Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies
Replication of SARS-CoV-2, the virus responsible for COVID-19, relies on a main protease (Mpro) to cleave viral proteins, making Mpro a key target for antiviral agents. Previously, we and others demonstrated that GC376, a bisulfite prodrug effective against coronaviruses in animals, is an efficient inhibitor of Mpro in SARS-CoV-2. In this study, we present structure-activity studies of enhanced GC376 derivatives with nanomolar affinities and therapeutic indices greater than 200. Crystallographic analyses of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, can accommodate the P3 position, influenced by polar P3 groups or a nearby methyl group. NMR and solubility studies of GC376 indicate that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Substituting its Na+ counterion with choline significantly improves solubility. The combined physical, biochemical, crystallographic, and cellular data suggest new directions for Mpro inhibitor design.