{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T22:18:39Z","timestamp":1791497919089,"version":"4.3.4"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T00:00:00Z","timestamp":1768176000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Pharmacol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Severe acute respiratory syndrome coronavirus 2 (SARS\u2010CoV\u20102) causes a highly infectious disease characterized by fever, acute respiratory illness, and pneumonia, known as coronavirus disease 2019 (COVID\u201019). SARS\u2010CoV\u20102 infects host cells through the interaction of its spike glycoprotein (S protein) with human angiotensin\u2010converting enzyme 2 (hACE2). Structural studies have shown that hACE2 interacts exclusively with the receptor\u2010binding domain (RBD) of the spike. A high binding affinity between spike and hACE2 has been linked to increased viral infection. Disrupting this interaction can reduce viral infectivity.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>This study aimed to assess infection using Omicron variant pseudovirus in a stable HEK\u2010293 cell line expressing hACE2 (HEK\u2010293\/ACE2), treated with bromhexine hydrochloride. First, immunofluorescence and Western blot confirmed the presence of hACE2 in the stable line. Then, bromhexine concentrations for treatment were determined by cytotoxicity assays. Next, infection was evaluated using Omicron pseudoviruses carrying GFP and luciferase reporter genes. Infection levels were measured through fluorescence or luciferase activity.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Bromhexine reduced infection with an IC\n                      <jats:sub>50<\/jats:sub>\n                      of 17.3 \u00b1 0.9 \u03bcM. About 40% inhibition was also observed against Alpha, Beta, and Delta variants at 40 \u03bcM. Computational docking followed by molecular dynamics simulations showed that bromhexine binds to the extracellular domain of hACE2, with recurrent contacts near Phe40, Phe390, and Asn394.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>Consistent with this model, our findings support an entry\u2010inhibition mechanism whereby bromhexine destabilizes the SARS\u2010CoV\u20102 spike\u2013ACE2 interface, preventing viral entry. Overall, these results suggest bromhexine as a potential repurposing candidate and support its inclusion in therapeutic strategies aimed at both current and emerging SARS\u2010CoV\u20102 variants.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fphar.2025.1745277","type":"journal-article","created":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T08:12:31Z","timestamp":1768205551000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms"],"prefix":"10.3389","volume":"16","author":[{"given":"Rafael","family":"Z\u00fa\u00f1iga","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Whitney","family":"Venturini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalia","family":"Gonz\u00e1lez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paulina","family":"Valenzuela-Hormaz\u00e1bal","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"S\u00e1nchez-Aros","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Ram\u00edrez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angel","family":"Cayo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cristian","family":"Vilos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leandro","family":"Z\u00fa\u00f1iga","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,1,12]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"952192","DOI":"10.3389\/fphar.2022.952192","article-title":"Network pharmacology reveals multitarget mechanism of action of drugs to be repurposed for COVID-19","volume":"13","author":"Alegr\u00eda-Arcos","year":"2022","journal-title":"Front. Pharmacol."},{"key":"B2","doi-asserted-by":"publisher","first-page":"450","DOI":"10.1038\/s41591-020-0820-9","article-title":"The proximal origin of SARS-CoV-2","volume":"26","author":"Andersen","year":"2020","journal-title":"Nat. Med."},{"key":"B3","doi-asserted-by":"publisher","first-page":"8801","DOI":"10.1128\/jvi.77.16.8801-8811.2003","article-title":"The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex","volume":"77","author":"Bosch","year":"2003","journal-title":"J. Virol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"1586","DOI":"10.1126\/science.abd4251","article-title":"Distinct conformational states of SARS-CoV-2 spike protein","volume":"369","author":"Cai","year":"2020","journal-title":"Science"},{"key":"B5","doi-asserted-by":"publisher","first-page":"221","DOI":"10.1080\/22221751.2020.1719902","article-title":"Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan","volume":"9","author":"Chan","year":"2020","journal-title":"Emerg. Microb. Infect."},{"key":"B6","doi-asserted-by":"publisher","first-page":"418","DOI":"10.1002\/jmv.25681","article-title":"Emerging coronaviruses: genome structure, replication, and pathogenesis","volume":"92","author":"Chen","year":"2020","journal-title":"J. Med. Virol."},{"key":"B7","doi-asserted-by":"publisher","first-page":"991369","DOI":"10.3389\/fchem.2022.991369","article-title":"In-silico molecular modelling, MM\/GBSA binding free energy and molecular dynamics simulation study of novel pyrido fused imidazo[4,5-c]quinolines as potential anti-tumor agents","volume":"10","author":"Dasmahapatra","year":"2022","journal-title":"Front. Chem."