Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS‐CoV‐2 Papain‐Like Protease
Lennart Brewitz, Jos J. A. G. Kamps, Petra Lukacik, Claire Strain‐Damerell, Yilin Zhao, Anthony Tumber, Tika R. Malla, Allen M. Orville, Martin Walsh, Christopher J. Schofield
Abstract
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.