Structural Basis for the Inhibition of SARS-CoV-2 M^pro D48N Mutant by Shikonin and PF-07321332

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.