qHTS Assay for Inhibitors of Firefly Luciferase
To aid in the interpretation of high-throughput screening (HTS) results derived from luciferase-based assays, we used quantitative HTS, an approach that defines the concentration-response behavior of each library sample, to profile the ATP-dependent luciferase from Photinus pyralis against more than 72 000 samples. ..more
BioActive Compounds: 1571
Depositor Specified Assays
To aid in the interpretation of high-throughput screening (HTS) results derived from luciferase-based assays, we used quantitative HTS, an approach that defines the concentration-response behavior of each library sample, to profile the ATP-dependent luciferase from Photinus pyralis against more than 72 000 samples.
Luciferase (PKLight, Cambrex Corporation) was assayed for its ability to generate light using ATP and luciferin as substrates. The ATP concentration in the assay (10 uM) was within the linear range of enzyme activity for the assay conditions used.
See following reference for further details:
 Auld DS, Southall NT, Jadhav A, Johnson RL, Diller DJ, Simeonov A, Austin CP, Inglese J. Characterization of chemical libraries for luciferase inhibitory activity. J Med Chem. 2008 Apr 24;51(8):2372-86.
 Auld DS, Zhang YQ, Southall NT, Rai G, Landsman M, Maclure J, Langevin D, Thomas CJ, Austin CP, Inglese J. A Basis for Reduced Chemical Library Inhibition of Firefly Luciferase Obtained from Directed Evolution. J. Med. Chem., 2009, 52 (5), pp 1450#1458
 Auld DS, Thorne N, Nguyen DT, Inglese J. A specific mechanism for nonspecific activation in reporter-gene assays. ACS Chem Biol. 2008 Aug 15;3(8):463-70.
 Auld DS, Thorne N, Maguire WF, Inglese J. Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression. Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3585-90.
 Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11473-8.
Four uL of substrate mix (final concentration, 50 mM imidazole pH 7.2, 50 mM KCl, 7 mM MgCl2, 10 uM ATP, 0.01% Tween 20, 0.05% BSA) was dispensed into white 1536 well plates. Twenty-three nL of compound was delivered by a pin tool and 2 uL of luciferase detector mix (PKLight, Cambrex) was added. Following 8-minute incubation at ambient temperature, luminescence was measured by a ViewLux (Perkin Elmer) at 5 seconds exposure/plate. Data were normalized to the controls for basal activity (DMSO only) and 100% inhibition (77 uM resveratrol) and curve fitting was performed using Hill equation.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)