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revised on 04.08.2020 and posted on 06.08.2020by Petr Kuzmic
Covalent (irreversible) enzyme inhibitors are an
important group of actual or potential therapeutics. For example, Aspirin is an
irreversible inhibitor of the cyclooxygenase enzyme. Evaluating covalent inhibitors in the drug discovery
is exceptionally challenging, because their overall inhibitory potency consists
of two separate but intertwined contributions: (1) initial binding affinity and
(2) chemical reactivity. It is
especially difficult to reliably asses the kinetic mechanism of
inhibition. This 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 [Copeland (2013) "Evaluation
of Enzyme Inhibitors in Drug Discovery", section 9.1]. 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. The proposed
statistical toolkit uses four independent methods to arrive at a reliable
mechanistic conclusion. The results are illustrated
by using recently published experimental data on the inhibition of two
different protein kinases by the experimental drugs ibrutinib (PCI-32765) and
acalabrutinib [Hopper et al. (2020) J. Pharm. Exp. Therap.372, 331–338].
1. Revised Figure 1 to clarify differences between Ki and KI.
2. Added 'Appendix A' with explanation of symbols.
3. Revised Discussion section, next to last paragraph.
4. Added reference .
5. Reformatted manuscript to single-column.