the MLSMR that are highly specific activators of TNAP using a luminescence-based assay. II) Test confirmed positives in the secondary assay with natural substrates of TNAP and check for specificity against other recombinant phosphatases. III) Test confirmed positives for their ability to increase calcification in osteoblast cultures. The novel chemical probes to be identified in this way may ultimately lead to the novel therapy for the growing number of osteoporosis patients. Within the past five years this laboratory, in association with several collaborating groups, has focused on the factors that control mineralization and it is now clear that a main player in this process is phosphate in its two major forms, i.e., as inorganic phosphate (P)i and as inorganic pyrophosphate (PP). We have clearly shown i that the maintenance of a properly controlled extracellular P/PPi ratio is of paramount importance in promoting i healthy bone mineralization. Alterations in this ratio, either by genetic or pharmacologic means, can either correct or cause a pathologic state. Osteoporosis is characterized by an imbalance of osteoblast-mediated bone formation and osteoclast-mediated bone degradation, which results in overall increased bone resorption. Current treatments of osteoporosis aim at either reducing osteoclastic activity or augmenting osteoblastic function. In this proposal we will test a novel hypothesis, i.e., that we will be able to identify activators of TNAP’s pyrophosphatase activity that will serve to promote degradation of PPi thus increasing the P/PPi ratio i to favor increased mineralization. We anticipate that this project will validate manipulating the P/PPi ratio as a i valuable therapeutic option to treating osteoporosis by affecting osteoblast-mediated mineral deposition. Such a strategy could be used as an alternative or as a complement to currently used drugs that decrease osteoclastic activity (bisphosphonate treatment) or increase osteoblasts numbers (PTH-like peptide treatment). -
Novel Chemotypes Against Influenza A Virus
PELLECCHIA, MAURIZIO
Burnham Institute for Medical Research
1 R03 DA024886-01
6
DESCRIPTION (provided by applicant): Strains of influenza A (H3N2) virus have specific mutations in the M2 proton channel that result in resistance to the antiviral drug amantadine, one of the very few drugs currently available against the virus. Recent years have witnessed a dramatic increase in resistance to amantadine in communities in Asia and North America due to the spread of the H3N2 strain despite the absence of sustained selective drug pressure. Another important problem that limits the use of amantadine is its interference with the human NMDA receptor, causing potentially severe side effects. For these reasons, we propose to use NMR- and fragment-based screening approaches to derive novel agents against H3N2 influenza A infections by directly targeting the mutant M2 variants. In addition, we propose to target an essential RNA viral promoter by using similar NMR- based approaches. The chemical structures, SAR data and range of biochemical activities of the resulting compounds will provide a framework onto which to develop potentially novel anti-influenza therapies. -
Chemical Inhibitors of ER Stress
REED, JOHN C
Burnham Institute for Medical Research
1 R03 DA024887-01
6
DESCRIPTION (provided by applicant): Chemical Inhibitors of ER stress. Disturbances in the normal functions of the endoplasmic reticulum (ER) lead to an evolutionarily conserved cell stress response, the unfolded protein response (UPR), which is aimed initially at compensating for damage, but eventually, can trigger cell death if ER dysfunction is severe or prolonged. The mechanisms by which ER stress leads to cell death remain enigmatic, with multiple potential participants described but little clarity about which specific death effectors dominate in particular cellular contexts. Important roles for ER-initiated cell death pathways have been recognized for several diseases, including hypoxia, ischemia-reperfusion injury, neurodegeneration, heart disease, and diabetes. The goal of this application is to identify chemical compounds that block cell death induced by ER stress. A cell-based assay has been devised which is simple and effective for identifying such compounds. Several supporting secondary assays have also been devised that inform compound characterization. Proof of concept data are presented that validate this chemical biology approach for generating compounds that will become useful research tools for interrogating mechanisms of ER stress-induced cell death and dysfunction. 1 Essentially all cells possess the ability to produce proteins that are either displayed on the cell surface for purposes of sensing changes in the environment or that are secreted from the cell. The organelle within the cell responsible for this function is called the “endoplasmic reticulum” (ER). Defects in protein folding in the ER trigger an evolutionarily conserved cellular response that can activate programs for cell death and thus destroy cells. This protein folding problem is termed “ER stress” and it has been associated with a wide range of diseases, including ischemia-reperfusion injury (particularly stroke), neurodegeneration, and diabetes. In this proposal, we seek to identify chemical compounds that inhibit cell death induced by ER stress. These chemicals will be useful research tools for dissecting mechanisms of ER stress in the laboratory, and may also serve as the starting point for generation of novel medicines that preserve brain cells during stroke or neurodegeneration, or that overcome the effects of ER stress responsible for worsening insulin-resistance, thus improving diabetes care. -
Screening for Inhibitors of the Integrated Stress Response
RON, DAVID
NYU School of Medicine
1 R03 MH082370-01
6
DESCRIPTION (provided by applicant): A large number of biologically important proteins, such as hormones, enzymes and cell surface receptors undergo early steps of their biogenesis in the endoplasmic reticulum (ER). Defects and variations in efficiency of this process are believed to contribute to important human diseases. For example, misfolding and degradation accounts for lack of enzymes in lysosomal storage diseases or membrane transporters in certain kidney diseases. The protein folding environment in the ER is regulated by signal transduction pathways that together constitute the ER unfolded protein response (UPR), which responds to misfolded protein stress in the organelle. Relatively crude genetic manipulation of these pathways has shown that modulating the protein folding environment in the ER can have important pathophysiological consequences: For example, evidence suggests that loosening the quality control in the ER might allow mildly misfolded proteins that are otherwise functional to escape ER retention and degradation and contribute to essential cellular functions and thereby ameliorate severe phenotypes of loss-of-function mutations. Other studies show that cancer cells from human tumors are particularly reliant on their UPR for survival, suggesting that UPR inhibitors may have selective toxicity against cancer. Therefore, availability of pharmacological probes to modulate signaling in the UPR will provide much needed tools to test the suitability of the pathway as a target for therapeutic intervention in diseases of protein misfolding and cancer. Phosphorylation of translation initiation factor 2a (eIF2a) by the ER stress activated protein kinase PERK is a well understood and potentially malleable arm of the UPR that is referred to as the integrated stress response (ISR). Robust cell-based assays for activity of the ISR have been developed. These entail measurements of the magnitude of translation repression attendant upon PERK activation and eIF2a phosphorylation and a complementary assay that reports on the activity of the gene expression program that is initiated by eIF2a phosphorylation. The assays in question have been miniaturized and converted to a homogenous format suitable for high throughput screens (HTS) for small molecules (“probes”) that when added to cells, would either block PERK activity, impair the downstream steps required for eIF2a phosphorylation or the conversion eIF2a phosphorylation signal to the activation of gene expression. Tertiary assays have been developed to pinpoint the site of action of any inhibitory molecules discovered by the HTS. An HTS campaign using these assays is expected to yield potent cell penetrant small molecules that inhibit the ISR at various points in vivo. Unlike the genetic approaches, which tend to produce relatively discontinuous dose responses, small molecule inhibitors are predicted to have continuous dose-response relationships with lengthy monotonic phases. These feature will be exploited by the research community to test the hypothesis that gentle and partial inhibition of the ISR might promote the secretion of otherwise misfolded proteins and selectively compromise the viability of tumor models. Our understanding of the processes by which proteins attain their proper structure has increased markedly in recent years and with that understanding come the prospects of intervening in the process of protein folding to therapeutic ends. This study focuses on one pathway by which cells regulate their capacity to fold proteins in the secretory compartment and is designed to identify drug-like compounds that modulate signaling in that pathway by inhibiting one of its key components, an enzyme called PERK. If successful, this study will tell us whether or not PERK inhibitors have potential utility in treating diseases of protein misfolding, such as lysosomal storage diseases and various cancers. -
Promiscuous and Specific Inhibitors of Cruzain
SHOICHET, BRIAN K.
UNIVERSITY OF CALIFORNIA San Francisco
1 R03 DA024891-01
6
DESCRIPTION (provided by applicant): High-throughput screening (HTS) is the dominant technique in early drug discovery and has had an important impact on human health. A key challenge in HTS is the occurrence of false-positive inhibitors, which can dominate hit-lists. Multiple mechanisms have been proposed to explain these artifacts, including chemical reactivity, oxidation, assay interference, and colloidal aggregation. In an earlier screen of the MLSMR, we developed a direct screen for aggregation-based inhibition, using a-lactamase as a reporter enzyme. Two startling results emerged from this study. First, of the 1274 inhibitors found, 96% were aggregators and only 1% acted covalently. No true, competitive inhibitors were found by HTS. Second, a docking screen of the same library did find two novel inhibitors. These had Ki values of about 40 fM and bound specifically to a- lactamase by x-ray crystallography. Here we ask how representative these results might be. Is covalent inhibition really only a minor issue in enzyme HTS, far overwhelmed by aggregation? Can docking reliably prioritize compounds for experimental testing? We investigate a second enzyme for promiscuous and covalent inhibition, and as a target for docking. The specific aims are: 1. To determine the ratio of promiscuous and covalent inhibitors in an HTS screen of the cysteine protease cruzain. As a cysteine protease, cruzain will be much more sensitive to reactive and compounds than a-lactamase. It thus provides a robust test of the provocative hypothesis that covalent artifacts are rare in screening. We will use the same protocol we developed for a-lactamase: compounds that inhibit cruzain in the absence but not the presence of detergent are aggregators. The aggregators that are found will be compared to those found in the a-lactamase screen, which will itself be interesting for this widely used collection. Even more interesting will be the ratio of aggregators to all other mechanisms of inhibition. We will re-test characteristic inhibitors in low throughput secondary assays here at UCSF, determining their mechanisms of action using DLS, enzyme counter-screens, mass spectroscopy, and x-ray crystallography. 2. To compare docking hit rates with HTS hit rates. The HTS assay affords us an opportunity to, at no extra cost, compare high-throughput with virtual screening. We will prospectively dock the same MLSMR library (120,000 compounds) against the x-ray structure of cruzain, and compare the docking hits to those subsequently found by HTS. As with the screen against a-lactamase, we will be at liberty to test high-scoring docking hits at higher concentrations of inhibitor and lower concentrations of substrates, allowing for mid- micromolar inhibitors that HTS might miss. We will ask: which hits does docking capture, what are the false positives and the false negatives for both techniques? Here too, we will determine x-ray structures of enzyme complex structures for specific inhibitors. We will pursue the SAR of interesting hits with the synthetic chemistry group at the NCGC. The most widely used technique to find new candidate drugs is high-throughput screening (HTS). A major challenge for HTS is the occurrence of false-positive inhibitors, which can dominate hit-lists. We test a rapid method to detect these artifacts, which should significantly advance this critical technique. -
Scaffolds for Synthesis of Probes Directed Against Class II HMG-CoA Reductases
STAUFFACHER, CYNTHIA V
Purdue University
1 R03 MH082373-01
6
DESCRIPTION (provided by applicant): It has recently been shown that a number of common human pathogens, including Enterococcus faecalis, Streptococcus pneumoniae and Staphylococcus aureus are dependent on enzymes in the mevalonate pathway for biosynthesis of IPP, including the central pathway enzyme HMG-CoA reductase. This raises the possibility that a targeted inhibitor of HMG-CoA reductase would be an anti-bacterial specific for these pathogens, with a unique point of attack that would leave this class of inhibitors unaffected by the resistance mechanisms that have developed against the more common cell-wall antagonists. This selective property would make mevalonate pathway inhibitors particularly attractive in burn unit or intensive care settings where sterilization of the normal bacterial flora make yeast over growth or colonization by resistant bacteria a common and severe complication of broad-spectrum antibiotic use. The goal of this proposal is to identify a set of molecular probes that selectively inhibit the activity of the Class II HMG-CoA reductase (HMGR) of pathogenic bacteria. This proposal seeks access to HTS resources provided by the Molecular Libraries Screening Center Network (MLSCN) where an HTS-ready assay can be employed to explore the differences in the structure activity relationships of the human and pathogenic bacterial HMGRs. A preliminary study using a spectrophotometric assay in high throughput format has demonstrated this assay is exceptionally robust, and identified seven compounds (0.18% hit rate) with significant inhibitory effects, and therefore is ready to be translated to a designated screening center. Secondary assays to be performed in the collaborating laboratories are proposed to confirm the potency and to further understand the molecular basis of the inhibition, including more detailed enzymology of the inhibitory effects selected compounds, bacterial toxicity assessments and detailed structural studies of these compounds in complexes with bacterial HMGRs. It has recently been shown that a number of common human pathogens, including Enterococcus faecalis, Streptococcus pneumoniae and Staphylococcus aureus are dependent on enzymes in the mevalonate pathway for biosynthesis of IPP, including the central pathway enzyme HMG-CoA reductase. This raises the possibility that a targeted inhibitor of HMG-CoA reductase would be an anti-bacterial specific for these pathogens, with a unique point of attack that would leave this class of inhibitors unaffected by the resistance mechanisms that have developed against the more common cell-wall antagonists. This selective property would make mevalonate pathway inhibitors particularly attractive in burn unit or intensive care settings where sterilization of the normal bacterial flora make yeast over growth or colonization by resistant bacteria a common and severe complication of broad-spectrum antibiotic use. -
IMAP-Based Fluorescent Palarization Assay for High Throughput Screening of Protei
WANG, QIMING JANE
University of Pittsburgh
1 R03 DA024898-01
6
DESCRIPTION (provided by applicant): Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by diacylglycerol. It regulates many fundamental cell functions including cell proliferation, survival, differentiation and protein trafficking, and plays important roles in pathological conditions such as cardiac hypertrophy and cancer in multiple organ systems. However, the mechanisms underlying these effects of PKD are not clearly understood, and the role of PKD in cancer and other diseases has not been fully defined. This is partly due to the lack of effective pharmacological tools that specifically target PKD in normal cellular processes and in pathological conditions. The immediate goal of this proposal is to demonstrate the feasibility of an IMAP-based fluorescent polarization (FP) assay for high throughput screening (HTS) of PKD inhibitors. The assay we are proposing is in a 384-well, small volume format and has been adapted for high throughput screening. The research plan has been designed to achieve an important overall objective, specifically the discovery of novel potent and selective small molecule inhibitors of PKD that will be helpful in understanding the biological relevance of PKD and have the potential for long-term therapeutic application. Our preliminary study provided strong data on assay optimization and quality controls. Our proposed primary assay has passed extensive logistical analysis and variability assessment, and has been used successfully to screen a small compound library in HTS mode. Based on this solid foundation of preliminary data, we propose a detailed primary screening assay protocol with a cost estimate and outline the secondary and tertiary screening assays to identify and validate novel selective small molecule inhibitors of PKD. Successful completion of this application will have a profound impact on the advancement of research on diacylglycerol signaling and on the treatment of diseases caused by the deregulation of this signaling network. PKD plays an important role in the pathogenesis of cardiac hypertrophy and cancer in multiple organ systems. Inhibitors of PKD will facilitate the understanding of the relevance of PKD to these pathological conditions and could serve as potential drugs for the treatment of the diseases. -
HTS for Identification of Inhibitors against the ERK Signaling Pathway using a Ho
ZHENG, WEI
NIH/ NHGRI
1 X01 MH082406-01
6
DESCRIPTION (provided by applicant): The ERK phosphorylation signaling pathway plays a variety of important roles in regulation of cellular functions. ERK phosphorylation can be activated by receptor tyrosine kinases (RTK) and many G- protein coupled receptors (GPCR). The identification of inhibitors and antagonists for the multiple targets in this signaling pathway can provide useful research tools to study the cellular functions both in physiological and pathological conditions such as cancer and neuronal disorders. We propose to use a newly developed cell-based ERK phosphorylation assay for high throughput screening of the MLSCN compound library. Small molecule probes for multiple targets in this signaling pathway can be identified from a single primary screening. The specific antagonists/inhibitors for certain targets will be characterized by the secondary screenings and the information of these probes identified from this screening will be loaded into PubChem available for other investigators. This ERK phosphorylation assay is the first homogenous cell-based assay for intracellular kinase. The ERK phosphorylation signaling pathway plays a variety of important roles in regulation of cellular functions. ERK phosphorylation can be activated by receptor tyrosine kinases (RTK) and many G-protein coupled receptors (GPCR). A screening of the ERK phosphorylation in the MLSCN compound library stimulated by difference signaling pathways will identify inhibitors and antagonists for the multiple targets. It can provide useful research tools to study the cellular functions both in physiological and pathological conditions such as cancer and neuronal disorders. -
HTS for Identifying the Cell-Membrane Permeable IMPase Inhibitors.
ZHENG, WEI
NIH/ NHGRI
1 X01 MH082413-01
6
DESCRIPTION (provided by applicant): Inositol monophosphatase (IMPase) is a potential drug target for the development of lithium mimetic agents for the treatment of bipolar disorder. The traditional enzyme-based assays have failed to discover effective and cell-membrane permeable IMPase inhibitors. Cell-based enzyme assays offer a new approach for identifying inhibitors of intracellular enzymes. Recently, a cell- based inositol-1-phosphate (IP1) assay in the HTRF assay format was developed for the measurement of the activity of Gq-coupled GPCRs. This assay has been converted to a homogenous cell-based IMPase assay since IP1 is the substrate of IMPase. The additional advantage of this cell-based IMPase assay is that the enzyme activity is measured in its native form and in its physiological environment. We propose here to use this cell-based IMPase assay for a MLSCN compound library screen to identify cell membrane permeable IMPase inhibitors. Lithium is an important therapeutic agent for Bipolar disorder which affects approximately 1% of population. But the lithium treatment has server side effects and often intolerable to many patients. It is of interest to search for the small molecule lithium replacement which may have fewer side effects and is more tolerable to patients. By high throughput screening of the MLSCN compound collection we anticipate to identify the cell membrane permeable inhibitors for inositol monophosphatase. This may help to develop the new generation of lithium mimetic drugs. -
High Throughput Screening for Small Molecule Inhibitors of Heparin-Induced Tau Fibril formation
BALLATORE, CARLO
UNIVERSITY OF PENNSYLVANIA
1 X01 MH083262-01
7
DESCRIPTION (provided by applicant): Protein misfolding, fibrillization, and aggregation have emerged as potential therapeutic targets to treat many neurodegenerative diseases. Importantly, recent advances in the development of in vitro fibrillization assays have enabled high throughput screenings (HTS) to be used for interrogating compound libraries to identify fibrillization inhibitors. The studies proposed in this X01 application (PAR-06-259) entail the HTS of the compound libraries of the Molecular Libraries Small Molecule Repository (MLSMR) in a heparin-induced tau K18PL protein fibrillization assay based on two separate readouts: fluorescence polarization (FP) and thioflavine T (ThT) fluorescence. The tau fibrillization assay proposed in this application has been extensively validated and tested in 1536 well plate format. The compounds meeting the criteria set for selection in the tau fibrillization assay will be further evaluated in a panel of assays designed (a) to confirm the initial results and (b) to segregate possible false positives arising from non-specific mechanisms or assay dependent artifacts. The overall goal of the proposed studies is to identify new chemical entities (hits) that could inhibit the tau fibril formation. Such compounds would hold promise as new therapeutic or diagnostic agents in the area of Alzheimer’s disease and related tauopathies. -
A qHTS FRET-Quench Assay for 4′-Phosphopantethienyltransferases
BURKART, MICHAEL D
UNIVERSITY OF CALIFORNIA SAN DIEGO
1 R03 MH083266-01
7
DESCRIPTION (provided by applicant): The goal of this project is to identify chemical probes that inhibit phosphopantetheinyl transferase (PPTase) of bacterial secondary metabolism. This enzyme governs a posttranslational modification event that is essential to activation of polyketide and non-ribosomal peptide biosynthetic machinery. Many examples within these natural product classes have been identified as bacterial virulence factors required for pathogenesis. A competitive inhibitor or inactivator of PPTase activity could serve as potential therapeutic lead against a variety of Gram negative and Gram positive pathogens, including, among others, Mycobacterium tuberculosis, Staphylococcus aureus, and Escherichia coli. The essential nature of PPTase to fatty acid biosynthesis has targeted it for antimicrobial therapeutic development, and several inhibitor classes have been identified in the literature. However, these molecules do not show activity against PPTases of secondary metabolic pathways, a major target for drug discovery. We have developed a FRET-quench HTS bioassay for PPTase activity that functions in microplate format. This fluorescence-based system meets the minimal liquid-handling requirements of the NCGC, and it has been demonstrated against a small panel of PPTase inhibitor analogs. When identified, hits elucidated from this screening effort will serve as leads for antimicrobial therapeutic discovery. Furthermore, these compounds will also serve to further our ongoing NIH-funded investigations into the biosynthetic mechanisms of modular synthases. -
Screening for Inhibitors of Runx and CBFb Translocation Products in Leukemia
BUSHWELLER, JOHN H
UNIVERSITY OF VIRGINIA CHARLOTTESVILLE
1 X01 MH083230-01
7
DESCRIPTION (provided by applicant): The protein-protein interaction between the subunits of the heterodimeric transcription factor CBF, core binding factor a (CBFa) and Runx1 (CBFa), plays a critical role in hematopoiesis. Chromosomal rearrangements that target the core-binding factor genes are some of the most common mutations in leukemia. RUNX1 (AML1) is disrupted by the t(8;21)(q22;q22), t(12;21)(p13;q22), t(3;21)(q26;q22), t(16;21)(q24;q22), t(1;21)(p36;q22), t(5;21)(q13;q22), t(12;21)(q24;q22), t(14;21)(q22;q22), t(15;21)(q22;q22), and t(17;21)(q11.2;q22), all of which are associated with myeloid and lymphocytic leukemia. The gene coding for the CBFa subunit (CBFB) is also the target of a common chromosomal translocation, inv(16). Based on the importance of the interaction between CBFa and Runx1 for normal CBF function, it is not surprising that this interaction also plays a critical role in the function of CBF translocations. Binding to Runx1 is essential for the function of the CBFa-SMMHC fusion protein, making this a valid target for inhibitor development. We have recently examined the role of CBFa binding for the (dys) function of AML1-ETO, product of the t(8;21) The introduction of point mutations into the Runt domain in AML1-ETO which abrogate CBFa binding 400-fold results in loss of the ability to immortalize lin- BM cells as well as a loss of leukemogenesis in a mouse model of AML1-ETO leukemia. These results validate this protein-protein interaction as an appropriate target for the development of a small molecule inhibitor which may have therapeutic usefulness for appropriate forms of leukemia. Based on this, we have developed 2 specific aims. Aim 1 proposes to use HTS to identify inhibitors of the protein-protein interaction between the RUNX1 Runt domain and CBFa, a potential therapeutic approach for leukemias with translocations of RUNX1 or CBFB. We propose to use a well-validated FRET assay to screen the MLSCN compound library for inhibitors of this protein-protein interaction. A secondary ELISA assay will be used to confirm the hits. Subsequently, NMR methods will be utilized to identify the protein target of individual compounds. After optimization, these compounds will be tested in appropriate leukemia cell lines for efficacy and, if feasible, in appropriate mouse models of the leukemias. Aim 2 proposes to use HTS to identify inhibitors of the protein-protein interaction between the RUNX1 Runt domain and CBFa-SMMHC, a potential therapeutic approach for inv(16) related leukemia. This screen has the potential to identify compounds which can selectively inhibit CBFa-SMMHC with limited effects on CBFa itself, allowing restoration of CBF function as there is still one copy of the normal CBFa present in the leukemic cells harboring the inv(16). A well-validated FRET assay will be used here as well with a secondary ELISA assay and NMR verification. Compounds will be further optimized and tested in cells harboring the inv(16) followed by mouse model studies, if feasible. -
High Throughput Screen for Novel Inhibitors of Platelet Integrin alphaIIb-beta3
COLLER, BARRY S
ROCKEFELLER UNIVERSITY
1 R03 MH083257-01
7
DESCRIPTION (provided by applicant): The long term goal of the proposed screening project is the identification of novel inhibitors of the integrin IIb 3 with more favorable characteristics than currently available agents. The first specific aim is the identification of additional novel antagonists via high throughput screening, the second is the assessment of their specificity for IIb 3, and the third is the characterization of their mechanisms of inhibition. The development of new inhibitors represents a significant clinical need because current IIb 3 antagonists have been shown to cause thrombocytopenia in some patients, and the orally active IIb 3 inhibitors paradoxically caused increased mortality in Phase III trials. All of the orally active agents, as well as the 2 FDA-approved small molecule parenteral agents, function by competitively blocking the ligand binding site of IIb 3; thus, these agents act as ligand-mimetics. It is known that the binding of ligand by IIb 3 results in conformational changes in the receptor extracellular structure, and that these changes expose neo-epitopes on the receptor known as ligand-induced binding sites (LIBS). These conformational changes may underlie both the thrombocytopenia and the paradoxical prothrombotic effects of the oral agents. Thus, IIb 3 antagonists that lack ligand-mimetic activity may improve both therapeutic safety and efficacy. Specific Aim 1: To identify novel inhibitors of platelet adhesion to fibrinogen using platelet adhesion to fibrinogen as the screening assay. We have already used this assay to screen more than 33,000 compounds. Specific Aim 2: To assess the specificity of any inhibitors identified in the primary screen by testing their effects on various other cell adhesion molecules including but not limited to: interaction of the highly homologous integrin V 3 with vitronectin, interaction of integrin 2 1 with collagen, and interaction of GPIb with von Willebrand factor. The effect of the compounds on cell viability will also be assessed. Specific Aim 3: To characterize the mechanism by which the compounds inhibit adhesion of platelets to fibrinogen. The following assays will be employed to determine whether compounds interact directly with IIb 3: 1) aggregation of platelets in plasma following the addition of exogenous activators; 2) binding of fibrinogen to purified IIb 3; 3) adhesion of cells expressing IIb 3 to immobilized fibrinogen; 4) binding of soluble fibrinogen to platelets following treatment with IIb 3 activators. Those compounds that interact directly with IIb 3 will be characterized further to assess their effects on the conformation of IIb 3 by 1) measuring the exposure of ligand-induced binding site (LIBS) epitopes on platelets, and 2) testing the ability of transient exposure of the compounds to “prime” purified IIb 3 so that it binds fibrinogen. Finally, compounds that inhibit IIb 3 but do not induce conformational changes in the receptor will be selected for additional in silico docking studies, in vivo testing in animal models of thrombosis and hemostasis, and chemical modifications as a prelude to potential therapeutic development. -
HTS Assays for Microtubule Stabilizers
DAY, BILLY W
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
1 R03 MH081275-01A1
7
DESCRIPTION (provided by applicant): Agents that perturb the dynamics of cellular microtubules are proven cell biology tools, antitumor agents, and antifungal agents. Some have also shown promise as inhibitors of angiogenesis. Microtubule dynamics can be altered by small molecules through a variety of mechanisms, including direct interaction with tubulin (typically leading to inhibition of proliferation into microtubules — aka tubulin polymerization inhibitors; e.g., colchicinoids and vinca alkaloids), direct interaction with microtubules (typically leading to microtubule stabilization; e.g., taxanes), and through interaction with tubulin/microtubule-associated proteins (e.g., estramustine) Although literally thousands of agents are known to inhibit tubulin polymerization, only a handful of agents stabilize microtubules. The taxanes paclitaxel and docetaxel have provided great advances in clinical outcomes, and one or more of the epothilones will soon become part of oncologists’ arsenal. All of the known microtubule stabilizers are structurally complex, synthetically challenging small molecules. It would be of great interest to determine if the rich small molecule scaffolds within the MLSCN repositories will provide less synthetically expensive microtubule stabilizers. Traditionally, microtubule stabilizers have been found by low throughput, temperature-controlled turbidimetry screens with bovine or porcine brain tubulin, followed by electron microscopic analysis of the polymer formed and confirmatory cell-based fluorescence microscopy analyses. We have developed a fluorescence cell-based high information content screen that can be performed reproducibly in microtiter plate-based format with robotic liquid handling. The assay provides excellent Z’ factors (> 0.8) with low coefficients of variation. Cells are plated on collagen I, allowed to attach overnight, and are then treated with vehicle or test agents for 21 h. After gentle permeabilization, chromatin (Hoechst dye) and microtubules (anti-alpha-tubulin) are labeled with different fluorophores, followed by a digital image analysis that allows for quantitation of the labeled structures in large numbers of cells, leading to excellent statistical measures. Hits will be verified by examination of a concentration range in the cell-based experiments, as well as by the traditional biochemical methods mentioned above. -
Therapeutic Inhibitors of Phosphomannose Isomerase
FREEZE, HUDSON H
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 R03 MH082386-01A1
7
DESCRIPTION (provided by applicant): Our goal is to find novel non-competitive inhibitors of phosphomannose isomerase (PMI) that can be used as a therapeutic for treating patients with Congenital Disorder of Glycosylation Type Ia (CDG-Ia). This is a rare autosomal recessive metabolic disorder with multisystemic symptoms. Patients have decreased activity of phosphomannomutase 2 (PMM2) that impair the conversion of Mannose-6-P (Man-6-P) to Mannose-1-P leading to defective N-glycosylation. There is no therapy for CDG-Ia patients and many die. CDG-Ib patients have PMI, and this enzyme mutated interconverts Man-6-P and fructose-6-P (F-6-P). However, dietary supplements of mannose completely reverse the symptoms of CDG-Ib patients, some of which overlap with the symptoms of CDG-Ia patients. Unfortunately, mannose does not help CDG-Ia patients because their normal PMI activity catabolizes mannose-derived Man-6-P; the ratio of PMI: PMM activity is simply too high to increase intracellular Man-6-P. We propose that CDG-Ia patients will benefit from dietary mannose if we simultaneously reduce PMI activity with a non-competitive inhibitor. This would allow a modest intracellular accumulation of Man-6-P and drive metabolic flux into the glycosylation pathway using the residual PMM2 activity. The purpose of this application is to identify PMI-specific, cell-permeable, non-competitive inhibitors of human PMI using the MLSCN facilities and resources, including the MLSMR compound collection. PMI assays are well-established, and will be adapted to fluorescent readout in 384-well plate format. Confirmed hits will be validated using live cell-based PMI assays that measure metabolic flux of mannose into glycosylation vs. glycolysis. A successful outcome would initially be the identification of a novel chemical probe, and ultimately to the development of potential therapeutics to be used in conjunction with mannose therapy for CDG-Ia patients, who have no other therapeutic options. -
High Through-Put Screening of Plasmodium Falciparum Aminopeptidases
GARDINER, DONALD L
QUEENSLAND INSTITUTE OF MEDICAL RESEARCH
1 R03 MH082342-01A1
7
DESCRIPTION (provided by applicant): Malaria remains a significant cause of morbidity and mortality worldwide with an estimated 2 million people dying from this disease every year. The majority are children under 5 living in Sub-Saharan Africa. Drug resistance to the commonly available drugs is widespread, and with a safe and effective vaccine still many years away, new chemotherapeutic agents are required to ensure that cheap and effective treatment can be maintained. Aminopeptidases are involved in the catabolism of proteins in all organisms studied to date. In the apicomplexan parasite of the genus Plasmodium the cause of malaria, we have shown that it is possible to selectively target these enzymes such that we can kill the parasite both in the laboratory and in a mouse model. We have generated recombinant proteins from two of these enzymes that are suitable for adaptation to high throughput screening to identify new and novel compounds that can target these enzymes. -
Identification of Modulators of the TRPML3 and TRPN1 ion Channels
HELLER, STEFAN
STANFORD UNIVERSITY
1 R03 MH083077-01
7
DESCRIPTION (provided by applicant): The main goal of this project lies in identifying agonists for two transient receptor potential (TRP) ion channels that are potentially involved in inner ear mechanosensation. One of the major obstacles of inner ear biology is the lack of specific pharmacological tools to investigate the process of mechanotransduction of inner ear sensory hair cells. Although the vertebrate hair cell transduction channel remains a mystery, a number of TRPs are connected to a potential physiological function in the inner ear. Particularly TRPML3 and TRPN1, the focus of this application, have been shown to be important for hearing and balance in mice and zebrafish. Identification of agonists and, in future studies prospective antagonists, would not only advance inner ear and sensory neurobiology, but could also provide us with potential drug candidates for the treatment of vertigo and tinnitus. -
Multiplex Screening for ABC Transporter Inhibitors
LARSON, RICHARD S
UNIVERSITY OF NEW MEXICO ALBUQUERQUE
1 R03 MH081228-01A1
7
DESCRIPTION (provided by applicant): The presence of residual tumor cells (MRD, minimal residual disease) after initial induction therapy is a key predictor of patient outcome. Several sets of observations suggest that a relatively small number of tumor cells either have or acquire resistance to chemotherapeutics during induction therapy. In order to improve patient outcome, there is a critical need to identify new targets and antagonists for pharmaceutical intervention. Furthermore, an increasing and persistent number of drug resistant cells correlate with patient outcome in a number of cancers. Recently, ABC gene products, including ABCB1 (P-glycoprotein, MDR-1), multidrug resistance protein ABCC1 (MRP1), ABCC2 (MRP2, cMOAT), and ABCG2 (BCRP, MXR, ABCP) have been described and been shown to influence oral absorption and disposition of a wide variety of drugs. As a result, the expression levels of these proteins on human cancer have important consequences for an individual’s susceptibility to certain drug-induced side effects, interactions, and treatment efficacy. One of the most widely studied multidrug resistance (MDR)-ABC transporter is ABCB1. This particular member of the ABC transporter proteins functions to remove lipids and drugs as they intercalate and diffuse through the cell membrane. While more than 48 members of the ABC transporter superfamily have been identified, a relatively few such as ABCB1, ABCC1 and lung resistance protein (LRP) appear to have the greatest clinical impact on therapy resistance. The goal of this proposal is to first screen for ABCB1 inhibitors in a developed flow cytometry based assay and then to develop and employ a multiplex assay that can screen for inhibitors of multiple members of the ABC family. -
High-Throughput Screening for Compounds Blocking Interaction Between CBFb-SMMHC and RUNX1
LIU, PU PAUL
U.S. NATIONAL HUMAN GENOME RESEARCH INST
1 X01 MH083259-01
7
DESCRIPTION (provided by applicant): The fusion gene CBFB-MYH11 is generated by a chromosome 16 inversion that is present in almost all cases of acute myeloid leukemia subtype M4Eo. Previous results from in vitro and in vivo studies have shown that CBFB-MYH11 is a key player in the pathogenesis of leukemia and that the encoded fusion protein, CBFa-SMMHC, binds to RUNX1 with high affinity and dominantly inhibits RUNX1 function in hematopoiesis. Consistent with this hypothesis, RUNX1 mutations and translocation fusion products involving RUNX1 are frequently found in human leukemias, and these mutations and translocation products also inhibit RUNX1 function. We therefore propose to identify compounds blocking the interaction between CBFa-SMMHC and RUNX1 for future studies of this important leukemia fusion protein. These compounds are likely to interrupt leukemogenesis caused by CBFB-MYH11 and thus potentially serve as starting points for the development of a new treatment for the leukemia patients with this fusion gene. -
High Throughput Screen for Hsp90 Inhibitors
MATTS, ROBERT L.
