The National Institute of Diabetes & Digestive & Kidney Diseases

Overview. Our primary research interests are three-fold, (i) to carry out multi-faceted studies of biologically active natural products, also known as secondary metabolites, (ii) to design peptide and protein inhibitors and probes of HIV-1 entry, and (iii) to discover and fully characterize novel carbohydrate binding proteins from understudied sources.

Natural products chemistry. We subscribe to the notion that through millions of years of evolutionary pressure to effect specific biological processes, natural products represent an ideal starting point for identifying new inhibitors of macromolecular receptors and biological processes. Ongoing projects, approaches and interests include isolation and complete structure elucidation of natural product inhibitors of mycobacterial enzymes and HIV-1 membrane fusion using multidimensional NMR and modern spectroscopic techniques; determination of mechanism of action using bioassays and biophysical techniques; determining the structural basis accounting for activity by NMR methods; and for some cases structure-guided design and synthesis of natural product mimics or analogs.

Peptide and protein inhibitors of HIV-1 entry. A simplistic but generally accepted model for the initial step of HIV infection, or HIV-1 Envelope-mediated membrane fusion, involves stepwise binding of the surface envelope glycoproteins gp120/gp41 to cellular receptors CD4 and CCR5 or CXCR4. Peptides and proteins derived from these receptors can block HIV-1 fusion, can provide valuable mechanistic probes for studying fusion events, and can elicit antibodies directed toward these molecules. Current projects include engineering stable trimeric gp41 N-helices as inhibitors and immunogens, chemical synthesis of post-translationally modified coreceptor-derived peptides and analogs, and high resolution structural studies of each.

Novel carbohydrate-binding proteins. Protein-carbohydrate interactions play a critical role in countless biological processes and recognition events ranging from sperm-egg interactions leading to fertilization, leukocyte homing during the course of inflammation, and trafficking of tumor cells during metastasis. Alternatively, the vast majority (if not all) of microorganisms and pathogens display specific glycan structures and/or carbohydrate binding proteins on their cell or membrane surfaces. A third ongoing project involves discovery of novel carbohydrate binding proteins isolated from under-studied sources such as cyanobacteria and eubacteria, and comprehensive studies of carbohydrate specificity and recognition for these proteins. This is accomplished through glycan profiling and biophysical techniques, evaluation of antimicrobial or antiviral activities and high-resolution structure determination by NMR or X-ray crystallography.

1. Louis JM, Bewley CA, Gustchina E, Aniana A, Marius Clore G Characterization and HIV-1 Fusion Inhibitory Properties of Monoclonal Fabs Obtained From a Human Non-immune Phage Library Selected Against Diverse Epitopes of the Ectodomain of HIV-1 gp41. J Mol Biol (353): 945-51, 2005. [Full Text/Abstract]

2. Williams DC Jr, Lee JY, Cai M, Bewley CA, Clore GM Crystal structures of the HIV-1 inhibitory cyanobacterial protein MVL free and bound to Man3GlcNAc2: structural basis for specificity and high-affinity binding to the core pentasaccharide from n-linked oligomannoside. J Biol Chem (280): 29269-76, 2005. [Full Text/Abstract]

3. Bewley CA, Ray S, Cohen F, Collins SK, Overman LE Inhibition of HIV-1 envelope-mediated fusion by synthetic batzelladine analogues. J Nat Prod (67): 1319-24, 2004. [Full Text/Abstract]

4. Bewley CA, Cai M, Ray S, Ghirlando R, Yamaguchi M, Muramoto K New carbohydrate specificity and HIV-1 fusion blocking activity of the cyanobacterial protein MVL: NMR, ITC and sedimentation equilibrium studies. J Mol Biol (339): 901-14, 2004. [Full Text/Abstract]

5. Fetterolf B, Bewley CA Synthesis of a bromotyrosine-derived natural product inhibitor of mycothiol-S-conjugate amidase. Bioorg Med Chem Lett (14): 3785-8, 2004. [Full Text/Abstract]

6. Louis JM Nesheiwat I Chang L Clore GM Bewley CA Covalent trimers of the internal N-terminal trimeric coiled-coil of gp41 and antibodies directed against them are potent inhibitors of HIV envelope-mediated cell fusion. J Biol Chem (278): 20278-85, 2003. [Full Text/Abstract]

7. Chang L, Whittaker NF, Bewley CA Crambescidin 826 and dehydrocrambine A: new polycyclic guanidine alkaloids from the marine sponge Monanchora sp. that inhibit HIV-1 fusion. J Nat Prod (66): 1490-4, 2003. [Full Text/Abstract]

8. Nicholas GM Eckman LL Newton GL Fahey RC Ray S Bewley CA Inhibition and kinetics of mycobacterium tuberculosis and mycobacterium smegmatis mycothiol-S-conjugate amidase by natural product inhibitors. Bioorg Med Chem (11): 601-8, 2003. [Full Text/Abstract]

9. Bewley CA Louis JM Ghirlando R Clore GM Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41. J Biol Chem (277): 14238-45, 2002. [Full Text/Abstract]

10. Kelley BS Chang LC Bewley CA Engineering an obligate domain-swapped dimer of cyanovirin-N with enhanced anti-HIV activity. J Am Chem Soc (124): 3210-1, 2002. [Full Text/Abstract]

11. Knapp S Gonzalez S Myers DS Eckman LL Bewley CA Shortcut to mycothiol analogues. Org Lett (4): 4337-9, 2002. [Full Text/Abstract]

12. Bewley CA Kiyonaka S Hamachi I Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9). J Mol Biol (322): 881-9, 2002. [Full Text/Abstract]

13. Nicholas GM Kovac P Bewley CA Total synthesis and proof of structure of mycothiol bimane. J Am Chem Soc (124): 3492-3, 2002. [Full Text/Abstract]

14. Nicholas GM Newton GL Fahey RC Bewley CA Novel bromotyrosine alkaloids: inhibitors of mycothiol S-conjugate amidase. Org Lett (3): 1543-5, 2001. [Full Text/Abstract]

15. Bewley CA Solution structure of a cyanovirin-N:Man alpha 1-2Man alpha complex: structural basis for high-affinity carbohydrate-mediated binding to gp120. Structure (Camb) (9): 931-40, 2001. [Full Text/Abstract]

16. Bewley CA Otero-Quintero S The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinity: implications for binding to the HIV envelope protein gp120. J Am Chem Soc (123): 3892-902, 2001. [Full Text/Abstract]