{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T10:34:34Z","timestamp":1790764474621,"version":"4.1.0"},"reference-count":57,"publisher":"International Union of Crystallography (IUCr)","issue":"10","license":[{"start":{"date-parts":[[2021,9,27]],"date-time":"2021-09-27T00:00:00Z","timestamp":1632700800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/legalcode"},{"start":{"date-parts":[[2021,9,27]],"date-time":"2021-09-27T00:00:00Z","timestamp":1632700800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/legalcode"}],"funder":[{"DOI":"10.13039\/100010661","name":"Horizon 2020 Framework Programme","doi-asserted-by":"publisher","award":["871037"],"award-info":[{"award-number":["871037"]}],"id":[{"id":"10.13039\/100010661","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010661","name":"Horizon 2020 Framework Programme","doi-asserted-by":"publisher","award":["731005"],"award-info":[{"award-number":["731005"]}],"id":[{"id":"10.13039\/100010661","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003407","name":"Ministero dell'Istruzione, dell'Universit\u00e0 e della Ricerca","doi-asserted-by":"publisher","award":["20177XJCHX"],"award-info":[{"award-number":["20177XJCHX"]}],"id":[{"id":"10.13039\/501100003407","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100015694","name":"Fondazione Cassa di Risparmio di Firenze","doi-asserted-by":"publisher","award":["CRF2020.1395"],"award-info":[{"award-number":["CRF2020.1395"]}],"id":[{"id":"10.13039\/501100015694","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["iucr.org","wiley.com","iucrj.org"],"crossmark-restriction":true},"short-container-title":["Acta Crystallogr D Struct Biol","Acta Crystallogr D","Acta Cryst D","Acta Cryst D Struct Biol","Acta Cryst Sect D","Acta Cryst Sect D Struct Biol","Acta Crystallogr Sect D","Acta Crystallogr Sect D Struct Biol","Acta Crystallogr D Biol Crystallogr","Acta Cryst D Biol Cryst"],"accepted":{"date-parts":[[2021,8,31]]},"published-print":{"date-parts":[[2021,10,1]]},"abstract":"<jats:p>Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained <jats:italic>in vitro<\/jats:italic>, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding.<\/jats:p>","DOI":"10.1107\/s2059798321009037","type":"journal-article","created":{"date-parts":[[2021,9,27]],"date-time":"2021-09-27T15:23:46Z","timestamp":1632756226000},"page":"1270-1281","update-policy":"https:\/\/doi.org\/10.1107\/cm_01","source":"Crossref","is-referenced-by-count":24,"title":["Determination of intracellular protein\u2013ligand binding affinity by competition binding in-cell NMR"],"prefix":"10.1107","volume":"77","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4183-4311","authenticated-orcid":true,"given":"Enrico","family":"Luchinat","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7457-490X","authenticated-orcid":true,"given":"Letizia","family":"Barbieri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matteo","family":"Cremonini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matteo","family":"Pennestri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessio","family":"Nocentini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4262-0323","authenticated-orcid":true,"given":"Claudiu T.","family":"Supuran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0562-5774","authenticated-orcid":true,"given":"Lucia","family":"Banci","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"329","published-online":{"date-parts":[[2021,9,27]]},"reference":[{"key":"qt5006_bb1","doi-asserted-by":"crossref","first-page":"412","DOI":"10.3390\/metabo10100412","volume":"10","author":"Angeli","year":"2020","journal-title":"Metabolites"},{"key":"qt5006_bb2","doi-asserted-by":"crossref","first-page":"1243","DOI":"10.1107\/S0907444906029799","volume":"62","author":"Aricescu","year":"2006","journal-title":"Acta Cryst. D"},{"key":"qt5006_bb3","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1016\/j.pep.2004.06.031","volume":"37","author":"Banerjee","year":"2004","journal-title":"Protein Expr. Purif."},{"key":"qt5006_bb4","unstructured":"Barbieri, L. & Luchinat, E. (2021). J. Vis. Exp., e62323."