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BioAssay: AID 2687

AlphaScreen 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(4)
 
 
Active(4)
 
 
 Tested Substances
 Tested Substances
All(4)
 
 
Active(4)
 
 
AID: 2687
Data Source: NCGC (JMJD220)
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: 4
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)
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
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
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). See AID 2147 for details on the primary screen.

The assay here is a secondary AlphaScreen screen used to validate follow-up compounds.

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
Assay Buffer: 50 mM HEPES pH7.5, 0.01% Tween-20, 0.1% BSA (Cohn Fraction V; Sigma A7030)

Peptide: biotin-ARTKQTARK(me3/2)STGGKA

Antibody: Abcam Ab1220, Anti-H3K9Me2

Final concentration of components in enzyme reaction:
JMJD2E 5 nM, Peptide 30 nM, 100 mM L-Ascorbic Acid, 1 mM Ferrous Ammonium Sulphate, 10 mM 2-OG, DMSO 0.1%

Alphascreen beads made up in the dark in assay buffer and pre-incubated for 1 hour in the dark.

Compounds counter-screened against 10 nM biotin-H3K9Me2 to check for interference of alphascreen chemistry.

Protocol Steps:
Step 1. Compount Stock = 100mM

Step 2. Dilute compound to 1 mM in 50 mM HEPES pH7.5 + 0.01 % Tween-20 (final DMSO = 1%)

Step 3. Serial dilution (1:3 using 50 mM HEPES pH7.5 + 0.01 % Tween-20 containing 1% DMSO)

Step 4. Addition of 1 ul of compound per well (triplicates for each concentration)

Step 5. Addition of 5 ul per well of assay buffer containing 10 nM JMJD2E, 200 uM L-Ascorbic Acid, 2 uM Ferrous Ammonium Sulphate

Step 6. Incubate for 15 minutes at room temperature

Step 7. Addition of 4 ul assay buffer containing 75 nM Biotin-H3K9Me3, 25 uM 2-OG

Step 8. Incubate for 25 minutes at room temperature

Step 9. Addition of 5 ul of assay buffer containing 30 mM EDTA

Step 10. Addition of 5 ul Alphascreen Beads (0.08 mg / ml + 8 nM Ab) leave for 1 hour at room temperature

Step 11. Read plate on BMG Labtech Pherastar FS
Comment
Keywords: JMJD2, JMJD2E, Human 2-Oxoglutarate Oxygenase, qHTS, AlphaScreen

Compound Ranking:
Compounds were ranked by taking the floor of the -Log_IC50*10. Compounds were 'active' if Potency < 100uM. If compound showed dose-dependent effect but had Potency > 100uM, it was considered 'inconclusive'. All other compounds were considered 'inactive'.
Result Definitions
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, IC50. Extrapolated IC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3Log_IC50The logarithm of the IC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
4HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
5Compound QCSource of compound QCString

* Activity Concentration.
Additional Information
Grant Number: U54 CDP

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