{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T11:20:36Z","timestamp":1792668036593,"version":"4.5.0"},"reference-count":41,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2020,11,25]],"date-time":"2020-11-25T00:00:00Z","timestamp":1606262400000},"content-version":"vor","delay-in-days":65,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_ccby_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100010661","name":"Horizon 2020 Framework Programme","doi-asserted-by":"publisher","award":["810856"],"award-info":[{"award-number":["810856"]}],"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":["871037"],"award-info":[{"award-number":["871037"]}],"id":[{"id":"10.13039\/100010661","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003407","name":"Ministero dell\u2019Istruzione, dell\u2019Universit\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\/501100003407","name":"Ministero dell\u2019Istruzione, dell\u2019Universit\u00e0 e della Ricerca","doi-asserted-by":"publisher","award":["2017XYBP2R"],"award-info":[{"award-number":["2017XYBP2R"]}],"id":[{"id":"10.13039\/501100003407","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2020,10,16]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Candidate drugs rationally designed in vitro often fail due to low efficacy in vivo caused by low tissue availability or because of unwanted side effects. To overcome the limitations of in vitro rational drug design, the binding of candidate drugs to their target needs to be evaluated in the cellular context. Here, we applied in-cell NMR to investigate the binding of a set of approved drugs to the isoform II of carbonic anhydrase (CA) in living human cells. Some compounds were originally developed toward other targets and were later found to inhibit CAs. We observed strikingly different dose- and time-dependent binding, wherein some drugs exhibited a more complex behavior than others. Specifically, some compounds were shown to gradually unbind from intracellular CA II, even in the presence of free compound in the external medium, therefore preventing the quantitative formation of a stable protein\u2013ligand complex. Such observations could be correlated to the known off-target binding activity of these compounds, suggesting that this approach could provide information on the pharmacokinetic profiles of lead candidates at the early stages of multitarget drug design.<\/jats:p>","DOI":"10.1021\/acschembio.0c00590","type":"journal-article","created":{"date-parts":[[2020,9,21]],"date-time":"2020-09-21T12:14:02Z","timestamp":1600690442000},"page":"2792-2800","source":"Crossref","is-referenced-by-count":33,"title":["Intracellular Binding\/Unbinding Kinetics of Approved\nDrugs to Carbonic Anhydrase II Observed by in-Cell NMR"],"prefix":"10.1021","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4183-4311","authenticated-orcid":true,"given":"Enrico","family":"Luchinat","sequence":"first","affiliation":[{"name":"Universita\u0300 degli Studi di Firenze , , Via Luigi sacconi 6 , ,","place":["Sesto Fiorentino, Italy, 50019"]},{"name":"Consorzio per lo Sviluppo dei Sistemi a Grande Interfase \u2212 CSGI , Via della Lastruccia 3 , ,","place":["Sesto Fiorentino, Italy, 50019"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Letizia","family":"Barbieri","sequence":"additional","affiliation":[{"name":"Universita\u0300 degli Studi di Firenze , , Via Luigi sacconi 6 , ,","place":["Sesto Fiorentino, Italy, 50019"]},{"name":"Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine , Via Luigi Sacconi 6 , ,","place":["Sesto Fiorentino, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matteo","family":"Cremonini","sequence":"additional","affiliation":[{"name":"Universita\u0300 degli Studi di Firenze , , Via Luigi sacconi 6 , ,","place":["Sesto Fiorentino, Italy, 50019"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3342-702X","authenticated-orcid":true,"given":"Alessio","family":"Nocentini","sequence":"additional","affiliation":[{"name":"Universita\u0300 degli Studi di Firenze , , Via Ugo Schiff 6 , ,","place":["Sesto Fiorentino, Italy, 50019"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4262-0323","authenticated-orcid":true,"given":"Claudiu