Currently, of the >2,000 assays listed in PubChem, bioluminescence and fluorescence represent the two most prominent detection strategies. NCI is the NCI-60 cell viability assay panel.

A. The dual luciferase assay protocol is illustrated. Cells are lysed with a detection reagent containing luciferase substrates (1), FLuc luminescence is measured (2), the FLuc reaction is then stopped along with the addition of the coelenterazine substrate for RLuc (3), and luminescence is again measured (4). In this case total luminescence is measured using a clear filter; the luminescence emission spectra of FLuc and RLuc are also shown (CPS, counts per second measured on a luminometer). B. CBLuc two-color dual-luciferase assay protocol is shown in which cell lysis and detection are performed with a single reagent addition (1) and green luminescence and red luminescence are then measured sequentially on a microtiter plate reader (2) using the appropriate optical filters. The luminescence emission maximums are noted and the luminescence emission spectra for both green and red CBLuc are shown below. The dotted lines on the emission spectra represent the optical filters used in (Davis et al., 2007). C. A live cell kinetic assay is depicted using a secreted luciferase (GLuc) that uses coelenterazine as the substrate. Cell culture supernatant containing the secreted GLuc is removed at different times (1) and upon mixing with the coelenterazine substrate (2) the total luminescence for each time point is measured using a clear filter (3). Also shown is the emission spectra for GLuc relative to RLuc (spectra adapted from (Tannous et al., 2005). The GLuc wavelength emission maximum is similar to RLuc, but GLuc shows brighter luminescence.

A. The light reaction catalyzed by FLuc (i). The substrates D-luciferin (D-LH₂) and ATP are used by FLuc to form a luciferyl-adenylate intermediate (LH₂-AMP). This intermediate then undergoes nucleophilic attack by molecular oxygen, and upon subsequent displacement of AMP, an unstable dioxetanone is formed which then spontaneously breaks down to form oxylucifein, and CO₂ with the emission of a photon (Marques and Esteves da Silva, 2009). (ii) Dark reactions catalyzed by FLuc are shown in the grey shaded area. One of these involves a side-reaction in which oxidation of LH₂-AMP occurs to form the potent inhibitor L-AMP which can undergo pyrophosphorolysis or thiolysis to yield less potent inhibitors, L or L-CoA, respectively (Fontes et al., 1997). B) FLuc has also been reported to use certain fatty acids as substrates yielding fatty acyl-CoA metabolites. Kinetic constants for the synthesis of lineoic acid-CoA are taken from Oba et al. (2003). C. The potent inhibition observed for the novel 3,5-diaryloxadiazole, PTC124, is due to exploitation of a dark reaction catalyzed by FLuc in which PTC124 forms an adduct with AMP via its m-carboxylate. Also shown is the potential FLuc-catalyzed thiolysis of PTC124-AMP by CoASH to yield a metabolite, PTC124-CoA.

A. The apo structure of FLuc as determined by Conti and colleagues (Conti et al., 1996) is shown (PDB: 1LCI) depicting the secondary α and β structural elements comprising the N- and C-terminal domains. B. Overlay of the apo FLuc structure (cyan) with the DLSA bound structure of LcrLuc (gold; PDB 2D1S). Closure of the cleft between the N- and C-terminal domains is observed in the DLSA-bound structure. The active site resides in the N-terminal domain near the cleft.

A. The LcrLuc:DLSA bound structure is shown and the three conserved motifs, originally proposed by (Conti et al., 1996) as likely candidates of the active site are shown in cyan. Several invariant residues that line the active site are highlighted (residue numbering based on LcrLuc). B. Overlay of the LcrLuc:DLSA bound structure to the FLuc:PTC124-AMP bound inhibitor complex. LcrLuc:DLSA (protein in gold; ligand in green), PTC124-AMP (protein in purple; ligand in yellow). Conserved motifs are again shown in cyan. The interactions and conformations of both ligands and invariant protein regions are highly conserved between the two structures.

A. Potency distribution (pAC₅₀) for four prominent chemotypes found to inhibit FLuc from profiling efforts. Shown are benzthiazoles (i), benzimidazoles (ii), benzoxazoles (iii), and 3,5-diaryl-oxadiazoles (iv). B. Comparison of FLuc and Ultra-Glo luciferase against a 2-phenylbenzothiazole luciferase inhibitor at multiple substrate concentrations. Graphs of ATP variation (main) or D-LH₂ variation (inset) are shown and were varied at 0.25, 2, 25, and 250 μM, resulting in four sets of CRCs. In each case, the constant substrate was present at 250 μM. The FLuc data is shown as solid circles and the Ultra-Glo luciferase is shown as open circles. Simple benzthiazoles appear to be purely competitive with D-LH₂ for either luciferase as seen from the right shift of the CRCs (decrease in potency). C. Difference in potency distribution between inhibitors identified in a commercial formulation of FLuc (PK-Light™) and a commercial formulation of Ultra-Glo (KinaseGlo™), illustrating how the source of luciferase reagent can affect in vitro assay interference. D. Comparison of FLuc and Ultra-Glo luciferase inhibition potencies for quinoline analogs assayed using K_M levels of substrates. CRCs with for quinolines i (squares) and ii (circles) are shown for FLuc (solid shapes and dotted lines) and Ultra-Glo luciferase (open shapes and solid lines). Selectivity between the two luciferases is observed for such quinolines. Figures adapted from (Auld et al., 2008a; Auld et al., 2009b).

A. Level of apparent reporter activation depends on properties of the luciferase inhibitor and the assay detection protocol used. Shown is the theoretically observed activity of the reporter enzyme in the presence of a FLuc inhibitor as determined in a biochemical assay in which substrate concentrations are held near their K_M’s (orange line). Inset graph, the theoretical increase in enzyme concentration in the cell-based assay occurring as a result of inhibitor-based stabilization. Theoretical reporter activity in the cell-based assay for a fully reversible FLuc inhibitor is shown (blue line). In this case addition of excess beetle luciferase substrates in detection reagents fully relieves inhibition by the competitive inhibitor, causing apparent activation of FLuc that shows compound concentration dependence. This type of CRC can also result when a reversible inhibitor is removed by washing cells prior to detection. The theoretical activity for an irreversible FLuc inhibitor is shown for both a biochemical and cell-based assay (red line). Here, only inhibition of the reporter activity is observed in the cell-based assay. B. Observed effect for a FLuc inhibitor (PTC124) using a no-wash protocol and Steady-Glo^® detection reagent (Promega). The top graph shows FLuc activity obtained from HEK293 cells transfected with the construct pGL3 (Promega; E1741) after treatment with PTC124 for 72 hrs (orange). The pGL3 construct contains a wild-type version of FLuc under the control of a SV40 promoter. A bell-shaped CRC is observed in the cell based assay due to the persistent inhibition of FLuc at high concentrations of compound. Also shown is the activity of PTC124 against purified FLuc assayed using K_M levels of substrate (black data; bottom graph). Data is from Auld et al., (2009a) C. Experimental results for the three regioisomers shown at right, for the FLuc^UGA cell-based assay (top) and the enzyme assay at K_M levels of substrate (bottom). Data is from Auld et al., (2010). Apparent activation in the cell-based assay and potency in the biochemical assay correlate with the reactivity of the carboxylate regioisomers for MAI formation (colors correspond to the structures shown).