OKLAHOMA STATE UNIVERSITY STILLWATER
1 X01 MH083240-01
7
DESCRIPTION (provided by applicant): Hsp90 functions as molecular chaperone that is ubiquitously expressed and required for the viability of all eukaryotic cells. Hsp90 has an obligatory role in facilitating the post-translational maturation of numerous proteins that regulate many signal transduction pathways in vivo. Of the known Hsp90-dependent clients, 48 are directly related to oncogenesis, with Hsp90-dependent clients being represented in all six hallmarks of cancer. Because so many oncogenic proteins are dependent upon Hsp90 to attain and maintain a functional conformational maturation, inhibitors of Hsp90 provide a combinatorial attack on multiple signaling pathways that are essential for malignant cell growth. Consequently, Hsp90 has emerged as an exciting new target for the development of anti-tumor agents. Additional observations suggest that Hsp90 inhibitors also have the potential to prevent or reverse the progression of neurodegenerative diseases characterized by the accumulation of misfolded protein aggregates. Inhibitors of Hsp90 based on the natural product, geldanamycin, have entered more than 20 clinical trials for the treatment of cancer. While initial results appear promising, the low solubility and hepato-toxicity of geldanamycin derivatives may limit its clinical potential. Because of the tremendous potential of Hsp90 inhibitors for the treatment of cancer, neurodegenerative disease and other disorders characterized by the accumulation of toxic protein aggregates, a robust, sensitive and simple high- throughput assay for identifying Hsp90 inhibitors has been developed and verified based on the Hsp90- dependent refolding of thermally denatured firefly luciferase in rabbit reticulocyte lysate [20]. The specific aims of this proposal are to: 1) implement this high-throughput assay to identify Hsp90-inhibitors at the Molecular Libraries Screening Centers Network; 2) eliminate false positives by using a secondary screen that detects compounds that act by directly inhibiting the activity of native luciferase; 3) identify the most promising candidate compounds for the follow-up research program by estimating an initial IC50 for the inhibitory activity of the compounds. In the follow-up research program previously developed protocols will be applied to: a) determine the potency (IC50) and the biochemical mechanism of inhibitory action of the selected compounds (i.e., effect of compounds on Hsp90 proteolytic fingerprints, and the co-chaperone composition of Hsp90 complexes); b) assay the ability of the compounds to inhibit the ATPase activity of Hsp90, the proliferation of cultured cancer cells, and deplete Hsp90-dependent oncogenic proteins from cultured cells; and c) synthesize derivatives of promising compounds to elucidate structure-activity relationships that may lead to the development easily modified scaffolds and more potent and less toxic Hsp90-inhibitors. Once obtained, these results will clearly provide a rational method for the preparation of improved analogues for the treatment of cancer and neurodegenerative disorders. -
Identification of Inhibitors of Trypanosoma Brucei Hexokinases
MORRIS, JAMES CULVIN
CLEMSON UNIVERSITY
1 R03 MH082340-01A1
7
DESCRIPTION (provided by applicant): Current therapies for African sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, are toxic and inefficacious. Glycolysis is a promising target in the fight against T. brucei, as the pathway is essential. Inhibitors of hexokinase (TbHK), a key glycolytic enzyme, have been shown to be toxic to trypanosomes. Since the trypanosomal HKs share little similarity with their human counterpart, they make a promising target for therapy development. We have recently cloned and expressed active recombinant TbHK, and have modified standard enzyme assays for high throughput identification of inhibitors. Here we propose to provide the Molecular Libraries Screening Centers Network with sufficient recombinant protein (and the methods used to monitor its activity under high throughput conditions) in order to identify inhibitors that may prove useful lead compounds as both anti-trypanosomal drugs and as tools for better understanding of the biochemistry of TbHKs. -
A High-Throughput Screen to Identify Small Molecule Compounds That Augment the spread of oncolytic G47D-HSV for prostate tumor therapy
PASSER, BRENT J
MASSACHUSETTS GENERAL HOSPITAL
1 R03 MH083258-01
7
DESCRIPTION (provided by applicant): Oncolytic HSV-based vectors selectively replicate in tumor cells causing direct killing i.e., oncolysis, while at the same time sparing normal cells. Although the use replication- competent HSV viruses for tumor therapy have proven to be effective to a certain degree, a number of issues remain that preclude its successful use in clinical trials. One major drawback is its limited capacity to spread intercellularly and kill neighboring cancer cells. The goal of this project is to identify small molecule compounds that augment the spread of the oncolytic HSV-based G47?? virus expressing the green fluorescent protein (G47?? -CMV-GFP) in prostate cancer cells using a simple “mix and measure” 384-well formatted cell-based fluorescent assay. PC3 prostate cancer cells will be inoculated with G47?? -CMV-GFP at a low multiplicity of infection (MOI) and augmented viral spread promoted by pretreatment with the chemical library will be quantified by fluorescence. The identification and validation of chemical amplifiers of G47? spread will permit the exploration of two long-term objectives: 1) to develop therapeutic agents not only for treating prostate cancer but also as “generalized” therapeutic enhancers of viral oncolysis for treating other forms of cancers and 2) to use as a “chemical toolbox” in order to dissect the molecular pathways involved in viral oncolysis. Understanding the molecular events that govern viral oncolysis will not only have direct implications in developing novel anti-cancer HSV therapeutics but it may also provide unexpected insights into the pathology of HSV. -
A Cell Based HTS Approach for the Discovery of New Inhibitors of RSV
SEVERSON, WILLIAM E
SOUTHERN RESEARCH INSTITUTE
1 R03 MH082403-01A1
7
DESCRIPTION (provided by applicant): Currently, there are no commercially available vaccines to protect humans against Respiratory syncytial virus (RSV) infection. RSV is associated with substantial morbidity and mortality and is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Since humans have no immunity against RSV infection, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. The existing therapies for the acute infection are ribavirin, which has inconsistent clinical results, and the prophylactic humanized monoclonal antibody (Synagis(r) from MedImmune) that is expensive and limited to use in high risk pediatric patients. The economic impact of RSV infections due to hospitalizations and indirect medical costs is approximately > $ 650 million annually. Thus, there is a critical need to discover novel antiviral drugs to supplement existing chemotherapeutics. A high throughput screening (HTS) approach provides an opportunity to screen large compound libraries in vitro. We have developed and validated a 384-well cell-based assay that measures CPE induced in HEp-2 cells by RSV infection, using a luminescent-based detection system for signal endpoint. This molecular HT molecular screen will provide the foundation for secondary dose response assays that will divulge critical antiviral potency and cytotoxicity information on active compounds and bestow the scientific community with assays that allow for the rapid identification of potential inhibitors of RSV by evaluating large compound libraries in vitro. -
HTS to Find Inhibitors of p47phox, a Regulatory Protein of Noxes
SMITH, SUSAN M.E.
EMORY UNIVERSITY
1 R03 MH083234-01
7
DESCRIPTION (provided by applicant): Numerous diseases are linked to inflammation and oxidative stress. Nox enzymes provide the majority of reactive oxygen species associated with oxidative stress and have recently been validated as targets for drugs that would prevent and treat these conditions. Technical problems have to date prevented the adaptation of Nox activity assays for high throughput screening (HTS). In collaboration with Dr. Haian Fu, Director of Emory’s MLSCN center, we have developed and optimized a novel HTS method with which we have successfully selected new candidate inhibitors of Noxes. Secondary activity screens that we developed and tested allowed us to identify bona fide Nox inhibitors that are candidates for lead drug compounds. Continuing our collaboration with Dr. Fu, we will use the HTS method to screen the MLSCN library at the Emory MLSCN center. Using hits from this screen, we will carry out secondary activity and counterscreens to determine the highest potency and most selective Nox1 and Nox2 inhibitors. Such inhibitors should provide valuable tools for research, and may serve as leads for drugs that can prevent and/or cure disease. Follow-on studies will focus on hit-to-lead development with the goal of identifying candidate drugs. -
Targeting HIV-1 Nef with Small Molecules
SMITHGALL, THOMAS EDWARD
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
1 R03 MH083223-01
7
DESCRIPTION (provided by applicant): The HIV-1 accessory protein Nef is essential for high-titer viral replication and AIDS progression. Nef has no known catalytic activity, and interacts with multiple host cell signaling proteins, including members of the Src protein-tyrosine kinase family. Previous work from our laboratory has shown that Nef binds and activates Hck, a Src family kinase (SFK) selectively expressed in macrophages, which are an essential HIV target cell and viral reservoir. Other work has established that knock-down of Hck expression blocks M-tropic HIV replication in primary human macrophages, and that homozygous-null hck mice show longer latency for Nef-induced AIDS-like disease. These studies strongly implicate Nef-Hck protein complexes in AIDS pathogenesis and as rational targets for anti-HIV drug discovery. Recently, we developed an in vitro fluorescence-based assay to identify inhibitors of a homogeneous Nef:Hck complex, and have validated this assay in 384-well plates. In a preliminary screen of a small library of compounds biased towards kinase inhibitors, we obtained three confirmed hits, all of which exhibited activity in a secondary assay against HIV replication. We believe that this assay is ideal for use in the Molecular Libraries Screening Center Network (MLSCN), and propose the following Specific Aims to be conducted in conjunction with the Center: Aim 1 – Transfer and implement our Nef:Hck HTS assay in the MLSCN; Aim 2 – Validate hits obtained in the primary screen in secondary assays with Hck and other SFKs in the presence and absence of Nef; Aim 3 – Evaluate the cytotoxicity and preliminary anti-HIV activity of the confirmed hits. Due to the emergence of HIV strains resistant to conventional anti-retroviral agents, there is an urgent need to identify and validate new HIV targets for the development of novel therapeutics. HIV accessory proteins in general, and the Nef protein in particular, have been suggested as possible therapeutic targets but to date no useful compounds have been reported that work via these proteins. Our hypothesis, supported by preliminary data, is that HIV Nef in complex with a host cell kinase (Hck) essential for disease progression represents a rational target for novel anti-HIV drug discovery. Our published work strongly suggests that Interaction with Nef may induce a novel conformation of Hck that may be addressable with small molecules. In addition, our assay has the potential to identify novel structures that interact directly with Nef itself, compromising its ability to recruit and activate Hck in the assay. Performing a screen in conjunction with the MLSCN is a very exciting prospect because of the wide range of novel structures available. In addition to the potential for new therapeutics, identification of compounds that selectively modify HIV Nef function will be invaluable tools to explore the function of this enigmatic protein. -
Luciferase Screen for MDM2 E3 Ligase Inhibitors
STOCKWELL, BRENT R
COLUMBIA UNIV NEW YORK MORNINGSIDE
1 R03 MH082369-01A1
7
DESCRIPTION (provided by applicant): The E3 ligases are involved in regulating other proteins by covalent ligation to the 76 amino acid protein ubiquitin. This post-translational modification can result in altered conformation, altered activity, or degradation of the substrate protein. Thus, E3 ligases are effectors of a major means of post-translational modification of proteins in many species, including mammals. Ubiquitin-protein ligation requires the action of an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme and an E3 ubiquitin ligase. In humans, there are thought to be two E1 enzymes, 36 E2 enzymes and up to 1000 E3 enzymes. Two main domains have been found to function as E3 ligases: HECT and RING domains; the majority of predicted E3 ligases have a RING domain rather than a HECT domain. The RING domain proteins can be further subdivided into the canonical RING domain and the RING-related domains known as U box and PHD finger. While HECT domain ligases contain a 350-residue ligase domain that directly binds the activated ubiquitin molecule, RING finger ligases serve as a scaffold for spatially coordinating the ubiquitin source and the target molecule. Although the RING finger motifs show little homology to each other, the core conserved structure for these domains is a pattern of cysteine and histidine residues coordinating two zinc ions. The dipeptide boronic acid bortezomib (PS-341, Velcade) is a potent proteasome inhibitor, has selective anticancer activity in tumor cells and in mice and was recently approved for clinical use in multiple myeloma. Selective E3 ligase inhibitors would be preferable because they should be more selective and less toxic. Unlike other large protein families, such as proteases and kinases, there are few small molecule tools for modulating the functions of E3 ligases. Specific inhibitors and activators are valuable for defining functions and substrates of proteins. RNA interference and cDNA expression can also assist in illuminating functions of proteins, but precise spatial and temporal regulation of proteins is optimally performed with small molecule modulators. In addition, E3 ligase domains are frequently found within the context of a large protein with other functions. Small molecules, in contrast to RNAi knockdown or cDNA expression, can readily reveal the function of a single domain in a large multi-domain protein, expressed at endogenous, physiological levels. In addition, small molecules can sometimes be developed into therapeutic agents. In this application, we propose to screen for small molecules that inhibit MDM2 E3 ligase activity. -
High-Content Cell-Based Screening for Modulators of Autophagy
YIN, XIAO-MING
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
1 R03 MH083154-01
7
DESCRIPTION (provided by applicant): Autophagy is a cellular process in which a part of cellular contents are sequestered by a double membrane structure, which are then degraded through the lysosome. This fundamental process is important for cell survival, organelle homeostasis, energy metabolism, and defense against intracellular pathogens. Autophagy has significant implications in the pathogenesis of neurodegenerative diseases, cancer and microbial infections. Studies have shown that modulating autophagy could improve cancer therapy, promote the resolution of infection and lessen neurodegeneration in animal models. However, there are still very much to be learnt about the molecular mechanism of autophagy and the regulation in these settings. We propose in this application a high content cell based screening for novel chemicals that can modulate autophagy. We have conducted preliminary studies to demonstrate that this approach is feasible and the assay conditions can be optimized. Our short-term goal is that through this screening we will be able to find unique compounds that are more potent, less toxic and more specific. Our immediate future goal is to define the structure of promising compounds in relationship with their biological effects. We would also like to determine the specific molecular targets to understand how the compound works. Our long term hope is that the knowledge and the materials developed through this study will be valuable for future clinical applications. -
Screening for Selective Ligands for GPR55
ABOOD, MARY E
CALIFORNIA PACIFIC MED CTR RES INSTITUTE
1 X01 DA026205-01
8
DESCRIPTION (provided by applicant): Marijuana is the most widely abused illegal drug and its spectrum of effects suggests that several receptors are responsible for its activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, and recently the orphan G protein coupled receptor, GPR55, was declared as one of the missing cannabinoid receptor subtypes. However, the validity of this assignment remains in question based upon the identification of another class of GPR55 ligands. As a consequence of the identification of GPR55 as a target for cannabinoid binding, it becomes important to identify GPR55-selective ligands that can define its role in addiction. The primary high throughput assays we propose for identifying GPR55 active compounds are based upon the ability to monitor the activation states of ligand-bound GPR receptors using high content imaging employing beta-arrestin green fluorescent protein biosensors. The specific aims of this proposal are: 1) To identify small molecule agonists of GPR55 by high content screening of MLSCN (Molecular Library Screening Center Network) libraries and 2) To identify small molecule antagonists of GPR55 by high content screening of MLSCN libraries. We propose to provide the MLSCN with cell lines expressing the receptors and beta-arrestin fluorescent protein biosensors. These studies will provide tools for delineating the biochemistry of GPR55 to enable an understanding of the molecular basis of cannabinoid addiction and treatment. -
High Throughput Screen for SMA
ANDROPHY, ELLIOT J
UNIV OF MASSACHUSETTS MED SCH WORCESTER
1 R03 MH084179-01
8
DESCRIPTION (provided by applicant): Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder characterized by progressive muscle weakness and death due to deterioration of motor neurons. SMA results from homozygous mutation of the SMN (survival motor neuron) 1 gene. A nearly identical copy, SMN2, fails to protect from development of SMA as its major mRNA undergoes alternative splicing that encodes for an unstable SMN protein. This alternative spliced product excludes exon 7. A small fraction of SMN2 transcripts include exon 7 and encode the identical SMN protein as does SMN1. We have re-designed and validated a cell-based reporter assay that combines three mechanisms to increase SMN protein: induction the SMN promoter, increased inclusion of exon 7, and stabilizing the SMN RNA or protein. The assay has already been established at the NIH NCGC and is fully operational. The first aim is to perform a HTS with the full MLSCN library. The second aim is to validate the most active compounds in secondary assays. The objective is to define structure activity relationships (SAR) for lead optimization and subsequently test available structural analogues to identify pharmacologically active drug-like compounds. -
Agonists and Antagonists for the Kappa Opioid Receptor
BARAK, LAWRENCE S.
DUKE UNIVERSITY
1 X01 DA026208-01
8
DESCRIPTION (provided by applicant): Addiction in its many forms is a major health concern. The recent realization that addictive behavior is a consequence of signaling disorders in the brain, and as such is a disease, provides a rational basis to expect that effective treatments can be designed. Many of the signaling pathways responsible for major components of drug addiction and addictive behaviors originate with the binding of drugs and transmitters to cell membrane receptors. A long term goal of our laboratory has been to unravel these pathways. We believe that the identification of small molecules, each able to block only a distinct receptor underlying an addiction will provide a means to untangle the many pathways resulting in addictive behavior and create detailed pharmacological maps for designing novel targeted treatments. This grant proposes screening a G protein-coupled receptor relevant to drug abuse and to the study and treatment of addiction. The specific aims of this application are: (1) To identify subtype specific small molecule agonists of the human kappa opioid receptor (KOR). (2) To identify subtype specific small molecule antagonists of the human kappa opioid receptor (KOR). We propose to provide the MLSCN with cell lines expressing the receptors and beta-arrestin fluorescent protein biosensors that in combination can be used in high-content, high throughput primary screens to identify high-affinity signaling pathway modulators. These compounds can be optimized to provide the pharmacological means to precisely control the signaling of their specific target receptor subtype. This work thus provides the addiction field both novel research tools and potential therapeutic scaffolds. -
Identification of SV40 T Antigen Inhibitors: A Route to Novel Anti-Viral Reagent
BRODSKY, JEFFREY L.
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
1 R03 MH084077-01
8
DESCRIPTION (provided by applicant): The Polyomaviruses (Py) normally form latent infections in the kidney but upon re-activation can trigger the onset of incurable diseases. For example, re-activation of JC virus (JCV) results in Progressive Multifocal Leukoencephalopathy, which kills ~5% of AIDS patients, and re-activation of BK virus (BKV) leads to BKV Associated Nephropathy, which is the leading cause of rejection in kidney transplant recipients. Another Py is SV40, whose natural hosts are monkeys, and ~10% of the human population is infected with SV40 because the first polio vaccines were inadvertently contaminated with the virus; the long-term effects of SV40 infection in humans are unclear, although SV40 has been linked to several cancers. Each member of the Py family encodes the large tumor antigen (TAg), which is essential for viral replication and tumorigenesis. Previous work in the PI’s laboratory contributed to the discovery that TAg possesses a chaperone-like domain, and that the interaction between this domain and the cellular Hsp70 chaperone is essential to orchestrate viral replication and cellular transformation. Furthermore, small molecule modulators that compromise TAg-Hsp70 interaction have been identified. One modulator (MAL2-11B) inhibits TAg’s endogenous ATPase activity, an activity that is also essential for viral replication and cellular transformation. Indeed, MAL2-11B inhibits SV40 DNA synthesis in vitro and viral replication in tissue culture cells. Through these efforts, a significant battery of in vitro methods have been developed that have been re-configured for a high throughput screen to identify new, more potent TAg and Py inhibitors. Specifically, TAg’s ATPase activity has recently been assessed in a 96-well format. Thus, the hypothesis underlying these studies is that chemical modulators of TAg ATPase activity might represent a new avenue to combat the catastrophic effects of Polyomavirus-associated diseases. The Specific Aims of this application are: (1) To perform high throughput screens to isolate TAg inhibitors using small molecule libraries at the resident University of Pittsburgh MLSCN, and (2) To utilize established assays as secondary and counter screens to assess the effects of “hits” from Aim 1. In both aims, MAL2-11B will serve as a positive control. Further tests will be conducted with the MLSCN and resident medicinal chemists to obtain more refined chemical probes. The Relevance of this study is underscored by the lack of approved and efficacious methods to treat diseases that arise from Polyomavirus infection. The described efforts employ a novel target, a positive control, and local collaborators, which will aid the completion of the stated goals. -
Chemical Genetic Analysis of Platelet Granule Secretion
FLAUMENHAFT, ROBERT C
BETH ISRAEL DEACONESS MEDICAL CENTER
1 R03 DA026209-01
8
DESCRIPTION (provided by applicant): Platelets are essential components of arterial thrombosis. Interference with platelet signal transduction can dramatically reduce morbidity and mortality of platelet-mediated thrombotic disease such as heart attack, stroke, and peripheral vascular disease. Identification of optimal therapeutic targets for inhibition of platelet activation has been slowed, however, because platelets are anucleate and not amenable to genetic manipulations frequently used to study signal transduction mechanisms in nucleated cells. In contrast, the ready availability of platelets and their ability to undergo profound phenotype change upon exposure to agonists has made them a popular cellular model for studies using small molecules. We have developed a high throughput assay to identify novel anti-platelet agents that inhibit platelet activation as monitored by dense granule secretion. The goal of this undertaking is to discover new molecular targets that control platelet granule secretion. In collaboration with a facility within the Molecular Library Screening Center Network (MLSCN), we will expand the number of compounds that we screen, improve the rate at which screening is performed, and optimize compounds with antiplatelet activity. We will evaluate granule secretion in two related assays. One will identify compounds that inhibit dense granule secretion induced by SFLLRN, which stimulates platelets via protease-activated receptor 1. The second assay will identify compounds that inhibit dense granule secretion induced by collagen I, which activates platelets via glycoprotein VI. This strategy will facilitate identification of compounds that inhibit platelet function by diverse mechanisms. Once identified, compounds will be tested in a previously established battery of platelet function tests and evaluated for antithrombotic potential in an in vivo murine model of thrombus formation. -
Identification of Inhibitors of the Plasmodium Falciparum M18 Metalloprotease
GARDINER, DONALD L
QUEENSLAND INSTITUTE OF MEDICAL RESEARCH
1 R03 MH084103-01
8
DESCRIPTION (provided by applicant): Malaria remains a significant cause of morbidity and mortality worldwide with an estimated 2 million people dying from this disease every year. The majority are children under 5 living in Sub-Saharan Africa. Drug resistance to the commonly available drugs is widespread, and with a safe and effective vaccine still many years away, new chemotherapeutic agents are required to ensure that cheap and effective treatment can be maintained. The M18 class of metalloproteases are involved in the catabolism of proteins. In the apicomplexan parasite of the genus Plasmodium the causative agent of malaria, this key enzyme is involved with hemoglobin digestion. Using an antisense transgenic approach we have shown that it is possible to selectively target this enzyme. This leads to a lethal phenotype. We have generated a recombinant protein to this enzyme that is suitable for adaptation to high throughput screening. This will lead to the identification of new and novel compounds that can target this enzyme. -
Identification of Small Molecule Antagonists for the Neuropeptide Receptor Using
HEILIG, MARKUS ALEXANDER
U.S. NATIONAL INST ALCOHOL AB/ALCOHOLISM
1 X01 DA026210-01
8
DESCRIPTION (provided by applicant): The MLSCN library will be screened for non-peptide antagonist with activity at the Neuropeptide S (NPS) receptor. NPS has recently been identified as a central nervous system neurotransmitter, and is the endogenous ligand for a previously orphan GPCR. The NPS receptor is coupled to Gq and Gs type G proteins. NPS has an unusual activity profile, in that its central administration to rodents is anxiolytic as well as arousing, while prototypical anxiolytics are all sedative. We have recently found that central NPS administration potentiates stress-induced reinstatement of alcohol seeking in rats. NPS is currently the only known ligand for the NPS receptor. Its peptide nature and agonist properties limit its utility for an analysis of the role played by endogenous NPS in alcohol related behaviors. Identification of small molecule NPS antagonists will therefore provide unique research tools to evaluate the role of NPS receptor function in alcohol-seeking behavior, and may facilitate development of new therapeutics for alcoholism. A screenable NPS receptor assay has been developed and passed robotic validation. Mouse neuropeptide S receptor was stably expressed in a CHO-K1 cell line (Invitrogen). This cell line has good attachment properties and is suitable for use in high throughput screening. The assay has been miniaturized into 1536-well format and validated on the Kalypsys system. Using a highly sensitive TR-FRET based cAMP assay and optimized conditions, a robust and reproducible activation of adenylate cyclase in response to NPS was found, with a signal-to- basal ratio of 3.6. The cAMP assay allows screening for ligands with agonist or, following agonist activation, antagonist activity at the NPS receptor. The primary objective of this project is to identify compounds with antagonist properties, to provide tools that will allow studies of the role played by endogenous NPS in alcohol seeking behavior. A large amount (1g) of neuropeptide S has been synthesized and will be used as agonist in the antagonist screen. Representative hits from candidate structure groups, as well as potent singletons, will be confirmed by titration in the same cAMP assay as above. Counter screen for false positives will be carried out in CHO-K1 cell line expressing the neuropeptide Y (NPY) Y1 receptor. False positive antagonists will be identified by their ability to inhibit adenylate cyclase activity stimulated by forskolin. An established receptor binding assay using iodinated NPS as ligand, and an intracellular calcium release assay will be performed for final characterization of leads. Optimization of leads will be carried out in collaboration with chemists at the NCGC, and optimized leads will be assessed for in vivo activity using blockade of NPS induced locomotion as a first level. Neuropeptide S is a recently found neurotransmitter. Administration of NPS has been shown to potentiate relapse to alcohol seeking behavior in experimental animals. Identification of small molecule ligands for NPS receptor will provide useful research tools to evaluate the role of endogenous NPS in relapse to alcohol seeking, and could ultimately lead to development of new therapeutics for alcoholism. -
High Throughput Chemical Genomics to Identify Novel Inhibitors of CHOP
KAUFMAN, RANDAL J.
