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Deciding Between One-Step and Two-Step Irreversible Inhibition Mechanisms on the Basis of “Kobs” Data: A Statistical Approach

preprint
revised on 04.08.2020 and posted on 06.08.2020 by 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].

History

Email Address of Submitting Author

petr.kuzmic@gmail.com

Institution

BioKin Ltd.

Country

USA

ORCID For Submitting Author

0000-0001-5250-8381

Declaration of Conflict of Interest

The author declares no conflict of interest.

Version Notes

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 [9]. 5. Reformatted manuscript to single-column.

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