AbstractThe papain-like protease (PLpro) in coronavirus is one of key cysteine proteases responsible for the proteolytic processing of viral polyproteins, and plays an important role in dysregulation of host immune response. PLprois a promising therapeutic target with a major challenge in inhibitor design due to the restricted S1/S2 sites for two consecutive glycine of substrates. Here we reported the discovery of two activators of the SARS-CoV-2 PLprofrom a biochemical screening, and the identification of the unique residue, C270, as an allosteric and covalent regulation site for the activators. This site was also specifically modified by glutathione oxidized, resulting in the S-glutathionylation and activation of the protease. Furthermore, one compound was found to allosterically inhibit the protease by covalent binding to this crucial site. Together, these results elucidated an unrevealed molecular mechanism for allosteric modulation of the protease’s activity, and provided a new strategy for discovery of allosteric inhibitors of the SARS-CoV-2 PLpro.