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BioAssay: AID 1865

Quantitative High-Throughput Screen for Regulators of Epigenetic Control

The Locus Derepression (LDR) assay detects the derepression or induction of a GFP reporter that is stably integrated in a genomic region of murine cells that is presumably silenced. Transcription of the GFP reporter is controlled by a CMV promoter, which normally is constitutively active. However, this cell line, termed LDR, was selected for lack of constitutive production of GFP. GFP production more ..
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 Tested Compounds
 Tested Compounds
All(272107)
 
 
Active(537)
 
 
Inactive(266805)
 
 
Inconclusive(4852)
 
 
 Tested Substances
 Tested Substances
All(276569)
 
 
Active(543)
 
 
Inactive(271143)
 
 
Inconclusive(4883)
 
 
 Related BioAssays
 Related BioAssays
AID: 1865
Data Source: NCGC (LDR883)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-07-10

Data Table ( Complete ):           Active    All
BioActive Compounds: 537
Depositor Specified Assays
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AIDNameTypeProbeComment
597qHTS Assay for Epigenetic Modulatorsconfirmatory
1653Quantitative High-Throughput Screen for Epigenetic Modulators: Summarysummary5
1043H358 Cell Viability Secondary Assay for qHTS Assay for Epigenetic Modulatorsconfirmatory
1042CDH13 Derepression/Induction Secondary Assay for qHTS Assay for Epigenetic Modulatorsother
1041p16 Derepression/Induction Secondary Assay for qHTS Assay for Epigenetic Modulatorsother
1039Normal 2 Cell Viability Secondary Assay for qHTS Assay for Epigenetic Modulatorsconfirmatory
1038Normal 1 Cell Viability Secondary Assay for qHTS Assay for Epigenetic Modulatorsconfirmatory
1037NSCLC 2 Cell Viability Secondary Assay for qHTS Assay for Epigenetic Modulatorsconfirmatory
1036NSCLC 1 Cell Viability Secondary Assay for qHTS Assay for Epigenetic Modulatorconfirmatory
1035HDAC Secondary Assay for qHTS Assay for Epigenetic Modulatorsother
990Parental Cell Counter Screen Assay for Epigenetic Modulatorsconfirmatory
890Confirmation Concentration-Response Assay for Epigenetic Modulatorsconfirmatory
493065Parental Cell Counter Screen Assay for Epigenetic Modulators: Hit Validationconfirmatory
493066HBEC Cell Viability Secondary Assay for qHTS Assay for Epigenetic Modulatorsconfirmatory
493067Confirmation Concentration-Response Assay for qHTS Assay for Epigenetic Modulatorsconfirmatory
493071HCC4017 Cell Viability Secondary Assay for qHTS Assay for Epigenetic Modulatorsconfirmatory
493074H358 Cell Viability Secondary Assay for qHTS Assay for Epigenetic Modulators: Round 2confirmatory
Description:
NIH Molecular Libraries Screening Centers Network [MLPCN]
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 (LDR) assay detects the derepression or induction of a GFP reporter that is stably integrated in a genomic region of murine cells that is presumably silenced. Transcription of the GFP reporter is controlled by a CMV promoter, which normally is constitutively active. However, this cell line, termed LDR, was selected for lack of constitutive production of GFP. GFP production can be induced by incubating the cells with histone deacetylase or DNA methyltransferase inhibitors (Martinez et al., 2006). Compounds that derepress or induce this reporter locus are identified by enumerating GFP positive cells using a laser-scanning microplate cytometer (Auld et al., 2006). This assay was screened against the MLSMR earlier (AID 597; (Johnson et al., 2008) and has been rescreened here against the expanded collection.
Protocol
NCGC Assay Protocol Summary:
Three hundred LDR 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.

References

Auld, DS, Johnson, RL, Zhang, YQ, Veith, H, Jadhav, A, Yasgar, A, Simeonov, A, Zheng, W, Martinez, ED, Westwick, JK, Austin, CP, Inglese, J (2006). Fluorescent protein-based cellular assays analyzed by laser-scanning microplate cytometry in 1536-well plate format. Methods Enzymol 414: 566-89.

Johnson, RL, Huang, W, Jadhav, A, Austin, CP, Inglese, J, Martinez, ED (2008). A quantitative high-throughput screen identifies potential epigenetic modulators of gene expression. Anal Biochem 375: 237-48.

Martinez, ED, Dull, AB, Beutler, JA, Hager, GL (2006). High-content fluorescence-based screening for epigenetic modulators. Methods Enzymol 414: 21-36.
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
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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 0.0000528111 uM (5.28111e-05μM**)% Activity at given concentration.Float%
15Activity at 0.0001179724 uM (0.000117972μM**)% Activity at given concentration.Float%
16Activity at 0.0005898618 uM (0.000589862μM**)% Activity at given concentration.Float%
17Activity at 0.00132 uM (0.00131982μM**)% Activity at given concentration.Float%
18Activity at 0.00297 uM (0.00296939μM**)% Activity at given concentration.Float%
19Activity at 0.00432 uM (0.00432097μM**)% Activity at given concentration.Float%
20Activity at 0.00701 uM (0.00701458μM**)% Activity at given concentration.Float%
21Activity at 0.015 uM (0.0147473μM**)% Activity at given concentration.Float%
22Activity at 0.033 uM (0.032985μM**)% Activity at given concentration.Float%
23Activity at 0.065 uM (0.0648048μM**)% Activity at given concentration.Float%
24Activity at 0.083 uM (0.082641μM**)% Activity at given concentration.Float%
25Activity at 0.144 uM (0.143643μM**)% Activity at given concentration.Float%
26Activity at 0.365 uM (0.364932μM**)% Activity at given concentration.Float%
27Activity at 0.510 uM (0.5102μM**)% Activity at given concentration.Float%
28Activity at 0.824 uM (0.824093μM**)% Activity at given concentration.Float%
29Activity at 1.910 uM (1.90984μM**)% Activity at given concentration.Float%
30Activity at 2.954 uM (2.95417μM**)% Activity at given concentration.Float%
31Activity at 4.995 uM (4.99536μM**)% Activity at given concentration.Float%
32Activity at 10.08 uM (10.0844μM**)% Activity at given concentration.Float%
33Activity at 16.87 uM (16.8673μM**)% Activity at given concentration.Float%
34Activity at 40.67 uM (40.6703μM**)% Activity at given concentration.Float%
35Activity at 53.88 uM (53.8826μM**)% Activity at given concentration.Float%
36Activity at 96.61 uM (96.611μM**)% Activity at given concentration.Float%
37Activity at 150.4 uM (150.369μM**)% Activity at given concentration.Float%
38Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 X01 MH079860-01

Data Table (Concise)
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