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

Vuong, Wayne; Fischer, Conrad; Khan, Muhammad Bashir; van Belkum, Marco J.; Lamer, Tess; Willoughby, Kurtis D.; Lu, Jimmy; Arutyunova, Elena; Joyce, Michael A.; Saffran, Holly A.; Shields, Justin A.; Young, Howard S.; Nieman, James A.; Tyrrell, D. Lorne; Lemieux, M. Joanne; Vederas, John C.

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (M(pro)) to cleave viral proteins. Consequently, M(pro) 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(pro) 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(pro) complexes reveal that an alternative binding pocket in M(pro), 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(pro) inhibitor design.