Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase <b>6</b>

Abstract

Histone deacetylase <b>6</b> (HDAC6) is an important drug target in oncological and non-oncological diseases. Most available HDAC6 inhibitors (HDAC6i) utilize hydroxamic acids as a zinc-binding group, which limits therapeutic opportunities due to its genotoxic potential. Recently, difluoromethyl-1,3,4-oxadiazoles (DFMOs) were reported as potent and selective HDAC6i but their mode of inhibition remained enigmatic. Herein, we report that DFMOs act as mechanism-based and essentially irreversible HDAC6i. Biochemical data confirm that DFMO <b>6</b> is a tight-binding HDAC6i capable of inhibiting HDAC6 via a two-step slow-binding mechanism. Crystallographic and mechanistic experiments suggest that the attack of <b>6</b> by the zinc-bound water at the sp² carbon closest to the difluoromethyl moiety followed by a subsequent ring opening of the oxadiazole yields deprotonated difluoroacetylhydrazide <b>13</b> as active species. The strong anionic zinc coordination of <b>13</b> and the binding of the difluoromethyl moiety in the P571 pocket finally result in an essentially irreversible inhibition of HDAC6.