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 state.