},{"key":"qt5006_bb5","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1038\/nprot.2016.061","volume":"11","author":"Barbieri","year":"2016","journal-title":"Nat. Protoc."},{"key":"qt5006_bb6","unstructured":"Bekei, B. (2013). Dissertation. Freie Universit\u00e4t Berlin, Germany."},{"key":"qt5006_bb7","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1021\/acs.biochem.8b00975","volume":"58","author":"Bouvier","year":"2019","journal-title":"Biochemistry"},{"key":"qt5006_bb8","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1021\/acs.biochem.7b00938","volume":"57","author":"Breindel","year":"2018","journal-title":"Biochemistry"},{"key":"qt5006_bb9","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1002\/anie.202007184","volume":"60","author":"Broft","year":"2021","journal-title":"Angew. Chem. Int. Ed."},{"key":"qt5006_bb10","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1016\/bs.mie.2019.02.029","volume":"621","author":"Burz","year":"2019","journal-title":"Methods Enzymol."},{"key":"qt5006_bb11","doi-asserted-by":"crossref","first-page":"1693","DOI":"10.1038\/s41467-018-04114-x","volume":"9","author":"Capper","year":"2018","journal-title":"Nat. Commun."},{"key":"qt5006_bb12","doi-asserted-by":"crossref","first-page":"2569","DOI":"10.1016\/j.bmc.2017.03.027","volume":"25","author":"Carta","year":"2017","journal-title":"Bioorg. Med. Chem."},{"key":"qt5006_bb13","doi-asserted-by":"crossref","first-page":"8094","DOI":"10.1021\/acs.jpcb.7b05679","volume":"121","author":"Cerofolini","year":"2017","journal-title":"J. Phys. Chem. B"},{"key":"qt5006_bb14","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1038\/nrd.2015.18","volume":"15","author":"Copeland","year":"2016","journal-title":"Nat. Rev. Drug Discov."},{"key":"qt5006_bb15","doi-asserted-by":"crossref","first-page":"7702","DOI":"10.1021\/ja020174b","volume":"124","author":"Dalvit","year":"2002","journal-title":"J. Am. Chem. Soc."},{"key":"qt5006_bb16","doi-asserted-by":"crossref","first-page":"1115","DOI":"10.1002\/cmdc.201900152","volume":"14","author":"Dalvit","year":"2019","journal-title":"ChemMedChem"},{"key":"qt5006_bb17","doi-asserted-by":"crossref","first-page":"733","DOI":"10.1021\/acschembio.7b00879","volume":"13","author":"DeMott","year":"2018","journal-title":"ACS Chem. Biol."},{"key":"qt5006_bb18","doi-asserted-by":"crossref","first-page":"2165","DOI":"10.1002\/anie.201712284","volume":"57","author":"Dzatko","year":"2018","journal-title":"Angew. Chem. Int. Ed."},{"key":"qt5006_bb19","doi-asserted-by":"crossref","first-page":"1688","DOI":"10.1021\/ja310928u","volume":"135","author":"Hamatsu","year":"2013","journal-title":"J. Am. Chem. Soc."},{"key":"qt5006_bb20","doi-asserted-by":"crossref","first-page":"1239","DOI":"10.1111\/j.1476-5381.2010.01127.x","volume":"162","author":"Hughes","year":"2011","journal-title":"Br. J. Pharmacol."},{"key":"qt5006_bb21","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1038\/nature07839","volume":"458","author":"Inomata","year":"2009","journal-title":"Nature"},{"key":"qt5006_bb22","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.chemolab.2014.10.003","volume":"140","author":"Jaumot","year":"2015","journal-title":"Chemom. Intell. Lab. Syst."},{"key":"qt5006_bb23","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1080\/10408340600970005","volume":"36","author":"Juan","year":"2006","journal-title":"Crit. Rev. Anal. Chem."},{"key":"qt5006_bb24","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1038\/nrd3373","volume":"10","author":"Kepp","year":"2011","journal-title":"Nat. Rev. Drug Discov."},{"key":"qt5006_bb25","doi-asserted-by":"crossref","first-page":"13281","DOI":"10.1021\/jacs.9b03031","volume":"141","author":"Krafcikova","year":"2019","journal-title":"J. Am. Chem. Soc."},{"key":"qt5006_bb26","doi-asserted-by":"crossref","first-page":"1208","DOI":"10.1002\/anie.201207243","volume":"52","author":"Kubo","year":"2013","journal-title":"Angew. Chem. Int. Ed."},{"key":"qt5006_bb27","doi-asserted-by":"crossref","first-page":"2292","DOI":"10.1021\/acs.jmedchem.7b01408","volume":"61","author":"Linkuvien\u0117","year":"2018","journal-title":"J. Med. Chem."},{"key":"qt5006_bb28","doi-asserted-by":"crossref","first-page":"e10","DOI":"10.1017\/S0033583518000082","volume":"51","author":"Linkuvien\u0117","year":"2018","journal-title":"Q. Rev. Biophys."