Substrate-Selective Inhibition of the SARS-CoV-2 Papain-Like Protease: Inhibition of Hydrolysis of Human Over Viral Substrates.

The SARS-CoV-2 papain-like protease (PLpro) and the main protease (Mpro) catalyze hydrolysis of the viral polyproteins pp1a/1ab into functional nonstructural proteins. PLpro and Mpro are medicinal chemistry targets, with Mpro inhibitors being used for COVID-19 treatment. PLpro also catalyzes hydrolysis of ubiquitin and interferon-stimulated gene 15 (ISG15) from post-translationally modified human proteins. Here we report how screening of reported deubiquitinase inhibitors using solid-phase extraction coupled to mass spectrometry assays with oligopeptide substrates based on pp1a/1ab and on an ISG15-modified human protein enabled the identification of substrate-selective PLpro inhibitors. The results reveal that the deubiquitinase inhibitor ML364 selectively inhibits the deISGylase activity of isolated PLpro over its pp1a/1ab-processing activity. Structure-activity relationship and computational studies support the assignment of ML364 and derivatives as substrate-selective PLpro inhibitors. The combined results provide proof-of-concept for developing substrate-selective inhibitors of PLpro and, by implication, related proteolytic enzymes, including deubiquitinases.