Improved SARS-CoV-2 M pro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (M ) to cleave viral proteins. Consequently, M is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of M in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-M complexes reveal that an alternative binding pocket in M , S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for M inhibitor design.