Irreversible covalent enzyme inhibitors, including targeted covalent inhibitors (TCIs) and mechanism-based enzyme inhibitors (MBEIs), play an increasingly important role in drug discovery. Their pharmacological behavior is governed by intrinsic inactivation parameters, typically described by the inactivation constant KI, the maximal inactivation rate constant kinact, and their ratio kinact/KI. However, no consensus exists regarding how these parameters should be experimentally determined and interpreted, particularly in high-throughput screening environments where IC50 values are often used as primary descriptors. This article presents a critical survey of the kinetic methodologies employed to characterize irreversible enzyme inhibition. Continuous progress-curve analysis, discontinuous end-point assays, IC50-based estimation strategies, direct mass-spectrometric monitoring of covalent modification, and numerical approaches required by pre-incubation protocols are examined and compared. Attention is given to the statistical robustness of parameter estimation under realistic experimental error, including bootstrap-based uncertainty analysis. For mechanism-based enzyme inhibitors, the kinetic consequences of branching between productive turnover and irreversible inactivation are analyzed, and limitations of classical half-life-based linearization methods are discussed. Intrinsic inactivation parameters are distinguished from protocol-dependent observables, and experimental conditions that may compromise reliable parameter extraction are identified. The objective is to clarify how irreversible inhibitors should be kinetically characterized when the goal is mechanistic understanding and rational drug design. By bridging classical enzymology with current discovery practices, this review provides practical guidance on what experimental data can legitimately support and where caution is required.
Jean Chaudière (Thu,) studied this question.