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

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

doi:10.1016/j.ejmech.2021.113584

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

Eur J Med Chem. 2021 Oct 15:222:113584. doi: 10.1016/j.ejmech.2021.113584. Epub 2021 May 30.

Authors

Wayne Vuong¹, Conrad Fischer¹, Muhammad Bashir Khan², Marco J van Belkum¹, Tess Lamer¹, Kurtis D Willoughby¹, Jimmy Lu³, Elena Arutyunova³, Michael A Joyce⁴, Holly A Saffran⁴, Justin A Shields⁴, Howard S Young², James A Nieman⁵, D Lorne Tyrrell⁴, M Joanne Lemieux³, John C Vederas⁶

Affiliations

¹ Department of Chemistry, University of Alberta, Edmonton AB, T6G 2G2, Canada.
² Department of Biochemistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton AB, T6G 2R3, Canada.
³ Department of Biochemistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton AB, T6G 2R3, Canada; Li Ka Shing Institute of Virology, University of Alberta, Edmonton AB, T6G 2E1, Canada.
⁴ Department of Medical Microbiology and Immunology, University of Alberta, Edmonton AB, T6G 2R3, Canada; Li Ka Shing Institute of Virology, University of Alberta, Edmonton AB, T6G 2E1, Canada.
⁵ Department of Medical Microbiology and Immunology, University of Alberta, Edmonton AB, T6G 2R3, Canada; Li Ka Shing Applied Virology Institute, University of Alberta, Edmonton AB, T6G 2E1, Canada.
⁶ Department of Chemistry, University of Alberta, Edmonton AB, T6G 2G2, Canada. Electronic address: john.vederas@ualberta.ca.

Abstract

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.

Keywords: COVID-19; Crystallography; GC376 analogs; Main protease; Protease inhibitor; Structure-guided design.

Publication types

Review

MeSH terms

Animals
Antiviral Agents / chemical synthesis
Antiviral Agents / metabolism
Antiviral Agents / pharmacology*
Binding Sites
Chlorocebus aethiops
Coronavirus 3C Proteases / antagonists & inhibitors*
Coronavirus 3C Proteases / chemistry
Coronavirus 3C Proteases / metabolism
Crystallography, X-Ray
Cysteine Proteinase Inhibitors / chemical synthesis
Cysteine Proteinase Inhibitors / metabolism
Cysteine Proteinase Inhibitors / pharmacology*
Humans
Micelles
Microbial Sensitivity Tests
Molecular Structure
Protein Binding
Pyrrolidines / chemical synthesis
Pyrrolidines / metabolism
Pyrrolidines / pharmacology*
SARS-CoV-2 / drug effects*
SARS-CoV-2 / enzymology
Solubility
Structure-Activity Relationship
Sulfonic Acids / chemical synthesis
Sulfonic Acids / metabolism
Sulfonic Acids / pharmacology*
Vero Cells

Substances

Antiviral Agents
Cysteine Proteinase Inhibitors
Micelles
Pyrrolidines
Sulfonic Acids
3C-like proteinase, SARS-CoV-2
Coronavirus 3C Proteases
GC376