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BioAssay: AID 597

qHTS Assay for Epigenetic Modulators

The Locus Derepression assay detects the derepression of a GFP reporter that is stably integrated in a region of the genome of murine c127i mammary cells that is presumably silenced. GFP transcription in this construct is controlled by a CMV promoter, which normally is strong and constitutively active. However, this line was selected for lack of constitutive expression of the GFP protein. GFP more ..
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 Tested Compounds
 Tested Compounds
All(67379)
 
 
Active(57)
 
 
Inactive(66987)
 
 
Inconclusive(343)
 
 
 Tested Substances
 Tested Substances
All(68401)
 
 
Active(59)
 
 
Inactive(67998)
 
 
Inconclusive(344)
 
 
 Related BioAssays
 Related BioAssays
AID: 597
Data Source: NCGC (LDRF188)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-02-20
Modify Date: 2009-03-25

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 57
Related Experiments
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AIDNameTypeProbeComment
990Parental Cell Counter Screen Assay for Epigenetic ModulatorsConfirmatory depositor-specified cross reference
1035HDAC Secondary Assay for qHTS Assay for Epigenetic ModulatorsOther depositor-specified cross reference
1036NSCLC 1 Cell Viability Secondary Assay for qHTS Assay for Epigenetic ModulatorConfirmatory depositor-specified cross reference
1037NSCLC 2 Cell Viability Secondary Assay for qHTS Assay for Epigenetic ModulatorsConfirmatory depositor-specified cross reference
1038Normal 1 Cell Viability Secondary Assay for qHTS Assay for Epigenetic ModulatorsConfirmatory depositor-specified cross reference
1039Normal 2 Cell Viability Secondary Assay for qHTS Assay for Epigenetic ModulatorsConfirmatory depositor-specified cross reference
1041p16 Derepression/Induction Secondary Assay for qHTS Assay for Epigenetic ModulatorsOther depositor-specified cross reference
1042CDH13 Derepression/Induction Secondary Assay for qHTS Assay for Epigenetic ModulatorsOther depositor-specified cross reference
1043H358 Cell Viability Secondary Assay for qHTS Assay for Epigenetic ModulatorsConfirmatory depositor-specified cross reference
1653Quantitative High-Throughput Screen for Epigenetic Modulators: SummarySummary3 depositor-specified cross reference
1865Quantitative High-Throughput Screen for Regulators of Epigenetic ControlConfirmatory depositor-specified cross reference
890Confirmation Concentration-Response Assay for Epigenetic ModulatorsConfirmatory same project related to Summary assay
493065Parental Cell Counter Screen Assay for Epigenetic Modulators: Hit ValidationConfirmatory same project related to Summary assay
493066HBEC Cell Viability Secondary Assay for qHTS Assay for Epigenetic ModulatorsConfirmatory same project related to Summary assay
493067Confirmation Concentration-Response Assay for qHTS Assay for Epigenetic ModulatorsConfirmatory same project related to Summary assay
493071HCC4017 Cell Viability Secondary Assay for qHTS Assay for Epigenetic ModulatorsConfirmatory same project related to Summary assay
493074H358 Cell Viability Secondary Assay for qHTS Assay for Epigenetic Modulators: Round 2Confirmatory same project related to Summary assay
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

MLSCN Grant: 1 X01 MH079860-01
Assay Provider: Elisabeth D. Martinez, University of Texas SW Medical Center

NCGC Assay Overview:
The Locus Derepression assay detects the derepression of a GFP reporter that is stably integrated in a region of the genome of murine c127i mammary cells that is presumably silenced. GFP transcription in this construct is controlled by a CMV promoter, which normally is strong and constitutively active. However, this line was selected for lack of constitutive expression of the GFP protein. GFP production can be induced by incubating the cells with histone deacetylase or DNA methyltransferase inhibitors. Compounds that cause derepression of the locus to express GFP are identified by enumerating GFP positive cells using a laser-scanning microplate cytometer.

For further details of this screen and follow-up characterization, see R.L. Johnson, W. Huang, A. Jadhav, C.P. Austin, J. Inglese, E.D. Martinez, A Quantitative High-Throughput Screen Identifies Potential Epigenetic Modulators of Gene Expression, Analytical Biochemistry (2008) 375(2):237-48.
Protocol
NCGC Assay Protocol Summary:
Two hundred and fifty cells in 5 uL per well were dispensed into black, clear-bottom 1536-well plates. Twenty-three nL compound was transferred to the assay plate and cells were incubated 30 hours at 37 C. Medium was removed, cells were washed two times with PBS, and GFP-positive cells were enumerated as fluorescent objects between the sizes of 20 to 120 um width and depth using an Acumen Explorer.

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, qHTS, HDAC, DNMT, HAT, DNA methyltransferases, histone acetyl transferases, histone deacetylases
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 Classification". 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. 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
1Activity DirectionIndicates direction of observed activity: inactive, decreasing, increasing.String
2Curve 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 signficant, but below 80% of control.String
3Log of AC50Log of AC50.Float
4Hill CoefficientHill slope of fitted curve.Float
5Curve R2R^2 fit value of curve. Closer to 1.0 equates to better Hill equation fit.Float
6Curve Infinite ActivityHill Equation Infinite ActivityFloat
7Curve Zero ActivityHill Equation Zero ActivityFloat
8Curve Excluded 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
9Curve ClassNumerical encoding of curve description for the fitted Hill equation.Float
10Activity at 0.59nM% Activity at given concentration.Float%
11Activity at 1.319nM% Activity at given concentration.Float%
12Activity at 2.95nM% Activity at given concentration.Float%
13Activity at 6.597nM% Activity at given concentration.Float%
14Activity at 14.751nM% Activity at given concentration.Float%
15Activity at 0.033uM% Activity at given concentration.Float%
16Activity at 0.074uM% Activity at given concentration.Float%
17Activity at 0.165uM% Activity at given concentration.Float%
18Activity at 0.369uM% Activity at given concentration.Float%
19Activity at 0.824uM% Activity at given concentration.Float%
20Activity at 1.844uM% Activity at given concentration.Float%
21Activity at 4.122uM% Activity at given concentration.Float%
22Activity at 9.217uM% Activity at given concentration.Float%
23Activity at 20.61uM% Activity at given concentration.Float%
24Activity at 46.08uM% Activity at given concentration.Float%
25Compound TypeNCGC designation for compound stage: 'qHTS ECL (Exploratory)', 'NIHSMR (MLSCN)', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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