AID	Name	Type	Comment
504402	qHTS Assay for Inhibitors of JMJD2A-Tudor Domain: Summary	summary

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

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).

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.

Grant Number: MH084681-02