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

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.