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BioAssay: AID 602478

FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assay (72 hour incubation)

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(28)
 
 
Inactive(69)
 
 
Inconclusive(54)
 
 
 Tested Substances
 Tested Substances
All(151)
 
 
Active(28)
 
 
Inactive(69)
 
 
Inconclusive(54)
 
 
AID: 602478
Data Source: NCGC (FLUC1009)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: Other
Deposit Date: 2012-03-22

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 28
Related Experiments
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AIDNameTypeComment
2309Probe Summary for Inhibitors and Stabilizers of Firefly LuciferaseSummarydepositor-specified cross reference
411qHTS Assay for Inhibitors of Firefly LuciferaseConfirmatorysame project related to Summary assay
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
588342qHTS profiling assay for firefly luciferase inhibitor/activator using purifed enzyme and Km concentrations of substrates (counterscreen for miR-21 project)Confirmatorysame 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
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:
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 compounds that enchanced translational read-through of a FLuc cDNA that contained a premature stop codon (Auld et al., 2009; Auld et al., 2010). Compounds that enhance translational read-through or compounds that stabilize FLuc intracellularly are identified as active compounds in the assay.
Protocol
NCGC Assay Protocol Summary:

Reagents: Transient transfection of FlucUGA construct: TransFast Transfection Reagent (Promega #E2431); FLucUGA construct (refer to Auld et al., 2009)
#GripTite 293 MSR Cell Line (Invitrogen #R795-07)
#Complete growth media: DMEM (Invitrogen #11965), 10% HIFBS, 0.1mM MEM NEAA, 5mL/L pen-strep (of 5000U/mL stock), 600ug/mL Geneticin
#Assay media: DMEM without phenol red (Invitrogen #21063), 5% charcoal-stripped HIFBS (Hyclone #SH30071.03), 0.1mM MEM NEAA
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)
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:

Transient transfection of FlucUGA construct: Day 1: GripTite cells were plated in T175 flasks at a density of 1.75 x 107 cells/T175 flask in complete growth media without Geneticin and allowed to grow ON in a 37 degrees Celsius incubator. Day 2: Cells in T175 flask were transfected - with 10ug of DNA and 30uL Transfast in 12mL complete growth media without serum and without Geneticin for 4 hours at 37 degrees Celsius. After incubation, cells were trypsinized, spun down, and resuspended to a concentration of 3.3 x 105 cells/mL in assay media. 6uL of cells in assay buffer 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 72 hours. After incubation, plates were washed three times with DPBS (7uL/well). After the third wash, 6uL of DPBS was added to each well. 3uL of 3X luciferase detection reagent was then added to each well using a flying reagent dispenser (FRD). 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
From ChEMBL:
Assay Format: Cell-based
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
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 0.0006480382 uM (0.000648038μM**)% Activity at given concentration.Float%
17Activity at 0.00195 uM (0.00195088μM**)% 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 (0.157572μM**)% 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 12.73 uM (12.7271μM**)% 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)
Data Table ( Complete ):     View Active Data    View All Data
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