Dataset Information

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

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.

SUBMITTER: Brewitz L

PROVIDER: S-EPMC9015526 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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

Brewitz Lennart L Kamps Jos J A G JJAG Lukacik Petra P Strain-Damerell Claire C Zhao Yilin Y Tumber Anthony A Malla Tika R TR Orville Allen M AM Walsh Martin A MA Schofield Christopher J CJ

ChemMedChem 20220217 9

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 i ...[more]

PMID: 35085423

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Project description:Inhibition of the papain-like protease (PLpro) of SARS-CoV-2 has been demonstrated to be a successful target to prevent the spreading of the coronavirus in the infected body. In this regard, covalent inhibitors, such as the recently proposed VIR251 ligand, can irreversibly inactivate PLpro by forming a covalent bond with a specific residue of the catalytic site (Cys111), through a Michael addition reaction. An inhibition mechanism can therefore be proposed, including four steps: (i) ligand entry into the protease pocket; (ii) Cys111 deprotonation of the thiol group by a Brønsted-Lowry base; (iii) Cys111-S- addition to the ligand; and (iv) proton transfer from the protonated base to the covalently bound ligand. Evaluating the energetics and PLpro conformational changes at each of these steps could aid the design of more efficient and selective covalent inhibitors. For this aim, we have studied by means of MD simulations and QM/MM calculations the whole mechanism. Regarding the first step, we show that the inhibitor entry in the PLpro pocket is thermodynamically favorable only when considering the neutral Cys111, that is, prior to the Cys111 deprotonation. For the second step, MD simulations revealed that His272 would deprotonate Cys111 after overcoming an energy barrier of ca. 32 kcal/mol (at the QM/MM level), but implying a decrease of the inhibitor stability inside the protease pocket. This information points to a reversible Cys111 deprotonation, whose equilibrium is largely shifted toward the neutral Cys111 form. Although thermodynamically disfavored, if Cys111 is deprotonated in close proximity to the vinylic carbon of the ligand, then covalent binding takes place in an irreversible way (third step) to form the enolate intermediate. Finally, due to Cys111-S- negative charge redistribution over the bound ligand, proton transfer from the initially protonated His272 is favored, finally leading to an irreversibly modified Cys111 and a restored His272. These results elucidate the selectivity of Cys111 to enable formation of a covalent bond, even if a weak proton acceptor is available, as His272.

Dataset Information

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

Publications

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

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