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

Ramos-Guzmán, Carlos A.; Andjelkovic, Milorad; Zinovjev, Kirill; Ruiz‐Pernía, J. Javier; Tuñón, Iñaki

doi:10.1039/d2sc06584c

Cited by 13 publications

(39 citation statements)

References 71 publications

(114 reference statements)

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“…74 The CHARMM36 force-field 75 was used in the MM subsystems. Sizes of QM subsystems in these calculations were somewhat smaller than in the QM/MM optimization, but considerably larger than, for example, in previous studies [25][26][27] of the M Pro -nirmatrelvir reaction. We performed umbrella sampling simulations with additional harmonic potentials centered at different collective variable values.…”

Section: System Preparation and Computational Protocolsmentioning

confidence: 73%

“…In this case, the IP intermediate is not clearly visible on the free energy surface; the free energy profile at the step resembles the features reported by Ramos-Guzmán et al in modeling other reactions of covalent inhibition of M Pro . [24][25][26]34 In contrast, the MC intermediate corresponds to the minimum-energy structure in both the QM/MM and QM/MM MD calculations. We note that the nature of the Meisenheimer complex in the SNAr reactions 45,76,77 is still debated, in particular, whether it represents a reaction intermediate or a transition state.…”

Section: Reaction Of M Pro With X77cmentioning

confidence: 99%

“…Ramos-Guzmán et al 25,26 computed the minimum free energy path for the nirmatrelvir covalent binding to M Pro using the adaptive string method with QM/MM potentials. The QM subsystem composed of the fragments of Cys145 and His41, a water molecule and the warhead of the inhibitor (about 50 atoms in total), was described at the B3LYP-D3/6-31+G* level in Ref.…”

Section: Reaction Of M Pro With Nirmatrelvirmentioning

confidence: 99%

“…25 and at the M06-2X-D3/6-31+G* level in Ref. 26. The path was determined through biased MD simulations using 7 collective variables that included the distances of all the bonds being broken, formed or whose formal order changed during the process.…”

Section: Reaction Of M Pro With Nirmatrelvirmentioning

confidence: 99%

“…QM/MM-based computer simulations provide insights into cysteine protease reaction mechanisms and can be used to predict novel compounds as prospective drugs. [22][23][24][25][26][27][28][29][30][31][32][33][34][35] Numerous studies investigated irreversible (or covalent) inhibitors of cysteine proteases (a class to which M Pro belongs). 12,[28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] The list of prospective inhibitors is growing, but mechanisms of their interaction with the enzyme are not yet fully elucidated.…”

Section: Introductionmentioning

confidence: 99%

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QM/MM Simulations of the Covalent Inhibition of the SARS-CoV-2 Main Protease: Four Compounds and Three Reaction Mechanisms

Grigorenko

Polyakov

Khrenova

et al. 2023

Preprint

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The search for efficient inhibitors of the SARS-CoV-2 enzymes is ongoing due to the continuing COVID-19 pandemic. 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 been shown experimentally the ability to inhibit MPro . Two other compounds, X77A and X77C, were designed computationally in this work, 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 efficient chemical reactions with the catalytic cysteine residue in the M Pro active site. The reaction mechanisms of the four molecules with M Pro were investigated by quantum mechanics/molecular mechanics (QM/MM) calculations using large quantum subsystems. First, at the QM/MM level, we optimized structures of stationary points on the potential energy surfaces corresponding to the reactants, products, intermediates, and transition states along the hypothesized reaction coordinates. Analysis of these structures has informed the selection of collective variables for the subsequent calculations of the Gibbs energy profiles using molecular dynamics simulations with QM/MM potentials (QM/MM MD). In these simulations, the QM part was treated by DFT with the PBE0 functional. 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 compounds with M Pro follow three distinct mechanisms. In all cases, the reaction is 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 SN Ar mechanism. The reaction of M Pro with nirmatrelvir, which has a reactive nitrile group, leads to the formation of the covalent thioimidate adduct with the thiolate of the Cys145 residue in the enzyme active site.

