Enhanced Properties of a Benzimidazole Benzylpyrazole Lysine Demethylase Inhibitor Mechanism of Action, Binding Site Analysis, and Activity in Cellular Models of Prostate Cancer

Jumonji domain containing lysine demethylase KDM enzymes are encoded by genes of the KDM superfamily. Activities of the KDM4 subfamily promote aggressive phenotypes associated with prostate cancer PCa . Previously, we discovered a benzimidazole pyrazole molecule that inhibited KDM4 isoforms with properties tractable for development. Here, we demonstrate that a benzyl substituted variant of this inhibitor exhibits improved potency in biochemical assays, is cell permeable, and kills PCa cells at low micromolar concentrations. By X ray crystallography and kinetics based assays, we demonstrate that the mechanism of inhibition is complex, proceeding via competition with the enzyme for binding of active site Fe2 and by populating a distal site on the enzyme surface. Furthermore, we provide evidence that the inhibitor s cytostatic properties arise from direct intracellular inhibition of KDM4 enzymes. PCa cells treated with the inhibitor exhibit reduced expression of genes regulated by the androgen receptor, an outcome accompanied by epigenetic maintenance of a heterochromatic stat