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The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5. Study of 1\u20136 shows that, according to molecular dynamics (MD) simulations and molecular mechanics Poisson\u2013Boltzmann surface area (MM\/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5, respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6. In the active M2 S31N blockers 1 and 6, the phenyl and isoxazolyl head groups achieve a deeper binding position and high kon\/low koff and high kon\/high koff rate constants, compared to inactive 2\u20135, which have much lower kon and higher koff. Compounds 1\u20135 block the M2 WT channel by binding in the longer area from V27\u2013H37, in the inward orientation, with high kon and low koff rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 1\u20135 or 1\u20134 and 6, respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2\u20134 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.<\/jats:p>","DOI":"10.1021\/acschembio.0c00553","type":"journal-article","created":{"date-parts":[[2020,8,12]],"date-time":"2020-08-12T10:06:57Z","timestamp":1597226817000},"page":"2331-2337","source":"Crossref","is-referenced-by-count":23,"title":["Chemical Probes for Blocking of Influenza A M2 Wild-type\nand S31N Channels"],"prefix":"10.1021","volume":"15","author":[{"given":"Christina","family":"Tzitzoglaki","sequence":"first","affiliation":[{"name":"National and Kapodistrian University of Athens , , Panepistimioupolis-Zografou, ,","place":["Athens, Greece, 15771"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kelly","family":"McGuire","sequence":"additional","affiliation":[{"name":"Brigham Young University , , , ,","place":["Provo, Utah, United States, 84602"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Panagiotis","family":"Lagarias","sequence":"additional","affiliation":[{"name":"National and Kapodistrian University of Athens , , Panepistimioupolis-Zografou, ,","place":["Athens, Greece, 15771"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Athina","family":"Konstantinidi","sequence":"additional","affiliation":[{"name":"National and Kapodistrian University of Athens , , Panepistimioupolis-Zografou, ,","place":["Athens, Greece, 15771"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anja","family":"Hoffmann","sequence":"additional","affiliation":[{"name":"Jena University Hospital , , Hans Knoell Str. 2 , ,","place":["Jena, Germany, D-07745"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalie A.","family":"Fokina","sequence":"additional","affiliation":[{"name":"Justus Liebig University , , Heinrich-Buff-Ring 17 , ,","place":["Giessen, Germany, 35392"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chulong","family":"Ma","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6974-2561","authenticated-orcid":true,"given":"Ioannis P.","family":"Papanastasiou","sequence":"additional","affiliation":[{"name":"National and Kapodistrian University of Athens , , Panepistimioupolis-Zografou, ,","place":["Athens, Greece, 15771"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3608-5515","authenticated-orcid":true,"given":"Peter R.","family":"Schreiner","sequence":"additional","affiliation":[{"name":"Justus Liebig University , , Heinrich-Buff-Ring 17 , ,","place":["Giessen, Germany, 35392"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9296-6026","authenticated-orcid":true,"given":"Santiago","family":"Va\u0301zquez","sequence":"additional","affiliation":[{"name":"Universitat de Barcelona , , Av. Joan XXIII, 27-31 , ,","place":["Barcelona, Spain, 08028"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michaela","family":"Schmidtke","sequence":"additional","affiliation":[{"name":"Jena University Hospital , , Hans Knoell Str. 2 , ,","place":["Jena, Germany, D-07745"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4845-4621","authenticated-orcid":true,"given":"Jun","family":"Wang","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David D.","family":"Busath","sequence":"additional","affiliation":[{"name":"Brigham Young University , , , ,","place":["Provo, Utah, United States, 84602"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6110-1903","authenticated-orcid":true,"given":"Antonios","family":"Kolocouris","sequence":"additional","affiliation":[{"name":"National and Kapodistrian University of Athens , , Panepistimioupolis-Zografou, ,","place":["Athens, Greece, 15771"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,8,12]]},"reference":[{"key":"2026081714190898200_cit1","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1099\/0022-1317-38-1-97","article-title":"On the\nMechanism of Inhibition of\nInfluenza Virus Replication by Amantadine Hydrochloride","volume":"38","author":"Skehel","year":"1978","journal-title":"J. 