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Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS‐CoV‐2 Papain‐Like Protease
Author(s) -
Brewitz Lennart,
Kamps Jos J. A. G.,
Lukacik Petra,
StrainDamerell Claire,
Zhao Yilin,
Tumber Anthony,
Malla Tika R.,
Orville Allen M.,
Walsh Martin A.,
Schofield Christopher J.
Publication year - 2022
Publication title -
chemmedchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.817
H-Index - 100
eISSN - 1860-7187
pISSN - 1860-7179
DOI - 10.1002/cmdc.202200016
Subject(s) - proteases , protease , chemistry , papain , biochemistry , enzyme
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.