Enzymological Studies on SARS-CoV-2 Main Protease (M^pro)-Ligand Interactions for the Optimization of M^pro Inhibitor Development and Characterization

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the coronavirus disease 2019 (COVID-19). The viral main protease (M^pro) is vital for the replication of the pathogen in host cells and is therefore the subject of extensive COVID-19-directed drug research. Several M^pro inhibitors have received market approval, underscoring the effectiveness of targeting this protease in the combat against the disease. Nevertheless, frequently emerging virus variants and challenging pharmacokinetic profiles of existing therapeutics necessitate the ongoing development of next-generation M^pro inhibitors. <br/> Microplate reader-based assays are a standard technique for the early kinetic investigation of M^pro inhibitor candidates. In this work, an in-depth literature review of such assays was conducted and guidelines for the selection of assay components, the design of assay protocols, and the kinetic evaluation of progress curves were derived. Aiming to optimize M^pro inhibitor assays, an active-site titrant was developed for the precise quantification of catalytically active M^pro in reaction mixtures. The titrant enabled the employed active-site concentrations to be adapted to specific assay conditions. In a consecutive study, structural motifs from an established fluorogenic M^pro substrate, utilized for the optical detection of enzymatic activity in protease assays, and from the approved high-potency M^pro inhibitor nirmatrelvir were combined to create a library of improved fluorogenic substrates. These compounds made the inhibitory properties of highly active, kinetically challenging M^pro inhibitors accessible. The previous tools and protocols were exploited for the characterization of newly developed M^pro-targeting agents. Fragment-based active-site scanning of an azanitrile library identified promising substituents for each position of a tetrapeptide scaffold. Macrocyclization rigidified a sterically favored inhibitor conformation and proved beneficial in terms of protease inactivation efficacy. A number of highly potent open-chain and cyclized M^pro inhibitors were obtained, some of which exhibited antiviral potency in the single-digit micromolar range. In a follow-up study, an initial series of small-molecule inhibitors were conceptualized by equipping identical scaffolds with eleven different warheads. The most effective M^pro-targeting electrophiles were subsequently attached to a favorable tetrapeptide from the previous study, resulting in a potent chloroacetohydrazide lead inhibitor. Taken together, this work aimed to provide a biochemical toolbox for the precise and comprehensive kinetic characterization of SARS-CoV-2 M^pro inhibitor candidates, elucidate structure-activity relationships, and advance the development of novel, highly effective M^pro-targeting drugs.