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

ABSTRACT The SARS‐CoV‐2 papain‐like protease (PL pro ) and the main protease (M pro ) catalyze hydrolysis of the viral polyproteins pp1a/1ab into functional nonstructural proteins. PL pro and M pro are medicinal chemistry targets, with M pro inhibitors being used for COVID‐19 treatment. PL pro 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 PL pro inhibitors. The results reveal that the deubiquitinase inhibitor ML364 selectively inhibits the deISGylase activity of isolated PL pro over its pp1a/1ab‐processing activity. Structure‐activity relationship and computational studies support the assignment of ML364 and derivatives as substrate‐selective PL pro inhibitors. The combined results provide proof‐of‐concept for developing substrate‐selective inhibitors of PL pro and, by implication, related proteolytic enzymes, including deubiquitinases.