A Perspective on the Kinetics of Covalent and Irreversible Inhibition

Abstract

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 IC value. In a biochemical assay, the IC 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/K. The k/K is a rate constant describing the efficiency of covalent bond formation resulting from the potency (K) of the first reversible binding event and the maximum potential rate (k) of inactivation. In this perspective, it is proposed that the k/K 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/K 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.

Lysine‐Targeting Covalent Inhibitors

“…[48] Alexeev et al studied the suicide-inhibition of a-oxamine synthases with the substrate mimic trifluoroalanine (14). Following imine formation of trifluoroalanine (14)w ith PLP (9), decarboxylative defluorination generates the reactive iminium intermediate 15,w hich then undergoes covalent bond formation with Lys236 to give iminium ion 16,w hich was confirmed by the protein crystal structure ( Figure 4B). Following imine formation of trifluoroalanine (14)w ith PLP (9), decarboxylative defluorination generates the reactive iminium intermediate 15,w hich then undergoes covalent bond formation with Lys236 to give iminium ion 16,w hich was confirmed by the protein crystal structure ( Figure 4B).…”

“…Thesecond-order rate constant k inact /K I ,which is the primary descriptor of the efficiencyo ft he process,i sd etermined by measuring the total occupancyo ft he target over time at different inhibitor concentrations ( Figure 1A). [14] Although the importance of describing irreversible inhibitors through their respective rate constants is well established, its application is limited. Ar ecent analysis by Strelow derived an equation that relates the second-order rate constant for covalent inhibition with the free exposure of the targeted covalent inhibitor (% covalent occupancy, Figure 1C).…”

“…[48] Alexeev et al studied the suicide-inhibition of a-oxamine synthases with the substrate mimic trifluoroalanine (14). [48] Alexeev et al studied the suicide-inhibition of a-oxamine synthases with the substrate mimic trifluoroalanine (14).…”

“…This allows better assessment of drug activity in vitro and in vivo and describes how these properties relate to free concentration and time. [14] Although the importance of describing irreversible inhibitors through their respective rate constants is well established, its application is limited. Focus is often only on the downstream consequences of covalent bond formation (usually ahalf maximal inhibitory concentration (IC 50 )value), rather than the rate of the covalent modification (k inact /K I ).…”

“…Serine palmitoyltransferase (SPT) is apyridoximine phosphate (PLP)-dependent enzyme that can be irreversibly inhibited by targeting lysine residues.U sing mass spectrometry,protein crystallography,and enzyme kinetics,Wadsworth et al demonstrated that myriocin (10)u ndergoes aldimine formation with PLP (9)togive intermediate 11,before being degraded through a"retro-aldol-like" cleavage mechanism to the C18 aldehyde 12.T his reactive product then acts as as uicide inhibitor to form imine adduct 13 with Lys265 (Scheme 2B). [48] Alexeev et al studied the suicide-inhibition of a-oxamine synthases with the substrate mimic trifluoroalanine (14). [49] Crystallographic studies demonstrated that ab inding-site lysine of 8-amino-7-oxononanoate synthase forms acovalent adduct with trifluoroalanine (14)t hrough ap roposed mechanism also involving the cofactor PLP (9; Figure 4A).…”