Early Kinetic Characterization of SARS-CoV-2 Main Protease Inhibitors: A Review and Guidance for Biochemical Assessments

Optical, microplate reader-based assays are a standard tool for the initial biochemical analysis of SARS-CoV-2 main protease (M pro ) inhibitor candidates. Such assays monitor M pro -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 M pro 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 M pro and of the assay medium to representative kinetic data is debated. Protocols for high-throughput screening, IC 50 investigation, elucidation of binding mode and modality, as well as for the determination of the kinetic parameters, K i, α K i, k on, k off, and k inac / K I, 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 M pro inhibitor candidates. Typical confounders and false conclusions are demonstrated, along with strategies to circumvent these pitfalls.