T.","family":"Supuran","sequence":"additional","affiliation":[{"name":"Universita\u0300 degli Studi di Firenze , , Via Ugo Schiff 6 , ,","place":["Sesto Fiorentino, Italy, 50019"]},{"name":"Universita\u0300 degli Studi di Firenze , , Via della Lastruccia 3 , ,","place":["Sesto Fiorentino, Italy, 50019"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0562-5774","authenticated-orcid":true,"given":"Lucia","family":"Banci","sequence":"additional","affiliation":[{"name":"Universita\u0300 degli Studi di Firenze , , Via Luigi sacconi 6 , ,","place":["Sesto Fiorentino, Italy, 50019"]},{"name":"Universita\u0300 degli Studi di Firenze , , Via della Lastruccia 3 , ,","place":["Sesto Fiorentino, Italy, 50019"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,9,21]]},"reference":[{"key":"2026090302082371300_cit1","doi-asserted-by":"publisher","first-page":"1531","DOI":"10.1126\/science.274.5292.1531","article-title":"Discovering high-affinity\nligands for proteins: SAR by NMR","volume":"274","author":"Shuker","year":"1996","journal-title":"Science"},{"key":"2026090302082371300_cit2","doi-asserted-by":"publisher","first-page":"738","DOI":"10.1038\/nrd2606","article-title":"Perspectives\non NMR\nin drug discovery: a technique comes of age","volume":"7","author":"Pellecchia","year":"2008","journal-title":"Nat. Rev. Drug Discovery"},{"key":"2026090302082371300_cit3","doi-asserted-by":"publisher","first-page":"137","DOI":"10.1021\/acsmedchemlett.7b00483","article-title":"HTS by NMR\nfor the Identification of Potent and Selective Inhibitors of Metalloenzymes","volume":"9","author":"Baggio","year":"2018","journal-title":"ACS Med. Chem. Lett."},{"key":"2026090302082371300_cit4","doi-asserted-by":"publisher","first-page":"106","DOI":"10.1038\/nature07839","article-title":"High-resolution\nmulti-dimensional\nNMR spectroscopy of proteins in human cells","volume":"458","author":"Inomata","year":"2009","journal-title":"Nature"},{"key":"2026090302082371300_cit5","doi-asserted-by":"publisher","first-page":"108","DOI":"10.1107\/S2052252516020625","article-title":"In-cell NMR: a topical\nreview","volume":"4","author":"Luchinat","year":"2017","journal-title":"IUCrJ"},{"key":"2026090302082371300_cit6","doi-asserted-by":"publisher","first-page":"2165","DOI":"10.1002\/anie.201712284","article-title":"Evaluation\nof the Stability of DNA i-Motifs in the Nuclei of Living Mammalian\nCells","volume":"57","author":"Dzatko","year":"2018","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026090302082371300_cit7","doi-asserted-by":"publisher","first-page":"1550","DOI":"10.1021\/acs.accounts.8b00147","article-title":"In-Cell NMR in Human\nCells: Direct Protein Expression Allows Structural\nStudies of Protein Folding and Maturation","volume":"51","author":"Luchinat","year":"2018","journal-title":"Acc.\nChem. Res."},{"key":"2026090302082371300_cit8","doi-asserted-by":"publisher","first-page":"7284","DOI":"10.1002\/anie.201900840","article-title":"High-Resolution Protein\n3D Structure Determination in Living Eukaryotic Cells","volume":"58","author":"Tanaka","year":"2019","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026090302082371300_cit9","doi-asserted-by":"publisher","first-page":"733","DOI":"10.1021\/acschembio.7b00879","article-title":"Potent Inhibitors\nof Mycobacterium tuberculosis Growth Identified by Using in-Cell NMR-based\nScreening","volume":"13","author":"DeMott","year":"2018","journal-title":"ACS Chem. Biol."},{"key":"2026090302082371300_cit10","doi-asserted-by":"publisher","first-page":"13281","DOI":"10.1021\/jacs.9b03031","article-title":"Monitoring\nDNA-Ligand Interactions in Living Human Cells Using NMR Spectroscopy","volume":"141","author":"Krafcikova","year":"2019","journal-title":"J. Am. Chem. Soc."},{"key":"2026090302082371300_cit11","doi-asserted-by":"publisher","first-page":"202","DOI":"10.1016\/j.jmr.2019.07.018","article-title":"Cells, drugs and NMR","volume":"306","author":"Siegal","year":"2019","journal-title":"J. Magn.\nReson."},{"key":"2026090302082371300_cit12","doi-asserted-by":"publisher","first-page":"139","DOI":"10.3390\/ijms20010139","article-title":"Applications\nof In-Cell NMR in Structural Biology and Drug Discovery","volume":"20","author":"Kang","year":"2019","journal-title":"Int. J. Mol. Sci."