A Perspective on the Kinetics of Covalent and Irreversible Inhibition

The clinical and commercial success of covalent drugs has prompted a renewed and more deliberate pursuit of covalent and irreversible mechanisms within drug discovery. A covalent mechanism can produce potent inhibition in a biochemical, cellular, or in vivo setting. In many cases, teams choose to focus on the consequences of the covalent event, defined by an IC₅₀ value. In a biochemical assay, the IC₅₀ may simply reflect the target protein concentration in the assay. What has received less attention is the importance of the rate of covalent modification, defined by k_inact/K_I. The k_inact/K_I is a rate constant describing the efficiency of covalent bond formation resulting from the potency (K_I) of the first reversible binding event and the maximum potential rate (k_inact) of inactivation. In this perspective, it is proposed that the k_inact/K_I should be employed as a critical parameter to identify covalent inhibitors, interpret structure-activity relationships (SARs), translate activity from biochemical assays to the cell, and more accurately define selectivity. It is also proposed that a physiologically relevant k_inact/K_I and an (unbound) AUC generated from a pharmacokinetic profile reflecting direct exposure of the inhibitor to the target protein are two critical determinants of in vivo covalent occupancy. A simple equation is presented to define this relationship and improve the interpretation of covalent and irreversible kinetics.