Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (M^pro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 M^pro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of M^pro, map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed M^pro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.

Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (M^pro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 M^pro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 M^pro at 2.5 µM, which is more potent than against SAR-CoV-1 M^pro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 M^pro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19.