TY - JOUR

T1 - Pre-Steady-State Kinetics of the SARS-CoV-2 Main Protease as a Powerful Tool for Antiviral Drug Discovery

AU - Zakharova, Maria Yu

AU - Kuznetsova, Alexandra A.

AU - Uvarova, Victoria I.

AU - Fomina, Anastasiia D.

AU - Kozlovskaya, Liubov I.

AU - Kaliberda, Elena N.

AU - Kurbatskaia, Inna N.

AU - Smirnov, Ivan V.

AU - Bulygin, Anatoly A.

AU - Knorre, Vera D.

AU - Fedorova, Olga S.

AU - Varnek, Alexandre

AU - Osolodkin, Dmitry I.

AU - Ishmukhametov, Aydar A.

AU - Egorov, Alexey M.

AU - Gabibov, Alexander G.

AU - Kuznetsov, Nikita A.

N1 - Funding Information: This research was supported by the RFBR Grant #20-04-60468 as well as by State Research Funding for FSASI “Chumakov FSC R&D IBP RAS” (Institute of Poliomyelitis): project “Development of inhibitors for SARS-CoV-2 main protease”. The part of the work with stopped-flow kinetics was specifically funded by Russian-State-funded project #121112900214-2 for Institute of Chemical Biology and Fundamental Medicine “Inhibitors of main protease of coronavirus SARS-CoV-2”. Publisher Copyright: Copyright © 2021 Zakharova, Kuznetsova, Uvarova, Fomina, Kozlovskaya, Kaliberda, Kurbatskaia, Smirnov, Bulygin, Knorre, Fedorova, Varnek, Osolodkin, Ishmukhametov, Egorov, Gabibov and Kuznetsov.

PY - 2021/12/6

Y1 - 2021/12/6

N2 - The design of effective target-specific drugs for COVID-19 treatment has become an intriguing challenge for modern science. The SARS-CoV-2 main protease, Mpro, responsible for the processing of SARS-CoV-2 polyproteins and production of individual components of viral replication machinery, is an attractive candidate target for drug discovery. Specific Mpro inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of Mpro-targeting molecules is urgently needed. Here, we propose a pre–steady-state kinetic analysis of the interaction of Mpro with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type Mpro and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of Mpro by boceprevir, telaprevir, GC-376, PF-00835231, or thimerosal was investigated. Detailed pre–steady-state kinetics of the interaction of the wild-type enzyme with the most potent inhibitor, PF-00835231, revealed a two-step binding mechanism, followed by covalent complex formation. The C145A Mpro mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A Mpro is still good enough to inhibit the enzyme. Therefore, our results suggest that even noncovalent inhibitor binding due to a fine conformational fit into the active site is sufficient for efficient inhibition. A structure-based virtual screening and a subsequent detailed assessment of inhibition efficacy allowed us to select two compounds as promising noncovalent inhibitor leads of SARS-CoV-2 Mpro.

AB - The design of effective target-specific drugs for COVID-19 treatment has become an intriguing challenge for modern science. The SARS-CoV-2 main protease, Mpro, responsible for the processing of SARS-CoV-2 polyproteins and production of individual components of viral replication machinery, is an attractive candidate target for drug discovery. Specific Mpro inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of Mpro-targeting molecules is urgently needed. Here, we propose a pre–steady-state kinetic analysis of the interaction of Mpro with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type Mpro and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of Mpro by boceprevir, telaprevir, GC-376, PF-00835231, or thimerosal was investigated. Detailed pre–steady-state kinetics of the interaction of the wild-type enzyme with the most potent inhibitor, PF-00835231, revealed a two-step binding mechanism, followed by covalent complex formation. The C145A Mpro mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A Mpro is still good enough to inhibit the enzyme. Therefore, our results suggest that even noncovalent inhibitor binding due to a fine conformational fit into the active site is sufficient for efficient inhibition. A structure-based virtual screening and a subsequent detailed assessment of inhibition efficacy allowed us to select two compounds as promising noncovalent inhibitor leads of SARS-CoV-2 Mpro.

KW - inhibitor binding

KW - main protease

KW - molecular docking

KW - pre-steady-state kinetics

KW - SARS-CoV-2

KW - stopped-flow

KW - substrate cleavage

UR - http://www.scopus.com/inward/record.url?scp=85121626068&partnerID=8YFLogxK

U2 - 10.3389/fphar.2021.773198

DO - 10.3389/fphar.2021.773198

M3 - Article

C2 - 34938188

AN - SCOPUS:85121626068

VL - 12

JO - Frontiers in Pharmacology

JF - Frontiers in Pharmacology

SN - 1663-9812

M1 - 773198

ER -