Bookmark and Share
BioAssay: AID 652016

qHTS Assay for Inhibitors of Firefly Luciferase from the GSK Published Protein Kinase Inhibitor Set

FLuc (firefly luciferase) is the prototypical bioluminescent reporter gene from Photinus pyralis.[1] The enzyme catalyzes the oxidation luciferin via luciferin-AMP to produce light. FLuc is used in cell-based reporter gene-based assays for screening small molecule chemical libraries [2]. However, small molecule luciferase inhibitors present in most large chemical libraries are often enriched in the output of high throughput screens using this reporter sometimes complicating interpretation and candidate selection [3]. ..more
_
   
 Tested Compounds
 Tested Compounds
All(360)
 
 
Active(35)
 
 
Inactive(222)
 
 
Inconclusive(104)
 
 
 Tested Substances
 Tested Substances
All(367)
 
 
Active(35)
 
 
Inactive(227)
 
 
Inconclusive(105)
 
 
AID: 652016
Data Source: NCGC (FLUC2000)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-02-05
Modify Date: 2013-02-12

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 35
Depositor Specified Assays
AIDNameTypeComment
2309Probe Summary for Inhibitors and Stabilizers of Firefly Luciferasesummary
Description:
FLuc (firefly luciferase) is the prototypical bioluminescent reporter gene from Photinus pyralis.[1] The enzyme catalyzes the oxidation luciferin via luciferin-AMP to produce light. FLuc is used in cell-based reporter gene-based assays for screening small molecule chemical libraries [2]. However, small molecule luciferase inhibitors present in most large chemical libraries are often enriched in the output of high throughput screens using this reporter sometimes complicating interpretation and candidate selection [3].

RLuc is a bioluminescent reporter gene luciferase from Renilla reniformis, also known as the sea pansy. RLuc uses the luciferin substrate coelenterazine. RLuc is often used in combination with the nonhomologous luciferase FLuc (firefly luciferease) as a trasfection or pathway normalization control [4]. Recently, non-homologous reporters have been described for high throughput screening 'coincidence' reporter gene strategies [5].

1. Fan F, Wood KV (2007) Bioluminescent assays for high-throughput screening. Assay Drug Dev Technol 5: 127-136.

2. Thorne N, Inglese J, Auld DS (2010) Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chem Biol 17: 646-657.

3. Thorne N, Shen M, Lea WA, Simeonov A, Lovell S, et al. (2012) Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter. Chem Biol 19: 1060-1072.

4. Michelini E, Cevenini L, Mezzanotte L, Coppa A, Roda A (2010) Cell-based assays: fuelling drug discovery. Anal Bioanal Chem 398: 227-238.

5. Cheng KC, Inglese J (2012) A coincidence reporter-gene system for high-throughput screening. Nat Methods 9: 937.
Protocol
Biochemical firefly luciferase enzyme assay
NCGC Assay Protocol Summary:

Reagents: 50mM Tris acetate, pH 7.5; 10mM Mg acetate; 10uM D-luciferin (Sigma #L9504); 10uM ATP; 0.01% Tween-20; 0.05% BSA; 10nM P. pyralis luciferase (Sigma #L9506)
Control compounds used were two known firefly luciferase inhibitors (compounds (2) and (5) in Auld et al., 2010), and DMSO.

Assay Summary:
Three microliters containing firefly luciferase substrates in buffer (final concentrations: 50mM Tris acetate, pH 7.5, 10mM Mg acetate, 0.01% Tween-20, 0.05% BSA, 10uM D-luciferin, and 10uM ATP) are dispensed into each well of a Greiner white, solid-bottom 1536-well format plate using a flying reagent dispenser (FRD). These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 7-point interplate titration of each compound to the assay plates (quantitative HTS), with final compound concentrations ranging from approximately 60uM to 300pM. One microliter of firefly luciferase in 50mM Tris-acetate buffer was then delivered by FRD to each well for a final enzyme concentration of 10nM. Luciferase activity was then measured using an Envision plate reader (PerkinElmer), with an average incubation time of 5 min.
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.
Result Definitions
Show more
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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0003244253 uM (0.000324425μM**)% Activity at given concentration.Float%
16Activity at 0.0009732759 uM (0.000973276μM**)% Activity at given concentration.Float%
17Activity at 0.00292 uM (0.00291983μM**)% Activity at given concentration.Float%
18Activity at 0.00876 uM (0.00875954μM**)% Activity at given concentration.Float%
19Activity at 0.026 uM (0.0262972μM**)% Activity at given concentration.Float%
20Activity at 0.056 uM (0.0561243μM**)% Activity at given concentration.Float%
21Activity at 0.079 uM (0.0788357μM**)% Activity at given concentration.Float%
22Activity at 0.112 uM (0.112249μM**)% Activity at given concentration.Float%
23Activity at 0.236 uM (0.236374μM**)% Activity at given concentration.Float%
24Activity at 0.449 uM (0.448994μM**)% Activity at given concentration.Float%
25Activity at 0.710 uM (0.709522μM**)% Activity at given concentration.Float%
26Activity at 0.898 uM (0.897989μM**)% Activity at given concentration.Float%
27Activity at 1.796 uM (1.79598μM**)% Activity at given concentration.Float%
28Activity at 2.129 uM (2.12857μM**)% Activity at given concentration.Float%
29Activity at 3.592 uM (3.59195μM**)% Activity at given concentration.Float%
30Activity at 6.394 uM (6.39381μM**)% Activity at given concentration.Float%
31Activity at 19.10 uM (19.0978μM**)% Activity at given concentration.Float%
32Activity at 28.74 uM (28.7356μM**)% Activity at given concentration.Float%
33Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
34Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
PageFrom: