{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T02:35:35Z","timestamp":1789180535945,"version":"build-2803163510"},"reference-count":46,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,19]],"date-time":"2026-01-19T00:00:00Z","timestamp":1768780800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"High-Performance Computer Center of Dali University"},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31960198"],"award-info":[{"award-number":["31960198"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31860243"],"award-info":[{"award-number":["31860243"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan","award":["2024KF003"],"award-info":[{"award-number":["2024KF003"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The clinical durability of SARS-CoV-2 main protease (Mpro) inhibitors depends on their resilience to emerging resistance mutations. Recent genomic surveillance and functional reports have highlighted substitutions at positions 49, 165, and 301, raising questions about the robustness of the noncovalent inhibitor WU-04 in variant backgrounds. Here, we combined \u03bcs-scale, triplicate molecular dynamics simulations with end-state binding free energy estimates and a network-rewiring inference (NRI) framework that maps long-range dynamical communication across the full protease dimer. We evaluated wild type (WT), single mutants M49K, M165V, S301P, and selected double mutants (M49K &amp; M165V, M49K &amp; S301P). Relative to WT, single substitutions produced reductions in computed binding affinity of up to ~12kcal\/mol, accompanied by loss or reshaping of the S2 subsite and altered ligand burial. Notably, the M49K\/S301P double mutant partially restored WU-04 engagement, narrowing the \u0394\u0394Grestore gap to within \u0394\u0394Grestore of WT and re-establishing key hydrophobic and hydrogen-bond contacts. NRI analysis revealed that distal residue 301 participates in a communication corridor linking the C-terminal helical domain to the active-site cleft; its substitution rewires inter-domain coupling that can compensate for local disruptions at residue 49. Together, these results identify structural hotspots and network pathways that may inform the design of next-generation Mpro inhibitors with improved mutation tolerance\u2014specifically by strengthening interactions that do not rely solely on the mutable S2 pocket and by engaging conserved backbone features near the 165\u2013166 region.<\/jats:p>","DOI":"10.3390\/ijms27021000","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T08:36:33Z","timestamp":1768898193000},"page":"1000","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Mutation-Induced Resistance of SARS-CoV-2 Mpro to WU-04 Revealed by Multi-Scale Modeling"],"prefix":"10.3390","volume":"27","author":[{"given":"Mengting","family":"Liu","sequence":"first","affiliation":[{"name":"College of Pharmacy, Dali University, Dali 671003, China"},{"name":"College of Agriculture and Biological Science, Dali University, Dali 671003, China"},{"name":"Key Laboratory of Bioinformatics and Computational Biology, Department of Education of Yunnan Province, Dali University, Dali 671003, China"},{"name":"Co-Innovation Center for Cangshan Mountain and Erhai Lake Integrated Protection and Green Development of Yunnan Province, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Derui","family":"Zhao","sequence":"additional","affiliation":[{"name":"College of Agriculture and Biological Science, Dali University, Dali 671003, China"},{"name":"Key Laboratory of Bioinformatics and Computational Biology, Department of Education of Yunnan Province, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Duan","sequence":"additional","affiliation":[{"name":"College of Agriculture and Biological Science, Dali University, Dali 671003, China"},{"name":"Key Laboratory of Bioinformatics and Computational Biology, Department of Education of Yunnan Province, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junyao","family":"Zhu","sequence":"additional","affiliation":[{"name":"College of Agriculture and Biological Science, Dali University, Dali 671003, China"},{"name":"Key Laboratory of Bioinformatics and Computational Biology, Department of Education of Yunnan Province, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liting","family":"Zheng","sequence":"additional","affiliation":[{"name":"College of Agriculture and Biological Science, Dali University, Dali 671003, China"},{"name":"Key Laboratory of Bioinformatics and Computational Biology, Department of Education of Yunnan Province, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nan","family":"Yuan","sequence":"additional","affiliation":[{"name":"College of Agriculture and Biological Science, Dali University, Dali 671003, China"},{"name":"Key Laboratory of Bioinformatics and Computational Biology, Department of Education of Yunnan Province, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3196-7065","authenticated-orcid":false,"given":"Yuanling","family":"Xia","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, School of Life Sciences, Yunnan University, Kunming 650091, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Sang","sequence":"additional","affiliation":[{"name":"College of Agriculture and Biological Science, Dali University, Dali 671003, China"},{"name":"Key Laboratory of Bioinformatics and Computational Biology, Department of Education of Yunnan Province, Dali University, Dali 671003, China"},{"name":"Co-Innovation Center for Cangshan Mountain and Erhai Lake Integrated Protection and Green Development of Yunnan Province, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liquan","family":"Yang","sequence":"additional","affiliation":[{"name":"College of Agriculture and Biological Science, Dali University, Dali 671003, China"},{"name":"Key Laboratory of Bioinformatics and Computational Biology, Department of Education of Yunnan Province, Dali University, Dali 671003, China"},{"name":"Co-Innovation Center for Cangshan Mountain and Erhai Lake Integrated Protection and Green Development of Yunnan Province, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,19]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2025, December 26). 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