SARS-CoV-2 Main Protease Inhibitors That Leverage Unique Interactions with the Solvent Exposed S3 Site of the Enzyme

The main protease (M^Pro) of SARS-CoV-2 is crucial for the virus’s replication and pathogenicity. Its active site is characterized by four distinct pockets (S1, S2, S4, and S1–3′) and a solvent-exposed S3 site for accommodating a protein substrate. During X-ray crystallographic analyses of M^Pro bound with dipeptide inhibitors containing a flexible N-terminal group, we often observed an unexpected binding mode. Contrary to the anticipated engagement with the deeper S4 pocket, the N-terminal group frequently assumed a twisted conformation, positioning it for interactions with the S3 site and the inhibitor component bound at the S1 pocket. Capitalizing on this observation, we engineered novel inhibitors to engage both S3 and S4 sites or to adopt a rigid conformation for selective S3 site binding. Several new inhibitors demonstrated high efficacy in M^Pro inhibition. Our findings underscore the importance of the S3 site’s unique interactions in the design of future M^Pro inhibitors as potential COVID-19 therapeutics.