UNIVERSITY OF MICHIGAN AT ANN ARBOR
1 R03 MH084182-01
8
DESCRIPTION (provided by applicant): Many genetic and environmental diseases result from defective protein folding within the secretory pathway. Aberrantly folded proteins are recognized by the cellular surveillance machinery and retained within the endoplasmic reticulum (ER). Under conditions of malfolded protein accumulation, the cell activates the Unfolded Protein Response (UPR) to clear the malfolded proteins and, if unsuccessful, initiates a cell death response. The proposed studies will identify and characterize drug-like small molecular probes that specifically inhibit the apoptotic arm of the UPR. These compounds will have potential therapeutic application to diverse disease states. The proposed studies will test the hypothesis that reversible inhibition of the UPR death program mediated by CHOP will be an effective strategy to facilitate recovery from pathophysiological stress in diseases characterized by chronic ER stress and protein misfolding. In preliminary studies we have employed complementary high throughput cell-based assays and identified small molecule inhibitors of pro-apoptotic CHOP and enhancers of the adaptive IRE1-XBP1 axis. We propose to extend these initial screens by interrogating larger more diverse chemical collections in collaboration with the Molecular Libraries Screening Centers initiative. Biochemical and genetic approaches will validate the ability of lead compounds to modulate specific UPR subpathways and to identify their molecular targets and mechanism(s) of action. Synthetic chemistry will determine structure activity relationships in search for compounds with higher affinity and specificity. Lead compounds will be tested for their ability to facilitate proper protein folding and limit pathological UPR responses in cell -based assays. Most promising candidates will be evaluated in vivo in several physiologically relevant model systems of human protein misfolding diseases including diabetes, hemophilia, lysosomal storage diseases, and a1-antitrypsin deficiency. The long-term objective of the proposed studies is to identify novel small molecule inhibitors of the UPR death response and characterize their potential as therapeutic agents for treating diseases caused by aberrant protein folding and/or trafficking. Small molecule UPR agonists/antagonists identified through the proposed studies will provide a convenient cost-effective means of treating patients suffering from diseases for which therapies are limited to minimally effective gene therapy or extensive costly enzyme replacement therapies. -
High Throughput Screen for JAK2V617F Mutant Selective Inhibitors
LEVINE, ROSS L
SLOAN-KETTERING INSTITUTE FOR CANCER RES
1 R03 DA026206-01
8
DESCRIPTION (provided by applicant): The identical gain-of-function JAK2V617F allele is present in the majority of patients with the myeloproliferative disorders (MPD) polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), and in vitro and in vivo data demonstrate the central role of this activating mutation in the pathogenesis of these disorders. Although small molecule inhibitors of JAK2 kinase activity are being developed, these compounds inhibit both wild-type and mutant JAK2 kinase activity. The central role of JAK2 signaling in a spectrum of cellular processes suggests these compounds may have hematopoietic and non-hematopoietic toxicities. The aims in this proposal are designed to implement a high throughput screen for mutant selective inhibitors of the JAK2V617F kinase, and to optimize the probes from this screen using secondary assays. The aims of this proposal are: A. Develop, transfer, and screen a HTS assay designed to identify inhibitors of the JAK2V617F mutant kinase. B. Perform a counter screen using Ba/F3-EPOR-JAK2 cells grown in the presence of EPO in order to identify compounds that are selective for the JAk2V617F mutant kinase. Test the compounds in secondary assays and optimize if necessary to generate a chemical probe suitable for Aim C. C. Characterization of JAK2V617F mutant select inhibitors using in vitro and in vivo assays, in order to demonstrate proof of concept for efficacy for mutant selective kinase inhibition, followed by optimization and preclinical development of JAK2V617F inhibitors that demonstrate activity in cell culture and animal model systems. The identification of mutant selective inhibitors is of biologic and clinical importance, as this would provide a novel approach to the design of molecularly targeted therapies and would offer a way to develop candidate compounds with an improved toxicity profile for MPDs and for human malignancies in general. -
Screen for Small Molecule Compounds that Modulate the Transcriptional Activity of
LITTMAN, DAN R
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
1 R03 DA026211-01
8
DESCRIPTION (provided by applicant): The orphan nuclear receptor ROR?t has a central role in the differentiation of Th17 cells, a subset of T helper cells that secrete the inflammatory cytokines IL-17, IL-17F, and IL-22. Th17 cells have recently been shown to have major pathogenic functions in a variety of murine autoimmune disease models and in graft versus host disease, and they have also been implicated in human autoimmune disease and asthma. ROR?t is induced in antigen-stimulated na ve T helper cells in the presence of TGF-??combined with IL-6, IL-21, or IL-23, and thereafter directs the expression of the Th17 lineage cytokines. We have shown that mice defective for expression of ROR?t lack Th17 cells and are resistant to autoimmune disease. A better understanding of how ROR?t functions in inducing the Th17 lineage is therefore likely to provide important information for developing new therapeutic approaches for inflammatory diseases. To study the mechanism of action of ROR?t, we have developed an insect cell-based assay to identify host factors involved in its transcriptional activity and to screen for small molecules in high throughput assays. Cells in which a Gal4-ROR?t fusion protein directs expression of luciferase under regulation of the Gal4 promoter have been used in genome-wide RNAi and small molecule compound screens to identify inhibitory or enhancing activities. The screening assay has been optimized for a 384-well format, and multiple candidate genes have been identified in the RNAi screen. A proof-of-principle screen with a limited small molecule compound library has also been successfully performed. We propose to collaborate with the NIH Molecular Libraries Screening Network to perform a high throughput screen to identify compounds that modulate the activity of ROR?t. Compounds that affect transcriptional activity in the primary screen will then be examined for activity in T cell differentiation assays and other secondary screens to determine if they act directly on ROR?t or if they affect other components of ROR?t-directed transcription. Compounds that specifically affect Th17 cell differentiation will be of great utility in dissecting the transcriptional program of these inflammatory T cells and will guide us in developing new therapeutic approaches for autoimmune disease and graft versus host disease. -
High Throughput Screening for Potassium Channel Modulators
MAKHINA, ELENA
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
1 R03 DA026212-01
8
DESCRIPTION (provided by applicant): Potassium (K+) channels are well recognized as a target in treatment of cardiovascular, neurological, renal and metabolic disorders. However, the number of ion channel drugs is strikingly small compared to the number of ion channel genes. Most of the known K+ channel modulators lack potency and specificity. The reason of slow development of K+ channel drugs lies largely in the low capacity and high cost of electrophysiology-based screening techniques. To enable high throughput screening for K+ channel drugs, we developed an assay based on K+ uptake in yeast via the ectopically expressed mammalian inwardly rectifying channel Kir2.1. The assay performed in 96-well plates consists of one “mix and measure” step and is suitable for automation (Z’>0.5). In the screening scheme high throughput yeast-based assay is followed by secondary K+ current measurements in mammalian cells. Thus, in a pilot screen of 10,000 small molecules we identified ~30 compounds that inhibit K+ channels with EC50=0.03-1 fM. Even performed on a limited scale, the screening yielded novel modulators that affect both K+ channel activity and cell biology. Based on the validation of the yeast-based assay, we propose to expand the screening using facilities of a Molecular Library Screening Center. Specifically we will: screen >50,000 small molecules for high affinity K+ channel modulators in yeast (Aim 1), and verify primary hits by measurements of K+ current in mammalian cells using 86 Rb+ as a tracer (Aim 2). The goal of the project is to discover both inhibitors and activators of Kir2.1 channel with EC50 < 1 fM. Based on our experience, we expect to identify diverse K+ channel modulators with novel mechanisms of action. Identified compounds will serve both for basic study of K+ channel physiology and as a basis for development of clinical K+ channel drugs. -
A Screen for Modulators of Human Rad51, a Key DNA Repair Protein
MAZIN, ALEXANDER V
DREXEL UNIVERSITY
1 R03 MH084119-01
8
DESCRIPTION (provided by applicant): Ionizing radiation (IR) and inter-strand cross-linking agents (ICL) induce DNA double-stranded breaks (DSB). DSB are the most harmful type of DNA damage, which cause genome instability, cancer, genetic diseases, and premature aging. The system of homologous recombination (HR) is responsible for the repair of DSB repair in humans. However, DSB-inducing agents, IR and ICL, are also used in anti-cancer therapy. In order to increase the efficiency of this therapy, we propose to suppress HR in cancer cells by using specific inhibitors against a key human HR protein, Rad51 (hRad51). These inhibitors will help to develop novel combination therapies that allow to reduce doses of IR and ICL thereby decreasing their cytotoxicity. Specific inhibitors and stimulators of hRad51 will also present a valuable tool in fundamental studies on the mechanisms of HR in human cells. Therefore, the Aim of this proposal is to identify specific inhibitors and stimulators of hRad51 using high throughput screening (HTS) of the molecular libraries of reagents. hRad51 possesses a unique activity, it promotes DNA strand exchange between homologous DNA molecules, a basic step of HR. We have developed a fluorimetric assay that allows to measure DNA strand exchange activity of hRad51 in vitro. We will use this assay for the primary HTS. The selected compounds will be validated using conventional radioactively-based DNA strand exchange assays (secondary screen).Using microbial hRad51 homologues and structurally unrelated hRad54 protein we will determine the selectivity and specificity of the selected compounds. In continuation of this grant, we will employ the selected compounds for analysis of the mechanisms of HR in humans. The therapeutic potential the prioritized compounds will be examined using reconstituted HR system in vitro, transformed human cells, and immuno-deficient mice with transplanted human xenografts (SCID-hu mice). -
A High Throughput Imaging Assay for Hepatic Lipid Droplet Formation
MCDONOUGH, PATRICK M
VALA SCIENCES, INC.
1 R03 DA026213-01
8
DESCRIPTION (provided by applicant): Non-alcoholic fatty liver disease (NAFLD) is prevalent (overall occurrence of ~20%) in the US adult population and is very commonly associated with obesity and diabetes (occurs in ~ 60% of diabetic patients). NAFLD presents as increased number and size of lipid droplets within the hepatocytes, and, if untreated, can progress to the inflammatory disease steatohepatitis and liver dysfunction. This proposal, in response to PAR-06-545 for the Molecular Libraries Screening Centers Network (MLSCN), proposes an assay that will enable high- throughput/high-content screening of chemical libraries to detect agents that alter hepatic lipid droplet formation. The assay employs AML-12 cells (murine hepatocyte) or HuH7 cells (derived from human hepatocytes). Cells plated in a high-throughput format, are exposed to oleic acid, which induces lipid droplet formation, fixed and stained for lipid droplets, and imaged utilizing a robotic fluorescence microscopy workstation. Automated image analysis software is utilized to quantify the number and size of lipid droplets. Our assay has an exceptionally high Z’ value for reproducibility (Z’ > 0.5 for the effect of oleic acid) and is inexpensive and rapid to perform. Assay efficacy was demonstrated using triacsin C, an inhibitor of long-chain acyl-CoA synthetase (ACSL), to block oleic acid-induced lipid droplet formation (Z’ > 0.5 for this effect). Thus, the assay will provide an excellent method to identify new agents that could be developed for treatment of NAFLD disease or used as chemical probes to identify proteins in the hepatic triglyceride ester synthesis and lipid droplet formation pathways. -
The Role of PHOSPHO1 in the Initiation of Skeletal Calcification
MILLAN, JOSE LUIS
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 R03 MH084086-01
8
DESCRIPTION (provided by applicant): Mineralization of cartilage and bone occurs by a series of physicochemical and biochemical processes that together facilitate the deposition of hydroxyapatite (HA) in specific areas of the extracellular matrix (ECM). Experimental evidence has pointed to the presence of HA crystals along collagen fibrils in the ECM and also within the lumen of chondroblast- and osteoblast-derived matrix vesicles (MVs). Our working model is that bone mineralization is first initiated within the lumen of MVs. In a second step, HA crystals grow beyond the confines of the MVs and become exposed to the extracellular milieu where they continue to propagate along collagen fibrils. Our recent data have indicated that tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role in restricting the concentration of extracellular inorganic pyrophosphate (PP), a mineralization inhibitor, to maintain a P/PPi ratio permissive for normal bone mineralization. Using a variety of single and double gene knockout experiments we have found that mice deficient in TNAP function, i.e., Akp2-/- mice, display osteomalacia due to an arrest in the propagation of HA crystals outside the MVs caused by an increase in extracellular PPi concentrations. Inside the MVs, however, HA crystals are still present in Akp2-/- mice. We hypothesize that a newly identified soluble phosphatase, PHOSPHO1, with specificity for phosphoethanolamine (PEA) and phosphoserine (PS) present in the MVs, is responsible for increasing the local concentration of Pi inside the MVs to change the P/PPi ratio to favor precipitation of HA seed crystals. We will test this hypothesis by affecting the first and second steps of MV-mediated mineralization using a genetic and pharmacological approach. Experimentally we will characterize the mineralization abnormalities and related metabolic changes in mice deficient in Phospho1 expression compared to Akp2-/- mice and assess the effect of the simultaneous inactivation of the Phospho1 and Akp2 genes on skeletal mineralization. We will also study the effects of ablating or inhibiting PHOSPHO1 and/or TNAP activity on the ability of osteoblast-derived MVs to initiate and propagate calcification in vitro. We have now established an assay and screened the LOPAC and the Spectrum libraries and identified 17 compounds capable of inhibiting recombinant PHOSPHO1 activity with IC50 values of 10 fM or less. Three of these 17 compounds were selected for further characterization, i.e., SCH 202676; Lansoprazole and Ebselen. Lansoprazole and SCH 202676 decreased the amount of liberated Pi using isolated osteoblast-derived MV further indicating the involvement of PHOSPHO1 in the initiation of skeletal calcification. In response to this “Solicitation of Assays for High Throughput Screening (HTS) in the Molecular Libraries Screening Centers Network, PAR-06-545, we are submitting this R03 proposal to screen additional libraries in order to identify/develop the best possible PHOSPHO1-specific inhibitors to help us clarify the role of this enzyme in skeletal mineralization. -
Novel Chemical Probes Targeting the FRB Domain of mTOR
PELLECCHIA, MAURIZIO
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 R03 MH084221-01
8
DESCRIPTION (provided by applicant): The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting simultaneously with its FKBP-Rapamycin-Binding (FRB) domain and the proline cis-trans isomerase FKBP12. Several attempts have been made to design molecules that separate the FKBP12 binding activity from the mTOR inhibitory activity of Rapamycin, but to date no such viable compounds have been reported that are efficient against mTOR. However, we found that Rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 albeit at much higher concentrations (low micromolar versus the low nanomolar inhibition observed in presence of FKBP12). Hence, we propose an unbiased NMR- fragment-based approach to develop novel high affinity chemical probes that interfere with the function of mTOR by targeting its FRB domain. These ligands could become very useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds. The chemical structures, SAR data and range of biochemical activities of the resulting compounds will provide a framework onto which to develop potentially novel anti-cancer therapies. -
Screen for RAS-Selective Lethal Compounds and VDAC Ligands
STOCKWELL, BRENT R
COLUMBIA UNIV NEW YORK MORNINGSIDE
1 R03 MH084117-01
8
DESCRIPTION (provided by applicant): We are searching for compounds and mechanisms that cause oncogene-selective lethality. Such compounds eliminate tumor cells harboring specific oncogenic mutations, but have minimal effects on normal cells lacking these mutations. In this project, we propose to screen for new compounds that are selectively lethal to tumor cells expressing the RAS oncogenes. It is important to note that these compounds do not directly inhibit RAS proteins or the RAS pathway, but rather cause cell death specifically in tumor cells containing oncogenic RAS proteins. Using high-throughput screening of 70,000 compounds in isogenic, engineered tumor cell lines, we discovered three compounds selectively lethal to oncogenic-RAS-expressing cells. With the tools of synthetic chemistry, molecular biology and proteomics, we discovered that voltage dependent anion channels (VDACs) are target proteins for two of these compounds. We found that these compounds act through mitochondrial VDAC proteins to cause an oxidative, non-apoptotic death. These compounds thus revealed that oncogenic RAS signaling causes increased VDAC levels and that VDACs are novel gain-of-function targets for cancer therapeutics. Our results suggest that VDAC ligands are potential chemotherapeutic agents for the treatment of cancers with activated RAS signaling. We propose to find new RAS-selective lethal compounds and determine whether they act through VDAC proteins or through new mechanisms. New VDAC ligands would be valuable in trying to translate these compounds into potential therapeutics. -
High Throughput Screening for Small Compounds to Inhibit the Expression of Strept
SUN, HONGMIN
UNIVERSITY OF MISSOURI-COLUMBIA
1 R03 DA026214-01
8
DESCRIPTION (provided by applicant): The widespread occurrence of antibiotic resistance among bacteria is causing increasing concern as a major public health threats. Current antibiotics cause death or growth arrest in the target bacteria. As a result, antibiotic use exerts strong selective pressure to favor antibiotic resistant strains. Novel antimicrobial reagents that suppress pathogen virulence without selecting for antibiotic resistance provide a promising alternative approach for treatment of infectious diseases. Group A Streptococcus (GAS) is an important human pathogen affecting millions of people globally each year. The streptokinase (SK) is a major GAS virulence factor that activates human plasminogen. In our previous studies, we have established the streptokinase/plasminogen interaction as a critical factor in GAS pathogenesis. We propose to take advantage of this observation and design novel antimicrobial reagents for the treatment of GAS infection. In the preliminary study, we have screened 55,000 small compounds for inhibitors of SK expression in GAS and 23 candidate hit compounds have been identified. An additional high throughput screen of up to 500,000 more small compounds for SK expression inhibitors is proposed by taking advantage of the NIH Molecular Libraries and Imaging roadmap initiative. A growth based screen will be optimized to use a GAS strain with kanamycin resistance gene under control of SK promoter to screen for small compounds that can inhibit kanamycin resistance expression, which will serve as lead compounds for SK expression inhibitors. In the future, global effects of candidate compounds on GAS gene expression will be studied to provide clues for identification of the targets. Two GAS two-component systems that have been demonstrated to regulate SK expression in published reports will be tested as prime candidate targets. In addition, a number of murine GAS infection models established in our previous studies will be used to elucidate the effects of candidate compounds on GAS virulence in vivo. Data collected from the above studies will further our understanding of the contribution of SK to GAS infection and identify small compounds that can inhibit GAS virulence. As a result, alternative approach to treat bacterial infection by interfering with GAS virulence without unduly introducing selection pressure for resistance can be explored to supplement antibiotic treatment. -
High Throughput Screen for Small Molecule Inhibitors of Colorectal Cancer Cell Pr
YANG, VINCENT W.
EMORY UNIVERSITY
1 R03 DA026215-01
8
DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is one of the leading causes of cancer mortality and morbidity in the United States. Studies indicate that CRC results from stepwise changes (mutations) in key genes with important cellular functions. These genes include tumor suppressor genes (TSGs) and oncogenes. For example, somatic or germline inactivation of TSGs such as adenomatous polyposis coli (APC) and p53; and oncogenic activation of KRAS and BRAF are crucial in the pathogenesis of CRC. However, despite this knowledge, therapies targeting to specific components of the altered signaling pathways in CRC remain at a relatively early stage. Our group previously demonstrated that a member of the Krppel-like factor (KLF) family of zinc finger transcription factors, KLF5, plays important roles in regulating proliferation of intestinal epithelial cells. KLF5 is predominantly expressed in the proliferating crypt cell compartment of the intestinal epithelium. Ectopic expression of KLF5 in transfected cells results in increased rates of proliferation and leads to anchorage independent growth. In addition, oncogenic activation of HRAS and KRAS in NIH3T3 and IEC6 cells, respectively, leads to transformation with a concomitant increase in KLF5 levels. Importantly, reduction of KLF5 by genetic or pharmacological means results in reduced rates of proliferation and anchorage-independent growth in oncogenic RAS-transformed cells. Moreover, CRC with activated KRAS are shown to contain high levels of KLF5. These results indicate that KLF5 is a key mediator for the pro-proliferative and transforming activities of activated KRAS, heretofore mutated in approximately 50% of CRC. Reduction of KLF5 expression in such CRC may offer a novel therapeutic approach in the treatment of CRC. The long-term GOAL of this research project is to understand the signaling pathways that modulate KLF5 expression in CRC. Our CENTRAL HYPOTHESIS is that KLF5 is a key mediator of proliferation of CRC containing activated KRAS. Our OBJECTIVE is to identify small molecule inhibitors of KLF5 expression in CRC cells using high throughput screening (HTS), with which to better understand the biological functions of KLF5 in modulating CRC proliferation and to develop potential therapeutic agents in the treatment of CRC. Using this R03 grant mechanism, we propose 2 SPECIFIC AIMS: (1) To perform HTS for small molecule inhibitors of KLF5 using cell-based luciferase reporter assays, and (2) To perform secondary and counter screening assays with which to validate the active compounds identified in specific aim 1. Although beyond the scope of this R03 grant, we will also attempt to develop strategies for further testing, in collaboration with the MLSCN center, to provide a final refinement of the structure and function of the active compounds. At the conclusion of the proposed project, we will be able to identify several highly active and specific inhibitors of KLF5 with which to further investigate the biological functions of KLF5 in mediating CRC proliferation. The identification of these compounds may also aid in the development of novel therapeutic approaches for CRC. -
A high throughput screening assay for the identification of SUMOylation inhibitor
CHEN, YUAN
CITY OF HOPE/BECKMAN RESEARCH INSTITUTE
1 R03 DA026556-01
9
DESCRIPTION (provided by applicant): The overall goal of this proposal is to perform high throughput screening assays (HTS) to identify inhibitors for SUMOylation. Protein modification by the SUMO (Small Ubiquitin-like MOdifier) family of proteins has recently been established as an important post- translational modification that plays an essential role in many functions including gene transcription, cell cycle progression, DNA repair, viral and bacterial infection, and the development of neurodegenerative diseases. Inhibitors of SUMOylation will serve as much needed reagents for investigating the role of SUMOylation in different cellular functions, which remain largely unclear. The inhibitors will also have therapeutic potential for diseases, such as cancer and viral infection. However, such inhibitors have not been reported and are not yet available to the scientific community. We have developed several assays to identify such inhibitors. The primary HTS assay uses the ALPHA screening technology. The secondary screening assay uses fluorescence resonance energy transfer (FRET) to eliminate false positive hits obtained in the ALPHA screen. Additionally, an ubiquitination assay will be used to identify the hits that are specific for inhibiting SUMOylation and not other ubiquitin-like modification systems. The immediate applications of the inhibitors identified in the screen are: 1) to probe the role of SUMOylation in different cellular functions, such as in the DNA repair pathways, 2) to test the inhibitors in cellular and animal models of diseases where SUMOylation has been shown to play important roles, such as in cancer and viral infections. Our long term goal is to develop potent SUMOylation inhibitors based on the structure-activity relationship studies of the hits identified from this study. Inhibitors of the homologous ubiquitin-proteosome pathway have been critical in dissecting the role of the ubiquitin-proteosome pathway in many cellular functions and are surprisingly successful as anti-cancer therapeutic agents. SUMOylation inhibitors hold similar promise in having a broad impact in research and could lead to the establishment of a new paradigm in developing therapeutic agents for devastating diseases, such as cancer and infectious diseases. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to perform high throughput screening assays (HTS) to identify inhibitors for SUMOylation. -
High-Throughput Assay for G-Protein Coupled Receptors in Pain Research
Civelli, Olivier (NOTHACKER, HANS-PETER )
UNIVERSITY OF CALIFORNIA IRVINE
1 R03 DA026557-01
9
DESCRIPTION (provided by applicant): Chronic pain is a debilitating condition that exerts a high social cost in terms of productivity, economic impact and quality of life. Currently available therapies yield limited success in treating such pain, suggesting the need for new insight into underlying mechanism(s). Opioid peptides modulate pain sensation by binding and activating the opioid receptors, but their usefulness for pain treatment is limited due to their peptidic properties i.e. lack of blood brain barrier crossing and fast inactivation by metabolic enzymes. The small molecule alkaloid morphine, on the other hand, is a powerful analgesic that can be easily administered and activates the same receptors. The G protein coupled receptor, GPR7, is a receptor that shares sequence similarities with opioid receptors. GPR7 is activated by the neuropeptides NPW and NPB which have been recently discovered. GPR7 is thought to regulate pain perception in the periphery. Most notably, while under normal conditions GPR7 is expressed at low levels in the spinal cord; its expression is dramatically increased in patients suffering from inflammatory/immune-mediated neuropathies. Presently there are no small molecules available which could be used to modulate the GPR7 system. We therefore propose to use our recently developed robust and simple assay system for GPR7 in a high-throughput screen of molecular libraries conducted by one of the centers of the MLSCN. Compounds identified through our screening assay will represent valuable basic research tools that will greatly facilitate the long term goal of this project to understand the involvement of GPR7 in chronic pain, its role in the mechanism of pain transmission and its interaction with other pain- related pathways. Small compounds will be in particular useful to study the system with regard to its role in inflammatory neuropathies where established animal models are available. PUBLIC HEALTH RELEVANCE: There is a significant medical need for novel compounds for treating chronic pain that are effective, long lasting and safe. The overall objective of the proposed research is to identify small molecule, selective agonist and antagonists for the opioid receptor related G protein-coupled receptor GPR7 using a high-throughput assay format. Compounds identified through our screening assay will represent valuable basic research tools that will greatly facilitate the long term goal of this project to understand the involvement of GPR7 in chronic pain. -
High Throughput Screening for Activators of the Ah Receptor
DENISON, MICHAEL STEVEN
UNIVERSITY OF CALIFORNIA DAVIS
1 X01 DA026558-01
9
DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AhR) is a unique basic-helix-loop-helix ligand-dependent transcription factor that regulates the expression of a large variety of genes in a ligand-dependent manner. Activation of the AhR results in a wide variety of biological and physiological effects in target cells. Recent studies have shown that Ah receptor has a promiscuous ligand binding pocket and can be bound and/or activated by a variety of synthetic and naturally-occurring chemicals whose structural and physiochemical properties are distinct from well established AhR agonists. The demonstration that activation of AhR can result in some antitumorigenic/anticarcinogenic activities, such as the inhibition of proliferation of diverse hormone- dependent and independent cancer cell types, as well as its ability to inhibit allergic lung inflammation, indicate that the AhR is a promising target for the development of novel therapeutic agents. While ligands for the AhR have been identified, few chemicals are suitable for development into useful therapeutic agents and as such, the identification of new and novel activators of the AhR and AhR signaling pathway are critically needed for the development of such agents. In order to identify AhR agonists, we have developed a simple, rapid and inexpensive 96-well multiplate cell bioassay system for high throughput chemical screening. This cell line contains a stably transfected Ah receptor-dependent green fluorescent protein reporter gene plasmid that responds to AhR agonists with the induction of green fluorescent protein in a time-, dose- and AhR- dependent manner. We propose to use this bioassay to screen the MLNSC library of chemicals to identify new agonists of the AhR as lead chemicals for the development of therapeutic agents and ligands for further in- depth analysis of the structure and function of the AhR in vitro and in vivo. PUBLIC HEALTH RELEVANCE: Ah receptor is a unique chemical-responsive nuclear factor that plays a key role in regulating a variety of endogenous and adaptive responses in mammals and it is an emerging target for development of novel therapeutic agents. Through this project, our cell-based fluorescent reporter gene assay combined with the robust high throughput screening facilities from MLPCN will dramatically speed up the process of identification of novel potent and nontoxic AhR agonists that can be utilized in the specific development of therapeutic and/or chemotherapeutic agents as well as for molecular probes for basic AhR biology research. -
Screen for Inhibitors of 2′ Phosphotransferase
HARDING, HEATHER P
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
1 R03 DA026554-01
9
DESCRIPTION (provided by applicant): The proposed screen is to identify inhibitors for the 2′ phosphotransferase Tpt1 a gene conserved throughout all phylogeny with well defined and essential roles in RNA end-joining reactions in yeast such as non-conventional splicing during the unfolded protein response (UPR) and tRNA splicing. The UPR is a feedback pathway by which cells regulates the levels of proteins involved in secretory pathway protein folding by inducing transcription of their genes in response to detected shortfalls in their levels. Non-conventional splicing of the transcription factor Hac1/XBP1 mRNA that regulates gene expression during the UPR is initiated by excision of an intron by the conserved endoribonuclease IRE1. In yeast the subsequent RNA end-joining reactions required the sequential actions of two essential genes tRNA ligase Trl1p and 2′ phosphotransferase Tpt1p. tRNA ligase joins the ends leaving a 2′PO42+ at the splice junction and Tpt1p removes the 2′PO42+ by transfer to NAD+ to form the unique by-product ADP-2′-3′cyclic ribose (Appr>p). However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin RNA cleaved by IRE1 or tRNA endonuclease. Remarkably, inactivation of the Trpt1 gene, encoding the mammalian homologue of Tpt1p, eliminates all detectable 2′-phosphotransferase activity from cultured mouse cells but has no measurable effect on splicing during the UPR or tRNA splicing indicating that their ligation proceeds by distinct pathways in yeast and mammals. Furthermore, young Trpt1 mutant mice are apparently healthy and fertile. This raises the question of whether 2′ phosphotransferase activity has been conserved throughout phylogeny for alternative processes such as production of Appr>p a potential signaling molecule, or new rolls in RNA metabolism a possibility suggested by the neurological degeneration seen in mice lacking CNP, encoding a functional domain provided by in tRNA ligase in yeast. The isolation of inhibitors will prove useful in three areas of research: creating reagents useful for structural studies studying Tpt1s unique reaction chemistry; exploration of 2′phosphotransferase function in mammals, and isolating lead compounds for new anti-fungal agents that take advantage of the differential requirement of 2′phosphotransferase for viability in fungi and mammals. We have developed robust homogenous fluorescence polarization based screening method that measures 2′ phosphotransferase activity in a HTS-ready 384 well plate format and eliminates false positives through secondary screens. The assay was validated through the screening the ~1900 compound NCI diversity library in which a IC50 ~100um inhibitor was identified. We propose to apply the screen to search the~200fold larger chemical libraries available at the MLPCN centers to identify additional higher affinity inhibitors. PUBLIC HEALTH RELEVANCE: The identification of new antifungal agents that may be used independently or in combination with existing therapies is needed to combat the growing problem of topical and invasive/systemic fungal infections due to the increased number of diseases and treatments that lead to an immuno-compromised state. The enzyme that will be targeted in the proposed molecular probe screen Tpt1 is essential in fungi yet dispensable in mammals suggesting it may be possible new drug target for antifungal therapy. The probes identified in the proposed screen will allow us to test this hypothesis and provide tools to further explore the Tpt1 catalytic mechanism. -
A SCREEN FOR SMALL MOLECULE INHIBITORS OF SHIGA TOXIN AND RICIN ACTIVITY
HASLAM, DAVID B.
