Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300

Davis, David A.; Bulut, Haydar; Shrestha, Prabha; Yaparla, Amulya; Jaeger, Hannah K.; Hattori, Shin-ichiro; Wingfield, Paul T.; Mitsuya, Hiroaki; Yarchoan, Robert

SARS-CoV-2 encodes main protease (M(pro)), an attractive target for therapeutic interventions. We show M(pro) is susceptible to glutathionylation leading to inhibition of dimerization and activity. Activity of glutathionylated M(pro) could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated M(pro) primarily exists as a monomer and that a single modification with glutathione is sufficient to block dimerization and loss of activity. Proteolytic digestions of M(pro) revealed Cys300 as a primary target of glutathionylation, and experiments using a C300S M(pro) mutant revealed that Cys300 is required for inhibition of activity upon M(pro) glutathionylation. These findings indicate that M(pro) dimerization and activity can be regulated through reversible glutathionylation of Cys300 and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of M(pro) may have relevance to human disease and the pathophysiology of SARS-CoV-2 in bats, which develop oxidative stress during flight.