},{"key":"B8","doi-asserted-by":"publisher","first-page":"10824","DOI":"10.1080\/07391102.2023.2259476","article-title":"In-silico screening and molecular dynamics simulation of quinazolinone derivatives as PARP1 and STAT3 dual inhibitors: a novel DML approaches","volume":"42","author":"Dilipkumar","year":"2024","journal-title":"J. Biomol. Struct. Dyn."},{"key":"B9","doi-asserted-by":"publisher","first-page":"e1","DOI":"10.1161\/01.RES.87.5.e1","article-title":"A novel angiotensin-converting enzyme\u2013related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9","volume":"87","author":"Donoghue","year":"2000","journal-title":"Circ. Res."},{"key":"B10","doi-asserted-by":"publisher","first-page":"1429180","DOI":"10.3389\/fmolb.2024.1429180","article-title":"Cryo-electron microscopy in the study of virus entry and infection","volume":"11","author":"Dutta","year":"2024","journal-title":"Front. Mol. Biosci."},{"key":"B11","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/978-1-4939-2438-7_1","article-title":"Coronaviruses: an overview of their replication and pathogenesis","volume":"1282","author":"Fehr","year":"2015","journal-title":"Methods Mol. Biol."},{"key":"B12","doi-asserted-by":"publisher","first-page":"12359","DOI":"10.1021\/acs.jmedchem.0c00606","article-title":"COVID-19: drug targets and potential treatments","volume":"63","author":"Gil","year":"2020","journal-title":"J. Med. Chem."},{"key":"B13","doi-asserted-by":"publisher","first-page":"536","DOI":"10.1038\/s41564-020-0695-z","article-title":"The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2","volume":"5","author":"Gorbalenya","year":"2020","journal-title":"Nat. Microbiol."},{"key":"B14","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1016\/j.coviro.2021.11.011","article-title":"A structural view of the SARS-CoV-2 virus and its assembly","volume":"52","author":"Hardenbrook","year":"2022","journal-title":"Curr. Opin. Virol."},{"key":"B15","doi-asserted-by":"publisher","first-page":"779","DOI":"10.1016\/j.molcel.2020.04.022","article-title":"A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells","volume":"78","author":"Hoffmann","year":"","journal-title":"Mol. Cell"},{"key":"B16","doi-asserted-by":"publisher","first-page":"271","DOI":"10.1016\/j.cell.2020.02.052","article-title":"SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor","volume":"181","author":"Hoffmann","year":"","journal-title":"Cell"},{"key":"B17","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1038\/s41580-021-00418-x","article-title":"Mechanisms of SARS-CoV-2 entry into cells","volume":"23","author":"Jackson","year":"2022","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"B18","doi-asserted-by":"publisher","first-page":"108201","DOI":"10.1016\/j.jmgm.2022.108201","article-title":"Inhibitory mechanism of ambroxol and bromhexine hydrochlorides as potent blockers of molecular interaction between SARS-CoV-2 spike protein and human angiotensin-converting Enzyme-2","volume":"114","author":"Kehinde","year":"2022","journal-title":"J. Mol. Graph. Model."},{"key":"B19","doi-asserted-by":"publisher","first-page":"450","DOI":"10.1038\/nature02145","article-title":"Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus","volume":"426","author":"Li","year":"2003","journal-title":"Nature"},{"key":"B20","doi-asserted-by":"publisher","first-page":"565","DOI":"10.1016\/S0140-6736(20)30251-8","article-title":"Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding","volume":"395","author":"Lu","year":"2020","journal-title":"Lancet"},{"key":"B21","doi-asserted-by":"publisher","first-page":"4291","DOI":"10.1021\/acs.jctc.1c00302","article-title":"OPLS4: improving force field accuracy on challenging regimes of chemical space","volume":"17","author":"Lu","year":"2021","journal-title":"J. Chem. Theory Comp."},{"key":"B22","doi-asserted-by":"publisher","first-page":"1310","DOI":"10.1158\/2159-8290.CD-13-1010","article-title":"The androgen-regulated protease TMPRSS2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis","volume":"4","author":"Lucas","year":"2014","journal-title":"Cancer Discov."},{"key":"B23","doi-asserted-by":"publisher","first-page":"1494","DOI":"10.1038\/s41598-023-28716-8","article-title":"Virtual screening and molecular dynamics simulations provide insight into repurposing drugs against SARS-CoV-2 variants spike protein\/ACE2 interface","volume":"13","author":"Pirolli","year":"2023","journal-title":"Sci. Rep."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1165","DOI":"10.1021\/ci1000407","article-title":"Virtual fragment docking by glide: a validation study on 190 protein\u2212fragment complexes","volume":"50","author":"S\u00e1ndor","year":"2010","journal-title":"J. Chem. Inf. Model."},{"key":"B25","doi-asserted-by":"publisher","first-page":"221","DOI":"10.1007\/s10822-013-9644-8","article-title":"Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments","volume":"27","author":"Sastry","year":"2013","journal-title":"J. Comput. Aided Mol. Des."