Pre-Steady-State Kinetics of the SARS-CoV-2 Main Protease as a Powerful Tool for Antiviral Drug Discovery

The design of effective target-specific drugs for COVID-19 treatment has become an intriguing challenge for modern science. The SARS-CoV-2 main protease, M-pro, responsible for the processing of SARS-CoV-2 polyproteins and production of individual components of viral replication machinery, is an attractive candidate target for drug discovery. Specific M-pro inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of M-pro-targeting molecules is urgently needed. Here, we propose a pre-steady-state kinetic analysis of the interaction of M-pro with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type M-pro and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of M-pro by boceprevir, telaprevir, GC-376, PF-00835231, or thimerosal was investigated. Detailed pre-steady-state kinetics of the interaction of the wild-type enzyme with the most potent inhibitor, PF-00835231, revealed a two-step binding mechanism, followed by covalent complex formation. The C145A M-pro mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A M-pro is still good enough to inhibit the enzyme. Therefore, our results suggest that even noncovalent inhibitor binding due to a fine conformational fit into the active site is sufficient for efficient inhibition. A structure-based virtual screening and a subsequent detailed assessment of inhibition efficacy allowed us to select two compounds as promising noncovalent inhibitor leads of SARS-CoV-2 M-pro.