1810 J. Med. Chem. 2010, 53, 1810–1818 DOI: 10.1021/jm901680b Selective Inhibitors of the JMJD2 Histone Demethylases: Combined Nondenaturing Mass Spectrometric Screening and Crystallographic Approaches† Nathan R. Rose,‡ Esther C. Y. Woon,‡ Guy L. Kingham,‡ Oliver N. F. King,‡ Jasmin Mecinovic,‡ Ian J. Clifton,‡ Stanley S. Ng,§ Jobina Talib-Hardy,‡ Udo Oppermann,§ Michael A. McDonough,‡ and Christopher J. Schofield*,‡ ‡The Department of Chemistry and the Oxford Centre for Integrative Systems Biology, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom, and §Structural Genomics Consortium, University of Oxford, Old Road Campus Roosevelt Drive, Headington OX3 7DQ, United Kingdom, The Botnar Research Centre, Oxford Biomedical Research Unit, Oxford OX3 7LD, United Kingdom Received November 13, 2009 Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of Nε-methylated lysine resi- dues 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. Introduction Histone lysine methylation is a dynamic post-translational modification, with sequence-specific N-methyltransferases and demethylases acting to add and remove methyl groups (Figure 1).1 The methylation (or demethylation) of specific lysine residues can enable both euchromatic or heterochro- matic states, thus transcriptionally activating or silencing genes. Histone demethylases (HDMsa) are implicated in dis- eases including leukemia, prostate cancer, squamous cell carcinoma, and X-linked mental retardation.2 The largest identified family of HDMs is the JmjC-domain-containing family, the members of which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases.3,4 The human JMJD2 HDM subfamily has six members (JMJD2A, 2B, 2C, 2D, 2E, and 2F), of which JMJD2A-C have been shown to catalyze demethylation of the methylated forms of histone 3 lysine 9 (H3K9) and histone 3 lysine 36 (H3K36) in vitro and in cells, while JMJD2D is selective for the demethylation of H3K9.4-7 JMJD2C has been implicated in prostate cancer, and inhibition of JMJD2C expression has been shown to decrease cancer cell proliferation.4 Although †PDB ID 2WWJ. *To whom correspondence should be addressed. Phone: þ44 (0) 1865 275 625. Fax: þ44(0)1865 285 022. E-mail: Christopher.schofield@ chem.ox.ac.uk. aAbbreviations: ESI-MS, electrospray ionization-mass spectrometry; 2OG, 2-oxoglutarate; siRNA, small-interfering ribonucleic acid; JMJD, Jumonji domain containing protein; PHD, prolyl hydroxylase domain containing protein; FIH, factor-inhibiting hypoxia-inducible factor; HIF-1R, hypoxia inducible factor-1R; SAHA, suberoylanilide hydro- xamic acid; DCMS, dynamic combinatorial mass spectrometry; FDH, formaldehyde dehydrogenase; CODD, C-terminal oxygen degradation domain; CAD, C-terminal transactivation domain; NOG, N-oxalylgly- cine; TCA, tricarboxylic acid; HDM, histone demethylase. pubs.acs.org/jmc Published on Web 01/20/2010 JMJD2E has been classified as a pseudogene,8 recombinant JMJD2E has HDM activity, and is amenable to inhibition studies.9 JMJD2F has not been characterized. The precise in vivo roles of individual HDMs in the regulation of gene expression are unclear or only beginning to emerge. A degree of redundancy likely exists, with different demethylases targeting the same histone methyllysines. Small molecule chemical probes may be useful for investigating the in vivo role of these demethylases,10 and the therapeutic potential of HDM inhibitors is being considered.11 To be useful as probes, HDM inhibitors should not inhibit at least some of the other Fe(II)/2OG dependent oxygenases, e.g., those involved in the hypoxic response pathway, including the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor-inhibiting HIF (FIH), and the human HIF prolyl hydroxylase domain 2 (PHD2), because the activity of these enzymes regulates a large array of human genes.12-14 Recently, we reported screening methods and several HDM inhibitor “scaffolds” for the JMJD2 HDM subfamily.9 Com- pounds analyzed included the known histone deacetylase inhibitors suberoylanilide hydroxamic acid (SAHA), trichos- tatin A (TSA), pyridine-dicarboxylic acids, and various other 2OG analogues. The latter included N-oxalyl-D-phenylala- nine 1d, which has previously been shown to be selective for the inhibition of FIH with respect to PHD2;15 this selectivity was achieved by exploiting a hydrophobic subpocket adjacent to the 2OG binding site present in FIH but not in PHD2. FIH has been regarded as part of the Jmj subfamily of 2OG dependent oxygenases6,16,17 and is more similar in sequence and structure to the JMJD demethylases than it is to other human 2OG oxygenase subfamilies.12 Crystallographic studies and modeling imply that the JMJD2s possess a similar subpocket to that observed for FIH, r 2010 American Chemical Society