{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T00:01:53Z","timestamp":1770854513259,"version":"3.50.1"},"reference-count":36,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T00:00:00Z","timestamp":1768262400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0"},{"start":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T00:00:00Z","timestamp":1770768000000},"content-version":"vor","delay-in-days":29,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0"}],"funder":[{"name":"Frontier Science Support Program by the Institute for Protein Research","award":["R1-2"],"award-info":[{"award-number":["R1-2"]}]},{"DOI":"10.13039\/501100005936","name":"Casio Science Promotion Foundation","doi-asserted-by":"publisher","award":["J41-42"],"award-info":[{"award-number":["J41-42"]}],"id":[{"id":"10.13039\/501100005936","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP23K05654"],"award-info":[{"award-number":["JP23K05654"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP21K06046"],"award-info":[{"award-number":["JP21K06046"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP20am0101072"],"award-info":[{"award-number":["JP20am0101072"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001700","name":"Ministry of Education, Culture, Sports, Science and Technology","doi-asserted-by":"publisher","award":["JPMXS0120330644"],"award-info":[{"award-number":["JPMXS0120330644"]}],"id":[{"id":"10.13039\/501100001700","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003382","name":"Core Research for Evolutional Science and Technology","doi-asserted-by":"publisher","award":["JPMJCR1672"],"award-info":[{"award-number":["JPMJCR1672"]}],"id":[{"id":"10.13039\/501100003382","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"DOI":"10.1038\/s41598-026-35354-3","type":"journal-article","created":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T17:46:02Z","timestamp":1768326362000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Investigation of the functional hot-spot residues of an enzyme by real-time monitoring of the enzymatic reaction using NMR and computational approaches"],"prefix":"10.1038","volume":"16","author":[{"given":"Toshihiko","family":"Sugiki","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tomoki","family":"Yoshida","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masaki","family":"Tsukamoto","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Koichiro","family":"Miyanishi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Akinori","family":"Kagawa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Natsuko","family":"Miura","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tomoto","family":"Ura","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jun","family":"Fukazawa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuko","family":"Hatanaka","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tsuyoshi","family":"Murata","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Toshimichi","family":"Fujiwara","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masahiro","family":"Kitagawa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yasushi","family":"Morita","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kumiko","family":"Sakai-Kato","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yoichi","family":"Takakusagi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nobutada","family":"Tanaka","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Makoto","family":"Negoro","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,1,13]]},"reference":[{"key":"35354_CR1","doi-asserted-by":"publisher","first-page":"418","DOI":"10.1038\/363418a0","volume":"363","author":"M von Itzstein","year":"1993","unstructured":"von Itzstein, M. et al. Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363, 418\u2013423. https:\/\/doi.org\/10.1038\/363418a0 (1993).","journal-title":"Nature"},{"key":"35354_CR2","doi-asserted-by":"publisher","first-page":"2451","DOI":"10.1021\/jm980162u","volume":"41","author":"CU Kim","year":"1998","unstructured":"Kim, C. U. et al. Structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors. J. Med. Chem. 41, 2451\u20132560. https:\/\/doi.org\/10.1021\/jm980162u (1998).","journal-title":"J. Med. Chem."