5 and 7.5 (Figures S3-5). This may account for lower docking scores for prohexadione and trinexapac stereoisomers, compared to N-oxalylglycine,

at both pH 5.5 and 7.5 ( Table 1). The docking scores of all the ligands were lower at pH 7.5 compared to pH 5.5. This could be due to the electrostatic award which was more favorable when the ligands were docked to the Jmjd2a protein prepared at pH 5.5. In general, docking scores and ligand efficiency (i. e. docking score/number of heavy atoms) were best for N-oxalylglycine followed by prohexadione (R-prohexadione–10.1 kcal/mol and–8.2 kcal/mol at pH 5.5 and 7.5, respectively; S-prohexadione–10.3 kcal/mol and–9.3 kcal/mol at pH 5.5 and 7.5, respectively) and then by trinexapac (R-trinexapac–10.2 kcal/mol and–8.3 kcal/mol at pH 5.5 and 7.5, respectively; S-trinexapac–9.4 kcal/mol and–7.6 kcal/mol at pH 5.5 and 7.5, respectively), ( Table 1). Our docking BGB324 in vivo experiments suggest that prohexadione and trinexapac, to a lesser extent, Gefitinib molecular weight may inhibit Jmjd2a, and possibly other KDMs, by directly binding at the 2OG binding site in the active site of KDMs. Jmjd2a demethylates

tri-methylated H3-K9 (H3-K9me3), H3-K9me2, and H3-K36me3 [11]. In order to test our hypothesis that prohexadione and trinexapac act as general inhibitors of recently identified KDMs, Jmjd2a was purified to homogeneity and assayed for the ability of different chemicals e.g. N-oxalylglycine, prohexadione, and trinexapac to inhibit the demethylation of H3-K9me3 into the dimethylated product, H3-K9me2. The results showed that in the absence of N-oxalylglycine and PGRs, Jmjd2a efficiently converted H3-K9me3 peptide into H3-K9me2 ( Figure 1a). However, in the presence of 1 mM N-oxalylglycine, a known inhibitor of iron (II), 2OG-dependent KDMs, no product formation was detected ( Figure 1b).

These results suggest that our assay conditions are suitable for inhibition studies using prohexadione and trinexapac. The presence of 1 mM prohexadione in the reaction mixture completely abrogated the conversion of H3-K9me3 peptide substrate into H3-K9me2 product ( Figure 1c). However, under the same assay condition Inositol monophosphatase 1 only a partial inhibition of Jmjd2a catalytic activity was observed by trinexapac ( Figure 1d). Although our studies were performed with the racemic mixture of trinexapac, which contains both R/S-stereoisomers, a limited inhibition by trinexapac could be due to poor the docking score, especially for the S-trinexapac (–7.6 kcal/mol) at pH 7.5, at which the enzymatic assays were performed. These results demonstrate that prohexadione, and trinexapac to some extent, directly inhibit the catalytic activity of Jmjd2a demethylase. Next, the effects of prohexadione and trinexapac on KDMs were evaluated using neurosphere cultures.