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Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2025,8,19]]},"abstract":"<jats:p>Influenza A virus (IAV) is a zoonotic pathogen responsible for seasonal and pandemic flu. The extensive genetic and antigenic diversity within and between IAV phylogenetic groups presents major challenges for developing universal vaccines and broad-spectrum antiviral therapies. Current interventions provide limited protection due to the virus\u2019s high mutation rate and capacity for immune evasion. Recent advancements in viral hemagglutinin (HA)-targeting small-molecule entry inhibitors offer a promising avenue to overcome these limitations. Here, we present structural and functional analyses of two group 2 HA-specific small-molecule inhibitors recently identified by our team. Cryogenic electron microscopy (cryo-EM) structures revealed that these inhibitors bind a conserved pocket within the HA stalk, likely interfering with the conformational rearrangements necessary for membrane fusion and viral entry. Structure-guided mutagenesis confirmed the critical roles of key interacting residues and uncovered distinct resistance profiles between the two compounds, as well as in comparison to Arbidol, a previously reported HA inhibitor. Notably, our structural analysis highlights intrinsic barriers to achieving cross-group inhibition with current small-molecule designs. To address this, we propose an alternative strategy for broadening antiviral coverage. Together, these findings provide mechanistic insights into IAV entry inhibition and a foundation for the rational design of next-generation anti-influenza therapeutics.<\/jats:p>","DOI":"10.1073\/pnas.2503899122","type":"journal-article","created":{"date-parts":[[2025,8,13]],"date-time":"2025-08-13T17:38:58Z","timestamp":1755106738000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":10,"title":["Mechanistic insights into the small-molecule inhibition of influenza A virus entry"],"prefix":"10.1073","volume":"122","author":[{"given":"Yan","family":"Xu","sequence":"first","affiliation":[{"name":"Department of Veterinary Biosciences, The Ohio State University","place":["Columbus"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5370-1092","authenticated-orcid":false,"given":"Varada","family":"Anirudhan","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of Illinois","place":["Chicago"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8668-3222","authenticated-orcid":false,"given":"Irina N.","family":"Gaisina","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences and UICentre, Retzky College of Pharmacy, University of Illinois","place":["Chicago"]},{"name":"Chicago BioSolutions Inc.","place":["Chicago"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haijuan","family":"Du","sequence":"additional","affiliation":[{"name":"Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH","place":["Bethesda"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0931-0529","authenticated-orcid":false,"given":"Saad","family":"Alqarni","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, College of Pharmacy, University of Ha\u2019il","place":["Hail, Saudi Arabia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5410-306X","authenticated-orcid":false,"given":"Terry W.","family":"Moore","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences and UICentre, Retzky College of Pharmacy, University of Illinois","place":["Chicago"]},{"name":"University of Illinois Cancer Center, University of Illinois","place":["Chicago"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Caffrey","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois","place":["Chicago"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1964-0967","authenticated-orcid":false,"given":"Balaji","family":"Manicassamy","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, College of Medicine, University of Iowa","place":["Iowa City"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tongqing","family":"Zhou","sequence":"additional","affiliation":[{"name":"Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH","place":["Bethesda"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3938-0445","authenticated-orcid":false,"given":"Lijun","family":"Rong","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of Illinois","place":["Chicago"]},{"name":"Chicago BioSolutions Inc.","place":["Chicago"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5412-6942","authenticated-orcid":false,"given":"Kai","family":"Xu","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biosciences, The Ohio State University","place":["Columbus"]},{"name":"Department of Microbial Infection and Immunity, The Ohio State University","place":["Columbus"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2025,8,13]]},"reference":[{"key":"e_1_3_4_1_2","first-page":"1","article-title":"Overview of influenza viruses","volume":"370","author":"Pleschka S.","year":"2013","unstructured":"S. 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