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

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

  • J Biol Chem. 2023 Jun 2;104886. doi: 10.1016/j.jbc.2023.104886.
Andrey Kovalevsky 1 Annie Aniana 2 Leighton Coates 3 Peter V Bonnesen 4 Nashaat T Nashed 2 John M Louis 5
Affiliations

Affiliations

  • 1 Neutron Scattering Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN, 37831, USA. Electronic address: kovalevskyay@ornl.gov.
  • 2 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892-0520, USA.
  • 3 Second Target Station, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN, 37831, USA.
  • 4 Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA.
  • 5 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892-0520, USA. Electronic address: johnl@niddk.nih.gov.
Abstract

The effect of mutations of the catalytic dyad residues of SARS-CoV-2 main Protease (MProWT) 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 decreases in free energy (ΔG) and thermal stability (Tm), respectively, relative to MProWT. The H41A mutation results in a 20-fold increase in the dissociation constant (Kd), and 1.7 kcal/mol and 1.4 °C decreases in ΔG and Tm, respectively. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MProH41A, whereas no significant change is observed in binding to MProWT. Structures of the four inhibitor complexes with MPro1-304/C145A show that the active site geometries of the complexes are nearly identical to that of MProWT 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 MProWT that is similar to NMV but differs in its thermodynamic signature from NMV. The binding of ESV to MProC145A also results in a significant, but smaller, increase in Kd and decrease in ΔG and Tm, relative to NMV.

Keywords

C145A/H41A mutations; SARS-CoV-2 main protease; inhibitor binding thermodynamics; main protease inhibitors; room-temperature X-ray crystallography; thermal stability.

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