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

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.