Contribution of the catalytic dyad of SARS-CoV-2 main protease to binding covalent and noncovalent inhibitors

Kovalevsky, Andrey Y.; Aniana, Annie; Coates, Leighton; Bonnesen, Peter V.; Nashed, Nashaat T.; Louis, John M.

doi:10.1016/j.jbc.2023.104886

Contribution of the catalytic dyad of SARS-CoV-2 main protease to binding covalent and noncovalent inhibitors

Journal Article · Fri Jun 02 00:00:00 EDT 2023 · Journal of Biological Chemistry

DOI:https://doi.org/10.1016/j.jbc.2023.104886· OSTI ID:1987791

^[1]; ^[2]; ^[3]; ^[4]; Nashed, Nashaat T. ^[2]; ^[2]

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Neutron Scattering Division
National Institutes of Health (NIH), Bethesda, MD (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Sciences (CNMS)

The effect of mutations of the catalytic dyad residues of SARS-CoV-2 main protease (MPro^WT) on the thermodynamics of binding of covalent inhibitors comprising nitrile [nirmatrelvir (NMV), NBH2], aldehyde (GC373), and ketone (BBH1) warheads to MPro is examined together with room temperature X-ray crystallography. When lacking the nucleophilic C145, NMV binding is ~400-fold weaker corresponding to 3.5 kcal/mol and 13.3 °C decrease in free energy (ΔG) and thermal stability (T_m), respectively, relative to MPro^WT. The H41A mutation results in a 20-fold increase in the dissociation constant (K_d), and 1.7 kcal/mol and 1.4 °C decreases in ΔG and T_m, respectively. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MPro^H41A, whereas no significant change is observed in binding to MPro^WT. Structures of the four inhibitor complexes with MPro^1-304/C145A show that the active site geometries of the complexes are nearly identical to that of MPro^WT with the nucleophilic sulfur of C145 positioned to react with the nitrile or the carbonyl carbon. These results support a two-step mechanism for the formation of the covalent complex involving an initial non-covalent binding followed by a nucleophilic attack by the thiolate anion of C145 on the warhead carbon. Noncovalent inhibitor ensitrelvir (ESV) exhibits a binding affinity to MPro^WT that is similar to NMV but differs in its thermodynamic signature from NMV. The binding of ESV to MPro^C145A also results in a significant, but smaller, increase in K_d and decrease in ΔG and T_m, relative to NMV.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1987791

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 7 Vol. 299; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
C145A/H41A mutations
SARS-CoV-2 main protease
inhibitor binding thermodynamics
main protease inhibitors
room-temperature X-ray crystallography
thermal stability

Contribution of the catalytic dyad of SARS-CoV-2 main protease to binding covalent and noncovalent inhibitors

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References (49)

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