WASHINGTON UNIVERSITY
1 R03 MH084834-01
9
DESCRIPTION (provided by applicant): Shiga toxin (Stx) and ricin, produced by certain E. coli strains and the plant Ricin communicus, respectively, are important causes of human disease and are potential agents of biowarfare. Therapeutic options for persons exposed to these toxins are limited. In particular, antibiotics have no role following ricin exposure and are contraindicated in patients exposed to Stx-producing E. coli. Similarly, there is no specific therapeutics for their inactivation, nor for inhibition of the cell death caused by these toxins. Our laboratory has recently undertaken a screen for small molecules that inhibit intracellular transport of shiga, ricin, and cholera toxins. We have identified several compounds that block transport of these toxins within host cells. These compounds are useful probes of the toxin transport pathway and some may hold potential as therapeutic agents. In tandem with the above screen, which is a cell-based assay and targets toxin transport, we have developed a biochemical assay for toxin enzymatic activity. This assay is highly sensitive and reproducible and has been adapted to high-throughput format. Through the Molecular Libraries Probe Production Centers Network (MLPCN), we propose to screen up to 200,000 compounds for their ability to inhibit Stx and ricin enzymatic activity. Each of these compounds will be verified in a secondary assay then characterized with respect to their potency, specificity, cytotoxicity, reversibility, and effect on related toxins. In future studies, in collaboration with Southern Research Institutes, compounds with greatest therapeutic potential will be subjected to structure-activity relationship (SAR)-guided optimization of potency, specificity, lack of toxicity, and drug-like properties. These will be tested in an animal model of toxin-mediated disease. PUBLIC HEALTH RELEVANCE: This work has major implications for public health. Stx causes several hundred thousand cases of colitis in the US annually and accounts for thousands of deaths. In the event of their intentional release as bioweapons, these toxins could significance morbidity and mortality. We propose to identify and optimize as these compounds as the first drugs that could be used to prevent or treat individuals exposed to these toxins. -
A Cell Based Assay for the Identification of Lead Compounds with Anti-Viral Activ
HEIL, MARINTHA L
SOUTHERN RESEARCH INSTITUTE
1 R03 MH084847-01
9
DESCRIPTION (provided by applicant): West Nile virus (WNV) is a mosquito borne infectious agent that causes febrile illness and occasionally encephalitis. Outbreaks had been reported in Africa, Asia and Europe since 1937. In 1999, the first case of WNV was detected in New York City. Since establishing in North America, the virus has spread rapidly westward and southward resulting in the largest epidemics of neuorinvasive WNV disease ever reported. Thus, there is a need for vaccines and antivirals agents. Currently, there is no vaccine for human use or antivirals that are available to treat WNV infections, and the care is mainly supportive. WNV belongs to the family a flavivirus. Members of this family include dengue virus, yellow fever, Japanese encephalitis virus, St. Louis encephalitis virus, and tick borne encephalitis virus. These infections have a similar clinical presentation, which makes diagnosis of the causative agent only possible with sophisticated laboratory analysis which is time consuming and costly. Therefore, an antiviral would ideally demonstrate broad antiviral activity against flaviruses. Antivirals with activity against WNV are in various states of clinical development. The identified chemical compounds are believed to target the virus polymerase, NTPase/helicase and protease. Interestingly, these lead compounds also have activity against other members of the family flaviridae. Our hypothesis is the high degree of sequence similarity amongst the flaviviruses results in a similar viral replication in the host cell. A corollary to this hypothesis is that compounds that have activity against one flavivirus, WNV, will have potential as a broad spectrum anti-viral against the flaviviruses. If the hypothesis is correct, then this would mean that the probes generated would be useful to dissect similar pathways and assist in additional target development. If the hypothesis is incorrect and some compounds are broadly active and others are not, then the probes can be used to discriminate difference between the flaviviruses and learn more about WNV. We have developed a primary HTS assay that has validated against a 12,000 compounds in duplicate for anti-viral activity against WNV. In this proposal, we propose to utilize this assay to screen compounds for activity against WNV. Specifically we will: (Aim 1) Perform a primary screen using the MLPCN library to identify lead compounds with antiviral activity against WNV and (Aim 2) Perform secondary screens to determine the extent to which the active leads demonstrate specificity and selectivity. We will (Subaim 2A) perform dose response studies to determine the extent of a compounds efficacy and toxicity; (Subaim 2B) confirm lead compounds specific antiviral activity in a plaque assay; (Subaim 2C) confirm lead compounds specificity and breadth by determining the extent of the antiviral activity against a flaviviruses yellow fever. (Subaim 2D) perform confirmatory chemistry. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify probes with anti-viral activity against West Nile virus (WNV) some of which will be broadly reactive against the flavivirdae viruses. The probes generated would be useful to dissect similar pathways and assist in additional target development. West Nile virus (WNV) is a mosquito borne infectious agent that causes febrile illness and occasionally encephalitis. Outbreaks had been reported in Africa, Asia and Europe since 1937. In 1999, the first case of WNV was detected in New York City. Currently, there is no vaccine for human use or antivirals that are available to treat WNV infections, and the care is mainly supportive. WNV belongs to the family a flavivirus. Members of this family include dengue virus, yellow fever, Japanese encephalitis virus, St. Louis encephalitis virus, and tick borne encephalitis virus. These infections have a similar clinical presentation, which makes diagnosis of the causative agent only possible with sophisticated laboratory analysis which is time consuming and costly. Therefore, an antiviral would ideally demonstrate broad antiviral activity against flaviruses. -
Identification of compounds that protect HERG from block by proarrhythmic agents
KUPERSHMIDT, SABINA
VANDERBILT UNIVERSITY
1 R03 MH084820-01
9
DESCRIPTION (provided by applicant): Identification of Compounds that Protect HERG from Block by Proarrhythmic Agents The acquired/drug induced Long QT Syndrome (aLQTS) is a threat to public health and a major stumbling block for drug development. It is most often caused through unintended block of the cardiac repolarizing potassium channel, IKr, encoded by the Human Ether-`-go-go Related Gene (HERG). HERG is susceptible to inhibition by even transient exposure to a diverse array of common therapeutic compounds, including antihistamines, antidepressants, antibiotics, antiemetics, and antiarrhythmics that in turn provokes the life-threatening cardiac arrhythmia torsades de pointes in predisposed individuals. Indeed, several successful drugs (e.g., cisapride, seldane) had to be withdrawn from the market due to arrhythmia development caused by this mechanism. Our previous studies have shown that IKr can be protected from block by known HERG antagonists through the activity of the K+ channel regulator 1 (KCR1) protein which we characterized as an enzyme that functions in the cellular glycosylation pathway. This finding led to our hypothesis that identification of a small molecule that mimics the activity of KCR1 and diminishes IKr block by known HERG inhibitors may lead to the development of a co-drug to protect against the development of the aLQTS. We will use established high throughput methods to: 1. Screen a library of 130,000 compounds for agents that increase the IC50 of the well- characterized HERG inhibitor, dofetilide, using a Tl+ flux assay. Our preliminary data show that the assay is highly suitable for this type of screening. 2. Determine if the isolated hits also affect the IC50s of a range of drugs with divergent chemical structures (moxifloxacin, clozapine, and droperidol) and clinical applications in the Tl+ flux assay. Our findings will be validated through standard patch-clamp assays. The development of a small molecule chemical agent that could be safely co- administered with clinically useful pharmaceuticals to protect susceptible patients from HERG inhibitors and the subsequent development of arrhythmias would improve public health and greatly facilitate the drug discovery process. PUBLIC HEALTH RELEVANCE: Potentially lethal cardiac arrhythmias can be the tragic consequences of treating patients with otherwise useful therapeutic agents for non-cardiac conditions. The most common cause of this unpredictable effect is the unintended block of a cardiac potassium channel, called HERG that is essential for maintaining the balanced electrical activity and rhythmic contraction of the heart. The goal of this proposal is to utilize high throughput screening methods to identify a chemical compound that diminishes unintended HERG block, which could eventually be co-administered and thereby reduce the risk of arrhythmia development. -
A high-throughput screen for pre-mRNA splicing modulators
MISTELI, TOM
U.S. NATIONAL CANCER INSTITUTE
1 R03 MH084827-01
9
DESCRIPTION (provided by applicant): The coding regions of most human genes are interrupted by non-coding intron regions. During gene expression introns are removed in a process called pre-mRNA splicing and the coding exon regions are joined. Inaccurate splicing leads to the production of faulty proteins and aberrant splicing has now been implicated in a large number of human diseases including cancer, muscular dystrophy, neuropathies and a pre-mature aging disorder. Correction of defective splicing has recently been recognized as a novel therapeutic strategy for these diseases. The outcome of the splicing process can be modulated by introduction into cells of oligonucleotides targeted against an aberrant splice site with the intention of blocking it and forcing use of a correct site. The use of oligonucleotides for correction of pre-mRNA splicing is, however, limited by the difficulty of delivering them effectively to target tissues. An alternative strategy is to identify small molecule compounds which modulate pre-mRNA splicing. The long term goal of this project is to identify small molecule modulators of pre-mRNA splicing. As a first step, the aberrant splicing of the lamin A/C (LMNA) gene will be used as a model system for the discovery of small molecule modulators of pre-mRNA splicing. The project comprises a) a high-throughput screen for modulators of LMNA using an established cell-based reporter system and b) validation of candidate compounds in available in vivo and in-vitro assays. These efforts rep- resent the first application of an unbiased high-throughput screen to pre-mRNA splicing and they will establish proof-of-principle for the use of screening methodology to splicing, including any of the disease-relevant splicing defects. Candidate compounds will be useful for the mechanistic investigation of pre-mRNA splicing since no modulators of the splicing reaction are currently available. In addition, since defective splicing of LMNA used in this approach causes the pre-mature aging disease Hutchinson-Gilford Progeria Syndrome any candidate compounds will have direct disease relevance and might lead to novel therapeutic strategy for this disease. PUBLIC HEALTH RELEVANCE: The proposal represents the first unbiased screen for modulators of pre-mRNA splicing. The project will establish proof-of-principle for the application of high-throughput screening for the identification of compounds that modulate pre-mRNA splicing. This approach will be applicable to any pre-mRNA splicing event including many of the disease related splicing defects. -
High throughput screening for polyadenylation inhibitors using the MLSCN library
MOORE, CLAIRE L; KUMAMOTO, CAROL A.
TUFTS UNIVERSITY BOSTON
1 R03 DA026555-01
9
DESCRIPTION (provided by applicant): Accompanying the development of advanced medical techniques, candidiasis has emerged as a significant nosocomial infection that causes considerable morbidity and mortality among immunocompromised patients. Candida blood stream infections are increasing in frequency and are associated with high mortality. Oral candidiasis is an extremely common opportunistic infection in AIDS patients. Despite the prevalence of these infections, treatment options are limited. With the exception of the newly developed echinocandins, the antifungal drugs currently in use are limited by toxicity and natural or acquired resistance. Therefore, development of new antifungal drugs is of great importance. Our long- term goal is to develop new drug therapies for fungal infections. The difficulty in achieving this goal is that fungi use mechanisms for gene expression and cell growth that are similar if not almost identical to those used by mammalian cells. An essential process shared by all eukaryotes is the modification of the 3′ ends of mRNAs by cleavage of longer precursor molecules and the subsequent addition of a tract of adenosine residues. Acquisition of this poly(A) tail is important for accumulation of mature mRNA, its export from the nucleus, its utilization in translation of protein, and its removal when the mRNA is no longer needed by the cell. In the last few years, our research and that of others has identified most, if not all, of the subunits of this processing complex and revealed a remarkable conservation between the yeast Saccharomyces. Cerevisiae and metazoans. However, we have also found significant species-specific differences, suggesting that inhibitors uniquely interfering with fungal mRNA 3′ end formation could be found. In this study, we will screen the MLSCN Small Molecule Repository for inhibitors of mRNA polyadenylation. This screen utilizes an assay in which defects in 3′ end processing in S. cerevisiae lead to production of a reporter required for cell growth. We have adapted this assay to a 384-well format that can be analyzed by an automated plate reader, and it gives robust performance in pilot screens. To assess the spectrum of activity of our hit compounds, we will test them for growth inhibition of mammalian cells and fungal pathogens. We will also use in vivo and in vitro assays for polyadenylation as secondary screens to confirm that hits are indeed targeting mRNA 3′ end formation. Finally, we will work with the MLPCN Center to design and synthesize derivatives with increased potency and specificity. We expect that this study will yield a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi. An added benefit will be the discovery of chemical probes to help us understand the molecular mechanism of eukaryotic mRNA polyadenylation. PUBLIC HEALTH RELEVANCE: Candida has recently emerged as a significant opportunistic pathogen that causes considerable morbidity and mortality in immunocompromised patients. Unfortunately, treatment options for fungal diseases are extremely limited, and compounding this problem, resistance to some of the best anti-fungal drugs is emerging. By taking advantage of certain differences in how fungi and human cells synthesize messenger RNA, we propose to conduct a high throughput screen for a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi. An additional benefit will be the discovery of chemical probes that will help us understand the molecular mechanism of eukaryotic mRNA polyadenylation. -
TR-FRET HTS Assay for Inhibitors of MEKK2-MEK5 PB1 Domain Interaction
NAKAMURA, KAZUHIRO
UNIVERSITY OF NORTH CAROLINA CHAPEL HILL
1 R03 MH084830-01
9
DESCRIPTION (provided by applicant): PB1 (Phox/Bem1p) domains function for specific protein-protein interactions by forming PB1-PB1 domain heterodimers. There are at least 20 human PB1 domain containing proteins. Different PB1 domains contribute to the formation of specific protein complexes for the control of critical biological responses including proliferation, apoptosis, cell polarity, and angiogenesis. These proteins include MEKK2, MEKK3, and MEK5 that are MAP3Ks and the MAP2K, respectively, which solely governs the ERK5 MAPK pathway involved in angiogenesis, cell growth and inhibition of apoptosis. Two additional serine-threonine protein kinases, the atypical PKCs PKC9 and PKC6 also encode PB1 domains. The PKC9 and PKC6 PB1 domains both dimerize with PB1 domains of the scaffold/adaptor proteins p62/Zip (sequestosome-1) and Par6. The p62/Zip adaptor protein is involved in the regulation of NF: B activation and Par6 is a critical scaffold for the control of cell polarization. In addition, p40phox and p67phox use their respective PB1 domains to heterodimerize with each as part of the activation of NADPH oxidase (Nox2), the enzyme complex that catalyzes the generation of reactive oxygen radicals in immune cells. This proposal is based on rapidly accumulating evidence that PB1 domain is a crucial interface for MEKK2-MEK5 interaction involved in various patho-physiological conditions. Inhibition of the PB1-PB1 interaction by small molecules is a unique pharmacological mechanism to selectively disrupt the activation of the EKR5 signaling network in cells. Inhibition of MEKK2-MEK5 PB1-PB1 domain dimerization would be an extremely useful and innovative therapeutic intervention to inhibit angiogenesis, tumorigenesis and potentially chronic inflammation. The aims of this proposal are the development and optimization of robust and cost feasible HTS homogenous TR-FRET screening platforms for identifying small molecule inhibitors of MEKK2-MEK5 PB1-PB1 domain heterodimerization. The initial focus will be to optimize donor- and acceptor- dye conjugated GST MEKK2 or MEK5 fusion proteins and optimization of a robust HTS for 384 or 1536-well format. A counterscreen will be developed with a second PB1-PB1 domain interaction (Par6-aPKCs, p62/Zip- aPKCs, or p40phox-p67phox) that has the potential to multiplexed in the same well with the MEKK2-MEK5 PB1- PB1 domain screen. To validate leads, a live cell FRET-based assay using high content microscopy and straightforward functional cell based pathway assays will be implemented. PUBLIC HEALTH RELEVANCE: Small molecule inhibitors of MEKK2-MEK5 PB1 domain interaction would represent a new target class of drugs for therapeutic intervention to modulate angiogenesis, tumorgenesis, and chronic inflammation. -
Interdicting Eukaryotic Translation Initiation Factor-RNA Interaction
PELLETIER, JERRY
MC GILL UNIVERSITY
1 R03 MH084835-01
9
DESCRIPTION (provided by applicant): Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. – insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, translation initiation control is usurped upon viral infection and is deregulated in many human cancers. Over-expression of certain translation factors can lead to malignant transformation and many of the components of the translational apparatus are over-expressed in human cancers. Several tumor suppressor genes directly influence the translation process and recently, chemoresistance in vivo has been linked to deregulated translation initiation. In a transformed setting, where translation can be inhibited by a small molecule modulator (e.g. rapamycin), decreased translation rates are associated with reversal of chemoresistance, possibly by inhibition of pro-survival pathways or resetting of pro-apoptotic program. These results validate translation initiation as a potential chemotherapeutic target. The Specific Aims of the current application are to implement a High Throughput Screen (HTS) at the MLSCN in order to identify small molecules that block translation initiation by targeting two important components of this pathway – eIF4H and poly(A) binding protein [PABP]. Both proteins require RNA binding to mediate their effects on translation initiation and our screens are designed to block this process. In addition, both screens can be used as counterscreens for each other to eliminate non-specific inhibitors. We have secondary nitrocellulose binding assays and tertiary crosslinking analyses that can detect the binding of initiation factors to mRNA as a means of confirming true “hits”. Follow-up studies with optimized compounds will be performed to characterize their biological properties in vitro and in vivo. The therapeutic potential of inhibitors of eIF4H and PABP that show activity in vivo will be assessed in a number of setting, including potential to curtail, or delay, Herpes Simplex Virus infection (where eIF4H has been implicated), as well as synergize with conventional therapies in a mechanism-based mouse cancer model. Specifically, we will test the ability of identified inhibitors to synergize with conventional “standard of care” agents in lymphomas of defined genotypes generated in the E5-myc mouse cancer model. PUBLIC HEALTH RELEVANCE: Compounds identified in this screen will affect targets involved in regulating translation initiation, a process that is frequently usurped upon viral infection of cells, as well as in cancer cells. Therefore these compounds have the potential to act as anti-viral or anti-cancer agents. In addition, since this target of these compounds resides downstream of the target of rapamycin (called TOR), these may be of use against rapamycin-resistant cancers. -
High Throughput Screening Assays for NOD1 and NOD2 Inhibitors
REED, JOHN C
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 R03 MH084844-01
9
DESCRIPTION (provided by applicant): The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-KB, a central regulator of immune response, inflammation, and apoptosis. Mutations in the NOD1 and NOD2 genes are associated with a number of human inflammatory disorders, including Crohn’s disease (CD), Blau syndrome, early-onset sarcoidosis, and atopic diseases, which characteristically cause constitutive NF-:B activation. Chemical inhibitors of NOD1 and NOD2 would provide powerful research tools for elucidating the roles of these proteins in primary cultured cells from humans and in animal models. In this proposal, we describe cell-based HTS assays that utilize NF-:B-mediated luciferase reporter gene activity as a measure of NOD1 and NOD2 modulation. We also describe a secondary assay to confirm compound selectivity towards NOD activity, by measuring secretion into culture supernatants of interleukin-8 (IL-8), an endogenous NF-:B target gene. When combined with insights provided by cheminformatics analysis, and a variety of additional downstream assays provided by the assay provider for deconvoluting hits, we expect to obtain candidate compounds for subsequent optimization by medicinal chemistry. PUBLIC HEALTH RELEVANCE: Our goal is to identify chemicals that inhibit NF-:B activity induced by NOD1 and NOD2 proteins. The NOD1 and NOD2 proteins are members of the NLR family, a large group of host defense proteins that contain a shared architecture of Nucleotide-binding NACHT domains and pathogen-sensing LRRs. Chemical inhibitors of these innate immunity proteins will provide powerful research tools for evaluating the roles of NLR-family members in host-pathogen responses, inflammatory diseases and autoimmunity. -
High throughput screen for the master regulator of obesity and diabetes p/CIP
WANG, ZHIYONG
SALK INSTITUTE FOR BIOLOGICAL STUDIES
1 R03 MH084852-01
9
DESCRIPTION (provided by applicant): Genetic studies have demonstrated that loss of the transcriptional coactivator p/CIP leads to resistance to obesity and diabetes, especially in extreme mouse obesity models. The goal of this research is to find small molecules that decrease p/CIP protein stability, thus providing new understanding and novel approaches to cure obesity and diabetes. In an in vitro white fat differentiation system, p/CIP is important for fat cell formation. A fluorescence-based primary assay employing this in vitro cellular model has been developed to screen for small molecules that destabilize a p/CIP-EGFP fusion protein. Individual compounds will be incubated with the stably transfected cell line containing this GFP (green fluorescence) fusion protein. Small molecules will be identified that only diminish the GFP signal from the p/CIP-EGFP fusion protein cells, but not from a control cell line with GFP protein alone. These candidate compounds therefore increase the fusion protein turnover, and will be examined further for their effects on the endogenous p/CIP protein levels in secondary assays. If the small molecule hits destabilize the endogenous p/CIP protein, they can be used as chemical probes to decipher kinase, as well as proteasome degradation pathways that are critical for controlling p/CIP protein turnover and therefore controlling obesity and diabetes. These compounds will be chemically optimized and tested in readily available mouse obesity models for their effects in vivo. Potential therapeutic drugs may come out of these studies to help alleviate extreme obesity in human patients. PUBLIC HEALTH RELEVANCE: This research will provide crucial new information about how to control obesity and diabetes, and will provide potential cure for the extremely obese human population. -
Identification of Activators and Inhibitors of alpha-Glucosidase as Potential Cha
ZHENG, WEI
U.S. NATIONAL HUMAN GENOME RESEARCH INST
1 R03 MH084841-01
9
DESCRIPTION (provided by applicant): Pompe disease, also called glycogen storage disease type II, is an autosomal recessive disorder. This disorder is caused by mutations in the gene encoding acid 1-glucosidase (GAA), a lysosomal enzyme that catalyzes the hydrolysis of glycogen. The deficiency of this enzyme results in lysosomal accumulation of glycogen, predominantly disturbing the intracellular architecture of skeletal muscle fibers and cardiomyocytes. All patients suffer from progressive muscle weakness affecting their mobility and respiratory function. Although the enzyme Replacement Therapy (ERT) has recently become available for the treatment of patients with Pompe disease, the mortality rate for treated patients was still very high due to disease complications, and only a small subset of patients achieved significant gains in physical performance. Chemical chaperone treatment, alternatively, is a potential therapeutic option for Pompe disease. Several GAA inhibitors have been tested for the chaperone activities and only deoxynojirimycin showed positive results. But the enhancement of enzyme activity by the inhibitor’s chaperone action must be balanced against the direct inhibition of the enzyme. Thus, an enzyme activator with chemical chaperone activity should have better therapeutic potential than an inhibitor. Currently, the small molecule GLA activators are not known. We propose here a high throughput screening against the MLSCN’s compound collection to identify the small molecule activators and inhibitors of GAA as the research probes for the chemical chaperone therapy of Pompe disease. PUBLIC HEALTH RELEVANCE: Pompe disease is an inherited genetic disorder aused by mutations in the gene encoding acid 1- glucosidase (GAA). The deficiency of this enzyme results in lysosomal accumulation of glycogen, predominantly disturbing the intracellular architecture of skeletal muscle fibers and cardiomyocytes. The patients with Pompe disease suffer from progressive muscle weakness affecting their mobility and respiratory function. Although the enzyme Replacement Therapy has recently become available for the treatment of patients with Pompe disease, the mortality rate for treated patients was still very high and only a small subset of patients achieved significant gains in physical performance. Alternatively, Chemical chaperone treatment is a potential therapeutic option for Pompe disease. Currently, only deoxynojirimycin, an enzyme inhibitor, showed positive chaperone activity. But the enhancement of enzyme activity by the inhibitor’s chaperone action may be compromised by the direct inhibition of the enzyme. Thus, an enzyme activator with chemical chaperone activity should have better therapeutic potential than an inhibitor. But the small molecule GLA activators are not known. We propose here a high throughput screening to identify the small molecule activators and inhibitors of GAA as the research probes for the chemical chaperone therapy of Pompe disease. -
HTS for Identifying Activators and Inhibitors of Alpha-Galactosidase A for the Po
ZHENG, WEI
U.S. NATIONAL HUMAN GENOME RESEARCH INST
1 R03 MH084842-01
9
DESCRIPTION (provided by applicant): Fabry disease is a hereditary X-linked lysosomal storage disorder with an estimated incidence of 1 in 40,000 males. The disorder is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (GLA), which results in the accumulation of the glycosphingolipid globotriasylceramide (Gb3) in different cells and organs, notably endothelial cells and smooth muscle cells of blood vessels throughout the body. Although the enzyme replacement therapy (ERT) has recently become available for the treatment of Fabry disease, it does not appear to reduce premature strokes in these patients. In addition, some patients receiving ERT develop immune responses to the infused enzyme. It has been reported that some enzyme inhibitors can serve as chemical chaperones in several lysosomal storage disorders. But the enhancement of enzyme activity by the inhibitor’s chaperone action must be balanced against the direct inhibition of the enzyme. If an enzyme activator can function as a chaperone by binding to the enzyme and helping to correct its folding and trafficking, it should have better therapeutic potential than an inhibitor. Currently, no small molecule GLA activators are known. Therefore the discovery and development of chemical activators may provide a new strategy regarding therapy. We propose here to use a GA enzyme assay for high throughput screening to identify novel activators and inhibitors as the chemical chaperones for the potential treatment of Fabry disease. PUBLIC HEALTH RELEVANCE: Fabry disease is a hereditary lysosomal storage disorder with an estimated incidence of 1 in 40,000 males. This disease is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A. Patients with Fabry disease manifest chronic neuronopathic pain, gastrointestinal disturbances, angiokeratomata, progressive renal impairment, cardiomyopathy, premature myocardial infarctions and stroke. Although the enzyme replacement therapy has recently become available for the treatment of patients with Fabry disease, the effectiveness of this therapy is in question because it does not reduce premature strokes in patients with Fabry disease. We propose here a compound screening assay to identify the chemical chaperone as a potential and alternative treatment option for Fabry disease. -
Antagonists for the Orphan Receptor GPR-35
BARAK, LAWRENCE S.
