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 ..
BioActive Compound: 1
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
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.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)