Article Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1815 Figure 7. Stereoviews from the JMJD2A:10a crystal structure. (a) Stereoview of the crystal structure of JMJD2A (transparent ribbons and green sticks) in complex with compound 10a (yellow sticks). Ni(II) (orange sphere) replaces Fe(II) in the active site; the structural Zn(II) is shown as a blue sphere. Highlighted residues (green) chelate active site metal and form part of the substrate/cosubstrate binding pocket. (b) Close-up stereoview of substrate/cosubstrate binding site with compound 10a, showing details of hydrogen bonding interactions between 10a, Lys206 and Tyr132 side chains, and the backbone amide nitrogen of Ala186. Figure 8. Electron density map (stereoview) showing compound 10a (yellow sticks) in the active site of JMJD2A (green sticks). The experimental 2Fo - Fc electron density, displayed as blue mesh, is shown for the metal (gray sphere) coordinating residues and ligands (contoured to 1σ). inhibitors: steric interaction in the case of 7i and 8b, and conformational flexibility in the case of 9h. Taken together, our results indicate that a combined ap- proach involving nondenaturing ESI-MS binding assays to identify hits followed by biochemical assays provides an efficient approach to lead identification of inhibitors for metallo-enzymes. The statistical correlation between the bind- ing strength of compounds to the enzyme under nondenatur- ing ESI-MS conditions and the ability of compounds to inhibit enzyme activity in biochemical assays validates the nondenaturing ESI-MS method although it is appreciated that binding strength under noncatalytic conditions does not necessarily reflect inhibitory potency. One advantage of a binding-type assay is that it can be used to identify (potential) inhibitors in the absence of a catalytic turnover assay which requires knowledge of the substrate (substrates are not yet known for many of the 2OG oxygenases).35 Our work has also identified inhibitors selective for inhibi- tion of the JMJD2 histone demethylase subfamily members over the HIF hydroxylase PHD2, in that the most potent inhibitors of JMJD2E did not inhibit PHD2 (within the limits of detection), as predicted from structural comparisons