DUKE UNIVERSITY
1 X01 MH085708-01
10
DESCRIPTION (provided by applicant): Addiction is a growing problem and a major health concern in this country. Addictive behavior results from the modification of central nervous system signaling pathways and can be influenced by the binding of drugs to specific cell membrane receptors. Thus, the identification of small molecules capable of blocking receptors associated with addictive behavior will provide tools for understanding the underlying molecular basis of addiction and modifying the physiological response associated with a particular signaling pathway. This proposal addresses the potential role that an orphan G protein-coupled receptor, GPR35, may play in addictive behavior. The specific aim of this application is to identify small molecule antagonists of human GPR35. We propose to provide the MLSCN with cell lines expressing the receptors and beta-arrestin fluorescent protein biosensors that in combination can be used in high-content, high throughput primary screens to identify high-affinity antagonists and inverse agonists of GPR35. This work will identify novel compounds that may be utilized to characterize GPR35 signaling in vitro and GPR35 mediated behavior in animal models of addiction. PUBLIC HEALTH RELEVANCE: Drug addiction is a major health problem that results from abnormalities of signaling in the brain. Addiction thus is a potentially treatable medical disorder. This project will identify compounds to block the signaling that underlies addictive behaviors and consequently provide a basis for designing medical therapies to treat these behaviors. -
qHTS to Identify Modulators of Lipid Storage
BELLER, MATHIAS
MAX PLANCK INSTITUTE FOR BIOPHYS CHEM
1 R03 MH085686-01
10
DESCRIPTION (provided by applicant): The primary goal of this project is to identify chemical probes that either increase or decrease the lipid content of cells. Most, and probably all cells are capable of storing energy rich lipids (mainly triacylglycerols), which are generated on basis of de novo synthesized fatty acids or non-esterified free fatty acids (NEFA) taken up from the environment. Storage lipids are deposited in specialized organelles, the so-called lipid droplets (LDs). LDs are thought to arise at the endoplasmic reticulum in a budding-like process. Once released into the cytoplasm, LD volume increases by targeted lipogenesis or fusion of existing droplets, suggesting extensive trafficking events. Remobilization of LD stores involves enzymatic breakdown of the storage lipids by lipases. Lipogenesis and lipolysis needs to be delicately fine-tuned, as diminished or increased lipid stores have dramatic consequences for the organism, as demonstrated in human diseases including lipodystrophy, atherosclerosis or obesity. There are only few drugs for treating metabolic diseases and a very limited number of chemical probes to study lipid storage in vitro. We found that also embryonic Drosophila S3 and Kc167 cells are capable of depositing LDs and developed an assay to measure lipid storage amounts by fluorescent staining of LDs and cells and microscopic or laser-scanning microplate cytometry based detection. In a genome-wide RNA interference (RNAi) screen, the assay identified about 500 gene-functions regulating lipid stores. The same assay was further optimized for small compound screening in S3 cells. Assay profiling confirmed its precision, robust response to known small-molecule modulators of lipid storage and suitability for miniaturization and HTS. Screening to date of 8,874 compounds has yielded a small number of actives, some of which are connected with protein targets previously associated with organismic lipid storage regulation. Further screening will increase the chance that potent compounds can be found with the potential of mechanistic insights and therapeutic development. Validation of candidate chemical probes during assay development in mouse AML12 hepatocytes demonstrated evolutionary conservation of our findings. Orthologous testing with RNAi-mediated knockdown experiments additionally provided epistasis information suitable to further characterize identified compounds. PUBLIC HEALTH RELEVANCE: The chemical probes yielded by the project should be useful tools in providing a better understanding of cellular and organismic lipid storage on a functional and evolutionary level. Furthermore, active substances might result in therapies treating emerging lipid storage associated diseases including atherosclerosis, diabetes or obesity. As well, the present assay will establish a profile of compounds within the MLSMR that modulate this ubiquitous area of biology. -
HTS Screen for Small Molecule Inhibitors of Mint-PDZ Domain
BEZPROZVANNY, ILYA B
UNIVERSITY OF TEXAS SW MED CTR/DALLAS
1 R03 MH085675-01
10
DESCRIPTION (provided by applicant): The broad, long-term objectives of the project is to identify and develop small molecules that inhibit the association between N-type calcium channel pore-forming subunit CaV2.2 and Mint-PDZ protein. In the previous biochemical experiments we discovered association of carboxy-terminal tail of CaV2.2 subunit with Mint1-PDZ domain and demonstrated that association of CaV2.2 with Mint1-PDZ is important for synaptic targeting and synaptic function of N-type Ca2+ channels. Based on these results we proposed that small molecule inhibitors of association between CaV2.2 carboxy-terminal and Mint1-PDZ1 can be used to treat pain. We also proposed that such molecules may serve as useful probes for studies of synaptic function of N- type Ca2+ channels. My laboratory previousy setup pilot high throughput screen (HTS) for a small molecule inhibitors of Mint-PDZ domain. The main goal of the current grant proposal is to perform a large scale HTS screen for Mint-PDZ inhibitors in collaboration with Molecular Libraries Probe Production Centers Network (MLPCN). Aim 1. In collaboration with UT Southwestern HTS core we previously developed 384-well homogeneous time-resolved fluorescence resonance energy transfer assay (HTRF) for inhibitors of association between bio- NC peptide and GST-Mint-PDZ protein. We will transfer this assay to MLPCN center and convert this assay to 1536-well format for HTS. We will provide bio-NC peptide and GST-Mint-PDZ protein to MLPCN center for screening of 300-500,000 compounds library. Aim 2. In collaboration MLPCN center we will perform a series of control HTRF experiments to filter out “false positives”. A number of potential sources of “false positives” has been identified in our pilot screen and several filters were developed by our laboratory to rule them out Aim 3. We will validate the efficacy of resulting “hits” using amplified luminescent proximity homogeneous assay (ALPHA) to screen for small-molecule inhibitors of association between bio-NC and GST-Mint1-PDZ. Aim 4. We will select the “hits” that passed both HTRF and ALPHA assay for evaluation in biochemical pull- down experiments with HA-NC4 (fragment of CaV2.2 carboxy-terminal) and GST-Mint-PDZ protein. In the future generated probes will be used in studies of synaptic transmission between cultured hippocampal neurons and DRG and DH neurons. The analgesic activity of resulting “leads” will be tested in the animal models of chronic pain. The identified compounds will serve as useful probes for studies of synaptic transmission and may potentially lead to development of novel treatment for chronic pain. If successful, our study may also provide a first example for development of small molecule inhibitors of PDZ domain-mediated interactions. PUBLIC HEALTH RELEVANCE: Chronic pain (neuropathic pain, inflammatory pain, cancer pain) is a major health problem. The costs of healthcare and lost productivity due to chronic pain have been estimated around $60 billion dollars a year in the USA alone. Opiate-based drugs, such as morphine and morphine derivatives, are the primary standard of care for the treatment of chronic pain. Unfortunately, patients develop tolerance to opiates due to desensitization of the opiate receptor. Thus, alternative anti-nociceptive (“pain killing”) agents need to be developed to treat chronic pain. The main goal of the proposed project is to meet this challenge by developing a small molecule that specifically targets synaptic N-type Ca2+ channels. The N-type Ca2+ channels in DRG neurons is a well validated target for chronic pain and the molecules that we propose to develop will consitute a novel and improved class of “pain-killers” for treatment of chronic pain. Generated molecules may also serve as useful probes for studies of synaptic function of N-type Ca2+ channels. -
Screen for Synthetic Lethality in Mycobacterium Tuberculosis
BISHAI, WILLIAM RAMSES
JOHNS HOPKINS UNIVERSITY
1 R03 MH084877-01A1
10
DESCRIPTION (provided by applicant): Tuberculosis has been declared a WHO global health emergency, and the recent emergence of multidrug (MDR) and extensively drug resistant (XDR) TB has underscored the urgent need for new TB drugs. We hypothesize that HTS will identify probes which chemically sensitize Mycobacterium tuberculosis (M. tb.) to the activity of existing 2-lactam and macrolide antibiotics. The identification of such probes could lead to combination antibiotics, similar to amoxicillin-clavulanate, which have potency against M. tb. This proposal seeks access to the HTS resources provided by the Molecular Libraries Screening Center Network (MLSCN) with the goal of identifying sensitizing probes, identifying their molecular targets, and finding candidate agents as potential lead structures for further study. An HTS-ready, fluorescence-based whole cell screening assay is proposed that will utilize virulent M. tb. in combination with optimized concentrations of imipenem (IMI, representing the 2-lactam class) and clarithromycin (CLA, representing the macrolide class). The following aims are proposed to conduct an efficient HTS evaluation for probes that are synthetically lethal against M. tb. in the presence of 2-lactam and macrolide antibiotics: to transfer the M. tb./adjuvant drug susceptibility protocol to the designated screening center to reproduce, miniaturize, and automate a corresponding HTS assay that will be used to screen the MLSCN compound library for sensitizing probes, to provide technical support, and to conduct secondary assays confirming the potency and specificity of identified probes. PUBLIC HEALTH RELEVANCE: Tuberculosis has been declared a WHO global health emergency, and the recent emergence of multidrug (MDR) and extensively drug resistant (XDR) TB has underscored the urgent need for new TB drugs; indeed, many antibiotics such as those from the penicillin-, erythromycin-, and tetracycline-families are not clinically useful in the treatment of TB either because of poor bacterial penetration or drug degradation by the microbe. Our preliminary studies reveal that specific mutations can sensitize Mycobacterium tuberculosis (M. tb.) to penicillin and erythromycin, thus supporting the hypothesis that new drugs may be identified that would also sensitize the microbes to the activity of these familiar classes of antibiotics. We have developed a microtiter plate assay to seek compounds which amplify the activity of key members of the penicillin- and the erythromycin-class against M. tb., and we propose to transfer this assay to an appropriate MLSCN laboratory for high-throughput screening to identify potential lead compounds. -
HTS For Inhibitors of NADPH Oxidase 1 (Nox1)
BOKOCH, GARY M
SCRIPPS RESEARCH INSTITUTE
1 R03 MH083264-01A1
10
DESCRIPTION (provided by applicant): We describe here a plan to implement a high throughput screen (HTS) for inhibitors of the Nox1 NADPH oxidase. Nox1 is a member of a recently identified 7 member family of NADPH oxidases that plays important roles in cell signaling, growth, motility, and host defense. Currently, no specific inhibitors of NADPH oxidases in general, nor of Nox1 specifically, have been identified. The identification of a small molecule inhibitor of Nox1 would be extremely useful for studies into the biology of the Nox enzymes. In addition, such an inhibitor could represent a novel therapeutic approach to colon cancer, hypertension, and inflammatory diseases. We have developed a cell- based assay of Nox1 function that is now ready for a large scale HTS in conjunction with the MLSCN. The Specific Aims for this proposal are: 1. Implement the Nox1 screen at the nearby MSLCN center. We will participate in transferring the assay to a screening center such that ~105 to 106 compounds in the MLSCN libraries can be screened. The cell-based assay, which uses luminol to detect Nox1- dependent superoxide formation, has been fully validated and adapted to use in 384-well plates. Statistical analysis of the Nox1 assay confirms that it has a useful signal window that exceeds requirements for a validated screen (Z factor of 0.68). 2. Validate the primary hits from the MSLCN screen with followup assays. A series of followup assays are in place with increasing levels of rigor for the primary hits that emerge from the screening. First, the hits will be verified with full scale cellular assay using transfected Nox1 in which ROS formation is determined using a homovannilic acid-based peroxide assay.. Dose-response curves will be obtained to insure specific saturable inhibitor activity. Third, inactivity against other oxidative enzymes will be verified. Fourth, hits will be tested against a series of cells expressing other Nox family members to evaluate the Nox family selectivity of the inhibitors. 3. Use the validated inhibitors to probe the biological function and regulation of Nox1 using biochemical and genetic tools in biological assays. The compounds that emerge as validated hits will be powerful investigative tools into the biology and regulation of Nox1 when used in combination with ongoing biochemical and functional analyses. The effect of inhibitors on the interactions of Nox1 with known cytosolic regulators will be studied. The action of Nox1 in inflammatory responses of the colon and in colon cancer will be studied. PUBLIC HEALTH RELEVANCE: It is known that NADPH oxidases (Nox) play important roles in health and disease, yet much more remains to be learned about Nox1 function in biology. We will use a cell-based screen to identify Nox1 inhibitors, followed by secondary screens to verify selectivity, efficacy and mechanism. Such inhibitors will be useful to investigate Nox1 biology, and as potential therapeutic agents in inflammatory diseases and cancer. -
High-Throughput Campaign to Identify Reversible Small Molecule Inhibitor of p97/C
DESHAIES, RAYMOND J
CALIFORNIA INSTITUTE OF TECHNOLOGY
1 R03 MH085687-01
10
DESCRIPTION (provided by applicant): p97 is an essential protein that plays a critical role in a number of different cellular processes. This enzyme uses the energy released by hydrolysis of ATP to take apart assemblages of proteins and unfold individual polypeptides. A key cellular process in which p97 plays a critical role is in the degradation of proteins by the ubiquitin- proteasome system (UPS). Although there are abundant data to indicate that p97 plays a critical role in the UPS, the exact mechanism by which it works in protein turnover and the identity of its substrates largely remain unknown. It is particularly difficult to study the role of p97 in the UPS because p97 is essential, abundant, and involved in multiple other biological processes. The main approach to study protein function in human cells – depletion by RNA silencing – is rendered difficult by the abundance of p97. Moreover, as p97 is depleted, multiple cellular processes go awry and the cell begins to die, complicating the interpretation of experiments. For these reasons, we seek to identify a chemical compound that can be used to rapidly and reversibly inhibit the ATP hydrolysis activity of p97. The availability of such an inhibitor will allow in-depth exploration of the functions of p97 in normal and diseased human cells. PUBLIC HEALTH RELEVANCE: The ubiquitin-proteasome system (UPS) has been validated as a good target of cancer drugs by FDA approval of the proteasome inhibitor bortezomib for the treatment of multiple myeloma. Given that p97 is an essential protein that plays an important role in the UPS and is overproduced in multiple cancers, it is an intriguing target for the development of novel cancer drugs. Identification of a chemical compound that inhibits p97 will not only enable a deeper understanding of the function of this protein in normal and diseased cells (thereby shedding light on mechanisms of disease pathogenesis), but may also serve as a lead for the development of novel cancer drugs. -
High Throughput Screen for Inhibitors of Influenza NS1 Protein Function
ENGEL, DANIEL A
UNIVERSITY OF VIRGINIA CHARLOTTESVILLE
1 R03 MH085680-01
10
DESCRIPTION (provided by applicant): A high-throughput screen is proposed for the identification of inhibitors of the influenza NS1 protein. Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill global needs. In addition their potential usefulness against newly emergent strains is not known. Efforts are needed to develop novel agents against influenza virus, including broad-spectrum agents. Identification of small molecules that inhibit NS1 function either directly or by interfering with specific cellular pathways may be a key to increasing our defense against the virus. A yeast-based assay for NS1 function will be used to screen for novel inhibitors. NS1 induces a pronounced slow-growth phenotype that can be specifically suppressed by small molecule inhibitors. The growth assay has been converted to a 96 well format and it has been shown to be of high quality. A small scale screen of 2000 compounds from the NCI Diversity Set identified several inhibitors of NS1 function that also inhibit influenza virus replication in cell culture. The inhibitors block the ability of NS1 to prevent activation of the interferon response. Some of them also reverse the ability of NS1 to interfere with nuclear-to- cytoplasmic transport of cellular RNA. The high-throughput screen proposed here is expected to identify compounds of higher potency and greater structural diversity than those identified from the small scale screen. This will shed light on the mechanisms of NS1 function and provide starting points for the development of clinically useful agents. PUBLIC HEALTH RELEVANCE: Influenza is a world-wide public health problem of major proportions. The likelihood of a devastating influenza pandemic during this century makes it essential to discover new drugs to combat the disease. The proposed high throughput screen for small molecule inhibitors of the NS1 protein from influenza virus will explore the possibility that this protein can be used as a target for clinically useful, broad spectrum therapeutics against influenza virus. -
Hepatitis C VIrus NS3 Helicase Inhibitor Discovery
FRICK, DAVID N
NEW YORK MEDICAL COLLEGE
1 R03 MH085690-01
10
DESCRIPTION (provided by applicant): About 2% of all Americans are chronically infected with the hepatitis C virus (HCV), which eventually causes cirrhosis and liver cancer. HCV is the leading cause of liver failure in the USA and most liver transplant recipients are HCV positive. HCV infection is particularly common in HIV patients, where it can accelerate AIDS onset and lead to a myriad of complications for the immunocompromised patient. Current HCV therapies are costly and produce debilitating side effects. My laboratory studies the enzymes involved in the replication of the hepatitis C virus with a goal of finding new antiviral therapies. Recently we have developed a new high-throughput assay to screen for inhibitors of the viral NS3 helicase, which is needed for HCV RNA replication in human cells. Our goal is to find potential new drugs by identifying HCV helicase inhibitors with this new assay. The 50 5l assay has been optimized in 384-well microplates with a Z< factor of 0.7. In the reaction, a substrate consisting of a Cy5-labeled molecular beacon annealed to a longer DNA strand is pre-incubated with the HCV NS3 helicase and inhibitor. Upon initiation with 0.5 mM ATP, fluorescence is monitored for 30 minutes, after which time, control reactions are complete as measured by a complete loss of fluorescence. In reactions containing helicase inhibitors, fluorescence changes more slowly or not at all. Hits in this primary screen will be analyzed with DNA binding and RNA unwinding secondary screens to rule out compounds acting non-specifically. Specificity of NS3 helicase inhibitors will be further confirmed by analyzing compound effects on the NS3 protease and ATPase activities. Probes will then be used to explore the mechanism of the helicase reaction, to understand the role of the helicase in viral replication, and as leads for new antiviral drugs. PUBLIC HEALTH RELEVANCE: This project seeks to find new drugs to treat the virus that causes hepatitis C (HepC). Almost one in fifty Americans has been exposed to the HepC virus and over time it will cause progressive disease leading to liver failure in millions of Americans. The goal here is to find chemicals that prevent the virus from replicating in human cells. -
Identification of Molecular Probes to Activate the Hypoxia Inducible Factor Pathw
GILBERT, SHAWN ROBERT
UNIVERSITY OF ALABAMA AT BIRMINGHAM
1 R03 MH082355-01A2
10
DESCRIPTION (provided by applicant): Adequate blood supply is critical to tissue repair and regeneration and is frequently lacking in disease or injury. Therefore, agents that stimulate improved blood supply may be useful in a wide variety of health conditions. A fundamental pathway engaged in response to oxygen and nutrient deficit is the hypoxia inducible factor (HIF) pathway. Under normoxia, HIF molecules are constitutively degraded by oxygen dependent prolyl hydroxylase enzymes. Exposure to hypoxia inhibits prolyl hydroxylation which causes HIF to accumulate in the nucleus where it transactivates hypoxic responsive genes. There has been substantial interest in manipulating the HIF pathway both to impair and induce angiogenesis. Although several HIF activating agents have been identified, additional compounds are needed as there are drawbacks associated with current agents including toxicity and high costs. In this proposal, we seek to access the Molecular Library Screening Centers Network resources to screen for compounds which activate HIF. A high throughput assay will be used in which a cell line has been stably transfected with a luciferase reporter driven by HREs. It is anticipated that the screen may identify novel molecular probes which may reveal additional mechanisms of HIF activation. Ultimately, the development of techniques to improve tissue survival during ischemia and improve angiogenesis could have a broad scope of clinical application. PUBLIC HEALTH RELEVANCE: Many disease processes and injuries have in common poor blood supply and/or lack of oxygen. The current proposal seeks to find agents that can activate the hypoxia inducible factor pathway which is responsible for initiating a response to low oxygen that includes metabolic adaptation and development of increased blood supply. These activators of the HIF pathway may be useful in a broad range of diseases such as heart disease and limb ischemia and in approaches to tissue regeneration. -
Screening for Inhibitors Targeting the Menin-MLL Interaction in MLL Related Leuke
GREMBECKA, JOLANTA (contact);CIERPICKI, TOMASZ ;
UNIVERSITY OF VIRGINIA CHARLOTTESVILLE
1 R03 MH084875-01A1
10
DESCRIPTION (provided by applicant): Translocations of the MLL (Mixed Lineage Leukemia) gene are frequently found in human leukemias affecting both children and adults. Fusion of MLL to one of more than 60 genes results in generation of oncogenic proteins upregulating Hox genes that are vital to blood cell development. Patients harboring fusion of the MLL gene suffer from aggressive leukemias and poorly respond to available therapies. All of the oncogenic fusion proteins have a preserved N-terminal fragment of MLL that has been identified to interact with menin. It has been recently discovered that association with menin is critical to the leukemogenic activity of the MLL fusion oncoproteins. Selective targeting of the menin-MLL interaction has been emphasized as an effective therapeutic approach for the MLL-related leukemias. We propose to use HTS to develop small molecule inhibitors of the menin-MLL interaction as a potential therapeutic approach for acute leukemias related to MLL-translocations. No attempts to develop small molecule inhibitors of the menin-MLL interaction were reported to date. As a step in this direction, we have developed a fluorescence polarization (FP) assay for efficient screening of the MLPCN compound library at 10 5M concentration to identify inhibitors of this protein-protein interaction. To eliminate “false-positives” and identify ligands targeting menin we have optimized the secondary screenings by application of HTRF and NMR saturation transfer difference (STD) experiments. In this proposal we delineate an efficient strategy to combine HTS based on FP, HTRF and STD assays to develop inhibitors of the menin-MLL interaction, and then optimize their activities by medicinal chemistry approaches. Initial screening of small library of compounds resulted in identification of several weak ligands, demonstrating feasibility of such an approach. The most potent compounds developed and optimized within this proposal will be tested using appropriate leukemia cell- lines harboring MLL translocations. We believe that we will initiate a successful collaborative project to develop chemical compounds that may constitute a novel approach for treatment of acute leukemias related to genetic disorders of MLL gene. Furthermore, this work will facilitate the understanding of biological consequences of inhibiting the menin-MLL interaction and its impact on the disease development. PUBLIC HEALTH RELEVANCE: We propose to use high throughput screening (HTS) to develop chemical compounds that may reverse the oncogenic function of proteins causing acute leukemia. Ultimately, such compounds could be used as highly specific drugs for treatment of patients with aggressive forms of leukemia, which poorly respond to current therapies. -
HTP Screen of Compounds that Inhibit Giardia Fructose-1,6-Bisphosphate Aldolase
HERZBERG, OSNAT
UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
1 R03 MH085699-01
10
DESCRIPTION (provided by applicant): Giardia lamblia is a human pathogen afflicting impoverished nations, and the most common cause of outbreaks of diarrhea in the United States. Giardia has been classified by the CDC as a category B bioterrorism organism. Giardiasis is treated with metronidazole or tinidazole, compounds that have undesirable side effects. Moreover, increasing resistance to drug regimes and recurrence are a concern. It is thus clear that alternative drug treatments are needed. We have identified the G. lamblia Class II fructose-1,6-bisphosphate aldolase (FBPA) as an excellent candidate for drug development and thoroughly characterized its kinetics, catalytic mechanism (requiring Zn2+ cofactor for electrophilic catalysis), and crystal structure. FBPA, a key glycolytic pathway enzyme, is the sole aldolase in Giardia. As the organism requires glucose for growth, FBPA is crucial for its survival. The human FBPA belongs to the Class I aldolases that have a distinctly different substrate binding site structure and a radically different catalytic mechanism (proceeding via a lysine-Schiff base intermediate). Thus, the probability of identifying an inhibitor via high throughput compound screening that impedes the glycolytic pathway in the pathogen but not in the human host is considered to be very high. The G. lamblia FBPA catalytic activity will be assayed using several different assays, which in combination will identify potential specific inhibitors and eliminate “false positives”. After filtering compounds with undesirable characteristics, the potential inhibitors will be evaluated to determine the type of inhibition and the inhibition constants. The best inhibitors will be examined for growth inhibition of Giardia trophozoites and cytotoxicity towards human cells. Synthetic chemistry will be employed to improve potential inhibitors, integrating SAR studies with crystal structure determination of enzyme/inhibitor complexes. PUBLIC HEALTH RELEVANCE: Giardia lamblia is a human pathogen afflicting billions of people annually and a category B bioterrorism organism. Giardiasis is treated by drugs that have undesirable side effects and that enable recurrence and increasing resistance. To facilitate discovery of better drugs, we have identified the G. lamblia Class II fructose- 1,6-bisphosphate aldolase as an excellent candidate for drug development because it is an essential enzyme for the survival of the organism and it functions by a different mechanism than its human counterpart. Here we propose a high throughput screening of the NIH compound library to identify and characterize new selective inhibitors of the Giardia fructose-1,6-bisphosphate aldolase. -
A Novel HTP Assay for Identification of Compounds that Reduce PNC Prevalence
HUANG, SUI
NORTHWESTERN UNIVERSITY
1 R03 MH082371-01A2
10
DESCRIPTION (provided by applicant): Although extensive changes have been known to take place during transformation, the mechanism and the biological bases that promotes and maintains the malignant phenotype remains unclear due to the complexity of metastasis. Our lab has shown that the perinucleolar compartment (PNC), a multicomponent subnuclear structure, is unique to cancer cells derived from multiple solid tissues, both in vitro and in vivo (Huang et al., 1997; Kamath et al., 2005, Norton et al, 2008). We have shown that PNC prevalence (percentage of cells with at least one PNC) increases in parallel with breast cancer progression and reaches nearly 100% in distant metastases. In addition, high PNC prevalence in primary tumors correlates with poor patient prognosis (Kamath et al., 2005). Preliminary investigations of 4 other types of epithelial carcinomas; colon, ovarian, prostate, and cervix, show similar results. These findings suggest that PNC formation reflects advanced cellular transformation and is a surrogate biomarker of cellular metastatic capabilities. Importantly, the PNC is absent in normal cells in vivo, including human and mouse embryonic stem cells. Although the precise function of the PNC remains to be identified, the close association of the PNC with the metastatic phenotype makes it a simple morphological marker that reflects the complex trait of cellular malignancy. We hypothesize that PNC elimination/reduction can be used as a phenotypic marker for of a positive response to intervention with bioactive small molecules, thus, allowing for the discovery of novel compounds that selectively target malignant behaviors. The rationale is that compounds that eliminate the PNC should shift cell behavior toward benign by inhibiting processes that convey metastatic capability as reflected by the presence of the PNC. In other words, molecules that eliminate/reduce the PNC, although may not be directly targeting the PNC structure itself, may induce desired changes in cellular behavior upon treatment since the PNC reflects cellular malignancy. Our preliminary screening of NCI small compound libraries and others identified a group of compounds that effectively disassemble the PNC. Chemical-biology studies using representative compounds reveal that the PNC is directly associated with DNA and the nucleation of the PNC is dependent upon the integrity of the DNA structure. These results encourage further exploration of compounds with novel mode of action to help understand the function of PNCs. We have now developed a one-step cell based assay amenable for HTS. Our goal of this proposal is to identify PNC disrupting compounds that are selectively against malignant cells and malignant behaviors with novel modes of action, and to use these compounds to investigate the function of the PNC and its contribution to the malignant behavior of cancer cells. These studies will help understand cancer biology from a different angle and will potentially provide cellular targets and compounds for cancer treatment. PUBLIC HEALTH RELEVANCE: Cancer is a complex metastastic disease with high fatality. Although extensive changes that take place during transformation, the mechanism and the biological bases that promotes and maintains the malignant phenotype remains unclear. Our lab focus on a unique cellular structure that is selectively associated with metastastic phenotype of the cancer cells. We propose to use this marker to identify compounds that selectively target malignant cells, which will help understand the role of the structure in malignant behaviors of cancer cells and will also generate potential novel and selective anti-cancer agents. -
The High Throughput Screening for Developing T Cell Immune Modulaotrs
HWANG, INKYU
SCRIPPS RESEARCH INSTITUTE
1 X01 MH085707-01
10
DESCRIPTION (provided by applicant): The ultimate goals of this project are to broaden our knowledge about the cellular and molecular basis for TCR-mediated LFA-1 activation by exploiting the tools of chemical genetics and to develop small molecules potentially useful for treatments of various immunologic diseases. The specific aims of this grant proposal are (i) to establish robotic (automatic) high- throughput screening platform with our cell-based assay and (ii) to gain access to the small molecule library (Molecular Libraries Small Molecule Repository, MLSMR) managed by Molecular Libraries Probe Production Center Network (MLPCN). PUBLIC HEALTH RELEVANCE: This project is to develop novel therapeutics for immunologic diseases such as inflammatory and autoimmune diseases and prevention of graft rejection. It will also broaden our scientific knowledge about integrin activation process which is a key to the controlled host immunity. -
Small Molecule Modulators for Redox Regulation in the Mitochondrial Intermembrane
KOEHLER, CARLA M.
