Structure-Based Engineering of Irreversible Inhibitors against Histone Lysine Demethylase KDM5A

The active sites of hundreds of human alpha-ketoglutarate (alpha KG) and Fe(II)-dependent dioxygenases are exceedingly well preserved, which challenges the design of selective inhibitors. We identified a noncatalytic cysteine (Cys481 in KDMSA) near the active sites of KDMS histone H3 lysine 4 demethylases, which is absent in other histone demethylase families, that could be explored for interaction with the cysteine-reactive electrophile acrylamide. We synthesized analogs of a thienopyridine-based inhibitor chemotype, namely, 2-((3-aminophenyl)(2-(piperidin-1-y)ethoxy)methyl)thieno[3,2-b]pyridine-7-carboxylic acid (N70) and a derivative containing a (dimethylamino)but-2-enamido)phenyl moiety (N71) designed to form a covalent interaction with Cys481. We characterized the inhibitory and binding activities against KDMSA and determined the cocrystal structures of the catalytic domain of KDMSA in complex with N70 and N71. Whereas the noncovalent inhibitor N70 displayed alpha KG-competitive inhibition that could be reversed after dialysis, inhibition by N71 was dependent on enzyme concentration and persisted even after dialysis, consistent with covalent modification.