Article Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1811 Figure 1. Reactions catalyzed by the JMJD2 histone demethylases. Each step is coupled to the conversion of O2 and 2-oxoglutarate to succinate and CO2. 2OG=2-oxoglutarate, succ=succinate. which is adjacent to the active site.15,18 However, the crystal- lographic analyses imply that the subpocket of the JMJD2s is significantly larger and more open than that of FIH and extends into the substrate binding groove. We set out to explore this subpocket by derivatization of N-oxalyl- D-phenylalanine 1d as a possible means of achieving selectivity toward the JMJD2 subfamily. We used a combined approach employing binding assays using nondenaturing mass spectro- metry, disulfide exchange based dynamic combinatorial chemistry, inhibition studies, and crystallographic analyses, which led to the identification of N-oxalyl-D-tyrosine deriva- tives as potent inhibitors of JMJD2E. Results Initially, we used nondenaturing electrospray ionization mass spectrometry (ESI-MS) to assess binding of potential inhibitors to JMJD2E.19 When the target protein is amenable to such assays, the stoichiometry of interaction and binding strength can be rapidly assessed. This method has been used in the study of metallo-enzyme inhibition (e.g., refs 20, 21) and enzyme-metal ion interactions.22 In these ESI-MS analyses, it is important to appreciate that different types of noncova- lent interaction survive the transition from solution to gas- phase differently (for review, see ref 6). However, in some cases, including metallo-enzymes,20-25 good agreement between results from nondenaturing ESI-MS binding data and solution-phase data can occur. Our starting points for the binding assay were the 2OG analogues that we had identified in the initial study aimed at identifying “template” inhibitors of JMJD2E suitable for deri- vatization, in particular N-oxalylglycine 1a (IC50=24 μM) and N-oxalyl-D-homophenylalanine 1h (IC50 = 39 μM).9 A set of N-oxalyl-D-amino acids 1b-g and their corresponding L-iso- mers 1i-n were assessed for potential binding to the JMJD2E 3 Fe(II) 3 Zn(II) complex (hereafter JMJD2E) (Figure 2) by non- denaturing ESI-MS screening. The results revealed that of the 13 compounds tested, N-oxalylglycine 1a and N-oxalyl- D-cysteine 1b bound to JMJD2E (41256 Da), with formation of new peaks at 41403 and 41448 Da, corresponding to JMJD2E 3 1a and JMJD2E 3 1b complexes, respectively. Rela- tively weak binding was observed for N-oxalyl-D-phenylalanine 1d (JMJD2E 3 1d: 41493 Da), N-oxalyl-D-valine 1f (JMJD2E 3 1f: 41445 Da), and N-oxalyl-D-alanine 1g (JMJD2E 3 1g: 41417 Da). These observations were in general agreement with the formaldehyde dehydrogenase (FDH) coupled assay inhibition results (measuring JMJD2E turnover with a histone H3 Lys9 trimethylated (H3K9me3) peptide fragment substrate) (Table S1, Supporting Information). It is notable that while some of the N-oxalyl-D-amino acids are active, none of the L-compounds 1i-n bind to JMJD2E, and all, with the exception of 1n (IC50 = 285 μM), are inactive as inhibitors in FDH coupled assays. The importance of the D-stereochemistry in the N-oxalyl amino acid series is demonstrated by the significantly greater potency of N-oxalyl-D-cysteine 1b (IC50 = 73 μM) compared Figure 2. Structures of potential inhibitors used in this study. Figure 3. Dynamic combinatorial mass spectrometry (DCMS) ap- proach. (A) The N-oxalyl group of support ligand 1b anchors the molecule into the active site of JMJD2E via interaction with the Fe(II) ion (magenta), leaving the thiol side chain free for disulfide formation with the thiol library in solution. (B) Selective formation of a JMJD2E-disulfide complex with the thiol member that fits best into the active site. to N-oxalyl-L-cysteine 1i (IC50>1 mM) for inhibition of JMJD2E. To explore the extent of the JMJD2E 2OG-substrate bind- ing pocket that is accessible to the N-oxalyl amino acid series, a disulfide exchange based dynamic combinatorial-mass spec- trometry (DCMS) screen21,24,26 was then performed. The