UNIVERSITY OF CALIFORNIA LOS ANGELES
1 R03 MH085683-01
10
DESCRIPTION (provided by applicant): Defects in mitochondrial biogenesis lead to a broad range of diseases including neurodegeneration, stroke, myocardial infarction, ischemia, and cancer; however, therapies to correct such diseases are not readily available. We propose to conduct a high throughput screen in the Molecular Libraries Production Center Network (MLPCN) identical to the one that, on a smaller screening scale, has already successfully identified inhibitors of a mitochondrial redox pathway for the import and assembly of proteins in the mitochondrial intermembrane space. Specifically, we will employ a straightforward in vitro enzymatic assay with recombinant Erv1, a sulfhydryl oxidase that functions in this pathway. We have developed a robust fluorescence assay to monitor Erv1 activity. The aims of this proposal are to (1) identify small molecules that alter Erv1 function and then develop analogs for structure activity relationship (SAR) studies to identify specific chemical compounds that modulate Erv1 and (2) utilize these tools in secondary assays and develop probes that we can use in vertebrate systems to probe mitochondrial function, including the link to apoptosis. These studies will result in validated chemical probes for mechanistic studies of mitochondrial import and for potentially inducing/abrogating apoptosis. Given our success, we are confident that many novel compounds will be identified that are pertinent for understanding mitochondrial assembly in vertebrates. The medical importance of events regulated by mitochondrial assembly, such as apoptosis, indicates that the chemical genetic approach may also lead to the identification and development of novel therapeutic agents for diseases affected by dysfunctional mitochondria. Identification of these novel compounds, tied with our expertise in finding targets and our ability to exploit them to more fully understand mechanism, justifies our request to expand this screen through the MLPCN. This study is relevant to public health because it may lead to the development of new therapeutics for degenerative muscular and neural diseases. PUBLIC HEALTH RELEVANCE: This project will develop small molecules as probes to investigate the cause of neurodegenerative and degenerative muscular diseases that are initiated by defects in mitochondrial function. The mitochondrion generates energy for the cell and is linked to a broad range of diseases. Long-term, this project may lead to the development of therapeutics that modulate mitochondrial function in these diseases. -
Toward Improved Therapy for Classic Galactosemia
LAI, KENT
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
1 R03 MH085689-01
10
DESCRIPTION (provided by applicant): Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disease called Classic Galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to haunt the surviving children to their adulthood. 85% of treated galactosemic females suffer premature ovarian failure, while many others are inflicted with neurological disorders and developmental delay. Several lines of evidence suggested that elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with galactosemia. We therefore hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To identify selective GALK inhibitors, we have purified large quantity of GALK and developed a robust high throughput (HTS) GALK assay, which will be used to screen over 300,000 chemical compounds of diverse structural scaffolds (Specific Aim 1). Positive hits identified in Aim 1 will be validated and characterized in Specific Aim 2 to assess their potency, selectivity and efficacy to reduce gal-1-p accumulation in galactose-intoxicated cells. PUBLIC HEALTH RELEVANCE: Classic Galactosemia is a potentially lethal metabolic disorder that is included in the newborn screening programs of all 50 states in the U.S.. Although a galactose-restricted diet, which is the current standard of care, can prevent the neonatal lethality of this disorder, many well-treated patients continue to develop debilitating complications such as mental retardation, premature ovarian failure and other neurological deficits. The long-term goal of this project is to develop more effective therapies for this disorder. -
HTS Assay for UBC13, a Mediator of Inflammatory Signaling
REED, JOHN C
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 R03 MH085677-01
10
DESCRIPTION (provided by applicant): Tumor Necrosis Factor (TNF)-family cytokine receptors, Toll-like Receptors (TLR), and other types of receptors involved in the recognition of pathogen-associated molecular patterns transduce their signals into cells via adapter proteins known as Tumor Necrosis Factor Receptor-Associated Factors (TRAFs). The TRAFs are a family of adapter proteins that bind an unusual ubiquitin-conjugating enzyme, Ubc13, which produces polyubiquitin chains linked at lysine 63 of ubiquitin rather than the canonical lysine 48. These lysine 63-linked ubiquitin polymers trigger changes in protein activity, rather than targeting proteins for proteasomal degradation. Ubiquitination by Ubc13 of TRAFs and the various protein kinases to which TRAFs bind is recognized as a critical step in signaling by TNFRs, TLRs, NLRs, and T-cell and B-cell antigen receptors (TCR/BCR) during innate and acquired immune responses. Since aberrant signaling by these receptor systems is linked to a wide variety of autoimmune, inflammatory, and infectious diseases, compounds that neutralize Ubc13 may prove useful as a novel type of immunosuppressive or anti-inflammatory agent. The goal of this proposal is to generate chemical inhibitors of Ubc13 for use as research tools for studying the role of lysine63-linked polyubiquitination in signal transduction, and as potential proof of concept reagents for exploring the suitability of Ubc13 as a possible drug target in animal models of autoimmunity and inflammation. To this end, we have devised a high throughput screening (HTS) assay that measures the biochemical activity of Ubc13 in vitro, based on the principal of time-resolved fluorescence resonance energy transfer (TR-FRET) using terbium-conjugated Ubiquitin as a donor and fluorescein-conjugated Ubiquitin as an acceptor. The HTS assay has been optimized for 384 well plate, with z’ > 0.7. Importantly, Ubc13′s activity is dependent on a cofactor protein Uev1a. The 3D- structure of the Ubc13-Uve1a complex suggests that the binding interface can potentially be disrupted by small molecule compounds, thus providing two sites at which to attack Ubc13: (a) active site; and (b) Uve1a interface. Downstream secondary assays have been devised that will assist with validation and characterization of hits. PUBLIC HEALTH RELEVANCE: Ubc13 is an enzyme that is required for the function of multiple mediators of inflammation and immunity. This proposal seeks to produce chemical inhibitors of Ubc13, which will be useful research tools for understanding more about the role of Ubc13 in immune cell function. These chemical inhibitors may also provide a starting point for developing future medicines aimed at ameliorating a wide variety of acute and chronic inflammatory and autoimmune disorders, including sepsis, inflammatory bowel diseases, arthritis, lupus, and organ rejection. -
New Drugs for Acute and Chronic Stages of Trypanosome Cruzi
RODRIGUEZ, ANA
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
1 R03 MH085673-01
10
DESCRIPTION (provided by applicant): Chagas disease is endemic to 18 Latin American countries, with 13 million people chronically infected. Approximately 30% of chronically infected patients will suffer from irreversible damage to the heart and digestive tract leading to death. There is a need to develop new drugs against Chagas disease because only two drugs with strong toxic effects are available for treatment of the initial stages of infection and no drug is available for treating the chronic stage. The causative agent of the disease is Trypanosoma cruzi, an obligated intracellular protozoan parasite that in the chronic stage of disease is found almost exclusively in the intracellular stage. High-throughput methods that can reliably detect T. cruzi are required to screen compounds with activity against this parasite. We propose to use two high-throughput screening (HTS) assays using a recombinant T. cruzi line expressing beta-galactosidase. Since these parasites can be easily cultured in vitro, we propose to perform two assays for HTS of drugs that interfere with 1) the free extracellular parasites and 2) the intracellular infection cycle of T. cruzi. Inhibition of extracellular parasites will most likely identify drugs that are active against the early stages of disease, when parasites are found frequently outside host cells. Interfering with the cycle of infection is more likely to identify drugs that inhibit intracellular parasites, which are almost the only form observed during chronic infections. Both assays are currently performed the laboratory in 96-well plate format and have optimal sensitivity and reproducibility. We intend to perform primary screenings to select positive hits that will be further selected using secondary assays to exclude compounds toxic for host cells. We will also run secondary assays to confirm positive hits and counter screenings using alternative methods of detection to eliminate false positives. Chemical optimization will be performed if necessary and a plan for animal testing and development of putative new inhibitors into medically available drugs is described. PUBLIC HEALTH RELEVANCE: We intend to use two different assays for high-throughput screening: the inhibition of free Trypanosoma cruzi trypomastigotes and the inhibition of the infection of host cells by this parasite, with the aim of finding new drugs that could be used to treat Chagas disease. We will use a recombinant T. cruzi expressing 2-galactosidase, which allows for highly sensitive and reliable assays for high throughput screening. -
Discovery of Specific Cell-Permeable Inhibitors of the Ras Converting Enzyme
SCHMIDT, WALTER K
UNIVERSITY OF GEORGIA (UGA)
1 R03 MH085698-01
10
DESCRIPTION (provided by applicant): The RAS genes encode small GTPase Ras proteins that are important for controlling cell growth and differentiation. Activating mutations of Ras are associated with approximately 20-30% of all human cancers. All Ras proteins have a C-terminal CaaX motif that is extensively post-translationally modified. These modifications are prerequisite for localization of Ras proteins to the inner leaflet of the plasma membrane where they must be to function properly. It is therefore hypothesized that interfering with the post-translational modification of Ras could be an anti-cancer strategy. The CaaX motif is sequentially modified by the farnesyltransferase (FTase), the Ras converting enzyme (Rce1p), and the isoprenylcysteine carboxylmethyltransferase (ICMT), making them all promising anti-cancer targets. Our long-term goal is to identify agents that interfere with Rce1p. We hypothesize that pharmacological inhibition of Rce1p will lead to Ras mislocalization in vivo. Indeed, we have identified several compounds that inhibit Rce1p in vitro in a species-independent manner (i.e., human, yeast, and trypanosome) and disrupt Ras localization in vivo. This proposal aims to increase the number of similar acting compounds by identifying novel and potent Rce1p-specific inhibitors by high throughput screening (AIM 1). The primary screening of compounds will be performed in collaboration with the Emory HTS facility using a mix and measure fluorescence-based assay and Rce1p as the target enzyme. The specificity of primary hits will be determined using secondary screens to measure effects on two other enzymes, Ste24p and trypsin. Ste24p has a partially overlapping function with Rce1p in that it is required for the post-translational modification of certain CaaX motifs, but it is not specifically required for Ras modification. The expected hit rate using the proposed approach is 0.1% based on preliminary screening of a small compound library (i.e. NCI Diversity Set). The secondary hits will be further characterized by the applicant’s laboratory (AIM 2). This analysis will include additional specificity assessments using a distinct reporter and assay system that is based on in vitro production of the yeast isoprenylated a-factor mating pheromone. In vitro approaches will be used to characterize compound in terms of their inhibition kinetics (e.g. KM, KI, vmax), potency (e.g. IC50), and biophysical properties. In vivo cell-based assays will be used to determine toxicity and cell permeability, the latter using a unique GFP-Ras2p localization assay that we have developed. We expect this study to yield several active compounds that will be valuable for understanding the enzymatic nature of the relatively uncharacterized Rce1p and for serving as lead compounds for anti-cancer therapeutics involving targeted inhibition of Rce1p. PUBLIC HEALTH RELEVANCE: The Ras converting enzyme (Rce1p) is an anti-cancer target. High throughput screening for Rce1p- specific inhibitors will be conducted in association with the Emory HTS facility using a direct mix and measure in vitro fluorescence-based primary assay and secondary assays to address target specificity. The identified compounds, representing lead compounds for anti-cancer therapeutics, will be evaluated for their toxicity profile and cell permeability using cell-based assays and will be further assessed for target specificity and kinetic parameters by in vitro methods. -
HTS Assay for Inhibitors of HCV Core Protein
STROSBERG, ARTHUR DONNY
SCRIPPS RESEARCH INSTITUTE
1 X01 MH085709-01
10
DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a major cause of liver disease, with about 170 million people infected world wide. No vaccine has been developed and the only treatment available today is a combination of pegylated interferon alpha and ribavirin. Progress in developing a specific treatment has been greatly hampered by the lack of suitable infectious HCV culture systems, but recently such a system was developed using an isolate from a HCV genotype 2a virus growing in human Hepatoma cells. Unfortunately most pathologic effects in Western countries and in the USA are due to HCV of genotype 1, which is markedly less sensitive to interferon treatment than the 2a virus. A major aim of this proposal is to characterize a HCV genotype 1 infectious culture system based on initial encouraging preliminary results. Another aim is to analyze why these two HCV genotypes differ in their sensitivity to interferon, not only in patients, but, again based on our preliminary results, also in culture. Finally, a third aim is to compare the two culture systems by focusing on the effect on viral assembly, of the inhibitors of dimerization of structural core protein. PUBLIC HEALTH RELEVANCE: Hepatitis C is often described as the stealth epidemic: worldwide there are 170 million carriers, 3 million of which live in the US. Every year 38,000 Americans are newly infected, mainly through contact with contaminated blood. Twenty percent of infected individuals will develop liver cirrhosis and up to 2.5 percent will develop hepatocellular carcinoma. There is no known general cure for hepatitis C, and no vaccine. The only current treatment, a combination of interferon and ribavirin, is effective for less than half of the patients with genotype 1, the most prevalent in Western countries. The aim of the proposed research is to develop specific curative treatments for all six HCV genotypes that would effectively lead to the inhibition of the virus. For this purpose, we will develop a culture system for infectious HCV of genotype 1, responsible for most pathogenic effects in Western countries, and will use this to screen for small molecule inhibitors of viral assembly. -
Small-Molecule Inhibitors of Vaccinia-H1-Related Phosphatase VHR
TAUTZ, LUTZ
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 R03 MH084230-01A1
10
DESCRIPTION (provided by applicant): Protein tyrosine phosphatases (PTPs) play vital roles in numerous cellular processes and are implicated in a growing number of human diseases, ranging from cancer to cardiovascular, immunological, infectious, neurological, and metabolic diseases. The Vaccinia H1-related (VHR) PTP is a dual-specific Erk and Jnk phosphatase, the loss of which causes specific cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells without detrimental effects on normal cells. Recent studies by collaborators and us suggest that VHR is upregulated in several cervix cancer cell lines, in squamous intraepithelial lesions, and squamous cell carcinomas of the uterine cervix. High throughput chemical library screening will be used to identify hit compounds, which will be then further developed into potent and specific inhibitors of VHR. These inhibitors will be useful chemical probes for basic research into the mechanisms of signal transduction and mitogen-activated protein (MAP) kinase regulation, and may also establish VHR as a novel and promising drug target for the treatment of cervical cancer. Besides a screening ready HTS assay, we also have a secondary assay, profiling assays, and cell-based assays in place to verify, characterize, and prioritize hits. We have milligram amounts of highly pure VHR in hand, so that HTS for VHR inhibitors could commence immediately. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to seek access to high-throughput chemical library screening to identify hit compounds for the protein tyrosine phosphatase (PTP) VHR, which we will then further develop into potent and specific inhibitors of VHR. PTPs have been recently implicated with numerous human diseases, including cancer. Recent studies by our lab and others clearly suggest that VHR is critical for the development of cervical cancer. -
High Throughput Screening for Identifying Lead Compounds Against 3CLpro
TOKARS, VALERIE L
UNIVERSITY OF ILLINOIS AT CHICAGO
1 R03 MH084162-01A1
10
DESCRIPTION (provided by applicant): Coronaviruses are emerging, evolving and newly discovered viruses that are posing a threat to public health. Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. NL63-CoV was recently identified as a second coronavirus associated with respiratory illness. Like SARS-CoV, NL63-CoV enters its target cell via angiotensin-converting enzyme 2. Unlike SARS-CoV, the mortality rate of people infected with NL63-CoV is higher in children. To combat the effects of these viruses, the development of antivirals is warranted. A logical target for antiviral drug development against these coronaviruses is the 3C-like protease (3CLpro). This enzyme is essential for viral replication, and has become the focus of numerous drug discovery efforts worldwide. Evaluation of inhibitory activity of several published lead compounds has proven limited due to cytotoxicity issues, loss of activity in the presence of reducing agent, or lack of specificity. We propose to identify novel chemical probes that inhibit SARS-3CLpro and NL63-3CLpro by performing high throughput screening (HTS) in collaboration with the Molecular Libraries Screening Centers Network (MLSCN). A highly sensitive assay was developed in our laboratory for routine analysis and high-throughput screening. The assay is a Fvrster resonance energy transfer (FRET) based assay that uses a peptide substrate labeled with HiLyte fluorTM488 and QXLTM520. The QXLTM520 quenches the fluorescence of HiLyte fluorTM488 that is released upon peptide cleavage. The assay provides a high Z-factor and is available for measuring 3CLpro activity from both SARS- 3CLpro and NL63-3CLpro. The primary FRET based assay for identification of novel chemical probes will be followed by secondary screening to determine IC50 values for lead probes. PUBLIC HEALTH RELEVANCE: Infectious diseases remain among the leading causes of death worldwide. The coronaviruses SARS-CoV and NL63-CoV are infectious agents responsible for life-threatening respiratory syndromes. Discovery of chemical probes and antivirals to confront these diseases remain a priority for public health. -
Discovery of Lead Compounds which Modulate Tumor Specific Pyruvate Kinase M2 Acti
VANDER HEIDEN, MATTHEW
DANA-FARBER CANCER INSTITUTE
1 R03 MH085679-01
10
DESCRIPTION (provided by applicant): Discovery of lead compounds that modulate tumor specific pyruvate kinase M2 activity Abstract: We propose to discover and optimize selective modulators of human pyruvate kinase M2 (PK-M2), a splice isoform of pyruvate kinase that is specifically expressed in tumor cells. The proposed research has the following specific aims: (1) To identify both activators and inhibitors of human PK-M2 using a quantitative high-throughput screening approach against the MLSMR containing 300,000 small molecules. (2) To confirm the potency and selectivity of these compounds in a panel of secondary assays. This will include assays to identify those compounds which do not modulate the human M1, L, or R isoforms of pyruvate kinase that, in contrast to PK-M2, are expressed in most differentiated tissues but not in tumor cells. We will also determine the impact of compounds we identify on cell metabolism and proliferation. We will use these results to further improve the potency of the most promising molecules using structure-based methods, analogue synthesis and medicinal chemical principles. (3) Beyond the granting period for this proposal, to test the most promising compounds for their ability to decrease tumor growth in mouse models of cancer. PUBLIC HEALTH RELEVANCE: The M2 isoform of pyruvate kinase is expressed in all cancer cells where it plays a critical role in glucose metabolism. Because most normal tissues express another isoform of pyruvate kinase, targeting pyruvate kinase M2 provides an opportunity to selectively disrupt glucose metabolism in cancer cells. This project aims to find specific small molecule modulators of pyruvate kinase M2 to test the feasibility of targeting this enzyme in cells, and may lead to the development of novel cancer drugs. -
Discovery of Lead Compounds Against Trypanosomiasis and Leishmaniasis Through Ind
WALKINSHAW, MALCOLM DOUGLAS
UNIVERSITY OF EDINBURGH
1 R03 MH085697-01
10
DESCRIPTION (provided by applicant): Previous proof-of-principle research will provide the foundation for the discovery and optimization of selective inhibitors of pyruvate kinase (PYK) of trypanosomatid parasites (Trypanosoma brucei, T. cruzi and Leishmania species). These pathogens cause serious, often fatal diseases of humans such as sleeping sickness, Chagas’ disease and kala-azar in tropical and subtropical countries primarily in Africa, Central and South America, and Asia where many millions live in areas where the diseases are endemic. Tragically, current drugs for their treatment are unsatisfactory because they are toxic and ineffective against some forms of the diseases, and resistance is becoming increasingly common. Glycolysis is essential in T. brucei and therefore a promising drug target. Inhibitors of glycolytic enzymes such as PYK may thus serve as lead compounds for the development of new drugs. The proposed research has as specific aims: (1) to exploit unique features of trypanosomatid PYK (for which detailed structural information is already available and which has been validated as drug target by RNAi) for the discovery of selective inhibitors of PYKs from L. mexicana (LmPYK) and from T. brucei (TbPYK) through quantitative high-throughput screening of the Molecular Library Small Molecule Repository (MLSMR) containing 300,000 small molecules; (2) To confirm the potency of these compounds in a panel of secondary assays and to test the selectivity against human PYK, and to further improve the potency of the most promising molecules thus obtained by structure-based methods, analogue synthesis and medicinal chemical principles; (3) To test compounds displaying the highest potency for their ability to inhibit growth of cultured cells representing pathogenic stages of the parasites. PUBLIC HEALTH RELEVANCE: Sleeping sickness in sub-Saharan Africa, Chagas’ disease in Central and South America; and kala-azar and related diseases in tropical and subtropical regions of the world cause severe public health and economic burdens on populations that are already caught in a tragic cycle of poverty, poor nutrition and disease. Millions of people worldwide are infected by these potentially fatal diseases and hundreds of millions are at risk. Existing treatments have developed little in the past 40 years, and suffer from toxicity, inefficiency and resistance; the goal of this project is to develop lead drugs that will be suitable for entry into pre-clinical trials. -
Multiplex Screening to Identify Dual Action Probes in a Cell Model of Huntington?
ZHENG, WEI
U.S. NATIONAL HUMAN GENOME RESEARCH INST
1 R03 MH084839-01A1
10
DESCRIPTION (provided by applicant): Huntington’s disease is a progressive, fatal neurodegenerative disorder that has been linked to a CAG polyglutamine (polyQ) expansion located 17 amino acids from the initiation codon in exon 1 of the Huntingtin (HTT) gene. The severity and time of onset of the disease are directly proportional to the length of the polyglutamine expansion. Currently, effective treatment is not available for this disease. Many experimental results including the use of animal models have indicated that the extended polyQ repeat in the exon-1 of HTT gene can cause neurodegenerative changes in related cells and tissues. Several assays have been reported for the compound screening to identify the small molecule inhibitors of protein aggregation or cytotoxicity in the Huntington cells. These methods examine the effect of compounds either on prevention of cytotoxicity or on aggregate formation associated with expression of the polyQ protein. But the useful leads for the drug development to treat Huntington’s disease have not been reported yet. We have developed a new multiplex assay for identifying active compounds that act on both cytotoxicity and protein aggregation in the same assay plate using a Q103 cell model of Huntington’s disease. This multiplex assay was miniaturized in 1536-well plates and a screen test was carried out against a small compound collection. One active compound identified from this testing screening showed the positive activity in other model systems. Therefore, the results indicate that this multiplex assay is valuable and suitable for screening large compound collections. We propose here a high throughput screening using this multiplex assay against the MLSCN’s compound collection for identifying dual action probes as research tools as well as a potential chemistry starting point for the drug development to treat Huntington’s disease. PUBLIC HEALTH RELEVANCE: Huntington’s disease is a progressive, fatal neurodegenerative disorder that has been linked to a polyglutamine (polyQ) expansion in exon 1 of the Huntingtin (HTT) gene. The severity and time of onset of the disease are directly proportional to the length of the polyglutamine expansion. Currently, effective treatment is not available for this disease. Although the HTT gene was identified 15 years ago, the exact function of protein is still not clear. Many experimental results including the use of animal models have indicated that the extended polyQ repeat in the exon-1 of HTT gene can cause neurodegenerative changes. Several assays have been previously used for the compound screening to identify the small molecule inhibitors of protein aggregation or cytotoxicity in the Huntington cells. But the useful leads for the drug development of Huntington’s disease have not been reported yet. We have developed a new method to sequentially screen both protein aggregation and cytotoxicity using a Huntington cell line. We propose here to identify potential dual action compounds to study the pathophysiology of this disease as well as to serve as a starting point of drug development for Huntington’s disease. -
A High-Throughput Screen for LuxS Quorum-Sensing Inhibitors
BASSLER, BONNIE L
PRINCETON UNIVERSITY
1 R03 MH086452-01
11
DESCRIPTION (provided by applicant): The long-term goal of this research is to explore the molecular mechanisms that bacteria use for cell-cell communication (i.e., quorum sensing). Until recently, the ability of bacteria to communicate was considered an anomaly that occurred only in a few marine vibrio species. It is now clear that cell-cell communication is the norm in the bacterial world and that understanding this process is fundamental to all of microbiology, including industrial and clinical microbiology, and ultimately to understanding the development of higher organisms. Here we propose an integrative chemical, structural, and biological study of a recently identified quorum-sensing circuit whose functioning depends on the production and detection of a small molecule signal called autoinducer-2 (AI-2). AI-2, unlike other known bacterial autoinducers, is a universal quorum-sensing signal that mediates communication among different bacterial species. We propose to collaborate with the MLPCN to carry out high-throughput screens to identify inhibitors of the AI-2 synthase, LuxS and the AI-2 receptor, LuxPQ. Follow up studies to further characterize the lead compounds identified from the screens will combine synthetic organic chemistry, bacterial genetics, biochemistry, and X-ray crystallography. Antagonists of the AI-2 signaling pathway identified through this work will provide lead compounds for the development of broad-spectrum antibacterial drugs designed to interfere with quorum sensing which could have enormous ramifications for bettering human health.
Inhibitors of Cell Surface uPA Generation
BUGGE, THOMAS H
NIH NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH
1 R03 MH086467-01
11
DESCRIPTION (provided by applicant): The overall objective of this proposal is to perform a quantitative high-throughput screen to identify novel compounds that inhibit the generation of cell surface urokinase activity by using a recently established assay for specific and non-invasive optical imaging the activity of cell surface urokinase (Hobson, J.P. et al. Nat. Methods, 3: 259-61, 2006). This objective of the proposal will be pursued through the following two Specific Aims: Specific aim 1: Perform automated quantitative high-throughput screening of chemical compound libraries to identify 10-40 novel inhibitors of the generation of cell surface urokinase activity with inhibitory constants less or equal to 1 μM. Specific aim 2: Determine the mechanism by which these compounds prevent the formation of active urokinase on the surface of cells. The proposed research will: 1) Provide novel candidate agents for inhibition of pathological cell surface plasminogen activation. 2) Generate important new tools for dissecting key unknown molecular features of the cell surface urokinase plasminogen activation cascade.
Screening for Inhibitors of MLL Translocation Proteins in Leukemia
BUSHWELLER, JOHN HACKETT
UNIVERSITY OF VIRGINIA
1 R03 DA027717-01A1
11
DESCRIPTION (provided by applicant): Chromosomal translocations that affect the proto-oncogene MLL (Mixed Lineage Leukemia) occur in aggressive human acute leukemias, both in children and adults. The normal MLL protein plays a key role in regulation of HOX gene expression, which is required for proper hematopoiesis (blood cell development). In leukemias, this function is destroyed by a fusion of MLL with one of 60 alternative partner genes to form a chimeric oncogenes. MLL fusion proteins upregulate HOX expression resulting in a blockage of blood cell differentiation that ultimately leads to acute leukemia. Patients with leukemia harboring MLL translocations have a very poor prognosis (20 % event free survival at 3 years) and it is clear that there is a need for new therapies to treat these leukemias.
Despite the heterogeneity of fusion partner, the N-terminus of MLL translocations is always invariant and contains the conserved CXXC domain. Deletion of this domain eliminates transactivation properties of oncogenic MLL fusions. The CXXC domain functions as a DNA binding motif that specifically recognizes unmethylated CpG sequences. Importantly, the interaction of the CXXC domain with DNA is absolutely required for leukemogenic function of MLL translocations. To inhibit the oncogenic potential of MLL-translocations we plan to develop small molecules that could specifically inhibit interaction of the CXXC domain with DNA. Here, we propose to use high throughput screening (HTS) to identify inhibitors of this protein-DNA interaction. We propose to use a well-validated fluorescence polarization (FP) assay to screen the MLPCN library of compounds and TR-FRET (time resolved fluorescence resonance energy transfer) as a secondary assay to validate hits obtained by initial screening. To address selectivity, active compounds will be tested by FP with the methyl-CpG DNA binding domain of the MBD2 protein, which binds specifically to methylated CpG DNA elements and not to unmethylated CpG elements. Direct binding of the most active compounds to the CXXC domain will be verified by NMR spectroscopy. The compounds will be subsequently optimized to increase binding affinity to the CXXC domain by structure-based drug design approaches combined with standard medicinal chemistry. The most potent compounds will be tested in appropriate leukemia cell-lines harboring MLL-translocations and, if feasible, in appropriate mouse models of leukemias.
HTP screening for inhibitors of ser/the protein phosphatase 5
HONKANEN, RICHARD E
UNIVERSITY OF SOUTH ALABAMA
1 R03 MH085702-01A1
11
DESCRIPTION (provided by applicant): Almost every cell in the body is constantly exposed to a variety of hormones, growth factors and other agents that influence the biological functions of cells. To integrate and interpret these external stimuli, complex signaling networks have evolved, which allow different types of cells to respond appropriately to their environment. In eukaryotic cells the reversible phosphorylation of proteins regulates many signaling networks that control cell growth, metabolism, differentiation, senescence, and apoptosis. Protein phosphorylation occurs principally on serine, threonine and tyrosine residues, and the phosphorylation reaction is catalyzed by a large family of protein kinases. To date many compounds that function as potent and highly selective inhibitors of “key” protein kinases have been identified, and these inhibitors have proven to be powerful tools to probe the biological and pathological actions of protein kinases. In contrast, much less is known about the biological roles of protein phosphatases. To a large extent this is due to a lack of specific or highly selective small molecule inhibitors. Having developed methods to produce large amounts of catalytically active ser/thr protein phosphatase 5 (PP5), a fluorescent assay to rapidly and reliably measure PP5 activity, and having solved the crystal structure of PP5 at high resolution (1.6 Å), we now have the tools in place to develop inhibitors of PP5. The objective of this proposal is to identify a specific or highly selective inhibitor of PP5. There are two specific aims.
Aim 1. Conduct a HTP-screen in conjunction with the Molecular Libraries Probe Production Centers Network (MLPCN) to identify compounds that inhibit the catalytic activity of PP5.
Aim 2. Back screen compounds identified in Aim 1 against the structurally related phosphatases to determine selectivity, characterize the inhibitors, and work with members of the MLPNC for probe optimization (e.g. increase potency/specificity). The compounds produced from this effort will server as powerful small molecule probes that will greatly aid the elucidation of the biological and pathological roles played by PP5. They may also serve as lead compounds for the development of new drugs for medical management of human cancer.
Allosteric Potentiators of the Oxytocin System for the Control of Social Motivati*
JARSTFER, MICHAEL B
UNIVERSITY OF NORTH CAROLINA CHAPEL HILL
1 R03 MH085678-01A1
11
DESCRIPTION (provided by applicant): The biochemical underpinnings of complex social behavior are poorly understood. As a result of this knowledge gap, diseases and syndromes that result in social deficits cannot be effectively treated and rational approaches toward establishing effective treatment are restricted. Recently, the neuropetide oxytocin, which has well established roles in parturition and lactation, has been implemented in a wide range of complex social behaviors. Results from several reports suggest that oxytocin can positively affect anxiety, depression, psychosis and addiction. Additionally, the potency of oxytocin in forming social attachments (mother-infant, monogamous pair bonds) and social memories suggests that increased oxytocin activity in the brain can ameliorate the profound social motivation and cognition deficits of patients with autism spectrum disorders, schizophrenia or severe personality disorders. Social deficits are particularly disabling features of these disorders for which we have no effective pharmacological treatments. The hypothesis of this research project is that oxytocin receptor agonist and positive allosteric modulators would serve as leads for pharmaceutical intervention in disease states characterized by social deficits. Our objective is to identify agonists and positive allosteric modulators through a high throughput screen. To achieve our object we propose three specific aims: 1. Identify selective allosteric potentiators and agonists of the human OTR through a high throughput screening campaign. 2. Conduct post screen secondary analysis to validate hits identified in our high throughput screen. In order to validate hits from the primary screen, several cell-based secondary assays will be conducted. We have developed each assay for this purpose. 3. Develop a plan for optimization of validated hits. We will develop a plan to optimize the more promising class of actives identified through our screening efforts. The completion of these aims will provide the scientific community with new chemical probes to study the roles of oxytocin in complex mammalian social behavior.
Development of Chemical Probes for KCNQ Potassium Channels
LI, MIN
JOHNS HOPKINS UNIVERSITY
1 R03 DA027716-01
11
DESCRIPTION (provided by applicant): Voltage-gated potassium channels are critical for neuronal function. There are estimated 160 genes encoding different but highly homologous potassium channels in humans. Because of the considerable sequence homology, gene-specific channel modulators are very rare but they are sought-after reagents both for investigating channel function and developing therapeutics. The KCNQ (or Kv7) channel family includes five members: KCNQ1 to KCNQ5. Evidence from patient studies and animal models has shown that a small change in KCNQ expression by as little as 25% can cause disease conditions such as epilepsy. Therefore, chemical probes would be powerful analytical tools to investigate the structure and function of voltage-gated potassium channels, and in the case of KCNQ, these compounds may be very valuable for therapeutic development. To perform a large-scale compound screen, KCNQ channel cell lines have been generated and an HTS-ready protocol has been developed and optimized. The present application seeks to conduct a large scale compound screen using the developed assay. The specific aims of the proposal are: 1. To effectively work with the NIH-assigned MLPCN center to perform a large
compound library (>100,000 compounds) and validate lead compounds that specifically ACTIVATE the heteromultimeric KCNQ2/3 potassium channel; 2. To conduct the secondary assays and counter screen against KCNQ1, 4 and 5; 3. To perform initial characterization to allow for selection of potent lead compounds for detail functional analyses. The success of this project will lead to identification of novel compounds that are useful for developing therapeutics and investigating M-current and KCNQ ion channel function.
