The impact of SARS-CoV-2 3CL protease mutations on nirmatrelvir inhibitory efficiency. Computational insights into potential resistance mechanisms† † Electronic supplementary information (ESI) available: Complete table with residue contributions to the MMGBSA binding free energy of nirmatrelvir and the peptide substrate;distribution of distances between 166 and N-terminal residues in wild type and E166V variants of 3CLpro;distribution of distances between residues 166 and the N-terminal in wild type and E166V mutants;TS structures for the acylation of a peptide substrate and nirmatrelvir in the active site of 3CLpro;B3LYPD3/MM free energy profiles for the reaction of nirmatrelvir with the wild type and E166V variants of 3CLpro;link to the coordinates from the trajectories of the TS for the reaction of nirmatrelvir. See DOI: https://doi.org/10.1039/d2sc06584c

The use of antiviral drugs can promote the appearance of mutations in the target protein that increase the resistance of the virus to the treatment. This is also the case of nirmatrelvir, a covalent inhibitor of the 3CL protease, or main protease, of SARS-CoV-2. In this work we show how the by-residue decomposition of noncovalent interactions established between the drug and the enzyme, in combination with an analysis of naturally occurring mutations, can be used to detect potential mutations in the 3CL protease conferring resistance to nirmatrelvir. We also investigate the consequences of these mutations on the reaction mechanism to form the covalent enzyme-inhibitor complex using QM/MM methods. In particular, we show that the E166V variant of the protease displays smaller binding affinity to nirmatrelvir and larger activation free energy for the formation of the covalent complex, both factors contributing to the observed resistance to the treatment with this drug. The conclusions derived from our work can be used to anticipate the consequences of the introduction of nirmatrelvir in the fitness landscape of the virus and to design new inhibitors adapted to some of the possible resistance mechanisms. The use of antiviral drugs can promote the appearance of mutations in the target protein that increase the resistance of the virus to the treatment.

The impact of SARS-CoV-2 3CL protease mutations on nirmatrelvir inhibitory efficiency. Computational insights into potential resistance mechanisms.

The use of antiviral drugs can promote the appearance of mutations in the target protein that increase the resistance of the virus to the treatment. This is also the case of nirmatrelvir, a covalent inhibitor of the 3CL protease, or main protease, of SARS-CoV-2. In this work we show how the by-residue decomposition of noncovalent interactions established between the drug and the enzyme, in combination with an analysis of naturally occurring mutations, can be used to detect potential mutations in the 3CL protease conferring resistance to nirmatrelvir. We also investigate the consequences of these mutations on the reaction mechanism to form the covalent enzyme-inhibitor complex using QM/MM methods. In particular, we show that the E166V variant of the protease displays smaller binding affinity to nirmatrelvir and larger activation free energy for the formation of the covalent complex, both factors contributing to the observed resistance to the treatment with this drug. The conclusions derived from our work can be used to anticipate the consequences of the introduction of nirmatrelvir in the fitness landscape of the virus and to design new inhibitors adapted to some of the possible resistance mechanisms.

Inhibition Mechanism of SARS-CoV-2 Main Protease with Ketone-Based Inhibitors Unveiled by Multiscale Simulations: Insights for Improved Designs*.

We present the results of classical and QM/MM simulations for the inhibition of SARS-CoV-2 3CL protease by a hydroxymethylketone inhibitor, PF-00835231. In the noncovalent complex the carbonyl oxygen atom of the warhead is placed in the oxyanion hole formed by residues 143 to 145, while P1-P3 groups are accommodated in the active site with interactions similar to those observed for the peptide substrate. According to alchemical free energy calculations, the P1' hydroxymethyl group also contributes to the binding free energy. Covalent inhibition of the enzyme is triggered by the proton transfer from Cys145 to His41. This step is followed by the nucleophilic attack of the Sγ atom on the carbonyl carbon atom of the inhibitor and a proton transfer from His41 to the carbonyl oxygen atom mediated by the P1' hydroxyl group. Computational simulations show that the addition of a chloromethyl substituent to the P1' group may lower the activation free energy for covalent inhibition.

A microscopic description of SARS-CoV-2 main protease inhibition with Michael acceptors. Strategies for improving inhibitor design.

