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