Comprehensive Understanding of the Kinetic Behaviors of Main Protease from SARS-CoV-2 and SARS-CoV: New Data and Comparison to Published Parameters.

The main protease (M^pro) is a promising drug target for inhibiting the coronavirus due to its conserved properties and lack of homologous genes in humans. However, previous studies on M^pro's kinetic parameters have been confusing, hindering the selection of accurate inhibitors. Therefore, obtaining a clear view of M^pro's kinetic parameters is necessary. In our study, we investigated the kinetic behaviors of M^pro from SARS-CoV-2 and SARS-CoV using both FRET-based cleavage assay and the LC-MS method, respectively. Our findings indicate that the FRET-based cleavage assay could be used for preliminary screening of M^pro inhibitors, while the LC-MS method should be applied to select the effective inhibitors with higher reliability. Furthermore, we constructed the active site mutants (H41A and C145A) and measured the kinetic parameters to gain a deeper understanding of the atomic-level enzyme efficiency reduction compared to the wild type. Overall, our study provides valuable insights for inhibitor screening and design by offering a comprehensive understanding of M^pro's kinetic behaviors. [ABSTRACT FROM AUTHOR]