Optical, microplate reader-based assays are a standard tool for the initial biochemical analysis of SARS-CoV-2 main protease (Mpro) inhibitor candidates. Such assays monitor Mpro-catalyzed substrate proteolysis in order to investigate the kinetic impact of inhibitors under examination on the catalytic reaction. This review outlines the numerous intricate considerations involved in establishing suitable assay protocols. Commonly employed Mpro substrates that exploit different mechanisms for the optical detection of the cleavage reaction are introduced and compared with respect to their suitability for specific assay applications. The contribution of native or tagged forms of Mpro and of the assay medium to representative kinetic data is debated. Protocols for high-throughput screening, IC50 investigation, elucidation of binding mode and modality, as well as for the determination of the kinetic parameters, Ki, αKi, kon, koff, and kinac/KI, are discussed with continuous reference to the underlying kinetic models. This review provides guidelines for the design and establishment of robust assays for precisely characterizing the kinetics of Mpro inhibitor candidates. Typical confounders and false conclusions are demonstrated, along with strategies to circumvent these pitfalls.
Voget et al. (2026) studied this question.