Multiscale Simulations of the Covalent Inhibition of the SARS-CoV-2 Main Protease: Four Compounds and Three Reaction Mechanisms.

We report the results of computational modeling of the reactions of the SARS-CoV-2 main protease (MPro) with four potential covalent inhibitors. Two of them, carmofur and nirmatrelvir, have shown experimentally the ability to inhibit MPro. Two other compounds, X77A and X77C, were designed computationally in this work. They were derived from the structure of X77, a non-covalent inhibitor forming a tight surface complex with MPro. We modified the X77 structure by introducing warheads capable of reacting with the catalytic cysteine residue in the MPro active site. The reaction mechanisms of the four molecules with MPro were investigated by quantum mechanics/molecular mechanics (QM/MM) simulations. The results show that all four compounds form covalent adducts with the catalytic cysteine Cys 145 of MPro. From the chemical perspective, the reactions of these four molecules with MPro follow three distinct mechanisms. The reactions are initiated by a nucleophilic attack of the thiolate group of the deprotonated cysteine residue from the catalytic dyad Cys145-His41 of MPro. In the case of carmofur and X77A, the covalent binding of the thiolate to the ligand is accompanied by the formation of the fluoro-uracil leaving group. The reaction with X77C follows the nucleophilic aromatic substitution SNAr mechanism. The reaction of MPro with nirmatrelvir (which has a reactive nitrile group) leads to the formation of a covalent thioimidate adduct with the thiolate of the Cys145 residue in the enzyme active site. Our results contribute to the ongoing search for efficient inhibitors of the SARS-CoV-2 enzymes.

How Reproducible Are QM/MM Simulations? Lessons from Computational Studies of the Covalent Inhibition of the SARS-CoV-2 Main Protease by Carmofur.

This work explores the level of transparency in reporting the details of computational protocols that is required for practical reproducibility of quantum mechanics/molecular mechanics (QM/MM) simulations. Using the reaction of an essential SARS-CoV-2 enzyme (the main protease) with a covalent inhibitor (carmofur) as a test case of chemical reactions in biomolecules, we carried out QM/MM calculations to determine the structures and energies of the reactants, the product, and the transition state/intermediate using analogous QM/MM models implemented in two software packages, NWChem and Q-Chem. Our main benchmarking goal was to reproduce the key energetics computed with the two packages. Our results indicate that quantitative agreement (within the numerical thresholds used in calculations) is difficult to achieve. We show that rather minor details of QM/MM simulations must be reported in order to ensure the reproducibility of the results and offer suggestions toward developing practical guidelines for reporting the results of biosimulations.

Theoretical insights into the effect of size and substitution patterns of azobenzene derivatives on the DNA G-quadruplex.

Introducing photoswitches into the DNA G-quadruplex provides excellent opportunities to control folding and unfolding of these assemblies, demonstrating their potential in the development of novel nanodevices with medical and nanotechnology applications. Using a quantum mechanics/molecular mechanics (QM/MM) scheme, we carried out a series of simulations to identify the effect of the size and substitution patterns of three azobenzene derivatives (AZ1, AZ2 and AZ3) on the excitation energies of the two lowest excited states of the smallest photoswitchable G-quadruplex reported to date. We demonstrated that the size and the substitution pattern do not affect the ultrafast cis-trans photoiomerization mechanism of the azobenzene derivatives significantly, in agreement with the experiment. However, molecular dynamics simulations revealed that while AZ2 and AZ3 G-quadruplexes are structurally stable during the simulations, the AZ1 G-quadruplex undergoes larger structural changes and shows two ground state populations that differ in the azobenzene backbone adopting two different conformations. AZ1, with para-para substitution pattern, provides more flexibility to the whole G-quadruplex structure compared to AZ2 and AZ3, and can thus facilitate the photoisomerization reaction between a nonpolymorphic, stacked, tetramolecular G-quadruplex and an unstructured state after trans-cis isomerization occurring in a longer time dynamics, in agreement with the experimental findings. The QM/MM simulations of the absorption spectra indicated that the thermal fluctuation plays a more crucial role in the main absorption band of the azobenzene derivatives than the inclusion of the G-quadruplex, implying that the influence of the G-quadruplex environment is minimal. We propose that the latter is attributed to the position of the azobenzene linkers in the G-quadruplexes, i.e. the edgewise loops containing the azobenzene moieties that are located above the G-quartets, not being fully embedded inside or involved in the stacked structure. Our theoretical findings provide support to a recent study of the photoresponsive formation of photoswitchable G-quadruplex motifs.