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Section: System Preparation and Computational Protocolsmentioning

confidence: 73%

Section: Reaction Of M Pro With X77cmentioning

confidence: 99%

Section: Reaction Of M Pro With Nirmatrelvirmentioning

confidence: 99%

Section: Reaction Of M Pro With Nirmatrelvirmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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QM/MM Simulations of the Covalent Inhibition of the SARS-CoV-2 Main Protease: Four Compounds and Three Reaction Mechanisms

Grigorenko

Polyakov

Khrenova

et al. 2023

Preprint

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Pomotrelvir and Nirmatrelvir Binding and Reactivity with SARS‐CoV‐2 Main Protease: Implications for Resistance Mechanisms from Computations

Schillings,

Ramos‐Guzmán,

Ruiz‐Pernía

et al. 2024

Angewandte Chemie

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We investigate the inhibition mechanism between pomotrelvir and the SARS‐CoV‐2 main protease using molecular mechanics and quantum mechanics / molecular mechanics simulations. Alchemical transformations where each Pi group of pomotrelvir was transformed into its counterpart in nirmatrelvir were performed to unravel the individual contribution of each group to the binding and reaction processes. We have shown that while a γ‐lactam ring is preferred at position P1, a δ‐lactam ring is a reasonable alternative. For the P2 position, tertiary amines are preferred with respect to secondary amines. Flexible side chains at P2 position can disrupt the preorganization of the active site, favouring the exploration of non‐reactive conformations. The substitution of the P2 group of pomotrelvir by that of nirmatrelvir resulted in a compound, named as C2, that presents better binding free energy and a higher population of reactive conformations in the Michaelis complex. Analysis of the chemical reaction to form the covalent complex has shown a similar reaction mechanism and activation free energies for pomotrelvir, nirmatrelvir and C2. We hope that these findings could be useful to design better inhibitors to fight present and future variants of SARS‐CoV‐2 virus.

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Molecular Mechanism of Labelling Functional Cysteines by Heterocyclic Thiones

Mihalovits,

Kollár,

Bajusz

et al. 2023

ChemPhysChem

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Heterocyclic thiones have recently been identified as reversible covalent warheads, consistent with their mild electrophilic nature. Little is known so far about their mechanism of action in labelling nucleophilic sidechains, especially cysteines. The vast number of tractable cysteines promotes a wide range of target proteins to examine; however, our focus was put on functional cysteines. We chose the main protease of SARS‐CoV‐2 harboring Cys145 at the active site that is a structurally characterized and clinically validated target of covalent inhibitors. We screened an in‐house, cysteine‐targeting covalent inhibitor library which resulted in several covalent fragment hits with benzoxazole, benzothiazole and benzimidazole cores. Thione derivatives and Michael acceptors were selected for further investigations with the objective of exploring the mechanism of inhibition of the thiones and using the thoroughly characterized Michael acceptors for benchmarking our studies. Classical and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were carried out that revealed a new mechanism of covalent cysteine labelling by thione derivatives, which was supported by QM and free energy calculations and by a wide range of experimental results. Our study shows that the molecular recognition step plays a crucial role in the overall binding of both sets of molecules.

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

Abstract: 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...

Cited by 13 publications

References 71 publications

QM/MM Simulations of the Covalent Inhibition of the SARS-CoV-2 Main Protease: Four Compounds and Three Reaction Mechanisms

QM/MM Simulations of the Covalent Inhibition of the SARS-CoV-2 Main Protease: Four Compounds and Three Reaction Mechanisms

Pomotrelvir and Nirmatrelvir Binding and Reactivity with SARS‐CoV‐2 Main Protease: Implications for Resistance Mechanisms from Computations

Molecular Mechanism of Labelling Functional Cysteines by Heterocyclic Thiones

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