},{"key":"qt5006_bb29","doi-asserted-by":"crossref","first-page":"1550","DOI":"10.1021\/acs.accounts.8b00147","volume":"51","author":"Luchinat","year":"2018","journal-title":"Acc. Chem. Res."},{"key":"qt5006_bb30","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1042\/ETLS20170136","volume":"2","author":"Luchinat","year":"2018","journal-title":"Emerg. Top. Life Sci."},{"key":"qt5006_bb31","doi-asserted-by":"crossref","first-page":"17433","DOI":"10.1038\/s41598-017-17815-y","volume":"7","author":"Luchinat","year":"2017","journal-title":"Sci. Rep."},{"key":"qt5006_bb32","doi-asserted-by":"crossref","first-page":"9997","DOI":"10.1021\/acs.analchem.0c01677","volume":"92","author":"Luchinat","year":"2020","journal-title":"Anal. Chem."},{"key":"qt5006_bb33","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1007\/s10858-021-00358-w","volume":"75","author":"Luchinat","year":"2021","journal-title":"J. Biomol. NMR"},{"key":"qt5006_bb34","doi-asserted-by":"crossref","first-page":"6535","DOI":"10.1002\/anie.201913436","volume":"59","author":"Luchinat","year":"2020","journal-title":"Angew. Chem. Int. Ed."},{"key":"qt5006_bb35","doi-asserted-by":"crossref","first-page":"2792","DOI":"10.1021\/acschembio.0c00590","volume":"15","author":"Luchinat","year":"2020","journal-title":"ACS Chem. Biol."},{"key":"qt5006_bb36","doi-asserted-by":"crossref","first-page":"2727","DOI":"10.1021\/acs.biochem.5b00036","volume":"54","author":"Majumder","year":"2015","journal-title":"Biochemistry"},{"key":"qt5006_bb37","doi-asserted-by":"crossref","first-page":"19","DOI":"10.3390\/metabo8010019","volume":"8","author":"Mboge","year":"2018","journal-title":"Metabolites"},{"key":"qt5006_bb38","doi-asserted-by":"crossref","first-page":"484","DOI":"10.1097\/COC.0000000000000691","volume":"43","author":"McDonald","year":"2020","journal-title":"Am. J. Clin. Oncol."},{"key":"qt5006_bb39","doi-asserted-by":"crossref","first-page":"204","DOI":"10.3109\/14756366.2014.908291","volume":"30","author":"Mork\u016bnait\u0117","year":"2015","journal-title":"J. Enzyme Inhib. Med. Chem."},{"key":"qt5006_bb40","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1021\/acscentsci.8b00787","volume":"5","author":"Nguyen","year":"2019","journal-title":"ACS Cent. Sci."},{"key":"qt5006_bb41","doi-asserted-by":"crossref","first-page":"1175","DOI":"10.1080\/17460441.2019.1651289","volume":"14","author":"Nocentini","year":"2019","journal-title":"Exp. Opin. Drug. Discov."},{"key":"qt5006_bb42","doi-asserted-by":"crossref","first-page":"10834","DOI":"10.1021\/ja904407w","volume":"131","author":"Ogino","year":"2009","journal-title":"J. Am. Chem. Soc."},{"key":"qt5006_bb43","doi-asserted-by":"crossref","first-page":"101102","DOI":"10.1016\/j.redox.2019.101102","volume":"21","author":"Polykretis","year":"2019","journal-title":"Redox Biol."},{"key":"qt5006_bb44","doi-asserted-by":"crossref","first-page":"3367","DOI":"10.1002\/1873-3468.13250","volume":"592","author":"Primikyri","year":"2018","journal-title":"FEBS Lett."},{"key":"qt5006_bb45","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/nature07814","volume":"458","author":"Sakakibara","year":"2009","journal-title":"Nature"},{"key":"qt5006_bb46","doi-asserted-by":"crossref","first-page":"8014","DOI":"10.1021\/ja051306e","volume":"127","author":"Schanda","year":"2005","journal-title":"J. Am. Chem. Soc."},{"key":"qt5006_bb47","doi-asserted-by":"crossref","first-page":"9646","DOI":"10.1074\/jbc.M609679200","volume":"282","author":"Shimahara","year":"2007","journal-title":"J. Biol. Chem."},{"key":"qt5006_bb48","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1016\/j.jmr.2019.07.018","volume":"306","author":"Siegal","year":"2019","journal-title":"J. Magn. Reson."},{"key":"qt5006_bb49","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1007\/s10858-019-00243-7","volume":"73","author":"Simon","year":"2019","journal-title":"J. Biomol. NMR"},{"key":"qt5006_bb50","doi-asserted-by":"crossref","first-page":"1725","DOI":"10.1073\/pnas.1518620113","volume":"113","author":"Smith","year":"2016","journal-title":"Proc. Natl Acad. Sci. USA"},{"key":"qt5006_bb51","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1038\/nrd2467","volume":"7","author":"Supuran","year":"2008","journal-title":"Nat. Rev. Drug Discov."