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BioAssay: AID 411

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
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 Tested Compounds
 Tested Compounds
All(71297)
 
 
Active(1571)
 
 
Inactive(69071)
 
 
Inconclusive(683)
 
 
 Tested Substances
 Tested Substances
All(72359)
 
 
Active(1577)
 
 
Inactive(70097)
 
 
Inconclusive(685)
 
 
AID: 411
Data Source: NCGC (luciferase_pklight)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-05-05
Modify Date: 2010-03-03

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1571
Related Experiments
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AIDNameTypeComment
773Counter Screen for Luciferase-based Assay PositivesConfirmatorydepositor-specified cross reference
1006Counter Screen for Luciferase-based Primary Inhibition AssaysScreeningdepositor-specified cross reference
1027Counter Screen for Luciferase-based Primary Stimulation AssaysScreeningdepositor-specified cross reference
2309Probe Summary for Inhibitors and Stabilizers of Firefly LuciferaseSummarydepositor-specified cross reference: Probe Summary for Inhibitors and Stabilizers of Firefly Luciferase
588342qHTS profiling assay for firefly luciferase inhibitor/activator using purifed enzyme and Km concentrations of substrates (counterscreen for miR-21 project)Confirmatorydepositor-specified cross reference
2228Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Presence of ATPScreeningsame project related to Summary assay
2229Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Absence of ATPScreeningsame project related to Summary assay
602357FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrateConfirmatorysame project related to Summary assay
602358FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrate and 500microM CoASHConfirmatorysame project related to Summary assay
602364FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM ATPConfirmatorysame project related to Summary assay
602365FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM D-luciferinConfirmatorysame project related to Summary assay
602474FLuc inhibitory activity for the follow-up compounds in a biochemical assay with a commercial detection reagent - BriteliteTM Plus (PerkinElmer)Confirmatorysame project related to Summary assay
602475FLuc inhibitory activity for the follow-up compounds in a biochemical assay with an in-house formulation of detection reagentConfirmatorysame project related to Summary assay
602476FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assayConfirmatorysame project related to Summary assay
602477FLuc inhibitory activity for the follow-up compounds in a cell-based assay to assess the activity of miR-21Confirmatorysame project related to Summary assay
602478FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assay (72 hour incubation)Confirmatorysame project related to Summary assay
624030Biochemical firefly luciferase enzyme assay for NPCConfirmatorysame project related to Summary assay
652016qHTS Assay for Inhibitors of Firefly Luciferase from the GSK Published Protein Kinase Inhibitor SetConfirmatorysame project related to Summary assay
Description:
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:

[1] 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.

[2] 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

[3] 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.

[4] 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.

[5] 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.
Protocol
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.
Comment
Compound Ranking:

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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0007000000 uM (0.0007μM**)% Activity at given concentration.Float%
15Activity at 0.00160 uM (0.0016μM**)% Activity at given concentration.Float%
16Activity at 0.00370 uM (0.0037μM**)% Activity at given concentration.Float%
17Activity at 0.00820 uM (0.0082μM**)% Activity at given concentration.Float%
18Activity at 0.018 uM (0.0182857μM**)% Activity at given concentration.Float%
19Activity at 0.041 uM (0.041μM**)% Activity at given concentration.Float%
20Activity at 0.091 uM (0.0914286μM**)% Activity at given concentration.Float%
21Activity at 0.204 uM (0.2045μM**)% Activity at given concentration.Float%
22Activity at 0.457 uM (0.457143μM**)% Activity at given concentration.Float%
23Activity at 1.022 uM (1.02286μM**)% Activity at given concentration.Float%
24Activity at 2.286 uM (2.28571μM**)% Activity at given concentration.Float%
25Activity at 5.111 uM (5.00079μM**)% Activity at given concentration.Float%
26Activity at 11.43 uM (11.4286μM**)% Activity at given concentration.Float%
27Activity at 25.56 uM (25.5429μM**)% Activity at given concentration.Float%
28Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
29Compound QCSource of compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: U54

Data Table (Concise)
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