Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.
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