Reversing Antifungal Drug Resistance
LINDQUIST, SUSAN L
WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH
1 R03 MH086456-01
11
DESCRIPTION (provided by applicant): Acquired drug resistance by medically relevant microorganisms poses a grave threat to human health and has enormous economic consequences. Fungal pathogens pose a particular challenge because they are more closely related to humans than bacteria and share many of the same mechanisms at the molecular level that support the growth and survival of the cells comprising their human hosts. The number of drug classes that have distinct targets in fungi is very limited and the usefulness of current antifungal drugs is compromised owing to either significant toxicity for the patient receiving them or the frequent emergence of high grade resistance. The objective of this project is to discover new chemical compounds capable of reversing fungal drug resistance, thereby illuminating the mechanisms responsible for drug resistance in disease-causing fungi and making currently available antifungals safer and more effective. To achieve this ambitious goal, a research plan designed to achieve the following specific aims will be pursued in collaboration with a designated center within the National Institutes of Health (NIH) Molecular Libraries Probe Production Center Network (MLPCN): Aim 1: Optimize and then execute a high throughput robotic screen of hundreds of thousands of individual chemicals to find compounds that can reverse the resistance of a fungal strain that was isolated from a patient receiving the very commonly used antifungal drug fluconazole; Aim 2: Evaluate the compounds identified in the primary screen by measuring their potency, their spectrum of activity against various types of fungus, their selectivity for human versus fungal cells and determine the general way in which they reverse antifungal drug resistance; Aim 3: Select the 10 most promising compounds and synthesize a panel of derivatives for each one to optimize their antifungal potency and specificity. This project will combine our long established expertise in studying the molecular biology of fungi using genetic and biochemical techniques with the outstanding resources of an MLPCN center and its expertise in high throughput screening technology and medicinal chemistry. In collaboration with the Broad Institute which was recently designated an MLPCN center, the essential primary screening assay has already been developed and validated. As a result, the deliverable outcome from this brief one year project is expected to be several highly useful chemical compounds with which to probe fungal biology. These probes will be invaluable to us and others for studying the mechanisms underlying fungal drug resistance. In addition to their basic research applications, their therapeutic relevance can be readily evaluated using established animal models in future work. By virtue of the way in which they will be discovered, many of these compounds are likely to act in previously unknown and unexploited ways that could prove uniquely effective in addressing the ever increasing problem of acquired drug resistance by disease-causing microorganisms that confronts our society.
Chemical Modulation of the Siah-1 Pathway
REED, JOHN C
BURNHAM INSTITUTE FOR MEDICAL RESEARCH
1 R03 MH086475-01
11
DESCRIPTION (provided by applicant): Proteasomal degradation typically requires post-translational modification of target proteins with K48-linked polyubiquitin chains. This process of protein proteolysis plays a key role in normal cellular function. The E3 ubiquitin ligase, Siah-1, facilitates the transfer of ubiquitin to its substrate proteins destined for degradation by way of its RING domain. Siah-1 is a member of a family of highly conserved RING domain proteins, which regulate a variety of cellular functions, including cell cycle arrest, tumor suppression, and apoptosis through the beta-catenin degradation pathway. Siah-1 has also been identified as a p53-inducible gene, functionally linking it to an important tumor suppressor. Chemical modulators of the Siah-1 pathway would provide powerful research tools for elucidating the roles of this signaling pathway in cancer development and progression. In this proposal, we describe the development of a High Throughput Screening (HTS) assay based upon fluorescence polarization, and utilizing a peptide ligand of Siah-family proteins with an attached flurochrome. This fluorescence polarization assay (FPA) forms the basis for a high-throughput competitive displacement assay that we have optimized for chemical library screening. We propose to screen the NIH compound library using this HTS assay. Additional downstream assays provided by the assay provider will be performed for deconvoluting hits. We expect to obtain candidate compounds for Structure Activity Relationship (SAR) studies to be performed for a prototypical RING-containing protein, Siah-1. Together, these efforts will result in validated chemical probes for studying the biology of Siah-family E3 ligases in a variety of biological settings.
Chemical Screen of TOR pathway GFP fusion proteins in S. cerevisiae
WERNER-WASHBURNE, MARGARET C.
UNIVERSITY OF NEW MEXICO
1 R03 MH086450-01
11
DESCRIPTION (provided by applicant): We propose an HTS multiplex screening project to use small molecules to probe a complex, highly conserved, biological pathway. The project takes advantage of the yeast GFP strain collection to probe the TOR pathway, a pathway with therapeutic implications in man [1]. The target of rapamycin, TOR, is an essential ser/thr protein kinase that functions in two distinct multiprotein complexes, TOR complexes 1 and 2. The structure and functions of these complexes have been conserved from yeast to man. TOR complex 1 is inhibited by rapamycin and is thought to couple growth cues to cellular metabolism; TOR complex 2 is not inhibited by rapamycin and appears to regulate spatial aspects of growth, such as cell polarity [2, 3]. Rapamycin, an antifungal compound isolated from a bacterium found in soil on the island of Rapa Nui [4], was the drug originally used to characterize the TOR pathway in budding yeast (S. cerevisiae) by identifying mutants that were rapamycin sensitive or resistant [5]. Rapamycin acts as a cytostatic agent in fungi, arresting cells in a G0-like state. As a drug it is widely used as an immunosuppressant and rapamycin and derivatives of this macrocyclic lactone are being evaluated for a number of clinical applications. Because of its pleiotropic effects, rapamycin is thought of as a “dirty drug” in the pharmaceutical industry. To identify more specific TOR inhibitors and activators, we propose a cell-based multiplex high throughput flow cytometry assay to screen the MLSMR to define chemicals that target specific proteins in the TOR pathway. A chemical screen should yield small molecules that modulate specific branches of this pathway and these molecules may have significant therapeutic potential and fewer side effects. We will screen the yeast GFP-fusion strain set (4,159 strains) [6] prior to and post-rapamycin treatment in multiplex format because the fusion library is highly amenable to fluorescence-based flow cytometric readout. Change in fluorescence indicates that a chemical has affected production of the target GFP-fusion protein(s). Chemicals we identify that mimic or inhibit rapamycin activation will likely have targets in other organisms because TOR pathway components are highly conserved. We have already successfully screened this collection under two different growth conditions and have the capacity to identify chemicals affecting one or more than one of the multiplexed targets. We will conduct primary screens to detect both agonists and antagonists of the TOR pathway. As secondary screens, we will: 1) compare the impact of the novel molecules on protein expression in the GFP collection and 2) evaluate the ability of the small molecules to impact the cytostatic potential of rapamycin through analysis of cell cycle and/or growth arrest. Expected Results: The screen in the absence of rapamycin will reveal molecules that mimic rapamycin or otherwise impact the TOR pathway. The screen in the presence of rapamycin will reveal molecules that antagonize rapamycin.
Identifying Small Molecules To Probe the Role of Heat Shock Factor 1 in Cancer
WHITESELL, LUKE J
WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH
1 R03 DA027713-01
11
DESCRIPTION (provided by applicant): The heat shock response (HSR) is a powerful transcriptional program which acts genome-wide, not only to restore the normal protein folding environment through the induction of heat shock proteins but as more recent work has shown to re-shape global cellular pathways controlling survival, growth and metabolism. In mammals, this response is regulated primarily by Heat Shock Factor 1 (HSF1), a transcription factor whose mode of action has been conserved in broad outline across all eukaryotes. Surprisingly, our recent work has shown that the many beneficial effects of HSF1 known to enhance the survival of organisms under stress come at the cost of facilitating the initiation and maintenance of cancers in mouse models and diverse human tumor lines driven by a variety of underlying oncogenic lesions. Acting at a global systems level, HSF1 function permits cells to survive the drastic imbalances in signaling and profound alterations in DNA, protein and energy metabolism that occur during malignant transformation. A novel therapeutic opportunity has been highlighted by our demonstration that knockdown of HSF1 expression using genetic techniques is well tolerated in normal cells and whole animals but that malignant cells display a profound dependence on this “non-oncogene.” Relatively limited efforts have been directed so far at discovering inhibitors of this innovative target. The few HSF1 inhibitors that have been reported demonstrate limited specificity with prominent effects on general transcription and translation and poorly defined mechanisms of action. To address this deficiency and develop potent and selective inhibitors of HSF1 function, this project will join our extensive experience in the heat shock response, experimental therapeutics and cancer biology with the expertise in high-throughput screening and chemical probe optimization now available through the NIH Molecular Libraries Probe Production Center Network to accomplish the following specific aims: Aim1: Identify specific inhibitors of HSF1 using an optimized high throughput cell-based dual reporter assay that will markedly reduce false positives during the primary screening process. Aim 2: Evaluate the potency, specificity and mode of action of compounds using secondary and counter-screening assays in order to guide the selection of screen hits for analog synthesis and support their optimization into useful chemical biological probes. To validate our approach, we have completed a pilot screen with the Broad Institute Chemical Biology Platform using a collection of 70,000 known bioactive, small drug-like and purified natural products. As proof of principle, this screen identified a family of natural products that specifically inhibit the activation of HSF1. The discovery of additional inhibitors that target HSF1 function at mechanistically distinct regulatory steps in its activation will shed light on its multifaceted role in cancer biology and will provide important leads for preclinical studies in the chemoprevention and chemotherapy of a wide range of human cancers.
High Throughput Screening to Identify Inhibitors of the Human Abasic Endonuclease
WILSON, DAVID M.
U.S. NATIONAL INSTITUTE ON AGING
1 R03 MH086444-01
11
DESCRIPTION (provided by applicant): DNA repair systems have evolved to protect cells from the deleterious consequences of both endogenous and exogenous DNA damaging-agents. An un-intended role of these processes is their involvement in dictating cellular responsiveness to clinical anti-cancer agents, many of which are DNA-interactive compounds. Recent efforts have focused on the strategic manipulation of DNA damage responses to (a) protect normal cells from the toxic effects of anti-cancer agents and (b) enhance the sensitivity or killing of tumor cells. One strategy has been to isolate potent inhibitors of target DNA repair proteins that can potentiate the lethality of clinical DNA-interactive drugs. The goal of this project is to identify small molecule, non-covalent chemical inhibitors of the major human abasic endonuclease APE1, which functions centrally in base excision repair, the predominant pathway for coping with oxidative and alkylative DNA lesions. Towards this end, we will employ an optimized high throughput screening assay that utilizes a fluorescence intensity modulation scheme with a fluorophore and quencher positioned opposite one another within a duplex deoxyoligonucleotide substrate that contains a single abasic cleavage site.
Small molecule agonists of the relaxin receptor
Agoulnik, Alexander I
Baylor College of Medicine
1 R03 MH085705-01A1
12
The relaxin hormone is involved in the variety of biological functions in health and disease conditions. The role of relaxin is well-established in female reproduction and parturition, mammary gland and endometrial development, maintenance of myometrial quiescence during pregnancy. Relaxin signaling through its G protein-coupled receptor RXFP1 results in extracellular matrix remodeling through regulation of collagen deposition, cell invasiveness, proliferation and overall tissue homeostasis. Significantly, the therapeutic effects of relaxin in the treatment of renal, cardiac, skin, lung fibrosis, inflammation, and wound healing in animal models are well-established. Other data strongly indicate on the significance of relaxin in progression of prostate, breast, thyroid, endometrial and other cancers. Several clinical trials study the therapeutic role of relaxin in treatment of scleroderma, cervical ripening, fibromyalgia, orthodontics and as a protective agent in congestive heart failure. The easy and reliable indication of relaxin receptor activation in cells expressing RXFP1 is an increase of cAMP production. No chemical/small molecules or non-peptide agonists/antagonists are known for relaxin receptor. The current application is designed to fill this gap through high throughput small molecule agonist screening. Using HEK293T cells stably transfected with relaxin receptor we have optimized commercially available cAMP assay for a 1536-well plate format and performed trial screens of validation library. The assay is suitable for high throughput screening. After primary screen the active compounds will be selected in a series of secondary screens to identify specific relaxin receptor agonists. The discovery of relaxin agonists will provide basis for their use as anti-fibrotic, proliferative, vasodilatory, and angiogenic agents.
A yeast HTS for caloric restriction mimetics that inhibit age-related superoxide
BURHANS, WILLIAM C
ROSWELL PARK CANCER INSTITUTE CORP
1 R03 MH087439-01
12
DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) and aberrant growth signaling are important factors in aging and contribute to a variety of age-related pathologies, including neoplastic, cardiovascular and neurodegenerative diseases. At early stages of cancer, aberrant growth signaling increases ROS and causes DNA replication stress (inefficient DNA replication). Both ROS and DNA replication stress lead to DNA damage and oncogene-induced senescence of preneoplastic cells that inappropriately arrest growth in S phase. DNA replication stress also likely occurs in postmitotic neurons downstream of the increased ROS and aberrant growth signaling that is characteristic of many neurodegenerative disorders. In recent years, studies of chronological aging in the model organism S. cerevisiae (budding yeast) have been employed to investigate factors that impact aging of differentiated, postmitotic cells in humans. Our recent studies show that aberrant growth signaling accompanied by increased ROS and DNA replication stress in cells that growth arrest in S phase shortens the chronological lifespan of this organism (defined as the length of time cells survive after undergoing growth arrest in stationary phase). Downregulation of growth signaling pathways by caloric restriction or by caloric restriction mimetics extends chronological lifespan by inhibiting the chronological age-dependent accumulation of ROS and promoting a more efficient stationary phase growth arrest in G1, where cells cannot undergo DNA replication stress. Caloric restriction also extends the lifespans of higher eukaryotes and protects against cancer and neurodegeneration. These effects likely involve similar inhibition of ROS and more efficient G1 arrest of postmitotic, differentiated cells. The primary goal of the proposed research is to implement a highthroughput chemical screen of stationary phase budding yeast cells to identify additional compounds that mimic these conserved effects of caloric restriction. Compounds that exert these effects will be identified by their ability to inhibit the accumulation of age-dependent superoxide anions in concert with a tighter stationary phase growth arrest in G1. These compounds will likely be useful as cancer chemopreventive agents and in the treatment of neurodegenerative and other age-related disorders.
High throughput screening of a general anesthetic binding site
ECKENHOFF, RODERIC G
UNIVERSITY OF PENNSYLVANIA
1 R03 MH084836-01A2
12
DESCRIPTION (provided by applicant): We propose a high throughput screen at NCGC for general anesthetics based on binding to a well characterized site in a GABAA mimic, apoferritin. The assay is based on competition of compounds with enhanced 1-aminoanthracene fluorescence. The assay has been taken to a 1536-well format, and shows excellent signal: background and signal: noise ratios, and when tested with positive control compounds, achieves Z’ factors better than 0.8. The LOPAC 1280 set has been screened which resulted in retrieval of the only know general anesthetic in the library, and several other compounds most of which were validated by ITC. After primary screen, a novel computational filter will be applied, followed by SAR, clustering and triage of actives in collaboration with NCGC. Validation screens will use ITC to confirm and extend the HTS IC50 values. In addition to providing several probe compounds for further development, this project is likely to provide useful laboratory reagents and inform protein-ligand chemistry.
Small-molecule inhibitors of the human RECQ1 and Bloom DNA helicases
GILEADI, OPHER
UNIVERSITY OF OXFORD
1 R03 MH087284-01
12
DESCRIPTION (provided by applicant): Survival of cells and the faithful propagation of the genome depend on elaborate mechanisms of detecting and repairing DNA damage. Treatment of advanced cancer relies on radiation therapy or chemotherapy, which kill cancer cells by causing extensive DNA damage. It is often found, that cancer cells develop resistance to therapy through enhanced activity of DNA repair functions; this has led to an increased interest in developing drugs that interfere with DNA repair, which could sensitise cancer cells to conventional therapy. This application involves two related human proteins, RECQ1 and BLM, which are important in resolving abnormal DNA structures formed during replication or homologous recombination. The BLM gene is defective in patients of Bloom’s syndrome, which is associated with an increased incidence of cancer. Shutting down the expression of RECQ1 or BLM leads to chromosomal instability and higher radiation sensitivity in cultured cells. Remarkably, some cancer cell lines, but not normal cells, exhibit reduced growth and an increase in cell death when RECQ1 expression is inhibited by RNAi. The goal of this project is to develop inhibitors of RECQ1 and BLM activity, which can be used in cell and animal models to examine the consequences of inhibition on the survival of cancer cells. Initial candidates will be identified by a quantitative high throughput screen of the MLSMR compound library, using a fluorescence-based in vitro biochemical assay that reveals inhibitors of RECQ1 and BLM DNA unwinding activity. The resulting compounds will then be subject to orthogonal, secondary biochemical assays, to triage the initial hits, to classify compounds based on mode of action, and to derive structure-activity relationships (SARs) of candidate effectors. SAR and protein structural information will be used in further chemical development to improve the potency and selectivity of the compounds. Cell-based assays will then be applied as the first step in utilizations of the verified inhibitors, examining their effects on cancer cell survival and sensitivity to radiation and chemotherapeutics. This project is based on extensive preliminary studies carried out by the applicants in collaboration with the NCGC. A fluorescence-based primary assay for helicase activity has been optimized for the 1536-well screening format. The screen has been validated against a 1280-compound library (LOPAC), and showed acceptable reproducibility, stability, and Zfactor. Significantly, the screen yielded only a handful of significant hits, indicating that the assay is not dominated by false-positives. Three secondary assays, all adopted for high throughput, have been implemented for verification and classification: ATP binding, DNA binding (both by fluorescence polarization), and protein stabilization (thermal denaturation). Standard biochemical assays and a battery of cell-based assays have been used to evaluate the effects of inhibition of RECQ1 and BLM expression, so provide the appropriate benchmarks for specific effects of chemical inhibition.
Discovery of inhibitors of anti-apoptotic protein A1
GOLUB, TODD R.
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
1 R03 DA028853-01
12
DESCRIPTION (provided by applicant): Dysregulated apoptotic mechanisms are central to the pathogenesis and maintenance of cancer, and are major barriers to effective treatment. The BCL2 protein family, comprised of both pro- and anti-apoptotic members, controls the activation of downstream caspases, which are the major effectors of apoptosis. Thus developing small molecule probes that selectively target each anti-apoptotic protein family member may have relevance for both basic research and future clinical applications. We propose to screen for inhibitors of the anti-apoptotic protein A1. We have discovered that A1 is essential for cell survival in distinct subsets of cancer, including diffuse large B-cell lymphoma and melanoma. To our knowledge no small molecule inhibitor of A1 has been developed. We propose to collaborate with the MLPCN to employ a novel, validated high-throughput cell- based screen to identify inhibitors of A1. This cell-based screen presents A1 in its native state and allows us to discover compounds that can potentially modulate A1 protein function through multiple modes of binding. These include interaction at the conserved hydrophobic BH3 binding groove and thereby disrupt the interaction between A1 and its BH3-only protein partners (see Figure 1 and Section B) and also at modulatory allosteric sites of the A1 protein not necessarily conserved among BCL2 family proteins. Thus compared to conventional in vitro biochemical assays focusing on identifying compounds that bind to the conserved hydrophobic BH3-binding groove of anti-apoptotic proteins, our unique assay may broaden the spectrum of hit selection, increase the specificity among BCL2 family proteins, and will identify such hits in a physiologically relevant context. Following the primary screen, we will perform several validated secondary assays to identify compounds that act on A1-specific mechanisms. Data from those assays will be used to chemically optimize the hit compounds. These A1 inhibitors will serve as tools to further dissect the role of A1 in cancer. We anticipate that the proposed MLPCN collaboration will identify anti-A1 probe compounds that will greatly accelerate the study of apoptosis mechanisms in cancer.
Elucidation of physiology of non-replicating, drug-tolerant Mycobacterium tubercu
PI:
HUNG, DEBORAH T
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
1 R03 MH087444-01
<
12
DESCRIPTION (provided by applicant): While anti-tuberculosis drugs can kill the culprit Mycobacterium tuberculosis (TB) bacilli in vitro, axenic culture in a few days, treatment of an infected human host takes on the order of 6 months. Treating clinically latent forms of TB requires even longer periods of time, ~ 9 months. The difference between the in vitro and in vivo requirement is thought to be due to TB bacilli existing in a different physiologic state in vivo that is potentially “drug tolerant” due to the assumption of a metabolically dormant, non-replicating state (1). Understanding what functions are essential in these dormant bacilli and the mechanisms by which they switch between non-replicating and replicating states would be invaluable to the development of more effective, potentially shorter course TB therapy. Because of the current limitations in TB genetics resulting in the inadequate ability to study essential gene functions, the identification and use of small molecule probes of the physiology of non-replicating bacteria will play an incredibly powerful role in the elucidation of this biology. The goal of this project is to develop potent, soluble small molecule probes that modulate survival of nonreplicating TB bacilli, with good selectivity for TB over host cell types. These probes will be used to elucidate the physiology of non-replicating TB and to understand the functions that regulate the generation and maintenance of this state.
Discovery of potent, and selective allosteric inhibitors of GSK-3b
PAN, JEN
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
1 R03 MH087442-01
12
DESCRIPTION (provided by applicant): The serine/threonine kinase glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for multiple cellular pathways that include insulin signaling and glycogen synthesis, neurotrophic factor signaling, and Wnt signaling. Consequently, this enzyme has a critical role in metabolism, transcription, development, and neuronal functions and has been implicated in multiple human disorders including Alzheimer’s disease, bipolar disorder, noninsulindependent diabetes mellitus, cardiac hypertrophy, and cancer. Precisely how GSK-3b maintains its pathway specificity efficiently at the crossroads of many cellular processes is unclear. This regulation may involve allosteric sites within distinct structural domains of this complex kinase. The majority of the existing chemical inhibitors compete for the ATP-binding site of GSK-3b and inhibit additional kinases, while the two known structural series of ATP-noncompetitive inhibitors have low potency and suboptimal pharmacological properties that limit their use. Small molecules targeting the allosteric sites of GSK-3b could have the potential to provide highly specific GSK-3b inhibitors that may help elucidate GSK-3b function and regulation in distinct cellular pathways. Hence we propose a primary screen and a cascade of secondary biochemical and cellular assays to identify ATP-non competitive, allosteric inhibitors of GSK-3b. The anticipated high specificity of such probes could lead to the selective modulation of distinct cellular pathways dependent on GSK-3b. This probe development plan includes: 1) A primary kinase activity assay that will identify ATP-competitive as well as ATP- noncompetitive inhibitors that affect GSK-3b kinase activity. 2) A secondary kinase activity assay based on time resolved fluorescence (HTRF) detection of ADP that will distinguish allosteric modulators and ATPcompetitive inhibitors. 3) Binding-based assays that will more precisely define a compound’s mode of action. 4) Importantly, we have established a number of cellular assays that will help differentiate allosteric inhibitors and identify pathway-specific GSK-3b inhibitors. Ultimately, we plan to optimize the pharmacokinetic properties of pathway-specific, allosteric GSK-3b inhibitors, and test them in the established rodent models of mood and memory.
Intein inhibitors as potential TB drugs
PAULUS, HENRY
BOSTON BIOMEDICAL RESEARCH INSTITUTE
1 R03 MH087438-01
12
DESCRIPTION (provided by applicant): The alarming world-wide increase in multidrug-resistant and extensively drug-resistant tuberculosis calls for new antimycobacterial drugs against new types of targets that are less likely to undergo mutations that lead to drug-resistance. The discovery that three genes of M. tuberculosis, recA, dnaB and sufB, are interrupted by inteins, which must be excised from inactive precursor proteins by the process of protein splicing to yield functional proteins, suggests protein splicing as a novel target for anti-TB drugs. Since two of these genes (dnaB and sufB) are essential for growth and the third (recA) predisposes to drug-resistance mutations, protein splicing inhibitors, which disable two separate vital functions and at the same time suppress mutation rates, would be unlikely to elicit drug-resistance. A robust, protein- based high-throughput screening system for protein splicing inhibitors has been developed and used for screening commercial small-molecule compound libraries, leading to nearly 60 confirmed positives with IC50 values in the low micromolar range. Most of the confirmed protein splicing inhibitors were electrophiles that inhibit protein splicing irreversibly by binding to its catalytic cysteine, but a few non-covalent inhibitors were also found. The same assay will be used to screen the MLPCN library, and the hits obtained will be reviewed for compound classes based on the likelihood of covalent modification. The hits will then be re-screened in the presence of a large excess of a thiol, which is known to abrogate the inhibition of protein splicing by the thiol-reactive compounds in order to discriminate between covalent and non- covalent inhibitors. Confirmed positives of both inhibitor classes will be screened for the inhibition of the growth of virulent strains of M. tuberculosis and for cytotoxicity using animal cells to identify candidates for lead optimization and further testing as antimycobacterial drugs.
Reverse the Epigenetic Suppression of BRM through High Throughput Screening
REISMAN, DAVID N
UNIVERSITY OF MICHIGAN AT ANN ARBOR
1 R03 DA028854-01
12
DESCRIPTION (provided by applicant): The long-term goal of this translational research program is to develop drugs that target the process of tumorigenesis in cancers Epigenetic silencing of gene expression is used by cells to regulate differentiation and development. During cancer development, epigenetic silencing can be aberrantly activated, causing key cellular genes to be inappropriately silenced. These genes are not irreversibly altered, however. Identifying compounds that specifically reverse this process and restore expression of silenced genes is an important strategy for developing targeted gene therapies for cancer. We have found that BRM, a subunit of the essential chromatin remodeling complex SWI/SNF, is not mutated but epigenetically silenced. BRM loss occurs in 20- 30% of tumor types related to secondhand smoke, including lung, colon, and bladder cancers. In BRMdeficient cell lines, BRM expression can be restored with the small molecule inhibitors butyrate or trichostatin. Significantly, however, currently available small molecular inhibitors, including those in our studies, are broad- acting, affecting many proteins. These compounds thus interfere with required intracellular processes, contributing to their side-effect profile and diminishing their utility as clinical agents. Indeed, we have found that a variety of these inhibitors, while effective in restoring BRM expression, interfere with multiple other aspects of SWI/SNF function. Their lack of specificity hence negates much of their potential anticancer effects by blocking the beneficial effects of restoring the expression of BRM and other such proteins. To remedy this drawback, we propose to identify novel compounds using high-throughput drug screening that can target and selectively antagonize the mechanism underlying the aberrant silencing of BRM. We will also identify the site of action of each of these compounds. Such compounds will not only be clinically beneficial for cancer patients by restoring BRM, but they will also likely reverse the silencing of many other important anticancer genes suppressed by this mechanism. We have a team that is uniquely poised to accomplish this research: a clinical scientist with expertise in oncology, translational researchers with experience in drug development, an expert medicinal chemist, and the Life Science Institute drug screen facility at the University of Michigan as well as the Michigan High Throughput Center. Our work in developing compounds that restore BRM is an important first step in translating basic science discoveries into therapies that can directly benefit patients-and thus has strong potential for benefiting patients with cancers related to tobacco smoke exposure.
A Nonisotopic HTS Assay to Elucidate Choline Transporter (CHT) Modulators
RUGGIERO, ALICIA M
VANDERBILT UNIVERSITY
1 R03 DA028852-01
12
DESCRIPTION (provided by applicant): In the brain, the chemical acetylcholine (ACh) exerts powerful modulatory control over arousal, motor and cognitive circuits, and has been found to be deficient in Alzheimer’s Disease (AD). The current drugs available to positively impact cognitive deficits in Alzheimer’s Disease (AD) and other dementias are the cholinesterase inhibitors. These prevent the breakdown of the neurotransmitter acetylcholine (ACh), and thus augment Ach function. Due to the limited utility of the cholinesterase inhibitors, alternative therapies to augment ACh deficits are critical in our aging population. Another vital protein, the hemicholinium-3 sensitive choline transporter (CHT) is believed to be responsible for the efficient uptake of choline by neurons to allow for ACh synthesis. We have developed an assay system for high throughput screening to identify compounds with high selectivity for CHT. It is anticipated that these compounds may lead to future cholinergic therapies in AD, and multiple other CNS diseases regulated by cholinergic signaling. Our research aims are to implement a high throughput screen for discovery of novel enhancers of high-affinity choline transporter uptake as mediated by CHT using voltage-sensitive fluorescent measurements; to perform secondary assays aimed at identifying novel modulators that act selectively at the choline transporter (CHT); and to use database mining and chemical synthesis to optimize HTS hits for use in biological assays. Through the development of high-throughput screening (HTS) methodologies that allow for the screening of large chemical libraries, we seek to identify CHT activators and allosteric modulators. These compounds may be able to enhance choline uptake and increase the levels of ACh produced in the neuron. Such reagents would allow greater ACh release from viable neurons not lost in the disease process and could offer advantages over cholinesterase therapy in targeting precursors rather than ACh itself.
Inhibitors of MBNL1 – poly(CUG)binding
THORNTON, CHARLES A
UNIVERSITY OF ROCHESTER
1 R03 MH087421-01
12
DESCRIPTION (provided by applicant): Myotonic dystrophy type 1 (DM1) is a dominantly inherited disorder that causes progressive disability and premature death. DM1 is the most common form of muscular dystrophy in most referral centers in North America and Europe. No treatment that prevents or slows the progression of DM1 has been identified. The genetic basis of DM1 is an expansion of CTG repeats in the 3′ untranslated region of DMPK, a gene encoding a protein kinase. Because the expanded repeat is unstable in germline and somatic cells, and tends to grow larger over time, most individuals with DM1 have several thousand CTG repeats at the DM1 locus in skeletal and cardiac muscle cells. These very large repeat expansions give rise to an unusual RNA dominant disease process in which transcripts from the mutant allele, which contain an expanded CUG repeat (CUGexp), accumulate in the nucleus and interfere with RNA processing for a specific group of genes, thus generating symptoms of DM1. The mechanism for this RNA gain-of-function involves, at least in part, the sequestration by CUGexp RNA of splicing factors in the Muscle blind-like (MBNL) family. MBNL proteins bind to expanded CUGexp RNA with high affinity, forming high molecular weight complexes in the nucleus (ribonuclear foci), and resulting in loss of MBNL function. Recently we have found that oligonucleotide inhibitors of CUGexp-MBNL binding have beneficial effects in a transgenic mouse model of DM1, causing dispersal of ribonuclear foci, restoration of MBNL activity, and improvement of the phenotype. As a first step to developing treatments for DM1, here we propose high throughput screening against the MLSCN’s compound collection to identify small molecules that inhibit CUG repeat RNA -MBNL protein binding.
