Preventing the spread of SARS-CoV-2 and its variants is crucial in the fight against COVID-19. Inhibition of the main protease (M^pro) of SARS-CoV-2 is the key to disrupting viral replication, making M^pro a promising target for therapy. PF-07321332 and shikonin have been identified as effective broad-spectrum inhibitors of SARS-CoV-2 M^pro. The crystal structures of SARS-CoV-2 M^pro bound to PF-07321332 and shikonin have been resolved in previous studies. However, the exact mechanism regarding how SARS-CoV-2 M^pro mutants impact their binding modes largely remains to be investigated. In this study, we expressed a SARS-CoV-2 M^pro mutant, carrying the D48N substitution, representing a class of mutations located near the active sites of M^pro. The crystal structures of M^pro D48N in complex with PF-07321332 and shikonin were solved. A detailed analysis of the interactions between M^pro D48N and two inhibitors provides key insights into the binding pattern and its structural determinants. Further, the binding patterns of the two inhibitors to M^pro D48N mutant and wild-type M^pro were compared in detail. This study illustrates the possible conformational changes when the M^pro D48N mutant is bound to inhibitors. Structural insights derived from this study will inform the development of new drugs against novel coronaviruses.

• Crystal structure of SARS-CoV-2 and SARS-CoV M^pro in complex with CCF0058981 were first solved. • CCF0058981 occupies key M^pro subsites through hydrogen bonds and hydrophobic interactions. • CCF0058981 maintains stable binding against M^pro mutants (M49I and V186F).