},{"key":"35354_CR3","doi-asserted-by":"publisher","unstructured":"Pinto, L. H. & Holsinger, L. J. & Lamb, R. A. Influenza virus M2 protein has ion channel activity. Cell 69, 517\u2013528. https:\/\/doi.org\/10.1016\/0092-8674(92)90452-i (1992).","DOI":"10.1016\/0092-8674(92)90452-i"},{"key":"35354_CR4","doi-asserted-by":"publisher","first-page":"5585","DOI":"10.1128\/JVI.67.9.5585-5594.1993","volume":"67","author":"C Wang","year":"1993","unstructured":"Wang, C., Takeuchi, K., Pinto, L. H. & Lamb, R. A. Ion channel activity of influenza A virus M2 protein: Characterization of the amantadine block. J. Virol. 67, 5585\u20135594. https:\/\/doi.org\/10.1128\/JVI.67.9.5585-5594.1993 (1993).","journal-title":"J. Virol."},{"key":"35354_CR5","doi-asserted-by":"publisher","first-page":"977","DOI":"10.1128\/AAC.46.4.977-981.2002","volume":"46","author":"Y Furuta","year":"2002","unstructured":"Furuta, Y. et al. In vitro and in vivo activities of anti-influenza virus compound T-705. Antimicrob. Agents Chemother. 46, 977\u2013981. https:\/\/doi.org\/10.1128\/AAC.46.4.977-981.2002 (2002).","journal-title":"Antimicrob. Agents Chemother."},{"key":"35354_CR6","doi-asserted-by":"publisher","first-page":"615","DOI":"10.1124\/mol.113.087247","volume":"84","author":"L Naesens","year":"2013","unstructured":"Naesens, L. et al. Role of human hypoxanthine guanine phosphoribosyltransferase in activation of the antiviral agent T-705 (favipiravir). Mol. Pharmacol. 84, 615\u2013629. https:\/\/doi.org\/10.1124\/mol.113.087247 (2013).","journal-title":"Mol. Pharmacol."},{"key":"35354_CR7","doi-asserted-by":"publisher","first-page":"501","DOI":"10.1016\/j.ijid.2020.10.069","volume":"102","author":"S Joshi","year":"2021","unstructured":"Joshi, S. et al. Role of favipiravir in the treatment of COVID-19. Int. J. Infect. Dis. 102, 501\u2013508. https:\/\/doi.org\/10.1016\/j.ijid.2020.10.069 (2021).","journal-title":"Int. J. Infect. Dis."},{"key":"35354_CR8","doi-asserted-by":"publisher","first-page":"981","DOI":"10.1128\/AAC.49.3.981-986.2005","volume":"49","author":"Y Furuta","year":"2005","unstructured":"Furuta, Y. et al. Mechanism of action of T-705 against influenza virus. Antimicrob. Agents Chemother. 49, 981\u2013986. https:\/\/doi.org\/10.1128\/AAC.49.3.981-986.2005 (2005).","journal-title":"Antimicrob. Agents Chemother."},{"key":"35354_CR9","doi-asserted-by":"publisher","first-page":"95","DOI":"10.1016\/j.antiviral.2009.02.198","volume":"82","author":"Y Furuta","year":"2009","unstructured":"Furuta, Y. et al. T-705 (favipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections. Antiviral Res. 82, 95\u2013102. https:\/\/doi.org\/10.1016\/j.antiviral.2009.02.198 (2009).","journal-title":"Antiviral Res."},{"key":"35354_CR10","doi-asserted-by":"publisher","unstructured":"Furuta, Y., Gowen, B. B., Takahashi, K., Shiraki, K., Smee, D. F. & Barnard, D. L. Favipiravir (T-705), a novel viral RNA polymerase inhibitor. Antiviral Res. 100, 446\u2013454. https:\/\/doi.org\/10.1016\/j.antiviral.2013.09.015 (2013).","DOI":"10.1016\/j.antiviral.2013.09.015"},{"key":"35354_CR11","doi-asserted-by":"publisher","unstructured":"Peng, Q. et al. Structural Basis of SARS-CoV-2 Polymerase Inhibition by Favipiravir. Innovation (NY) 2, 100080. (2021). https:\/\/doi.org\/10.1016\/j.xinn.2021.100080","DOI":"10.1016\/j.xinn.2021.100080"},{"key":"35354_CR12","doi-asserted-by":"publisher","first-page":"26955","DOI":"10.1073\/pnas.2014441117","volume":"117","author":"SJF Kaptein","year":"2020","unstructured":"Kaptein, S. J. F. et al. Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity. Proc. Natl. Acad. Sci. U S A. 117, 26955\u201326965. https:\/\/doi.org\/10.1073\/pnas.2014441117 (2020).","journal-title":"Proc. Natl. Acad. Sci. U S A"},{"key":"35354_CR13","doi-asserted-by":"publisher","first-page":"11022","DOI":"10.1038\/s41598-021-90551-6","volume":"11","author":"S Hassanipour","year":"2021","unstructured":"Hassanipour, S. et al. The efficacy and safety of favipiravir in treatment of COVID-19: A systematic review and meta-analysis of clinical trials. Sci. Rep. 11, 11022. https:\/\/doi.org\/10.1038\/s41598-021-90551-6 (2021).","journal-title":"Sci. Rep."},{"key":"35354_CR14","doi-asserted-by":"publisher","first-page":"2489","DOI":"10.1007\/s40121-021-00517-4","volume":"10","author":"M Shinkai","year":"2021","unstructured":"Shinkai, M. et al. Efficacy and safety of favipiravir in moderate COVID-19 pneumonia patients without oxygen therapy: A Randomized, phase III clinical trial. Infect. Dis. Ther. 10, 2489\u20132509. https:\/\/doi.org\/10.1007\/s40121-021-00517-4 (2021).","journal-title":"Infect. Dis. Ther."