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Chem."},{"key":"2026081714190898200_cit15","doi-asserted-by":"publisher","first-page":"17987","DOI":"10.1021\/ja508461m","article-title":"Flipping in the Pore: Discovery of\nDual Inhibitors That Bind in Different Orientations to the Wild-Type\nversus the Amantadine-Resistant S31N Mutant of the Influenza a Virus\nM2 Proton Channel","volume":"136","author":"Wu","year":"2014","journal-title":"J. Am. Chem. Soc."},{"key":"2026081714190898200_cit16","doi-asserted-by":"publisher","first-page":"1111","DOI":"10.1021\/acsmedchemlett.8b00336","article-title":"Structure-Property Relationship Studies\nof Influenza A Virus AM2-S31N Proton Channel Blockers","volume":"9","author":"Hu","year":"2018","journal-title":"ACS Med. Chem. Lett."},{"key":"2026081714190898200_cit17","doi-asserted-by":"publisher","first-page":"1074","DOI":"10.1021\/acs.jmedchem.7b01536","article-title":"In Vitro Pharmacokinetic\nOptimizations of AM2-S31N Channel Blockers Led to the Discovery of\nSlow-Binding Inhibitors with Potent Antiviral Activity against Drug-Resistant\nInfluenza A Viruses","volume":"61","author":"Wang","year":"2018","journal-title":"J. Med. Chem."},{"key":"2026081714190898200_cit18","doi-asserted-by":"publisher","first-page":"9885","DOI":"10.1021\/ja4041412","article-title":"Drug-Induced Conformational and Dynamical\nChanges of the S31N Mutant\nof the Influenza M2 Proton Channel Investigated by Solid-State NMR","volume":"135","author":"Williams","year":"2013","journal-title":"J. Am. Chem. 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A."},{"key":"2026081714190898200_cit22","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1021\/acsmedchemlett.6b00311","article-title":"Affinity of\nRimantadine Enantiomers\nagainst Influenza A\/M2 Protein Revisited","volume":"8","author":"Drakopoulos","year":"2017","journal-title":"ACS\nMed. Chem. Lett."},{"key":"2026081714190898200_cit23","doi-asserted-by":"publisher","first-page":"3727","DOI":"10.1021\/acs.jmedchem.6b01758","article-title":"Slow but Steady\nWins the Race: Dissimilarities among New Dual Inhibitors of the Wild-Type\nand the V27A Mutant M2 Channels of Influenza A Virus","volume":"60","author":"Barniol-Xicota","year":"2017","journal-title":"J. Med. Chem."},{"key":"2026081714190898200_cit24","doi-asserted-by":"publisher","first-page":"198","DOI":"10.1021\/acsmedchemlett.7b00458","article-title":"Unraveling the Binding, Proton Blockage,\nand Inhibition of Influenza M2 WT and S31N by Rimantadine Variants","volume":"9","author":"Drakopoulos","year":"2018","journal-title":"ACS Med. Chem. Lett."},{"key":"2026081714190898200_cit25","doi-asserted-by":"publisher","first-page":"726","DOI":"10.1021\/acsinfecdis.6b00130","article-title":"Discovery of Potent\nAntivirals against Amantadine-Resistant Influenza A Viruses by Targeting\nthe M2-S31N Proton Channel","volume":"2","author":"Li","year":"2016","journal-title":"ACS Infect. Dis."},{"key":"2026081714190898200_cit26","doi-asserted-by":"publisher","first-page":"154","DOI":"10.1021\/j150560a005","article-title":"Application of the\nTheory of Diffusion-Controlled Reactions\nto Enzyme Kinetics","volume":"62","author":"Alberty","year":"1958","journal-title":"J. Phys. 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B"},{"key":"2026081714190898200_cit31","doi-asserted-by":"publisher","first-page":"577","DOI":"10.1016\/0092-8674(92)90219-3","article-title":"Unpacking\nthe Incoming Influenza Virus","volume":"69","author":"Helenius","year":"1992","journal-title":"Cell"}],"container-title":["ACS Chemical Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acschembio.0c00553","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/15\/9\/2331\/9223000\/cb0c00553.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/15\/9\/2331\/9223000\/cb0c00553.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T18:19:52Z","timestamp":1786990792000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acbcct\/article\/15\/9\/2331\/865762\/Chemical-Probes-for-Blocking-of-Influenza-A-M2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,8,12]]},"references-count":32,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2020,8,12]]},"published-print":{"date-parts":[[2020,9,18]]}},"URL":"https:\/\/doi.org\/10.1021\/acschembio.0c00553","relation":{"has-preprint":[{"id-type":"doi","id":"10.26434\/chemrxiv.11494170.v1","asserted-by":"object"}]},"ISSN":["1554-8929","1554-8937"],"issn-type":[{"value":"1554-8929","type":"print"},{"value":"1554-8937","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,8,12]]}}}