Dataset Information

Selective inhibitors of the JMJD2 histone demethylases: combined nondenaturing mass spectrometric screening and crystallographic approaches.

ABSTRACT: Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of N(epsilon)-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of N-oxalyl-d-tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds.

SUBMITTER: Rose NR

PROVIDER: S-EPMC2825117 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Selective inhibitors of the JMJD2 histone demethylases: combined nondenaturing mass spectrometric screening and crystallographic approaches.

Rose Nathan R NR Woon Esther C Y EC Kingham Guy L GL King Oliver N F ON Mecinović Jasmin J Clifton Ian J IJ Ng Stanley S SS Talib-Hardy Jobina J Oppermann Udo U McDonough Michael A MA Schofield Christopher J CJ

Journal of medicinal chemistry 20100201 4

Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of N(epsilon)-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of N-oxalyl-d-tyrosine derivat ...[more]

PMID: 20088513

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Project description:Multicellular tumor spheroids (MCTS) are valuable in vitro tumor models frequently used to evaluate the penetration and efficacy of therapeutics. In this study, we evaluated potential differences in epigenetic markers, i.e., histone post-translational modifications (PTMs), in the layers of the HCT116 colon carcinoma MCTS. Cells were grown in agarose-coated 96 well plates, forming reproducible 1-mm-diameter MCTS. The MCTS were fractionated into three radially concentric portions, generating samples containing cells from the core, the mid and the external layers. Using mass spectrometry (MS)-based proteomics and EpiProfile, we quantified hundreds of histone peptides in different modified forms; by combining the results of all experiments, we quantified the abundance of 258 differently modified peptides, finding significant differences in their relative abundance across layers. Among these differences, we detected higher amounts of the repressive mark H3K27me3 in the external layers, compared to the core. We then evaluated the epigenetic response of MCTS following UNC1999 treatment, a drug targeting the enzymes that catalyze H3K27me3, namely, the polycomb repressive complex 2 (PRC2) subunits enhancer of zeste 1 (EZH1) and enhancer of zeste 2 (EZH2). UNC1999 treatment resulted in significant differences in MCTS diameter under drug treatment of varying duration. Using matrix-assisted laser desorption/ionization (MALDI) imaging, we determined that the drug penetrates the entire MCTS. Proteomic analysis revealed a decrease in abundance of H3K27me3, compared to the untreated sample, as expected. Interestingly, we observed a comparable growth curve for MCTS under constant drug treatment over 13 days with those treated for only 4 days at the beginning of their growth. We thus demonstrate that MS-based proteomics can define significant differences in histone PTM patterns in submillimetric layers of three-dimensional (3D) cultures. Moreover, we show that our model is suitable for monitoring drug localization and regulation of histone PTMs after drug treatment.

Dataset Information

Selective inhibitors of the JMJD2 histone demethylases: combined nondenaturing mass spectrometric screening and crystallographic approaches.

Publications

Selective inhibitors of the JMJD2 histone demethylases: combined nondenaturing mass spectrometric screening and crystallographic approaches.

Similar Datasets

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