Molecular mechanisms of SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir is a specific antiviral targeting the main protease (M pro ) of SARS-CoV-2, and has been approved to treat COVID-19 1,2 . As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir but some result in a loss of viral replicative fitness, which in turn are compensated by additional mutations 3 . The molecular mechanisms for this observed resistance are yet unknown. Here we combined biochemical and structural methods to demonstrate that mutations at the substrate binding pocket of the M pro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of 14 complex structures of M pro mutants with drugs or substrate revealed that mutations at the S1 and S4 subsites significantly decreased inhibitor binding, while mutations at the S2 and S4' subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, whereas the latter compensated for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness as we have observed previously 3 . Such a profile was also observed for ensitrelvir, another clinically relevant M pro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation M pro inhibitors.