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 decrease 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.
| Original language | English |
|---|---|
| Article number | 104886 |
| Journal | Journal of Biological Chemistry |
| Volume | 299 |
| Issue number | 7 |
| DOIs | |
| State | Published - Jul 2023 |
Funding
This work was funded by NIDDK (Project number: DK075166-01) to J. M. L. This research used resources at the Spallation Neutron Source, the High Flux Isotope Reactor, and the Center for Nanophase Material Sciences for the synthesis of BBH1 and NBH2, which are DOE Office of Science User Facilities operated by the Oak Ridge National Laboratory. The Office of Biological and Environmental Research supported research at ORNL's Center for Structural Molecular Biology (CSMB), a DOE Office of Science User Facility. ORNL is managed by UT-Battelle LLC for DOE's Office of Science, the single largest supporter of basic research in the physical sciences in the United States. We thank John Lloyd and the NIDDK mass spectrometry core facility. This work was supported by the Intramural Research Program of National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, United States. A. Y. K and J. M. L. Conceptualization; A. Y. K. J. M. L. A. A. L. C. P. V. B. and N. T. N. Methodology; A. Y. K. J. M. L. A. A. L. C. P. V. B. and N. T. N. investigation; A. Y. K and J. M. L. Writing – original draft. A. A. L. C. P. V. B. and N. T. N. writing – review and editing. This work was funded by NIDDK (Project number: DK075166-01) to J. M. L. This research used resources at the Spallation Neutron Source, the High Flux Isotope Reactor, and the Center for Nanophase Material Sciences for the synthesis of BBH1 and NBH2, which are DOE Office of Science User Facilities operated by the Oak Ridge National Laboratory. The Office of Biological and Environmental Research supported research at ORNL's Center for Structural Molecular Biology (CSMB), a DOE Office of Science User Facility. ORNL is managed by UT-Battelle LLC for DOE’s Office of Science, the single largest supporter of basic research in the physical sciences in the United States. We thank John Lloyd and the NIDDK mass spectrometry core facility. This work was supported by the Intramural Research Program of National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, United States .
Keywords
- C145A/H41A mutations
- SARS-CoV-2 main protease
- inhibitor binding thermodynamics
- main protease inhibitors
- room-temperature X-ray crystallography
- thermal stability
