Structure-Guided Design of Potent and Selective Covalent Inhibitors Targeting the SARS-CoV-2 Papain-like Protease

The COVID-19 pandemic led to numerous initiatives to create antiviral medications and vaccines for the treatment and prevention of infections. However, the need remains for new therapies with distinct mechanisms of action from current treatment of COVID-19 infections as well as for future pandemic preparedness. SARS-CoV-2 papain-like protease (PLpro) is a cysteine protease that cleaves the viral polyprotein and possesses deubiquitylase (DUB) and deISGylase activity that can act on host proteins. Here, we report the structure-guided development of covalent inhibitors of SARS-CoV-2 PLpro that possess low nanomolar to subnanomolar antiviral activity in cell assays and inhibit viral replication in a mouse model of SARS-CoV-2 infection. The most potent inhibitors contain N-propargylamide electrophiles, a relatively inert warhead not typically featured in covalent protease inhibitors. These findings provide a foundation for further discovery and optimization of covalent PLpro inhibitors that could lead to future antiviral therapeutics.