},{"key":"qt5006_bb52","doi-asserted-by":"crossref","first-page":"2023","DOI":"10.1042\/BCJ20160115","volume":"473","author":"Supuran","year":"2016","journal-title":"Biochem. J."},{"key":"qt5006_bb53","doi-asserted-by":"crossref","first-page":"1233","DOI":"10.1042\/CS20210040","volume":"135","author":"Supuran","year":"2021","journal-title":"Clin. Sci."},{"key":"qt5006_bb54","doi-asserted-by":"crossref","first-page":"7284","DOI":"10.1002\/anie.201900840","volume":"58","author":"Tanaka","year":"2019","journal-title":"Angew. Chem. Int. Ed."},{"key":"qt5006_bb55","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/nature16531","volume":"530","author":"Theillet","year":"2016","journal-title":"Nature"},{"key":"qt5006_bb56","doi-asserted-by":"crossref","first-page":"5758","DOI":"10.1002\/anie.201811714","volume":"58","author":"Vasa","year":"2019","journal-title":"Angew. Chem. Int. Ed."},{"key":"qt5006_bb57","doi-asserted-by":"crossref","first-page":"2662","DOI":"10.3390\/ijms10062662","volume":"10","author":"Zubrien\u0117","year":"2009","journal-title":"Int. J. Mol. Sci."}],"container-title":["Acta Crystallographica Section D Structural Biology"],"original-title":[],"link":[{"URL":"http:\/\/journals.iucr.org\/d\/issues\/2021\/10\/00\/qt5006\/qt5006.pdf","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"http:\/\/journals.iucr.org\/d\/issues\/2021\/10\/00\/qt5006\/qt5006.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,1,13]],"date-time":"2025-01-13T10:40:19Z","timestamp":1736764819000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.iucr.org\/paper?S2059798321009037"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,9,27]]},"references-count":57,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2021,10]]}},"alternative-id":["S2059798321009037"],"URL":"https:\/\/doi.org\/10.1107\/s2059798321009037","relation":{"is-supplemented-by":[{"id-type":"doi","id":"10.1107\/S2059798321009037\/qt5006sup1.pdf","asserted-by":"subject"}]},"ISSN":["2059-7983"],"issn-type":[{"value":"2059-7983","type":"print"}],"subject":[],"published":{"date-parts":[[2021,9,27]]},"assertion":[{"value":"Acta Crystallographica Section D: Structural Biology","order":0,"name":"journal","label":"Publication","explanation":{"URL":"http:\/\/journals.iucr.org\/d"},"group":{"name":"general","label":"General"}},{"value":"research papers","order":1,"name":"content_type","label":"Content type","explanation":{"URL":"https:\/\/journals.iucr.org\/d\/services\/notesforauthors.html"},"group":{"name":"general","label":"General"}},{"value":"Available","URL":"https:\/\/scripts.iucr.org\/cgi-bin\/citedin?qt5006","order":2,"name":"article_metrics","label":"Article metrics","group":{"name":"general","label":"General"}},{"value":"Yes","order":0,"name":"peer_reviewed","label":"Peer reviewed","group":{"name":"peer_review","label":"Peer review"}},{"value":"Single blind","order":1,"name":"review_process","label":"Review process","group":{"name":"peer_review","label":"Peer review"}},{"value":"3 June 2021","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication history"}},{"value":"31 August 2021","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication history"}},{"value":"27 September 2021","order":2,"name":"published_online","label":"Published online","group":{"name":"publication_history","label":"Publication history"}},{"value":"This article has supporting information","name":"supplementary_materials","label":"Supplementary materials","explanation":{"URL":"https:\/\/scripts.iucr.org\/cgi-bin\/sendsup?qt5006"}},{"value":"\u00a9 2021 Enrico Luchinat et al.","order":0,"name":"copyright","label":"Copyright","explanation":{"URL":"http:\/\/journals.iucr.org\/services\/copyrightpolicy.html"},"group":{"name":"copyright_and_licencing","label":"Copyright and licencing"}},{"value":"Creative Commons Attribution (CC-BY)","order":1,"name":"license","label":"Licence","explanation":{"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/legalcode"},"group":{"name":"copyright_and_licencing","label":"Copyright and licencing"}}]}}