Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS‐CoV‐2 Papain‐Like Protease

Abstract

The two SARS-CoV-2 proteases, i. e. the main protease (M^pro ) and the papain-like protease (PL^pro ), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL^pro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PL^pro inhibitors and selected M^pro inhibitors on PL^pro catalysis. The results reveal that some, but not all, PL^pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M^pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL^pro . Less selective M^pro inhibitors, e. g. auranofin, inhibit PL^pro , highlighting the potential for dual PL^pro /M^pro inhibition. MS-based PL^pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.