Screening for Inhibitors of DNA Replication in Gram-Positive Bacteria
DALLMANN, H. GARRY
UNIVERSITY OF COLORADO AT BOULDER
1 R03 MH089672-01
13
DESCRIPTION (provided by applicant): Using a model Gram (+) organism, Bacillus subtilis, we propose to transfer to the MLPCN, a robust high throughput screening assay targeting the DNA Polymerase III holoenzyme. Small molecules discovered via screening of this target will be prioritized using appropriate biochemical specificity and microbiological activity assays to select inhibitors that have the potential to be developed into antibacterial and step-specific perturbants of DNA replication pathways. This model organism is closely related to most common Gram (+) human pathogens such as S. aureus, S. pyogenes and the biodefense category A organism, Bacillus anthracis. Over the last three decades only 2 new chemical classes of antibiotics have been approved by the FDA and it is widely recognized that bacterial resistance to existing classes of antibiotics is increasing. Presently there are no antibacterial targeting the essential process of DNA replication in bacteria. Bacterial DNA replication is performed by the cellular replicase, DNA polymerase III holoenzyme that has the processivity to replicate the entire chromosome without dissociation. This process employs at least 5 different essential proteins and enzymatic activities. These protein targets and the essential interactions that occur between them provide attractive targets for the development of antibacterial, and will also serve as an ideal system for developing chemical genetic approaches to perturb the various interactions and reaction stages.
HTP chemical genomic screens to identify positive regulators of the Unfolded Prot
KAUFMAN, RANDAL J.
UNIVERSITY OF MICHIGAN AT ANN ARBOR
1 R03 MH089782-01
13
DESCRIPTION (provided by applicant): Tremendous advances in our understanding of pathologic mechanisms have recently revealed that defective protein processing within the secretory pathway is an integral component of many genetic and environmental diseases. Diverse disease states ranging from diabetes, Alzheimer’s disease, and Parkinson’s disease, to hemophilia and lysosomal storage diseases have all been characterized by folding defects or impaired transport from the endoplasmic reticulum (ER). Very importantly, it has recently come to light that deregulation of protein synthesis is likely a key component in the pathogenic metastasis of a variety human cancers. When misfolded protein accumulates in the ER lumen, the cell activates the Unfolded Protein Response (UPR) to clear the malfolded proteins and restore homeostatic protein processing. When a stress is prolonged or robust, the UPR employs a genetic pathway that results in cell death. These proposed studies are focused to identify and characterize drug-like small molecule regulators of these two distinct arms of the UPR. We will investigate the hypotheses that: 1) Activation of the IRE1-XBP1 (adaptive) arm of the UPR will facilitate a prolonged recovery opportunity in pathologies where cells are burdened with an accumulation of mis-folded or poorly secreted proteins; and 2) Specific and robust activation of the PERK-eIF21-CHOP (apoptotic) arm will exacerbate the UPR and lead to cell death in malignant cells with hyper-active secretory machinery. In our preliminary studies, we have successfully screened a 66,000 compound library with Chinese Hamster Ovary (CHO) cell lines stably expressing luciferase constructs that report individually on the IRE1-XBP1 or PERK-eIF21-CHOP arms of the UPR. Having worked with a team of expert Medicinal, Organic/Synthetic and Computational Chemists we have identified some hits that productively modulate the UPR. We now propose to expand our probe database by repeating the screen with libraries of greater chemical diversity and complexity with the goal of identifying more specific and potent UPR-regulating probes.
HTS for Filo- and Arena-virus Entry Mechanisms
DAVEY, ROBERT A
UNIVERSITY OF TEXAS MEDICAL BR GALVESTON
1 R03 MH086850-01A1
13
DESCRIPTION (provided by applicant): Most antiviral drugs in the clinic today target virus components related to viral replication. These drugs must be able to penetrate into the cell and be of sufficient potency to stop an already active infection. Recently, the step of virus entry has begun to be exploited for drug discovery. Virus entry is the first and committal step toward establishing a virus infection. Compounds that interfere with entry can affect binding of virus to receptor(s), uptake of virus into endosomes and finally membrane fusion of virus and endosomal membranes. Each of these steps can be targeted by drugs that do not need to permeate the cell membrane as each occurs at the cell surface or within endosomes that contain fluid sampled from outside of the cell. Aside from those against HIV and Influenza A, few other antiviral drugs have been identified that target entry. In this project we aim to identify and characterize compounds that inhibit the entry of filoviruses and arenaviruses. Each family of viruses cause hemorrhagic fever and are classified as NIH category 1 biothreat and emerging infectious agents. We will use qHTS at NCGC to identify compounds that inhibit infection of Marburg (a filovirus related to Ebola virus) and Lassa Fever virus (an arenavirus). For both families little is known about the entry mechanism and no useful drugs are available for therapy. We have developed a robust assay platform for identification of virus entry inhibitors for both. The system is based on virus pseudotypes and so can be used at BSL2. Screens can be safely performed at BSL2 and the assay has been optimized at NCGC to give signal-to-noise ratios >25-fold and Z’ of >0.5 in the 1536-well format. Readout is by firefly luciferase and shows good dose-response linearity of signal. We will also perform a set of secondary assays designed to identify the step (binding, uptake and membrane fusion) at which virus entry was blocked. These assays have been previously published by our lab for both virus types. Each compound will also be tested for efficacy against wild type virus. We will then have the opportunity to identify agent- specific or broad-spectrum antiviral compounds. Future work will involve development of the best compounds into potentially useful drugs and use of each as probes to better understand the mechanism of virus entry for each virus and the pathways taken to infect the cell.
Screen for inhibitors of STK-33 kinase activity
GOULD, ROBERT
BROAD INSTITUTE, INC.
1 R03 MH089854-01
13
DESCRIPTION (provided by applicant): Mutations in the RAS family of small GTPases are responsible for approximately 30% of human tumors. Despite intense activity over the last 25 years, the identification of direct inhibitors of RAS has proven challenging, necessitating alternative approaches to modulating RAS activity. One such approach takes advantage of the apparent dependency of mutant RAS-dependent cells on non-oncogenic cellular factors. For instance, the serine/threonine kinase, STK33, has recently been shown by RNAi techniques to be required for the survival and proliferation of mutant KRAS-dependent cancer cells, but not KRAS-independent cells. These results indicate a codependency between mutant KRAS and STK33 that results in a synthetic lethal interaction upon suppression of STK33. The overall goal of this proposal is to identify a set of direct STK33 kinase activity inhibitors that can recapitulate the exquisite cell-based specificity of RNAi for inducing apoptosis in mutant KRAS-dependent cells without discernable effects on KRAS-independent cells. The specific aims of this proposal are to (1) identify STK33 kinase activity inhibitors in a robust, sensitive, previously validated primary screen, (2) utilize a panel of secondary cell-based assays to select for those STK33 inhibitors that specifically target mutant KRAS-dependent cells, and (3) implement a critical path to facilitate probe optimization and development. The longer term objective of this research program is to utilize these STK33- specific probes in conjunction with a comprehensive proteomics approach to elucidate the molecular basis of RAS-dependent regulation and mutant KRAS-dependent tumorigenesis with the goal of translating this knowledge into a therapeutic opportunity.
Identification of broad-spectrum antifungal efflux pump inhibitors
CANNON, RICHARD D
UNIVERSITY OF OTAGO
1 R03 MH087406-01A1
13
DESCRIPTION (provided by applicant): Fungal infections, exemplified by oral and invasive candidiasis, cause considerable morbidity and mortality in an ever-increasing human population: the immunocompromised. Treatment of patients with fungal infections is severely hampered by the development of antifungal drug resistance. The goal of our research is to address this health need by discovering novel therapeutic agents that overcome clinical drug resistance by inhibiting a major cause of antifungal resistance, the drug efflux pump molecules in the fungal cell membrane. Lead compounds will be identified by high throughput screening (HTS) of the Molecular Libraries Small Molecule Repository (MLSMR) using a multiplex assay system developed by Professor Sklar at the University of New Mexico Center for Molecular Discovery (UNMCMD) and a panel of strains of the model yeast Saccharomyces cerevisiae, developed in our laboratory. The strains express a range of individual fungal efflux pumps that will be the targets for the proposed HTS. The specific aims are to: 1. Undertake a primary screen of the MLSMR using a multiplex flow cytometry assay: Proof of principle preliminary experiments has indicated that the assay will be applicable to the panel of yeast strains expressing efflux pumps from the following clinically relevant fungi: Candida albicans, Candida glabrata, Candida krusei and Cryptococcus neoformans. The yeast cell-based multiplex approach developed involves the use of three ‘sentinel’ strains in an HTS which measures efflux of the fluorescent pump substrate Nile Red and follow-up microtitre fluorescent assays with the other panel strains to identify pump inhibitors. 2. Screen the hits obtained with a panel of three secondary screens: The hits obtained in the primary screen will confirmed using secondary screens previously developed in our laboratory. The secondary assays will confirm that the targets are fungal efflux pumps, the extent of inhibitory activity, and elucidate the nature of the inhibitor/target interaction. The compounds identified will guide further research to develop drugs that overcome antifungal resistance.
A high-throughput in vivo screen for small molecule inhibitors of SUMOylation
PRELICH, GREGORY
YESHIVA UNIVERSITY
1 R03 MH089779-01
13
DESCRIPTION (provided by applicant): Post-translational modification of proteins by ubiquitin and ubiquitin family members such as SUMO (Small Ubiquitin-like Modifier) are highly conserved processes in eukaryotes that are essential for cell viability. The roles of ubiquitin in signaling and in targeting proteins for proteosome-mediated degradation have been well-documented. SUMO is not as thoroughly understood as ubiquitin, largely due to the lack of small molecule inhibitors of the pathway. The goal of this proposal is to identify small molecules that inhibit the SUMOylation pathway. This will be accomplished in two stages: (1) by performing a large-scale chemical genetic screen for compounds that inhibit the SUMO pathway in vivo, and (2) by performing secondary assays on the hits from the primary chemical genetic screen to identify those molecules that directly inhibit SUMOylation. Inhibitors identified through this project will serve as reagents for studying the varied roles of SUMO in vivo, with strong potential for broader application in elucidating the roles of SUMO in human health and disease.
Cell-based Screen for RGS Modulators
NEUBIG, RICHARD R
UNIVERSITY OF MICHIGAN AT ANN ARBOR
1 R03 MH087441-01A1
13
DESCRIPTION (provided by applicant): Signal transduction processes are major targets of drug discovery with G protein-coupled receptors being a primary site of action of many current therapeutic agents. Recent work, however, has shown that signaling pathways are not just linear chains of information but are webs of interacting regulatory molecules in which protein scaffolding, intracellular proximity, and inhibitory control are major determinants of signaling efficacy and specificity. The twenty Regulator of G protein Signaling (RGS) protein family members which inhibit G protein signaling represent a novel site of pharmacologic intervention but: 1) their physiological functions remain incompletely understood and 2) there are no reported small molecule inhibitors of RGS function. The identification of selective RGS inhibitors would provide both: 1) tools for the study of RGS function in cells and in vivo and 2) a starting point for therapeutic drug development. We have recently developed a robust cell- based functional assay for assessing RGS4 activity. This will be utilized in highthroughput screening in the MLPCN to identify compounds that selectively inhibit the activity of RGS4.The ultimate aim of this project is the identification of selective small molecule modulators of RGS action. This will provide important chemical tools and accelerate the development of novel therapeutics.
High-throughput screening for small molecules with specific toxicity for breast c
LANDER, ERIC S
BROAD INSTITUTE, INC.
1 R03 MH089663-01
13
DESCRIPTION (provided by applicant): There is an important unmet need to identify small-molecules that can target breast cancer stem cells (CSCs). CSCs are responsible for driving tumor growth and metastasis. CSCs are also known to be resistant to most conventional therapies, including chemo- and radiation-therapy, clearly underscoring the need for therapies that target CSCs. The objective of the proposed study is to identify small-molecule probes that are selectively toxic to breast CSCs. Such molecules would be immensely useful to probe the biology of CSCs and would also serve as promising therapeutic drug candidates. We propose to screen for small molecules that target breast CSCs using a novel method we have developed that takes advantage of a recently discovered connection between breast CSCs and epithelial-mesenchymal transdifferentiation. In preliminary experiments, we have conducted a proof-of-principle pilot high-throughput screen that successfully led to the identification of a novel compound with specific toxicity for breast CSCs. The screen assay is robust, sensitive, and straightforward. Importantly, the secondary assay for the screen uses a cell-line reagent that is essentially isogenic with the cell line-reagent used from the primary screening assay. This minimizes the likelihood of find spurious compound hits unrelated to the process of interest, thereby providing a very useful advantage not present in most screens. The success of the pilot screen gives a very high degree of confidence that the proposed screen, if funded, would provide useful reagents for basic research and potential therapies targeted breast CSCs.
HTS for Enhancer of Glycosylation of Alpha Dystroglycan
WU, XIAOHUA
CAROLINAS MEDICAL CENTER
1 R03 MH085693-01A1
13
DESCRIPTION (provided by applicant): The long term goal of our research is to discover effective treatments for muscular dystrophies. A number of muscular dystrophies (MD) are associated with abnormal O-mannosylation of alpha dystroglycan (1-DG) that plays a central role in linking the extracellular matrix (ECM) to the actin-based cytoskeleton in muscle. Hypoglycosylation of 1-DG impairs its ability to bind to the ECM proteins such as laminins and results in pathogenesis in the muscle and nerve systems and often causes devastating diseases in humans. Currently, there is no specific treatment for any form of MD. Increasing evidence suggests that over-expression of glycosyltransferases enhancing the glycosylation of 1-DG can restore the functions of 1-DG in MD mouse models. We hypothesize that cells can enhance glycosylation of 1-DG in response to certain chemical stimuli. We recently developed a cell-based assay for determination of O-mannosylation of alpha-DG (ADOMA). With ADOMA, we are able to quantitatively monitor the levels of O-mannosylation of 1-DG and its functional binding to the ligands, laminins, on the cell surface with remarkable reliability and reproducibility. This assay has great potential to become a high throughput screening (HTS) platform. A pilot screening of 2000 bioactive compounds with ADOMA has been conducted to test our hypothesis and the performance of ADOMA under HTS conditions. The result has shown that the performance of the pilot screen is acceptable for HTS. Furthermore, we have identified several positive hits which are able to enhance the functional binding of laminins to the cell surface, thus proved our hypothesis. However, the current ADOMA screening procedure requires multiple washing steps and need to be reconfigured to HTS friendly format for large scale HTS campaign. In this project, thus, we will focus on the specific aim: Reconfiguration of ADOMA into a homogeneous, miniaturizable, and wash-free HTS assay. We plan to integrate homogeneous time-resolved fluorescence (HTRF) methodology into our HTS assay to develop HTRF based HTS assay.
Small Molecule Agonsists for the Neurotensin 1 Receptor
BARAK, LAWRENCE S
DUKE UNIVERSITY
1 R03 MH089653-01
13
DESCRIPTION (provided by applicant): This application addresses an important area of interest to the National Institute on Drug Abuse (NIDA): The utilization of molecular targets for the treatment of drug addiction disorders. The specific aims of the proposal are: 1. To identify small molecule agonists of the Neurotensin 1 Receptor (NTR1) using a -arrestin high throughput, high content primary assay, and confirming hits using a variety of secondary assays. 2. To optimize two to three distinct lead compounds for NTR1 agonist activity via aim 1. This proposal will identify novel small molecule neurotensin receptor agonists that form the foundation for developing drugs to treat and prevent methamphetamine abuse. It addresses an immediate goal of the National Institute on Drug Abuse that new approaches are needed for treating methamphetamine addiction (NIDA Research Report Series, 2006). We propose to identify novel small molecule neurotensin receptor agonists by high content screening of large libraries of compounds using a primary assay that is cell based and exploit the ability of an arrestin-green fluorescent protein reporter to directly recognize the activated state of the NTR1. Lead optimized NTR1 agonists from within this discovery program will be applicable for translational studies aimed towards the development of novel medical therapies to treat amphetamine abuse.
HTS of Rab9 promoter activators
IOANNOU, YIANNIS A
MOUNT SINAI SCHOOL OF MEDICINE OF NYU
1 R03 MH089537-01
13
DESCRIPTION (provided by applicant): Treatment modalities for lysosomal stage diseases (LSDs) with neuropathology such as Niemann-Pick Type C Disease (NPC) are currently non-existent due to the severe obstacles associated with accessing the central nervous system with proteins or genes. Also, these rare orphan disorders do not attract the interest of pharmaceutical companies, further contributing to the lack of prospects for any form of treatment of therpy. We have developed a new paradigm, termed “Orphan Receptor Bypass Therapy” (ORByT), to address these disorders. This paradigm posits the existence of endogenous “suppressor” proteins whose expression can dramatically improve LSD pathogenesis. The goal is the identification/discovery of small chemical compounds that can modulate the expression of these proteins and provide a new treatment modality for these devastating disorders. We have previously shown that the small GTPase protein Rab9 meets this requirement and can act as suppressor of the NPC phenotype. Thus, we propose to first carry out: 1) High throughput screening for identification of small molecules that upregulate Rab9 expression, utilizing the MLSCN (Molecular Libraries Screening Center Network) compound collection for druglike small chemical molecules, and 2) Characterize and confirm positive hits, using in vitro assay, for their therapeutic potential in the treatment of NPC1 disease, and 3) These studies will result in the identification of candidate compounds that will ultimately be evaluated in vivo in an NPC mouse model to determine their therapeutic potential and future drug development.
APOBEC3G DNA Deaminase Inhibitor High Throughput Screen
HARRIS, REUBEN S
UNIVERSITY OF MINNESOTA TWIN CITIES
1 R03 MH089432-01
13
DESCRIPTION (provided by applicant): The human APOBEC3G (A3G) protein is a DNA deaminase that has potent activity against HIV-1 and a variety of other retroelements. Although HIV encodes a natural inhibitor of A3G called Vif, we hypothesize that the virus still benefits from the mutagenic activity of A3G (immune escape and drug resistance). As an essential step toward testing this hypothesis in vivo, we have adapted, optimized and miniaturized a fluorescence-based DNA cytidine deaminase assay to high throughput format. Preliminary studies have identified 2 candidate inhibitors in the 1280 compound LOPAC library. Here, we propose two specific aims. First, we will collaborate with an NIH Molecular Libraries Probe Production Center in a full high-throughput screen for additional A3G activity inhibitors. Second, we will implement a variety of secondary screens to determine the specificity of the lead actives. We anticipate that the successful completion of the proposed studies will result in the identification and validation of approximately 50 active DNA deaminase inhibitors. A subset of these small molecules will undoubtedly become tools for many future experiments and further development.
Identification of ERalpha/ERbeta Heterodimer-specific Ligands by HTS
XU, WEI
UNIVERSITY OF WISCONSIN MADISON
1 R03 MH089442-01
13
DESCRIPTION (provided by applicant): One out of every eight women in the United States will develop breast cancer during her lifetime. The biological actions of estrogens are transduced by its binding to two estrogen receptors (ERs), ERa and ERb, which have a “Ying and Yang” relationship in regulating estrogen action (i.e., ERa promotes while ERb inhibits estrogen-dependent cell growth). ERb is thought to counteract the stimulatory effects of ERa through heterodimerization of the two receptors, and these heterodimers have been proposed to regulate sets of genes distinct from those regulated by either homodimer. However, the mechanism by which ERb functions in concert with ERa as a heterodimer in breast cancer has not been studied in detail due to the lack of specific ERa/b heterodimer ligands. We have established highly robust and reproducible Bioluminescent Resonance Energy Transfer (BRET) assays which can distinguish ligands with ability to induce ERa homodimers, ERb homodimers and ERa/b heterodimers. This proposal outlines three specific aims that focus on high throughput screening (HTS) for identification of ERa/b heterodimer specific ligands. In Aim 1, we take advantage of an ERE-reporter stably integrated cell line that is amenable to HTS. This T47D-KBLuc cell line expresses both ERa and ERb, making it feasible to identify compounds transactivating all forms of ER dimers including ERa and ERb homodimers as well as ERa/b heterodimers. In Aim 2, estrogenic compounds identified from this primary screening will be subjected to the HTS using BRET. In Aim 3, the identified hits will be tested for their abilities to inhibit growth of breast cancer cells co- expressing ERa and ERb. Development of a novel system by which ERa/b heterodimers may be studied will provide a means for direct evidence for heterodimer formation in vivo. Furthermore, these tools will allow preliminary exploration of heterodimer function in breast cancer via cell-based assays.
HTS for Identifying Potential Drug Treatment of Ataxia-Telangiectasia
ZHENG, WEI MB
U.S. NATIONAL HUMAN GENOME RESEARCH INST
1 R03 MH089030-01
13
DESCRIPTION (provided by applicant): Ataxia-telangiectasia (A-T) is a rare inherited disease that affects several body systems and causes severe disability. The disease is characterized by selective, progressive neurodegeneration leading to severe motor dysfunction, immunodeficiency, cancer predisposition, genomic instability, and radiation sensitivity. Patient life expectancy is 20-25 years. Currently, there is no cure for A-T. It is caused by null mutations in the ATM gene, which encodes the nuclear protein kinase ATM, the primary transducer of cellular responses to double strand breaks (DSBs) in the DNA. ATM is activated following DSB induction and in turn phosphorylates key players in the broad DSB response network. We propose to conduct high-throughput screening (HTS) of small molecule collections in search of a potential drug that will slow down the relentless progression of A-T. Such drug is expected to allow cultured A-T cells to mount the ATM- mediated DSB response, even partially, despite ATM’s absence. The biological rationale of such a search is potential enhancement of protein kinases redundant to ATM. To this end we developed a cell-based assay that measures the phosphorylation of KAP-1, a major ATM target, and adapted it to the HTS AlphaScreen format. We tested this assay by screening the LOPAC collection of small molecules. The biological significance of molecules identified in the proposed screen will be examined in depth using a variety of assays.
Quantitative High-throughput discovery of human tyrosyl-DNA Phosphodiesterase
MARCHAND, CHRISTOPHE
NATIONAL CANCER INSTITUTE
1 R03 MH089814-01
13
DESCRIPTION (provided by applicant): Tdp1 is a key enzyme for the repair of topoisomerase Iinduced DNA damage. Studies from several laboratories including ours have demonstrated the importance of Tdp1 for cellular response to camptothecins in yeast, murine models and human cells with genetic Tdp1 deficiencies (SCAN1). However, there are only a small number of inhibitors. All have been discovered in our laboratory. Yet, they still lack potency and selectivity. We propose to discover novel Tdp1 inhibitors based on biochemical assays previously set up in our laboratory with recombinant Tdp1 and oligonucleotide-based substrates. To do so, we have adapted, optimized and validated our Tdp1 assays for high throughput screening in the NCGC robotic set up. We have also developed secondary and tertiary assays and set up a network of collaborators to select the most selective and mechanistically interesting chemical families.
Discovery of Small Molecule Probes for Influenza NS1A
JONSSON, COLLEEN B
UNIVERSITY OF LOUISVILLE
1 R03 MH089536-01
13
DESCRIPTION (provided by applicant): Influenza viruses are negative-sense, single-stranded RNA viruses that infect the upper and lower respiratory tracts and cause substantial morbidity and mortality annually. In the United States, approximately 36,000 deaths are attributed to influenza or its complications each year. Influenza A viruses, which also infect a wide number of avian and mammalian species, pose a considerable public health burden with epidemic and pandemic potential. Mechanisms. The proposed research effort seeks to identify small molecule inhibitors for the RNA binding activity of NS1A. The NS1A protein of influenza is a highly conserved, multifunctional viral protein which interacts with host RNA and proteins. The influenza viral protein NS1A is regarded as an important viral factor by which these viruses antagonize host cell anti-viral defense. We have developed an AlphaScreen(R) (Perkin Elmer) based 1536-well high throughput screen (HTS) for NS1A protein to identify small molecule inhibitors of the NS1A double-stranded (ds) RNA binding activity. The Z-value for the assay ranges from 0.82-0.89. Identification of probes for this activity would provide valuable tools for probing its dsRNA binding function within the cell. Theoretically, this domain could also serve as a target for antiviral drug discovery. For this effort we propose two Aims: (1) Aim 1. Assist the MLPCN in transferring the validated HTS assay for Influenza NS1A to the appropriate screening center for primary screening and provide technical support for implementation of secondary assays of hits to remove false positives and validate biological relevance of the screening hits and (2) Aim 2. Collaborate with the MLPCN Chemistry Center in analyses of the hits acquired in the NS1A HTS with tertiary assays that will be used for the purpose of hit-to-probe development and probe characterizations (e.g. selectivity, specificity, cell and tissue penetration, and mechanisms of action).
High throughput screen for inhibitors of the mdm2/mdmx interaction
WAHL, GEOFFREY MYLES
SALK INSTITUTE FOR BIOLOGICAL STUDIES
R03 MH089489-01
13
DESCRIPTION (provided by applicant): Cancer is the second leading cause of death in the United States: 1 in every 4 deaths is attributable to the disease. Genetic and molecular profiling of tumor samples has lead to the identification of proteins that may be targeted by small molecules for therapeutic benefit. One such example is the p53 tumor suppressor pathway. The p53 gene remains wild type, yet the protein is functionally inactivated in approximately 50% of all human cancers. In these cases, overexpression of mdm2 and mdmx, the major negative regulators of p53, is frequently observed. Mdm2 is an E3 ubiquitin ligase, and catalyzes the transfer of ubiquitin from E2 ubiquitin conjugating enzyme(s) to p53. This in turn leads to proteasome-dependent degradation of p53. Many drug discovery efforts have focused on disruption of the mdm2/p53 interaction in order to increase p53 activity in tumors. However, current mdm2-specific antagonists cannot disrupt the interaction of mdmx with p53. Furthermore, recent data indicate that the interaction of mdm2 with mdmx, via so-called RING domains, engenders the most effective E3 ligase activity. Therefore, the identification of small molecules that can inhibit the mdm2/mdmx interaction may provide a new approach to p53 stabilization and activation in cancer. The Specific Aims of this proposal describe a cell-based screen of the MLCPN small molecule library to discover small molecule inhibitors of the mdm2/mdmx RING-RING interaction. Additional follow-up studies are presented that will validate the specificity of ‘hits’, and their efficacy in tumor cells that express wild type p53. The successful completion of these studies would provide proof-of- principle that small molecules can inhibit the mdm2/mdmx interaction, leading to stabilization and activation of p53. The chemical modification of primary hits in this screen will generate valuable chemical probes for basic scientific research, as well as potential lead compounds that may be taken forward for drug development.
qHTS for Small Molecule Agonists and Allosteric Enhancers of Human TRH Receptor
GERSHENGORN, MARVIN C
U.S. NATIONAL INST DIABETES/DIGST/KIDNEY
1 R03 MH089816-01
13
DESCRIPTION (provided by applicant): This project aims to discover small drug-like molecules that will activate (agonists) or synergize with thyrotropin-releasing hormone (TRH) (allosteric enhancers) in activating the human TRH receptor (TRHR), a member of the 7 transmembrane-spanning receptor (7TMR) (or G protein- coupled receptor, GPCR) family of drug-accessible cell surface receptors. Many studies have linked TRH and TRHR to psychiatric disorders. For example, individuals with major depression exhibit a blunted thyrotropin (thyroid-stimulating hormone, TSH) response to TRH, absence of the nocturnal TSH surge, and decreased TRH gene expression in the hypothalamus. TRH and TRHR in the limbic system and cortex are believed to mediate the endogenous and exogenous effects of TRH on affect, mood and arousal but it has been notoriously difficult to assign specific functions and behaviors to extrahypothalamic TRH. Small molecule TRHR agonists or allosteric TRHR enhancers that are metabolically stable and able to cross the blood-brain barrier might serve as probes to elucidate the roles of TRHRs in the brains in animal models and might be valuable as lead compounds for the development of drugs to treat patients with several central nervous system (CNS) disorders. It is important to note that there are no known nonpeptidic agonists of TRHR and there are no known modulators that enhance TRH action at TRHR. To identify small ligands active at TRHR, quantitative high-throughput screening (qHTS) will be performed at the NIH Chemical Genomics Center (NCGC) using a library of over 300,000 compounds. The qHTS assay to detect agonists has been validated at the NCGC and uses fluorescent dyes to detect compound-mediated increases in intracellular free calcium in cells engineered to express TRHR. A second qHTS for allosteric enhancers will seek to identify compounds that enhance the calcium transient mediated by a suboptimal concentration (EC20) of native ligand TRH. Active compounds will be confirmed in a secondary assay measuring generation of inositol phosphate second messengers following TRHR activation. Selectivity of active compounds will be tested using several signaling assays in cell lines expressing other 7TMRs. Compounds with the highest confirmed potency and efficacy will undergo chemical modification in an effort to improve potency and/or efficacy. Chemical modifications will in part be guided by computational docking of compounds into the binding pocket of a TRHR structural model. The most effective agonists and allosteric enhancers will be used in future in vitro studies of TRHR function and as useful probes of the biology of TRHRs in the CNS by comparing their effects in intact, TRH-R1-deficient- and TRH-R2- deficient mice.
