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BioAssay: AID 2677

Confirmation qHTS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)

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(138)
 
 
Active(128)
 
 
Inactive(2)
 
 
Inconclusive(8)
 
 
 Tested Substances
 Tested Substances
All(140)
 
 
Active(130)
 
 
Inactive(2)
 
 
Inconclusive(8)
 
 
AID: 2677
Data Source: NCGC (JMJD280)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 128
Related Experiments
AIDNameTypeComment
2147qHTS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)
2421Probe Summary for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)Summarydepositor-specified cross reference: Probe Summary for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)
493212Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E): 8HQs - Round 1Confirmatorydepositor-specified cross reference
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
687009qHTS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E): Counterscreen with LSD1Confirmatorysame 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.

References:
Inglese, J., Auld, D., Jadhav, A., Johnson, R., Simeonov, A., Yasgar, A., Zheng, W. and Austin, C. Proc Natl Acad Sci USA 2006, 103, 11473-11478.

N. R. Rose, S. S. Ng, J. Mecinovic, B. M. Lienard, S. H. Bello, Z. Sun, M. A. McDonough, U. Oppermann and C. J. Schofield, J Med Chem, 2008, 51, 7053-7056.

Sakurai, M., Rose, N., Schultz, L., Quinn, A., Jadhav, A., Ng, S., Oppermann, U., Schofield, C.J. and Simeonov, A. Mol BioSystems 2009, in press.


S. Michael, D. Auld, C. Klumpp, A. Jadhav, W. Zheng, N. Thorne, C. Austin, J. Inglese and A. Simeonov, Assay Drug Dev Technol, 2008, 6, 637-657.

Assay Provider: Structural Genomics Consortium [SGC]
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
Protocol
Reagents and Controls:
Ferrous ammonium sulfate, (+)-sodium L-ascorbate, beta-nicotinamide adenine dinucleotide hydrate (NAD+), Tween-20, formaldehyde dehydrogenase from P. putida (FDH), and disodium 2-oxoglutarate were from Sigma (St. Louis, MO). Black solid-bottom 384-well or 1,536-well assay plates were from Greiner Bio-One (Monroe, NC).

Enzyme was supplied by Prof. Udo Oppermann, SGC-Oxford. The catalytic domain of human JMJD2E (residues 1-337) was produced as an N-terminally His6-tagged protein in E. coli, and purified by Ni-affinity chromatography and size-exclusion chromatography, and stored in HEPES 50 mM/ NaCl 500 mM pH 7.5, as reported in Rose 2008.

The trimethylated histone peptide substrate ARK(me3)STGGK was synthesized and HPLC-purified by the Tufts University Core Facility (Boston, MA). Substrate solution (Sakurai 2009) contained 50 uM ARK(me3)STGGK, 0.00025 U/uL FDH, 50 uM 2-oxoglutarate, 250 uM NAD+, 1 mM ascorbate, and 10 uM ferrous ammonium sulfate.

Controls and plate map: Buffer (150 mM HEPES pH 7.5 containing 0.01% Tween-20) in columns 3 and 4 as negative control (no enzyme) and 100 nM JMJD2E final concentration in columns 1,2, and 5-48.


Assay Steps:
Enzyme and buffer solutions (3 uL) were dispensed into a 1,536-well Greiner black solid-bottom assay plate. The library compounds (23 nL) were transferred using a Kalypsys pintool equipped with 1,536-pin array. The plate was incubated at room temperature (15 min), and then a 1 uL aliquot of substrate solution was added to initiate the reaction. The plate was transferred to ViewLux imager where an initial reading using standard UV optics (Ex 340 nm, Em 450 nm) was obtained. The plate was then removed from the reader, incubated for 30 minutes at room temperature, and returned to the reader for a second fluorescence reading. A fully automated robotic screening system (Kalypsys Inc, San diego, CA) was used to perform the above steps as described previously (Michael 2008). Compound plates containing DMSO as a vehicle-only control were included at regular interval throughout the screen to monitor any systematic trend in the assay signal associated with reagent dispenser variation or decreases in enzyme specific activity. For activity calculations, percent values were computed as the difference in fluorescence intensity between last and first time points. The percentage activity was calculated from the median values of the catalyzed, or neutral control, and the uncatalyzed, or 100% inhibited, control, respectively, using in-house software.
Comment
Keywords: JMJD2, JMJD2E, Human 2-Oxoglutarate Oxygenase, qHTS

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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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 0.0000096336 uM (9.63357e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000272478 uM (2.72478e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000544957 uM (5.44957e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0001089913 uM (0.000108991μM**)% Activity at given concentration.Float%
18Activity at 0.0002179827 uM (0.000217983μM**)% Activity at given concentration.Float%
19Activity at 0.0003613535 uM (0.000361353μM**)% Activity at given concentration.Float%
20Activity at 0.0009261301 uM (0.00092613μM**)% Activity at given concentration.Float%
21Activity at 0.00175 uM (0.00174986μM**)% Activity at given concentration.Float%
22Activity at 0.00311 uM (0.00311098μM**)% Activity at given concentration.Float%
23Activity at 0.00798 uM (0.00797776μM**)% Activity at given concentration.Float%
24Activity at 0.014 uM (0.0140534μM**)% Activity at given concentration.Float%
25Activity at 0.027 uM (0.0268555μM**)% Activity at given concentration.Float%
26Activity at 0.068 uM (0.0677942μM**)% Activity at given concentration.Float%
27Activity at 0.113 uM (0.112671μM**)% Activity at given concentration.Float%
28Activity at 0.230 uM (0.230177μM**)% Activity at given concentration.Float%
29Activity at 0.581 uM (0.581128μM**)% Activity at given concentration.Float%
30Activity at 0.905 uM (0.905213μM**)% Activity at given concentration.Float%
31Activity at 1.972 uM (1.9718μM**)% Activity at given concentration.Float%
32Activity at 5.534 uM (5.53379μM**)% Activity at given concentration.Float%
33Activity at 14.21 uM (14.2081μM**)% Activity at given concentration.Float%
34Activity at 22.56 uM (22.5551μM**)% Activity at given concentration.Float%
35Activity at 56.82 uM (56.8166μM**)% Activity at given concentration.Float%
36Compound QCSource of compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: U54 CDP

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