Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS-CoV-2 Papain-Like Protease |
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| Authors: | Dr. Lennart Brewitz Dr. Jos J. A. G. Kamps Dr. Petra Lukacik Claire Strain-Damerell Yilin Zhao Dr. Anthony Tumber Tika R. Malla Dr. Allen M. Orville Dr. Martin A. Walsh Prof. Dr. Christopher J. Schofield |
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| Affiliation: | 1. Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, University of Oxford, 12 Mansfield Road, OX1 3TA Oxford, UK;2. Diamond Light Source Ltd., Harwell Science and Innovation Campus, OX11 0DE Didcot, UK Research Complex at Harwell, Harwell Science and Innovation Campus, OX11 0FA Didcot, UK;3. Diamond Light Source Ltd., Harwell Science and Innovation Campus, OX11 0DE Didcot, UK |
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| Abstract: | The two SARS-CoV-2 proteases, i. e. the main protease (Mpro) and the papain-like protease (PLpro), 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 PLpro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PLpro inhibitors and selected Mpro inhibitors on PLpro catalysis. The results reveal that some, but not all, PLpro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing Mpro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PLpro. Less selective Mpro inhibitors, e. g. auranofin, inhibit PLpro, highlighting the potential for dual PLpro/Mpro inhibition. MS-based PLpro 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. |
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| Keywords: | Nucleophilic cysteine protease PF-07321332/nirmatrelvir SARS-CoV-2 papain-like protease/PLpro SARS-CoV-2 main protease/Mpro viral protease inhibition |
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