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BioAssay: AID 687009

qHTS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E): Counterscreen with LSD1

The fine interplay among methylation states of several lysine residues on the tails of histone proteins is a major determinant of the transcriptional state of the associated DNA coding regions and is commonly referred to as the histone code. Histone lysine demethylases catalyze the removal of methyl groups from methylated lysine sidechains on histones H3 and H4, thus antagonizing the reactions more ..
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 Tested Compounds
 Tested Compounds
All(29)
 
 
Active(1)
 
 
Inactive(10)
 
 
Inconclusive(18)
 
 
 Tested Substances
 Tested Substances
All(29)
 
 
Active(1)
 
 
Inactive(10)
 
 
Inconclusive(18)
 
 
AID: 687009
Data Source: NCGC (JMJ700)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-05-28
Hold-until Date: 2013-09-06
Modify Date: 2013-09-06

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
AIDNameTypeComment
2421Probe Summary for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)Summarydepositor-specified cross reference: Summary AID
2147qHTS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)Confirmatorysame project related to Summary assay
2677Confirmation qHTS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)Confirmatorysame project related to Summary assay
2680Counterscreen for JMJD2E Inhibitors: qHTS Assay for Inhibitors of Formaldehyde Dehydrogenase (FDH)Confirmatorysame project related to Summary assay
2687AlphaScreen Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)Confirmatorysame project related to Summary assay
2688MALDI-TOF-MS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)Confirmatorysame project related to Summary assay
493212Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E): 8HQs - Round 1Confirmatorysame project related to Summary assay
Description:
The fine interplay among methylation states of several lysine residues on the tails of histone proteins is a major determinant of the transcriptional state of the associated DNA coding regions and is commonly referred to as the histone code. Histone lysine demethylases catalyze the removal of methyl groups from methylated lysine sidechains on histones H3 and H4, thus antagonizing the reactions catalyzed by histone lysine methyltransferases. The quest to define the biological roles of the multiple epigenetic modulator enzymes includes the identification and use of small molecules that selectively inhibit individual histone-modifying enzymes/enzyme subfamilies. In search for novel inhibitors of JMJD2E demethylase, a member of the largest set of histone demethylases belonging to the Fe(II) and 2-oxoglutarate oxygenase (2OG) superfamily, we performed a quantitative high-throughput screen (Inglese 2006) by using an assay which utilizes a trimethylated peptide substrate corresponding to a fragment of histone H3 (sequence ARKme3STGGK) with detection of the formaldehyde co-product in real time by a formaldehyde dehydrogenase (FDH) coupled reaction. FDH catalyzes oxidation of formaldehyde to formic acid with the concomitant reduction of the non-fluorescent beta-nicotinamide adenine dinucleotide hydrate (NAD+) cofactor to the fluorescent NADH co-product.

Compounds that were validated hits were screened against the histone LSD1 histone demethylase. This was a counterscreen, hence, the desired outcome was inactivity.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH084681
Assay Submitter (PI): Udo Oppermann, Structural Genomics Consortium, University of Oxford
Protocol
The assay was performed in a buffer containing 10 mM HEPES, Na pH 7.5, 5 mM MgCl2, 50 mM KCl, 0.01% Nonidet P-40, and 0.1% BSA. LSD1 was sourced from BPS Bioscience (Cat. No. 50100, San Diego, CA) and the substrate peptide was synthesized and purified by Tufts University Core Facilities (Boston, MA). Reagents (3 microL) containing either buffer-only (inhibited control) or LSD1 (120 nM) were dispensed into a 1,536-well Aurora black solid bottom cycloolefin plate (see Table 1 for protocol steps). Compounds (23 nL) were transferred via Kalypsys pintool equipped with a 1,536-pin array. The plate was incubated for 10 min at room temperature, followed by the addition of 1 microL 4 X substrate (10 mM HEPES, pH 7.5, 8 U/mL HRP, 800 muM Amplex Red, and 200 microM H3K4Me2 peptide [Sequence H-ARTXQTARKSTGGKAPRKQLA-NH2, where X = Nepsilon,Nepsilon-dimethyl-L-lysine]) to start the reaction. The plate was then centrifuged at 1,000 rpm for 15 seconds, and the fluorescence intensity was recorded on a ViewLux High-throughput CCD imager (Perkin-Elmer) using standard TAMRA optics (525 nm excitation and 598 nm emission). The plate was then incubated for 30 min at room temperature, and a second read on the ViewLux was performed. The fluorescence intensity difference over the 30 min was used to calculate the respective reaction rate for each well.
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
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 0.0009677143 uM (0.000967714μM**)% Activity at given concentration.Float%
16Activity at 0.00290 uM (0.00290314μM**)% Activity at given concentration.Float%
17Activity at 0.00871 uM (0.00870949μM**)% Activity at given concentration.Float%
18Activity at 0.026 uM (0.0261284μM**)% Activity at given concentration.Float%
19Activity at 0.078 uM (0.0783853μM**)% Activity at given concentration.Float%
20Activity at 0.235 uM (0.235156μM**)% Activity at given concentration.Float%
21Activity at 0.705 uM (0.705467μM**)% Activity at given concentration.Float%
22Activity at 2.116 uM (2.1164μM**)% Activity at given concentration.Float%
23Activity at 6.349 uM (6.34921μM**)% Activity at given concentration.Float%
24Activity at 19.05 uM (19.0476μM**)% Activity at given concentration.Float%
25Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
26Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

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