{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T12:38:29Z","timestamp":1781613509441,"version":"3.54.5"},"reference-count":31,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2025,9,20]],"date-time":"2025-09-20T00:00:00Z","timestamp":1758326400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100010031","name":"Postdoctoral Research Foundation of China","doi-asserted-by":"publisher","award":["2023M744315"],"award-info":[{"award-number":["2023M744315"]}],"id":[{"id":"10.13039\/501100010031","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82404415"],"award-info":[{"award-number":["82404415"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background\/Objectives: This study identifies novel dihydroorotate dehydrogenase (DHODH) inhibitors exhibiting potent broad-spectrum antiviral agents, particularly against influenza A virus (A\/PR\/8\/34(H1N1)) and SARS-CoV-2. Methods: Structure-based virtual screening of 1.6 million compounds (ChemDiv and TargetMol databases) yielded 10 candidates, with compounds 6, 9, and 10 demonstrating significant anti-influenza activity (IC50 = 4.85 \u00b1 0.58, 7.35 \u00b1 1.65, and 1.75 \u00b1 0.28 \u03bcM, respectively). Building on these, molecular hybridization principles and scaffold hopping principles were applied to design and synthesize six novel compounds (11\u201316) through cyclization, coupling, and carboxylate deprotection. Prior to subsequent biological assays, the molecular structures of each compound were elucidated by NMR spectroscopy and MS. Their antiviral activities were subsequently assessed against both influenza virus and SARS-CoV-2. The compound 11, demonstrating the most potent antiviral activity, was further subjected to surface plasmon resonance (SPR) analysis to assess its binding affinity for human DHODH. Results: Compound 11 emerged as the most potent DHODH inhibitor (KD = 6.06 \u03bcM), exhibiting superior broad-spectrum antiviral activities (IC50 = 0.85 \u00b1 0.05 \u03bcM, A\/PR\/8\/34(H1N1); IC50 = 3.60 \u00b1 0.67 \u03bcM, SARS-CoV-2) to the reported DHODH inhibitor (Teriflunomide, IC50 = 35.02 \u00b1 3.33 \u03bcM, A\/PR\/8\/34(H1N1); IC50 = 26.06 \u00b1 4.32 \u03bcM, SARS-CoV-2). Mechanistic evaluations via 100 ns MD simulations and QM\/MM calculations revealed stable binding interactions, particularly hydrogen bonds with GLN47 and ARG136, while alanine scanning mutagenesis confirmed these residues\u2019 critical roles in binding stability. Conclusions: This work identifies compound 11 as a potent broad-spectrum antiviral compound, offering a promising strategy for broad-spectrum antiviral therapy against RNA viruses by depleting pyrimidine pools essential for viral replication.<\/jats:p>","DOI":"10.3390\/ph18091416","type":"journal-article","created":{"date-parts":[[2025,9,22]],"date-time":"2025-09-22T08:37:39Z","timestamp":1758530259000},"page":"1416","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Comprehensive Identification and Mechanistic Evaluation of Novel DHODH Inhibitors as Potent Broad-Spectrum Antiviral Agents"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0166-7873","authenticated-orcid":false,"given":"Chao","family":"Zhang","sequence":"first","affiliation":[{"name":"State Key Laboratory of National Security Specially Needed Medicines, Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shiyang","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of National Security Specially Needed Medicines, Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huiru","family":"Xie","sequence":"additional","affiliation":[{"name":"State Key Laboratory of National Security Specially Needed Medicines, Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongzhao","family":"Ding","sequence":"additional","affiliation":[{"name":"State Key Laboratory of National Security Specially Needed Medicines, Beijing 100039, China"},{"name":"Key Laboratory of Structure-Based Drug Design Discovery, Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2919-2870","authenticated-orcid":false,"given":"Chun","family":"Hu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Structure-Based Drug Design Discovery, Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jialin","family":"Guo","sequence":"additional","affiliation":[{"name":"State Key Laboratory of National Security Specially Needed Medicines, Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junhai","family":"Xiao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of National Security Specially Needed Medicines, Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1582","DOI":"10.1016\/j.jmb.2012.12.019","article-title":"Three sites and you are out: Ternary synergistic allostery controls aromatic amino acid biosynthesis in Mycobacterium tuberculosis","volume":"425","author":"Blackmore","year":"2013","journal-title":"J. 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