Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E): 8HQs - Round 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 more ..
BioActive Compounds: 112
Depositor Specified Assays
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.
This assay is a following of SAR around 8-hydroxyquinoline-based inhibitors of JMJD2E.
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]
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.
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.
Keywords: JMJD2, JMJD2E, Human 2-Oxoglutarate Oxygenase, qHTS
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)