The irreversible inhibition of the main protease of SARS-CoV-2 by a Michael acceptor known as N3 has been investigated using multiscale methods. The noncovalent enzyme-inhibitor complex was simulated using classical molecular dynamics techniques and the pose of the inhibitor in the active site was compared to that of the natural substrate, a peptide containing the Gln-Ser scissile bond. The formation of the covalent enzyme-inhibitor complex was then simulated using hybrid QM/MM free energy methods. After binding, the reaction mechanism was found to be composed of two steps: (i) the activation of the catalytic dyad (Cys145 and His41) to form an ion pair and (ii) a Michael addition where the attack of the Sγ atom of Cys145 to the Cß atom of the inhibitor precedes the water-mediated proton transfer from His41 to the Cα atom. The microscopic description of protease inhibition by N3 obtained from our simulations is strongly supported by the excellent agreement between the estimated activation free energy and the value derived from kinetic experiments. Comparison with the acylation reaction of a peptide substrate suggests that N3-based inhibitors could be improved by adding chemical modifications that could facilitate the formation of the catalytic dyad ion pair.

Multiscale Simulations of SARS-CoV-2 3CL Protease Inhibition with Aldehyde Derivatives. Role of Protein and Inhibitor Conformational Changes in the Reaction Mechanism.

We here investigate the mechanism of SARS-CoV-2 3CL protease inhibition by one of the most promising families of inhibitors, those containing an aldehyde group as a warhead. These compounds are covalent inhibitors that inactivate the protease, forming a stable hemithioacetal complex. Inhibitor 11a is a potent inhibitor that has been already tested in vitro and in animals. Using a combination of classical and QM/MM simulations, we determined the binding mode of the inhibitor into the active site and the preferred rotameric state of the catalytic histidine. In the noncovalent complex, the aldehyde group is accommodated into the oxyanion hole formed by the NH main-chain groups of residues 143 to 145. In this pose, P1-P3 groups of the inhibitor mimic the interactions established by the natural peptide substrate. The reaction is initiated with the formation of the catalytic dyad ion pair after a proton transfer from Cys145 to His41. From this activated state, covalent inhibition proceeds with the nucleophilic attack of the deprotonated Sγ atom of Cys145 to the aldehyde carbon atom and a water-mediated proton transfer from the Nε atom of His41 to the aldehyde oxygen atom. Our proposed reaction transition-state structure is validated by comparison with X-ray data of recently reported inhibitors, while the activation free energy obtained from our simulations agrees with the experimentally derived value, supporting the validity of our findings. Our study stresses the interplay between the conformational dynamics of the inhibitor and the protein with the inhibition mechanism and the importance of including conformational diversity for accurate predictions about the inhibition of the main protease of SARS-CoV-2. The conclusions derived from our work can also be used to rationalize the behavior of other recently proposed inhibitor compounds, including aldehydes and ketones with high inhibitory potency.

Unraveling the SARS-CoV-2 Main Protease Mechanism Using Multiscale Methods.

We present a detailed theoretical analysis of the reaction mechanism of proteolysis catalyzed by the main protease of SARS-CoV-2. Using multiscale simulation methods, we have characterized the interactions established by a peptidic substrate in the active site, and then we have explored the free energy landscape associated with the acylation and deacylation steps of the proteolysis reaction, characterizing the transition states of the process. Our mechanistic proposals can explain most of the experimental observations made on the highly similar ortholog protease of SARS-CoV. We point to some key interactions that may facilitate the acylation process and thus can be crucial in the design of more specific and efficient inhibitors of the main protease activity. In particular, from our results, the P1' residue can be a key factor to improve the thermodynamics and kinetics of the inhibition process.

Computational simulations on the binding and reactivity of a nitrile inhibitor of the SARS-CoV-2 main protease.

We present a detailed computational analysis of the binding mode and reactivity of the novel oral inhibitor PF-07321332 developed against the SARS-CoV-2 3CL protease. Alchemical free energy calculations suggest that positions P3 and P4 could be susceptible to improvement in order to get a larger binding strength. QM/MM simulations unveil the reaction mechanism for covalent inhibition, showing that the nitrile warhead facilitates the recruitment of a water molecule for the proton transfer step.