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BioAssay: AID 602477

FLuc inhibitory activity for the follow-up compounds in a cell-based assay to assess the activity of miR-21

Firefly (Photinus pyralis) luciferase is one of the most commonly used transcriptional reporters. It is considered one of the more dynamically responsive reporters - that is, responsive to changes in reporter transcription - as it requires no post-translational modifications and is enzymatically active directly after protein synthesis (Wood, 1998). With no endogenous expression in mammalian cells and without the requirement of external excitation, it offers very little background signal, thus generally providing excellent signal to background for most assays. ..more
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 Tested Compounds
 Tested Compounds
All(151)
 
 
Active(87)
 
 
Inactive(32)
 
 
Inconclusive(32)
 
 
 Tested Substances
 Tested Substances
All(151)
 
 
Active(87)
 
 
Inactive(32)
 
 
Inconclusive(32)
 
 
AID: 602477
Data Source: NCGC (FLUC1007)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: Other
Deposit Date: 2012-03-22

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 87
Depositor Specified Assays
AIDNameTypeComment
2309Probe Summary for Inhibitors and Stabilizers of Firefly Luciferasesummarysummary AID
Description:
NCGC Assay Overview:

Firefly (Photinus pyralis) luciferase is one of the most commonly used transcriptional reporters. It is considered one of the more dynamically responsive reporters - that is, responsive to changes in reporter transcription - as it requires no post-translational modifications and is enzymatically active directly after protein synthesis (Wood, 1998). With no endogenous expression in mammalian cells and without the requirement of external excitation, it offers very little background signal, thus generally providing excellent signal to background for most assays.

Previous profiling work to determine the prevalence of compounds that affect FLuc enzymatic activity (PubChem AID 411) identified that ~3% of the MLSMR library (then at ~72K) inhibited FLuc (Auld et al., 2008). The MLSMR library was again profiled at a later date (when the library contained significantly more compounds - ~350K) for FLuc activity in a biochemical assay using purified FLuc in the presence of KM concentrations of substrates (D-LH2 and ATP), in an effort to identify compounds in the library that may act as competitive inhibitors. Of the compounds identified in this profile that inhibited FLuc, 151 compounds were selected for further analysis, in an effort to better understand their mode of inhibiting FLuc. The assay described here was to determine the activity of the compounds in a cell-based firefly luciferase reporter gene assay originally designed to identify antagonists of miR-21. Compounds that inhibit miR-21 or compounds that stabilize FLuc intracellularly are identified as active compounds in the assay.
Protocol
NCGC Assay Protocol Summary:

Reagents: In-house detection reagent: 300mM Tris-HCl, 300mM Trizma base, 300mM MgCl, 1% Triton-X100, 5mM DTT, 500uM Coenzyme A trilithium salt (Sigma #C3019),450uM ATP (Sigma #A7699), 1mM D-luciferin sodium salt monohydrate (Biosynth #L8240)
HeLa cells stably expressing firefly luciferase (FLuc) under the control of a CMV promoter (upstream) and a miR-21 binding sequence directly downstream of the gene. These cells endogenously express miR-21.
Control compounds used were two known firefly luciferase inhibitors (3-(5-(3-chlorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid and 5-(2-fluorophenyl)-3-phenyl-1,2,4-oxadiazole), and DMSO.

Assay Summary:
6uL of HeLa miR-21-FLuc cells in assay buffer (DMEM, Invitrogen #21063-029; 10% FBS, HyClone #SH30071.03) were dispensed at 2000cells/well (3.3 x 105 cells/mL) into Greiner solid-white TC-treated 1536-well plates using a Multidrop Combi (Thermo Fisher Scientific). Plates were incubated for one hour at 37 degree Celsius, 95% humidity, 5% CO2. These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 12-point titration of each compound to the assay plate, with a final compound concentration ranging from approximately ~38uM to 0.2nM. Assay plates were then incubated for 48 hours. After incubation, plates were allowed to come to RT for one hour prior to delivery of 3uL of 3X luciferase detection reagent. After a 15 minute incubation to allow for cell lysis, luciferase activity was measured using a ViewLux CCD imager (PerkinElmer), with an average exposure time of 2-12 seconds.
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 activators are ranked higher than compounds that showed apparent inhibition.
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
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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
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 0.0002162846 uM (0.000216285μM**)% Activity at given concentration.Float%
16Activity at 0.0006477581 uM (0.000647758μM**)% Activity at given concentration.Float%
17Activity at 0.00195 uM (0.00195236μM**)% Activity at given concentration.Float%
18Activity at 0.00584 uM (0.00583969μM**)% Activity at given concentration.Float%
19Activity at 0.00935 uM (0.00935405μM**)% Activity at given concentration.Float%
20Activity at 0.018 uM (0.0175191μM**)% Activity at given concentration.Float%
21Activity at 0.037 uM (0.0374162μM**)% Activity at given concentration.Float%
22Activity at 0.053 uM (0.0525572μM**)% Activity at given concentration.Float%
23Activity at 0.158 uM (0.157538μM**)% Activity at given concentration.Float%
24Activity at 0.473 uM (0.473015μM**)% Activity at given concentration.Float%
25Activity at 0.599 uM (0.598659μM**)% Activity at given concentration.Float%
26Activity at 1.419 uM (1.41904μM**)% Activity at given concentration.Float%
27Activity at 2.395 uM (2.39464μM**)% Activity at given concentration.Float%
28Activity at 4.257 uM (4.25713μM**)% Activity at given concentration.Float%
29Activity at 12.71 uM (12.712μM**)% Activity at given concentration.Float%
30Activity at 38.31 uM (38.3142μM**)% Activity at given concentration.Float%
31Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
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