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 .
【저자키워드】 SARS-CoV-2, main protease, molecular docking, pre-steady-state kinetics, substrate cleavage, inhibitor binding, stopped-flow, 【초록키워드】 COVID-19, Treatment, Efficacy, Drug discovery, peptide, Virtual screening, drug, SARS-CoV-2 main protease, viral replication, cleavage, mutant, inhibitor, Quantitative, platform, binding, Interaction, conformational change, enzyme, complex, Compound, M pro, wild-type, component, binding mechanism, Specific, polyprotein, kinetic analysis, effective, responsible, examined, subsequent, investigated, inhibit, less, interact, conformational, induce, inactive, binding constant, catalytically, kinetic mechanism, 【제목키워드】 drug, Kinetics, the SARS-CoV-2,