},{"key":"35354_CR15","doi-asserted-by":"publisher","first-page":"489","DOI":"10.1186\/s12879-021-06164-x","volume":"21","author":"T Manabe","year":"2021","unstructured":"Manabe, T., Kambayashi, D., Akatsu, H. & Kudo, K. Favipiravir for the treatment of patients with COVID-19: A systematic review and meta-analysis. BMC Infect. Dis. 21, 489. https:\/\/doi.org\/10.1186\/s12879-021-06164-x (2021).","journal-title":"BMC Infect. Dis."},{"key":"35354_CR16","doi-asserted-by":"publisher","first-page":"148","DOI":"10.3390\/molecules23010148","volume":"23","author":"T Sugiki","year":"2018","unstructured":"Sugiki, T., Furuita, K., Fujiwara, T. & Kojima, C. Current NMR techniques for structure-based drug discovery. Molecules 23, 148. https:\/\/doi.org\/10.3390\/molecules23010148 (2018).","journal-title":"Molecules"},{"key":"35354_CR17","doi-asserted-by":"publisher","first-page":"333","DOI":"10.1038\/s41592-019-0334-x","volume":"16","author":"A Boeszoermenyi","year":"2019","unstructured":"Boeszoermenyi, A. et al. Aromatic 19F-13C TROSY: A background-free approach to probe biomolecular structure, function, and dynamics. Nat. Methods. 16, 333\u2013340. https:\/\/doi.org\/10.1038\/s41592-019-0334-x (2019).","journal-title":"Nat. Methods"},{"key":"35354_CR18","doi-asserted-by":"publisher","first-page":"229","DOI":"10.1038\/s41467-020-20512-6","volume":"12","author":"R Mashiach","year":"2021","unstructured":"Mashiach, R. et al. In situ NMR reveals real-time nanocrystal growth evolution via monomer-attachment or particle-coalescence. Nat. Commun. 12, 229. https:\/\/doi.org\/10.1038\/s41467-020-20512-6 (2021).","journal-title":"Nat. Commun."},{"key":"35354_CR19","doi-asserted-by":"publisher","first-page":"e4888","DOI":"10.1002\/nbm.4888","volume":"36","author":"T Sugiki","year":"2023","unstructured":"Sugiki, T. et al. Real-time monitoring of enzyme-catalyzed phosphoribosylation of anti-influenza prodrug favipiravir by time-lapse nuclear magnetic resonance spectroscopy. NMR Biomed. 36, e4888. https:\/\/doi.org\/10.1002\/nbm.4888 (2023).","journal-title":"NMR Biomed."},{"key":"35354_CR20","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1186\/s12859-020-3513-y","volume":"21","author":"MD Olp","year":"2020","unstructured":"Olp, M. D., Kalous, K. S. & Smith, B. C. ICEKAT: An interactive online tool for calculating initial rates from continuous enzyme kinetic traces. BMC Bioinform. 21, 186. https:\/\/doi.org\/10.1186\/s12859-020-3513-y (2020).","journal-title":"BMC Bioinform."},{"key":"35354_CR21","doi-asserted-by":"publisher","first-page":"3700","DOI":"10.1021\/bi9616007","volume":"36","author":"Y Xu","year":"1997","unstructured":"Xu, Y., Eads, J., Sacchettini, J. C. & Grubmeyer, C. Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: Rapid phosphoribosyl transfer chemistry. Biochemistry 36, 3700\u20133712. https:\/\/doi.org\/10.1021\/bi9616007 (1997).","journal-title":"Biochemistry"},{"key":"35354_CR22","doi-asserted-by":"publisher","first-page":"170","DOI":"10.1016\/j.jmb.2005.05.061","volume":"351","author":"DT Keough","year":"2005","unstructured":"Keough, D. T., Brereton, I. M., de Jersey, J. & Guddat, L. W. The crystal structure of free human hypoxanthine-guanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle. J. Mol. Biol. 351, 170\u2013181. https:\/\/doi.org\/10.1016\/j.jmb.2005.05.061 (2005).","journal-title":"J. Mol. Biol."},{"key":"35354_CR23","doi-asserted-by":"publisher","first-page":"e1005659","DOI":"10.1371\/journal.pcbi.1005659","volume":"13","author":"P Eastman","year":"2017","unstructured":"Eastman, P. et al. OpenMM 7: Rapid development of high performance algorithms for molecular dynamics. PLoS Comput. Biol. 13, e1005659. https:\/\/doi.org\/10.1371\/journal.pcbi.1005659 (2017).","journal-title":"PLoS Comput. Biol."},{"key":"35354_CR24","unstructured":"Schr\u00f6dinger, L. L. C. The PyMOL Molecular Graphics System, Version 2.5, Schr\u00f6dinger (LLC, 2021)."},{"key":"35354_CR25","doi-asserted-by":"publisher","first-page":"221","DOI":"10.1007\/s10822-013-9644-8","volume":"27","author":"GM Sastry","year":"2013","unstructured":"Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R. & Sherman, W. Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 27, 221\u2013234. https:\/\/doi.org\/10.1007\/s10822-013-9644-8 (2013).","journal-title":"J. Comput. Aided Mol. Des."