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.

Substrate specificity of SARS-CoV and SARS-CoV-2 M pro s. To determine the SARS-CoV M pro and SARS-CoV-2 M pro substrate preferences, we applied a HyCoSuL approach. The library consists of

In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. 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 which would be facilitated by an understanding of its substrate specificity. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. We found that the P2 site of SARS-CoV-2-PLpro is highly specific for Gly, the P3 site exhibits a high degree of promiscuity, and the P4 site exhibits a preference for amino acids with hydrophobic side chains. We also demonstrate that SARS-CoV-2-PLpro harbors deubiquitinating activity. Both the substrate binding profile and deubiquitinating activity are shared with the highly related SARS-CoV-PLpro which harbors near identical S4-S2 binding pockets. On the scaffold of best hits from positional scanning we have designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. Altogether 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 repositioning.