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BioAssay: AID 504339

qHTS Assay for Inhibitors of JMJD2A-Tudor Domain

JMJD2A is a jumonji-domain-containing lysine demethylase that uses Fe(II) together with 2-OG to effect oxidative demethylation of histones. Methylation is one of many post-transcriptional modification that affect epigenetic transcription. Hence, cross-talk between histonemarks and histone modifying enzymes exists, whereby enzymes that transfer/remove histone marks also contain reader domains. more ..
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 Tested Compounds
 Tested Compounds
All(384521)
 
 
Active(16919)
 
 
Inactive(338945)
 
 
Inconclusive(29098)
 
 
 Tested Substances
 Tested Substances
All(388413)
 
 
Active(17028)
 
 
Inactive(342184)
 
 
Inconclusive(29201)
 
 
AID: 504339
Data Source: NCGC (JMJD2845)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-02-22

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 16919
Depositor Specified Assays
AIDNameTypeComment
504402qHTS Assay for Inhibitors of JMJD2A-Tudor Domain: Summarysummary
Description:
JMJD2A is a jumonji-domain-containing lysine demethylase that uses Fe(II) together with 2-OG to effect oxidative demethylation of histones. Methylation is one of many post-transcriptional modification that affect epigenetic transcription. Hence, cross-talk between histonemarks and histone modifying enzymes exists, whereby enzymes that transfer/remove histone marks also contain reader domains. JmjD2A has been shown to specifically bind to trimethyl H3K4 and H4K20 marks [1] It is proposed that the functions of JmjD2A binding to the histones, is to maintain marks after they are installed and to spread the modifications by positioning the enzyme for methylation of adjacent regions, establishing a self-propagating cycle for rapid methylation over a large area of chromatin. Due to the combinatorial nature of the histone code and the cross-talk between regulatory proteins, the value of a probe directed against a given target can be enhanced by possessing knowledge about the probes' interactions with additional epigenetic proteins. Thus, small molecule targeting of the JMJD2A-tudor domain interaction with its target methylhistone mark is expected to enable sophisticated experiments probing the fine regulatory pathways leading to selective demethylation of a given methyllysine locus based on the methylation state of adjacent histone marks. A quantitative high throughput [2,3] AlphaScreen has been developed to identify small molecule inhibitors based on a protocol published by NCGC and SGC [4].

Although AlphaScreen has significant advantages with its utility in a variety of epigenetic target assays, the primary screening data provided in this deposition should be used with caution due to the prevalence of screening artifacts [5]. The top compound concentration tested was high (>100uM) in this assay, which increases number of actives and potential artifacts. An AlphaScreen counterscreen is highly recommended to be run against any putative actives to eliminate non-specific artifacts. Alternatively, one can use other AlphaScreen assays in PubChem to filter out promiscuous hits.

[1] Lee, J. et al. Distinct binding modes specify the recognition of methylated histone H3K4 and H4K20 by JMJD2A-tudor. Nat Struct Mol Biol. 15(1); 109-11. 2008. PMID: 18084306

[2] Yasgar, et al. Compound Management for Quantitative High-Throughput Screening. JALA. 2008 Apr;13(2):79-89. PMID: 18496600

[3] Inglese, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci. 1;103(31):11473-8. 2006. PMID: 16864780

[4] Quinn et al. A homogeneous method for investigation of methyl-dependent protein-protein interactions in epigenetics. Nucleic Acids Res., 38(2):e11. 2010. PMID: 18084306

[5] Baell,Holloway. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem. 53(7):2719-40. 2010. PMID: 20131845

NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
Structural Genomics Consortium (SGC)
NIH Grant: 5U54 MH084681-02
Protocol
To each well of a 1,536w plate, 2ul of JMJD2A-Tudor (final 100nM) and 1ul biotinylated histone peptide methylated at Lys4 [b-H3(1-18)K4me3] (final 50nM) were added in 1x PBS buffer, pH 7.4, containing 0.01% Tween-20, using a nanoliter dispenser. Formation of a protein-peptide complex proceeded at room temperature for 30 min. A Kalypsys pin-tool transferred 23nl of library compound solution in DMSO to each well. Following a 30 min incubation of the protein-peptide complex with compounds at room temperature, a mixture of 40 g/mL each streptavidin-coated donor and nickel chelate acceptor AlphaScreen beads (final 10 g/mL) was added in a 1ul dispense for a final volume of 4ul, followed by a brief centrifugation (1000 rpm, 1 min). Plates were incubated protected from the light for 20 min at room temperature. Microplates were then read on an EnVision multilabel plate reader using the 1,536 plate HTS AlphaScreen aperture (excitation time 80 ms, measurement time 240 ms).
Comment
Keywords: JMJD2A-T, JMJD2A-Tudor Domain, JMJD2, Jumonji domain containing 2A, Lysine-specific demethylase 4A, KDM4A, JHDM3A, KIAA0677, Oxidoreductase, EC: 1.14.11.-

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
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 0.114 uM (0.114μM**)% Activity at given concentration.Float%
15Activity at 0.229 uM (0.229μM**)% Activity at given concentration.Float%
16Activity at 0.351 uM (0.350542μM**)% Activity at given concentration.Float%
17Activity at 0.457 uM (0.45657μM**)% Activity at given concentration.Float%
18Activity at 0.627 uM (0.627293μM**)% Activity at given concentration.Float%
19Activity at 0.914 uM (0.914μM**)% Activity at given concentration.Float%
20Activity at 1.140 uM (1.14μM**)% Activity at given concentration.Float%
21Activity at 1.600 uM (1.6μM**)% Activity at given concentration.Float%
22Activity at 2.286 uM (2.2855μM**)% Activity at given concentration.Float%
23Activity at 2.696 uM (2.69591μM**)% Activity at given concentration.Float%
24Activity at 3.270 uM (3.26994μM**)% Activity at given concentration.Float%
25Activity at 4.922 uM (4.92217μM**)% Activity at given concentration.Float%
26Activity at 8.000 uM (8μM**)% Activity at given concentration.Float%
27Activity at 10.30 uM (10.2971μM**)% Activity at given concentration.Float%
28Activity at 11.41 uM (11.4126μM**)% Activity at given concentration.Float%
29Activity at 16.20 uM (16.1981μM**)% Activity at given concentration.Float%
30Activity at 22.90 uM (22.9μM**)% Activity at given concentration.Float%
31Activity at 28.60 uM (28.6μM**)% Activity at given concentration.Float%
32Activity at 45.70 uM (45.6968μM**)% Activity at given concentration.Float%
33Activity at 57.07 uM (57.0746μM**)% Activity at given concentration.Float%
34Activity at 80.69 uM (80.6924μM**)% Activity at given concentration.Float%
35Activity at 113.8 uM (113.831μM**)% Activity at given concentration.Float%
36Activity at 162.0 uM (161.981μM**)% Activity at given concentration.Float%
37Activity at 229.0 uM (229μM**)% Activity at given concentration.Float%
38Compound QCSource of Compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084681-02

Data Table (Concise)
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