{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T22:36:50Z","timestamp":1785191810215,"version":"3.55.0"},"reference-count":38,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,5,29]],"date-time":"2025-05-29T00:00:00Z","timestamp":1748476800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2021YFD1800303"],"award-info":[{"award-number":["2021YFD1800303"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["32473044"],"award-info":[{"award-number":["32473044"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Natural Science Foundation of China","award":["2021YFD1800303"],"award-info":[{"award-number":["2021YFD1800303"]}]},{"name":"National Natural Science Foundation of China","award":["32473044"],"award-info":[{"award-number":["32473044"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Background: The 2C protein of foot-and-mouth disease virus (FMDV), a member of helicase superfamily 3 (SF3), drives viral genome replication and serves as a critical target for antiviral drug development. Methods: A fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) platform was developed to identify 2C helicase inhibitors. Primary screening evaluated 4424 compounds for helicase inhibition. Molecular docking analyzed inhibitor interactions with the N207 residue within the catalytic core and helicase inhibition assays classified the inhibitor type (mixed, competitive, noncompetitive). Differential scanning fluorimetry (nanoDSF) quantified 2C thermal destabilization. Antiviral activity was assessed via indirect immunofluorescence, RT-qPCR, and plaque reduction assays. Results: Six compounds inhibited 2C helicase activity at &gt;620 \u03bcM. Molecular docking revealed hydrogen bonding, hydrophobic interactions, and \u03c0-cation stabilization at the catalytic core. 2-MPO and MPPI were classified as mixed-type inhibitors, 5-TzS\u2212 and 2-PyOH as competitive, and DCMQ\/Spiro-BD-CHD-dione as noncompetitive. NanoDSF showed a \u0394Tm \u2265 1.5 \u00b0C (2.5 mM compounds), with reduced destabilization in N207A mutants. Antiviral assays identified 2-MPO and MPPI as optimal inhibitors. MPPI achieved effective FMDV suppression at 160 \u03bcM, exhibiting two orders of magnitude higher potency than 2-MPO (400 \u03bcM). Conclusions: The established FRET-based HTS platform targeting 2C helicase facilitates anti-FMDV lead discovery, while 2C inhibitors may serve as an effective therapeutic strategy against other picornaviruses.<\/jats:p>","DOI":"10.3390\/v17060785","type":"journal-article","created":{"date-parts":[[2025,5,30]],"date-time":"2025-05-30T08:07:58Z","timestamp":1748592478000},"page":"785","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Discovery of Small Molecules Against Foot-and-Mouth Disease Virus Replication by Targeting 2C Helicase Activity"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-3253-0957","authenticated-orcid":false,"given":"Saisai","family":"Zhou","sequence":"first","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suyu","family":"Mu","sequence":"additional","affiliation":[{"name":"Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuqi","family":"Yu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Tian","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sijia","family":"Lu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhen","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaying","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huanchun","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4975-739X","authenticated-orcid":false,"given":"Shiqi","family":"Sun","sequence":"additional","affiliation":[{"name":"Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunfeng","family":"Song","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1016\/j.cvfa.2024.01.001","article-title":"Foot-and-Mouth Disease","volume":"40","author":"Arzt","year":"2024","journal-title":"Vet. 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