Characterization and noncovalent inhibition of the K63-deubiquitinase activity of SARS-cov-2 PLpro

SARS-CoV-2 papain-like protease (PLpro) could facilitate viral replication and host immune evasion by respectively hydrolyzing viral polyprotein and host ubiquitin conjugates, thereby rendering itself as an important antiviral target. Yet few noncovalent PLpro inhibitors of SARS-CoV-2 have been reported with improved directed towards pathogenic deubiquitinating activities inhibition. Herein, we report that coronavirus PLpro proteases have distinctive substrate bias and are conserved to deubiquitylate K63-linked polyubiquitination, thereby attenuating host type I interferon response. We identify a noncovalent compound specifically optimized towards halting the K63-deubiquitinase activity of SARS-CoV-2 PLpro, but not other coronavirus (CoV) counterparts or host deubiquitinase. Contrasting with GRL-0617, a SARS-CoV-1 PLpro inhibitor, SIMM-036 is 50-fold and 7-fold (half maximal inhibitory concentration (IC50)) more potent to inhibit viral replication during SARS-CoV-2 infection and restore the host interferon-β (IFN-β) response in human angiotensin-converting enzyme 2 (hACE2)-HeLa cells, respectively. Structure-activity relationship (SAR) analysis further reveals the importance of BL2 groove of PLpro, which could determine the selectivity of K63-deubiquitinase activity of the enzyme. •PLpros are CoV conserved K63-deubiquitating Proteases that could antagonize the host type I interferon response.•Compound SIMM-036 specifically optimized to inhibit the K63-deubiquitinase activity of SARS-CoV-2 PLpro.•BL2 groove (residues 265–280) of PLpro could determine the selectivity of K63-deubiquitinase activity of the enzyme.