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.
Keywords: Nucleophilic cysteine protease; PF-07321332/nirmatrelvir; SARS-CoV-2 main protease/Mpro; SARS-CoV-2 papain-like protease/PLpro; viral protease inhibition.
【저자키워드】 Nucleophilic cysteine protease, PF-07321332/nirmatrelvir, SARS-CoV-2 main protease/Mpro, SARS-CoV-2 papain-like protease/PLpro, viral protease inhibition., 【초록키워드】 SARS-CoV-2, mass spectrometry, protease, in vitro, inhibitors, SARS-CoV-2 main protease, non-structural proteins, MPro, Papain-like protease, viral replication, auranofin, targets, Proteases, mechanisms, PF-07321332, PLPro, inhibitor, utility, Nirmatrelvir, cysteine, M pro, clinical development, Papain, selective, MONITOR, protease inhibition, selected, reported, assays, inhibit, applied, functional, highlighting, 【제목키워드】 inhibition, profile, mass, the SARS-CoV-2,