H172Y mutation perturbs the S1 pocket and nirmatrelvir binding of SARS-CoV-2 main protease through a nonnative hydrogen bond
Vinicius De Oliveira, Mohamed Ibrahim, Xinyuanyuan Sun, Rolf Hilgenfeld, Jana Shen
doi:10.21203/rs.3.rs-1915291/v1
Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID-19 patients. Although the prevalent Mpro mutants in the SARS-CoV-2 Variants of Concern (e.g., Omicron) are still susceptible to nirmatrelvir, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir's inhibitory activity. As the selective pressure of antiviral therapy may favor resistance mutations, there is an urgent need to understand the effect of the H172Y mutation on Mpro's structure, function, and drug resistance. Here we report the molecular dynamics (MD) simulations as well as the measurements of stability, enzyme kinetics of H172Y Mpro, and IC 50 value of nirmatrelvir. Simulations showed that mutation disrupts the interactions between the S1 pocket and N terminus of the opposite protomer. Intriguingly, a native hydrogen bond (H-bond) between Phe140 and the N terminus is replaced by a transient H-bond between Phe140 and Tyr172. In the ligand-free simulations, strengthening of this nonnative H-bond is correlated with disruption of the conserved aromatic stacking between Phe140 and His163, leading to a partial collapse of the oxyanion loop. In the nirmatrelvir-bound simulations, the nonnative H-bond is correlated with the loss of an important H-bond between Glu166 and nirmatrelvir's lactam nitrogen at P1 position. These results are consistent with the newly reported X-ray structures of H172Y Mpro and suggest a mechanism by which the H172Y substitution perturbs the S1 pocket, leading to the decreased structural stability and binding affinity, which in turn explains the drastic reduction in catalytic activity and antiviral susceptibility.
Supplemental Materials Available Supporting Information contains additional analysis of the trajectories and X-ray structures.
Supplementary Files This is a list of supplementary les associated with this preprint. Click to download.
MproH172YSI.pdf
References
Case, Ben-Shalom, Brozell, Cerutti, Cheatham et al., None
Cruzeiro, Darden, Duke, Ghoreishi, Gilson et al., None
Darden, York, Pedersen, Particle Mesh Ewald: An W Log(N) Method for Ewald Sums in Large Systems, J. Chem. Phys
Elbe, Buckland-Merrett, Data, Disease and Diplomacy: GISAID's Innovative Contribution to Global Health, Glob. Chall
Greasley, Noell, Plotnikova, Ferre, Liu et al., Structural Basis for the in Vitro Efficacy of Nirmatrelvir against SARS-CoV-2 Variants, J. Biol. Chem
Hammond, Leister-Tebbe, Gardner, Abreu, Bao et al., Oral Nirmatrelvir for High-Risk, Nonhospitalized Adults with Covid-19, N. Engl. J. Med
Harris, Shen, GPU-Accelerated Implementation of Continuous Constant pH Molecular Dynamics in Amber: pKa Predictions with Single-pH Simulations, J. Chem. Inf. Model
He, Man, Yang, Lee, Wang, A Fast and High-Quality Charge Model for the next Generation General AMBER Force Field, J. Chem. Phys
Henderson, Verma, Harris, Liu, Shen, Assessment of Proton-Coupled Conformational Dynamics of SARS and MERS Coronavirus Papain-like Proteases: Implication for Designing Broad-Spectrum Antiviral Inhibitors, J. Chem. Phys
Hu, Lewandowski, Tan, Morgan, Zhang et al., Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir, bioRxiv
Jorgensen, Chandrasekhar, Madura, Impey, Klein, Comparison of Simple Potential Functions for Simulating Liquid Water, J. Chem. Phys
Lamb, Nirmatrelvir Plus Ritonavir: First Approval, Drugs
Liu, Kati, Chen, Tripathi, Molla et al., Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction, Anal. Biochem
Maier, Martinez, Kasavajhala, Wickstrom, Hauser et al., ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB, J. Chem. Theory Comput
Sacco, Hu, Gongora, Meilleur, Kemp et al., The P132H Mutation in the Main Protease of Omicron SARS-CoV-2 Decreases Thermal Stability without Compromising Catalysis or Small-Molecule Drug Inhibition, Cell Res
Ullrich, Ekanayake, Otting, Nitsche, Main Protease Mutants of SARS-CoV-2 Variants Remain Susceptible to Nirmatrelvir, Bioorg. Med. Chem. Lett
Verma, Henderson, Shen, Proton-Coupled Conformational Activation of SARS Coronavirus Main Proteases and Opportunity for Designing Small-Molecule Broad-Spectrum Targeted Covalent Inhibitors, J. Am. Chem. Soc
Wang, Wolf, Caldwell, Kollman, Case, Development and Testing of a General Amber Force Field, J. Comput. Chem
Webb, Sali, Comparative Protein Structure Modeling Using MODELLER, Current Protocols in Bioinformatics
Yang, Yang, Ding, Liu, Lou et al., The Crystal Structures of Severe Acute Respiratory Syndrome Virus Main Protease and Its Complex with an Inhibitor
