1814 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 Rose et al. Figure 6. Screening for JMJD2E inhibitors. Compounds were tested as JMJD2E inhibitors (100 μM) using the FDH assay, and the percentage activities relative to the DMSO control are shown. Measurements were made in duplicate and shown as averages with standard errors of the mean. these compounds, 7f and 7c (IC50 values for JMJD2E of 5.4 and 8.6 μM, respectively), inhibited FIH with IC50 values of 60.2 and 37.2 μM, respectively. Note that direct comparisons of IC50 values determined for JMJD2E and FIH should be treated with caution because the assay used for FIH relies on measure- ment of hydroxylation after a significantly longer incubation period rather than those used in JMJD2E FDH coupled assay. However, the results suggest that, at least for compounds 7f and 7c, selectivity of inhibition with regard to FIH was relatively low as might be expected based on the similarity of its 2OG binding subpocket to that of the JMJD2s (Figure S2, Supporting Information). Crystallization of the most potent N-oxalyl-D-tyrosine inhibitors (Table 1) together with JMJD2A was then attemp- ted, and a crystal structure was obtained for compound 10a in complex with JMJD2A to 2.4 A˚ resolution (Figure S3 and Table S2, Supporting Information), confirming the predicted binding mode for N-oxalyl-D-tyrosinyl derivatives. 10a was observed to be bound to the active site of both the JMJD2A molecules that are present in the asymmetric unit. The oxalyl carbonyl oxygen and one of the carboxylic acid oxygens of 10a chelate the active site metal in a manner analogous to 2OG and N-oxalylglycine 1a (avg. 2.2 A˚ ) (Figures 7, 8). The other carboxylic acid of 10a forms a salt bridge with Lys206 Nε (2.8 A˚) and a hydrogen bond with Tyr132 OH (2.4 A˚). Apparent hydrophobic interactions between the tyrosinyl side chain of 10a and JMJD2A include side chains of residues Ile71, Tyr132, Tyr177, Phe185, and Lys241 (methylenes). The tyrosinyl side-chain oxygen of 10a is positioned to make a weak hydrogen bond (3.7 A˚) with the backbone amide nitro- gen of Ala186. All residues involved in binding 10a are conserved between JMJD2A (used for crystallographic ana- lysis) and JMJD2E (presently more amenable to biochemical inhibition assays), and the most potent JMJD2E inhibitors displayed activity against JMJD2A in a MS-based turnover assay (Table 2). Comparison of the JMJD2A:10a crystal structure with the JMJD2A-H3K9me3 substrate complex (PDB ID 2OQ6) revealed little difference in the conformation of the active site residues (Supporting Information Table S5). When the JMJD2A:H3K9me3 substrate complex structure is Table 1. IC50 Values for the Most Potent Compounds Tested (7f, 7c, 7e, 10k, 7d, 8m, 10e, 6d, 10a), and for Three Less Potent Compounds (7i, 8b, 9h) (JMJD2E Concentration = 2 μM) compd 7f 7c 7e 10k 7d 8m 10e 6d 10a 7i 8b 9h IC50 (μM) 5.4 8.6 11.7 14.7 14.7 14.8 16.6 20.1 37.1 82.9 185.1 326.5 ratio of JMJD2E-inhibitor complex to free JMJD2E (by ESI-MS) 4.3 poor spectrum poor spectrum 3.1 5.7 2.0 3.6 3.5 1.7 1.0 0.9 0.9 superimposed on the JMJD2A:10a structure (Figure 9), no overlap is observed between the inhibitor side chain and the substrate K9me3 side chain (Supporting Information Figure S3). However, the benzyl ether moiety of 10a is observed to partially occupy the same space as Thr11 and Gly12 of the H3K9me3 substrate, indicating that the binding of 10a (and probably the other potent inhibitors) to JMJD2A likely interferes with binding of the H3K9me3 substrate in addition to competing with 2OG. To investigate the rationale for the increased potency of the sulfonates 7c-f relative to the other derivatives, inhibitors 7f, 10k, 6d, and 8m were manually modeled into the active site of the JMJD2A:10a structure by superimposing their structures with 10a and altering the position of each tyrosine substituent to reduce steric clashes and to maximize hydrogen bonding interactions (Figure S4 and Table S3, Supporting Information). All compounds were constrained to form a hydrogen bond between the tyrosinyl oxygen and the backbone amide nitrogen of Ala186, as observed in the JMJD2A:10a structure (Figure S4). In all of these compounds, additional hydrogen bonds were predicted to occur compared to those observed in the JMJD2A:10a crystal structure. Modeling studies also suggest reasons why 7i, 8b, and 9h are less potent