Deciding between one-step and two-step irreversible inhibition mechanisms on the basis of “kobs” data: A statistical approach

Abstract

AbstractThis paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition from kinetic experiments based on the standard “kobs” method. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular step. Recently published experimental data [Hopperet al.(2020)J. Pharm. Exp. Therap.372, 331–338] on the irreversible inhibition of Bruton tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) by ibrutinib (PCI-32765) and acalabrutinib are used as an illustrative example. The results show that the kinetic mechanism of inhibition was misdiagnosed in the original publication for at least one of the four enzyme/inhibitor combinations. In particular, based on the availablekobsdata, ibrutinib behaves effectively as a one-step inhibitor of the TEC enzyme, which means that it is not possible to reliably determine either the inhibition constantKior the inactivation rate constantkinact, but only the covalent efficiency constantkeff=kinact/Ki. Thus, the published values ofKiandkinactfor this system are not statistically valid.