AID	Name	Type	Comment
2147	qHTS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)	confirmatory	qHTS Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)
2421	Probe Summary for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)	summary	Probe Summary 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 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 MALDI-TOF-MS (Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry) confirmatory screen used to qualify hit series for further optimization but not to drive structure activity relationships (SAR).

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]

For the MS assays, JMJD2E (2 uM), FAS (10 uM) and SA (100 uM) in HEPES buffer, 50 mM, pH 7.5, were incubated with inhibitor (stock solutions in DMSO, final in-assay concentration varied, but final DMSO concentration was 5% of assay mix) for 15 min at room temperature. Disodium 2OG (10 uM) and peptide (10 uM) were added, and the mixture was incubated for 30 min at 37 oC, before 1:1 quenching with methanol followed by addition of four volumes of 20 mM triammonium citrate. The diluted assay mixture (1 uL) was then mixed with alpha-cyano-4-hydroxycinnamic acid (the MALDI-TOF-MS matrix, 1uL) and spotted onto a MALDI-TOF-MS plate before analysis. The relative intensities of different methylation states observed in the mass spectra were then used to calculate percentage demethylation, and IC50s were calculated from the variation in percentage demethylation at different inhibitor concentrations.

Keywords: JMJD2, JMJD2E, Human 2-Oxoglutarate Oxygenase, qHTS, MALDI

Compound Ranking:
Compounds were ranked by taking the floor of the -Log_IC50*10. Compounds with Potency < 100uM were considered 'active'. Other compounds that showed any dose dependent effect but had Potency > 100uM were considered 'inconclusive'. Remaining compounds were considered 'inactive'.

Grant Number: U54 CDP