Article inhibitors compared to the support compound 1b (IC50 = 73 μM). This difference could be due to the differences in interactions in solution and in nondenaturing ESI-MS condi- tions or the inability of the carbon compounds to mimic the S-S bond in disulfides. Nonetheless, the DCMS method revealed that larger side-chains than those of N-oxalyl- D-cysteine could likely be accommodated in subpocket of the JMJD2E active site. It was noted that disulfide 2a binds slightly more strongly to JMJD2E than 2b, indicating that the presence of the methoxy group may improve inhibitory activity. This led us to consider using N-oxalyl-D-tyrosine 1c as a lead for further optimization of the aromatic ring. Consequently, a set of N-oxalyl-D-tyrosine derivatives (5a-12, Figure 4) substituted at the tyrosinyl-OH position with a variety of substituents, including benzoate esters (5a-j), other aliphatic/aromatic esters (6a-k), sulfonate esters (7a-j), aliphatic ethers (9a-i), β-ketoethers (8a-m, 11a-c, 12) and benzyl ethers (10a-q) were analyzed for binding to, and inhibition of, JMJD2E. The N-oxalyl-D-tyrosine derivatives (5a-12, Figure 4) were initially screened for binding affinity to JMJD2E by the nondenaturing ESI-MS method. The binding affinity of each compound was classified into one of five rank classes. Com- petition experiments (data not shown) showed that the top 11 compounds all bound more tightly than either N-oxalylgly- cine 1a or N-oxalyl-D-phenylalanine 1d. The N-oxalyl-D-tyrosinyl derivatives were then tested (at 100 μM) for activity against JMJD2E using the FDH based coupled assay (Figure 6),9 and the compounds were ranked by inhibitory potency based on the reduction in initial demethy- lation rate observed. IC50s were then determined for nine of the most potent compounds 7f, 7c, 7e, 10k, 7d, 8m, 6d, 10e, 10a, and three of the least potent compounds 7i, 8b, and 9h as a control (Table 1). The most potent compound in the FDH coupled assay was 7f, with an IC50 of 5.4 μM; other potent compounds also had IC50s in the low micromolar range (8-40 μM). This represents an improvement of potency with respect to N-oxalylglycine 1a (IC50 = 24 μM), N-oxalyl- D-phenylalanine 1d (IC50 = 320 μM), and N-oxalyl-D-homo- phenylalanine 1h (IC50 = 39 μM).9 The rankings of binding affinity as determined by nonde- naturing ESI-MS and inhibitory potency by the FDH coupled assay were compared using Kendall’s rank correlation (τB = 0.58, p<0.0001) and Spearman’s rank correlation (F=0.72, p < 0.0001). These results indicate a statistically significant positive correlation between the inhibitor rankings by non- denaturing ESI-MS and by FDH coupled assay activity (Figure 5b). Because there are differences in the physical interactions between ligand and enzyme in the gas phase as opposed to solution phase (in gas phase, ionic interactions and hydrogen bonding interactions are believed to be preserved more efficiently than hydrophobic interactions),19 inhibitors which rely on nonionic interactions for binding to the enzyme in solution may show relatively weaker binding affinity in the mass spectrometric screen. Nonetheless the results reveal that screening for binding by nondenaturing ESI-MS is an efficient and useful method for ranking affinity, which is amenable to medium-throughput analyses and displays a strong correla- tion to the FDH coupled assay inhibition data. The most potent inhibitors identified by these screens (7f, 7c, and 7e, as well as previously described inhibitors 1a and 1d) were also screened against PHD2 by nondenaturing ESI-MS binding affinity assays and biochemical activity assays (hydroxylation of CODD peptide substrate by Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1813 Figure 5. Correlation of nondenaturing ESI-MS analyses and in- hibition results for JMJD2E inhibitors. (a) Compounds were grouped in five ranking sets reflecting the strength of binding to the JMJD2E 3 Fe(II) 3 Zn(II) complex (E). Rank 1: ∼1:4 ratio un- bound:bound; rank 2: ∼1:2 unbound:bound; rank 3: ∼1:1 un- bound:bound; rank 4: ∼4:1 unbound:bound, and rank 5: ∼10:1 unbound:bound. The MS spectra show examples of data for representative compounds from each ranking set. Some samples (11 of the 73 compounds tested) were not considered to produce spectra of sufficient quality for classification and were thus excluded from the ranking. (b) Initial rates of all compounds tested as JMJD2E inhibitors (100 μM) binding rank as determined by ESI- MS, demonstrating correlation between the two data sets. Kendall’s τB = 0.58 (p < 0.0001), Spearman’s F = 0.72 (p < 0.0001). PHD2, analyzed by MALDI-TOF MS27). No inhibitory activity toward PHD2 was observed in the biochemical assay for the three N-oxalyl-D-tyrosine derivatives 7f, 7c, and 7e or for 1d; N-oxalylglycine 1a, however, was a potent inhibitor of PHD2 as previously reported.28-31 Some binding was ob- served between these inhibitors and PHD2 by nondenaturing ESI-MS. However, nondenaturing ESI-MS binding assays in the presence of the known potent inhibitor 1a showed binding of both 1a and either 7f, 7c, and 7e simultaneously, suggesting that binding of these N-oxalylamino acids was not occurring in the 2OG binding site of the enzyme. Thus, these inhibitors are selective for the JMJD2 HDMs over the human prolyl hydroxylase PHD2. This result is consistent with the lack of a large pocket adjacent to the 2OG side chain binding site in PHD2 as opposed to that observed for JMJD2A.18,32,33 Compounds 7f and 7c were also screened against FIH using a MALDI-TOF assay as reported.34 Dose-response curves representing the extent of hydroxylation as a function of inhibitor concentration were used to determine IC50s. Both of