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Updated: Jun 13, 2026

Procedures for the Identification of SARS-CoV-2 Entry Inhibitors as Potential Antivirals using MLV-Based Pseudoviruses
Published on: February 27, 2026
Substrate-Selective Inhibition of the SARS-CoV-2 Papain-Like Protease: Inhibition of Hydrolysis of Human Over Viral
Sakshi Sharma1, Peter A C Wing2, Wojtek Trede1
1Chemistry Research Laboratory and the Ineos Oxford Institute for Antimicrobial Research, University of Oxford, Oxford, UK.
Abstract:
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.
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