},{"key":"35354_CR26","unstructured":"Schr\u00f6dinger, R., Epik, Schr\u00f6dinger, L. L. C., New York, N. Y., Impact & Prime Protein Preparation Wizard;, 2023;Schr\u00f6dinger, LLC, New York, NY; Schr\u00f6dinger, LLC, New York, NY (2023). (2023)-4."},{"key":"35354_CR27","doi-asserted-by":"publisher","first-page":"597","DOI":"10.1016\/s0022-2836(02)00470-9","volume":"320","author":"MP Jacobson","year":"2002","unstructured":"Jacobson, M. P., Friesner, R. A., Xiang, Z. & Honig, B. On the role of the crystal environment in determining protein side-chain conformations. J. Mol. Biol. 320, 597\u2013608. https:\/\/doi.org\/10.1016\/s0022-2836(02)00470-9 (2002).","journal-title":"J. Mol. Biol."},{"key":"35354_CR28","doi-asserted-by":"publisher","first-page":"351","DOI":"10.1002\/prot.10613","volume":"55","author":"MP Jacobson","year":"2004","unstructured":"Jacobson, M. P. et al. A hierarchical approach to all-atom protein loop prediction. Proteins 55, 351\u2013367. https:\/\/doi.org\/10.1002\/prot.10613 (2004).","journal-title":"Proteins"},{"key":"35354_CR29","unstructured":"Schr\u00f6dinger Release 2023-4: Prime, Schr\u00f6dinger (LLC, 2023)."},{"key":"35354_CR30","doi-asserted-by":"crossref","unstructured":"Bowers, K. J. et al. Scalable algorithms for molecular dynamics simulations on commodity clusters, Proc. ACM\/IEEE Conf. Supercomput. (SC06) Tampa, Florida, November 11\u201317 (2006).","DOI":"10.1145\/1188455.1188544"},{"key":"35354_CR31","unstructured":"Schr\u00f6dinger Release 2019-1: Desmond Molecular Dynamics System, Shaw, D. E. & Research New York, NY, Maestro-Desmond Interoperability Tools, Schr\u00f6dinger, New York, NY (2019). (2019)."},{"key":"35354_CR32","doi-asserted-by":"publisher","unstructured":"Gowers, R. J. et al. MDAnalysis: A Python package for the rapid analysis of molecular dynamics simulations. In Proceedings of the 15th Python in Science Conference 98\u2013105. (2016). https:\/\/doi.org\/10.25080\/majora-629e541a-00e","DOI":"10.25080\/majora-629e541a-00e"},{"key":"35354_CR33","doi-asserted-by":"publisher","first-page":"2319","DOI":"10.1002\/jcc.21787","volume":"32","author":"N Michaud-Agrawal","year":"2011","unstructured":"Michaud-Agrawal, N., Denning, E. J., Woolf, T. B. & Beckstein, O. MDAnalysis: A toolkit for the analysis of molecular dynamics simulations. J. Comput. Chem. 32, 2319\u20132327. https:\/\/doi.org\/10.1002\/jcc.21787 (2011).","journal-title":"J. Comput. Chem."},{"key":"35354_CR34","doi-asserted-by":"publisher","first-page":"95","DOI":"10.1093\/jb\/mvp152","volume":"147","author":"R Takahashi","year":"2010","unstructured":"Takahashi, R. et al. Structure and reaction mechanism of human nicotinamide phosphoribosyltransferase. J. Biochem. 147, 95\u2013107. https:\/\/doi.org\/10.1093\/jb\/mvp152 (2010).","journal-title":"J. Biochem."},{"key":"35354_CR35","doi-asserted-by":"publisher","first-page":"101285","DOI":"10.1016\/j.mex.2021.101285","volume":"8","author":"W Escobedo-Hinojosa","year":"2021","unstructured":"Escobedo-Hinojosa, W., Wissner, J. L. & Hauer, B. A real-time 31P-NMR-based approach for the assessment of glycerol kinase catalyzed monophosphorylations. MethodsX 8, 101285. https:\/\/doi.org\/10.1016\/j.mex.2021.101285 (2021).","journal-title":"MethodsX"},{"key":"35354_CR36","doi-asserted-by":"publisher","unstructured":"Smithers, G. W.  &  O'Sullivan, W. J. 31P nuclear magnetic resonance study of phosphoribosyldiphosphate and its interaction with magnesium ions. J. Biol. Chem. 257, 6164\u20136170. https:\/\/doi.org\/10.1016\/S0021-9258(29)65121-3 (1982).","DOI":"10.1016\/S0021-9258(29)65121-3"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-026-35354-3","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-026-35354-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-026-35354-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T23:02:53Z","timestamp":1770850973000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-026-35354-3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,13]]},"references-count":36,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,12]]}},"alternative-id":["35354"],"URL":"https:\/\/doi.org\/10.1038\/s41598-026-35354-3","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,13]]},"assertion":[{"value":"4 July 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"5 January 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"13 January 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors declare no competing interests.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"5896"}}