Zhang, Lin, Sun, Curth, Drosten et al., Crystal Structure of SARS-CoV-2 Main Protease Provides a Basis for Design of Improved α-Ketoamide Inhibitors, Science
Zhao, Fang, Zhang, Zhang, Zhao et al., Crystal Structure of SARS-CoV-2 Main Protease in Complex with Protease Inhibitor PF-07321332, Protein Cell
DOI record:
{
"DOI": "10.21203/rs.3.rs-1915291/v1",
"URL": "http://dx.doi.org/10.21203/rs.3.rs-1915291/v1",
"abstract": "<jats:title>Abstract</jats:title>\n <jats:p>Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID- 19 patients. Although the prevalent Mpro mutants in the SARS-CoV-2 Variants of Concern (e.g., Omicron) are still susceptible to nirmatrelvir, a rare mutation, H172Y, was found to significantly reduce nirmatrelvir’s inhibitory activity. As the selective pres- sure of antiviral therapy may favor resistance mutations, there is an urgent need to understand the effect of H172Y mutation on Mpro’s structure, function, and drug re- sistance. Here we report the molecular dynamics (MD) simulations as well as the measurements of stability, enzyme kinetics of H172Y Mpro, and IC50 value of nir- matrelvir. Simulations showed that mutation disrupts the interactions between the S1 pocket and N terminus of the opposite protomer. Intriguingly, a native hydrogen bond (H-bond) between Phe140 and the N terminus is replaced by a transient H- bond between Phe140 and Tyr172. In the ligand-free simulations, strengthening of this nonnative H-bond is correlated with disruption of the conserved aromatic stacking between Phe140 and His163, leading to a partial collapse of the oxyanion loop. In the nirmatrelvir-bound simulations, the nonnative H-bond is correlated with the loss of an important H-bond between Glu166 and nirmatrelvir’s lactam nitrogen at P1 position. These results are consistent with the newly reported X-ray structures of H172Y Mpro and suggest a mechanism by which the H172Y substitution perturbs the S1 pocket, leading to the decreased structural stability and binding affinity, which in turns explains the drastic reduction in catalytic activity and antiviral susceptibility.</jats:p>",
"accepted": {
"date-parts": [
[
2022,
7,
31
]
]
},
"author": [
{
"affiliation": [
{
"name": "University of Maryland School of Pharmacy"
}
],
"family": "de Oliveira",
"given": "Vinicius",
"sequence": "first"
},
{
"affiliation": [
{
"name": "University of Luebeck"
}
],
"family": "Ibrahim",
"given": "Mohamed",
"sequence": "additional"
},
{
"affiliation": [
{
"name": "University of Luebeck"
}
],
"family": "Sun",
"given": "Xinyuanyuan",
"sequence": "additional"
},
{
"affiliation": [
{
"name": "University of Luebeck"
}
],
"family": "Hilgenfeld",
"given": "Rolf",
"sequence": "additional"
},
{
"ORCID": "http://orcid.org/0000-0002-3234-0769",
"affiliation": [
{
"name": "University of Maryland School of Pharmacy"
}
],
"authenticated-orcid": false,
"family": "Shen",
"given": "Jana",
"sequence": "additional"
}
],
"container-title": [],
"content-domain": {
"crossmark-restriction": false,
"domain": []
},
"created": {
"date-parts": [
[
2022,
8,
9
]
],
"date-time": "2022-08-09T16:51:06Z",
"timestamp": 1660063866000
},
"deposited": {
"date-parts": [
[
2022,
8,
9
]
],
"date-time": "2022-08-09T16:51:07Z",
"timestamp": 1660063867000
},
"group-title": "In Review",
"indexed": {
"date-parts": [
[
2022,
8,
10
]
],
"date-time": "2022-08-10T04:13:59Z",
"timestamp": 1660104839594
},
"institution": [
{
"name": "Research Square"
}
],
"is-referenced-by-count": 0,
"issued": {
"date-parts": [
[
2022,
8,
9
]
]
},
"license": [
{
"URL": "https://creativecommons.org/licenses/by/4.0/",
"content-version": "unspecified",
"delay-in-days": 0,
"start": {
"date-parts": [
[
2022,
8,
9
]
],
"date-time": "2022-08-09T00:00:00Z",
"timestamp": 1660003200000
}
}
],
"link": [
{
"URL": "https://www.researchsquare.com/article/rs-1915291/v1",
"content-type": "text/html",
"content-version": "vor",
"intended-application": "text-mining"
},
{
"URL": "https://www.researchsquare.com/article/rs-1915291/v1.html",
"content-type": "unspecified",
"content-version": "vor",
"intended-application": "similarity-checking"
}
],
"member": "8761",
"original-title": [],
"posted": {
"date-parts": [
[
2022,
8,
9
]
]
},
"prefix": "10.21203",
"published": {
"date-parts": [
[
2022,
8,
9
]
]
},
"publisher": "Research Square Platform LLC",
"reference-count": 0,
"references-count": 0,
"relation": {},
"resource": {
"primary": {
"URL": "https://www.researchsquare.com/article/rs-1915291/v1"
}
},
"score": 1,
"short-title": [],
"source": "Crossref",
"subtitle": [],
"subtype": "preprint",
"title": "H172Y mutation perturbs the S1 pocket and nirmatrelvir binding of SARS-CoV-2 main protease through a nonnative hydrogen bond",
"type": "posted-content"
}