},{"key":"B26","doi-asserted-by":"publisher","first-page":"11727","DOI":"10.1073\/pnas.2003138117","article-title":"Cell entry mechanisms of SARS-CoV-2","volume":"117","author":"Shang","year":"2020","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B27","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.biochi.2017.07.016","article-title":"TMPRSS2: a potential target for treatment of influenza virus and coronavirus infections","volume":"142","author":"Shen","year":"2017","journal-title":"Biochimie"},{"key":"B28","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1038\/nrmicro775","article-title":"SARS \u2014 beginning to understand a new virus","volume":"1","author":"Stadler","year":"2003","journal-title":"Nat. Rev. Microbiol."},{"key":"B29","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1097\/MNH.0b013e328341164a","article-title":"Angiotensin-converting enzyme 2 is a key modulator of the renin-angiotensin system in cardiovascular and renal disease","volume":"20","author":"Tikellis","year":"2011","journal-title":"Curr. Opin. Nephrol. Hypertens."},{"key":"B30","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1016\/j.cell.2020.02.058","article-title":"Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein","volume":"181","author":"Walls","year":"2020","journal-title":"Cell"},{"key":"B31","doi-asserted-by":"publisher","first-page":"470","DOI":"10.1016\/S0140-6736(20)30185-9","article-title":"A novel coronavirus outbreak of global health concern","volume":"395","author":"Wang","year":"","journal-title":"Lancet"},{"key":"B32","doi-asserted-by":"publisher","first-page":"587269","DOI":"10.3389\/fcimb.2020.587269","article-title":"SARS-CoV-2: structure, biology, and structure-based therapeutics development","volume":"10","author":"Wang","year":"","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"B33","doi-asserted-by":"publisher","first-page":"894","DOI":"10.1016\/j.cell.2020.03.045","article-title":"Structural and functional basis of SARS-CoV-2 entry by using human ACE2","volume":"181","author":"Wang","year":"","journal-title":"Cell"},{"key":"B34","doi-asserted-by":"publisher","first-page":"1522","DOI":"10.7150\/ijms.46695","article-title":"A comprehensive investigation of the mRNA and protein level of ACE2, the putative receptor of SARS-CoV-2, in human tissues and blood cells","volume":"17","author":"Wang","year":"","journal-title":"Int. J. Med. Sci."},{"key":"B35","doi-asserted-by":"publisher","first-page":"1260","DOI":"10.1126\/science.abb2507","article-title":"Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation","volume":"367","author":"Wrapp","year":"2020","journal-title":"Science"},{"key":"B36","doi-asserted-by":"publisher","first-page":"325","DOI":"10.1016\/j.chom.2020.02.001","article-title":"Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China","volume":"27","author":"Wu","year":"2020","journal-title":"Cell Host Microbe"},{"key":"B37","doi-asserted-by":"publisher","first-page":"202","DOI":"10.1038\/s41594-020-00549-3","article-title":"A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent","volume":"28","author":"Xiao","year":"2021","journal-title":"Nat. Struct. Mol. Biol."},{"key":"B38","doi-asserted-by":"publisher","first-page":"1444","DOI":"10.1126\/science.abb2762","article-title":"Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2","volume":"367","author":"Yan","year":"2020","journal-title":"Science"},{"key":"B39","doi-asserted-by":"publisher","first-page":"685","DOI":"10.1038\/s41579-021-00630-8","article-title":"Structural biology of SARS-CoV-2 and implications for therapeutic development","volume":"19","author":"Yang","year":"2021","journal-title":"Nat. Rev. Microbiol."},{"key":"B40","doi-asserted-by":"publisher","first-page":"60","DOI":"10.3390\/v11010060","article-title":"Advances in MERS-CoV vaccines and therapeutics based on the receptor-binding domain","volume":"11","author":"Zhou","year":"2019","journal-title":"Viruses"},{"key":"B41","doi-asserted-by":"publisher","first-page":"270","DOI":"10.1038\/s41586-020-2012-7","article-title":"A pneumonia outbreak associated with a new coronavirus of probable bat origin","volume":"579","author":"Zhou","year":"","journal-title":"Nature"},{"key":"B42","doi-asserted-by":"publisher","first-page":"867","DOI":"10.1016\/j.chom.2020.11.004","article-title":"Cryo-EM structures of SARS-CoV-2 spike without and with ACE2 reveal a pH-dependent switch to mediate endosomal positioning of receptor-binding domains","volume":"28","author":"Zhou","year":"","journal-title":"Cell Host Microbe"},{"key":"B43","doi-asserted-by":"publisher","first-page":"727","DOI":"10.1056\/NEJMoa2001017","article-title":"A novel coronavirus from patients with pneumonia in China, 2019","volume":"382","author":"Zhu","year":"2020","journal-title":"N. Engl. J. Med."}],"container-title":["Frontiers in Pharmacology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphar.2025.1745277\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T08:12:34Z","timestamp":1768205554000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphar.2025.1745277\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,12]]},"references-count":43,"alternative-id":["10.3389\/fphar.2025.1745277"],"URL":"https:\/\/doi.org\/10.3389\/fphar.2025.1745277","relation":{},"ISSN":["1663-9812"],"issn-type":[{"value":"1663-9812","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,12]]},"article-number":"1745277"}}