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.