},{"key":"2026090302082371300_cit13","doi-asserted-by":"publisher","first-page":"6535","DOI":"10.1002\/anie.201913436","article-title":"Drug Screening in Human Cells by\nNMR Spectroscopy Allows the Early Assessment of Drug Potency","volume":"59","author":"Luchinat","year":"2020","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026090302082371300_cit14","doi-asserted-by":"publisher","first-page":"168","DOI":"10.1038\/nrd2467","article-title":"Carbonic\nanhydrases: novel therapeutic applications for inhibitors and activators","volume":"7","author":"Supuran","year":"2008","journal-title":"Nat. Rev. Drug Discovery"},{"key":"2026090302082371300_cit15","doi-asserted-by":"publisher","first-page":"2023","DOI":"10.1042\/BCJ20160115","article-title":"Structure\nand function of carbonic anhydrases","volume":"473","author":"Supuran","year":"2016","journal-title":"Biochem.\nJ."},{"key":"2026090302082371300_cit16","doi-asserted-by":"publisher","first-page":"4421","DOI":"10.1021\/cr200176r","article-title":"Multiple binding\nmodes of inhibitors to carbonic anhydrases: how to design specific\ndrugs targeting 15 different isoforms?","volume":"112","author":"Alterio","year":"2012","journal-title":"Chem.\nRev."},{"key":"2026090302082371300_cit17","doi-asserted-by":"publisher","first-page":"1175","DOI":"10.1080\/17460441.2019.1651289","article-title":"Advances\nin the structural annotation of human carbonic\nanhydrases and impact on future drug discovery","volume":"14","author":"Nocentini","year":"2019","journal-title":"Expert Opin. Drug Discovery"},{"key":"2026090302082371300_cit18","volume-title":"A Study of SLC-0111 and Gemcitabine for Metastatic Pancreatic Ductal\nCancer in Subjects Positive for CAIX - Full Text View","year":"2020"},{"key":"2026090302082371300_cit19","doi-asserted-by":"publisher","first-page":"484","DOI":"10.1097\/COC.0000000000000691","article-title":"A Phase 1 Study of SLC-0111, a Novel\nInhibitor of Carbonic Anhydrase IX, in Patients With Advanced Solid\nTumors","volume":"43","author":"McDonald","year":"2020","journal-title":"Am. J. Clin. Oncol."},{"key":"2026090302082371300_cit20","doi-asserted-by":"publisher","first-page":"550","DOI":"10.1021\/jm030912m","article-title":"Unexpected nanomolar inhibition of\ncarbonic anhydrase by COX-2-selective celecoxib: new pharmacological\nopportunities due to related binding site recognition","volume":"47","author":"Weber","year":"2004","journal-title":"J. Med. Chem."},{"key":"2026090302082371300_cit21","doi-asserted-by":"publisher","first-page":"437","DOI":"10.1016\/j.bmcl.2005.09.040","article-title":"Carbonic anhydrase\ninhibitors: Valdecoxib binds to a different active site region of\nthe human isoform II as compared to the structurally related cyclooxygenase\nII \u201cselective\u201d inhibitor celecoxib","volume":"16","author":"Di Fiore","year":"2006","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"2026090302082371300_cit22","doi-asserted-by":"publisher","first-page":"2567","DOI":"10.1016\/j.bmcl.2008.03.051","article-title":"Carbonic anhydrase\ninhibitors. Interaction of indapamide and related diuretics with 12\nmammalian isozymes and X-ray crystallographic studies for the indapamide-isozyme\nII adduct","volume":"18","author":"Temperini","year":"2008","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"2026090302082371300_cit23","doi-asserted-by":"publisher","first-page":"322","DOI":"10.1021\/jm801386n","article-title":"Carbonic anhydrase\ninhibitors. Comparison of chlorthalidone and indapamide X-ray crystal\nstructures in adducts with isozyme II: when three water molecules\nand the keto-enol tautomerism make the difference","volume":"52","author":"Temperini","year":"2009","journal-title":"J. Med. Chem."},{"key":"2026090302082371300_cit24","doi-asserted-by":"publisher","first-page":"2499","DOI":"10.1039\/b800767e","article-title":"Carbonic anhydrase\ninhibitors. Sulfonamide diuretics revisited--old leads for new applications?","volume":"6","author":"Temperini","year":"2008","journal-title":"Org. Biomol. Chem."},{"key":"2026090302082371300_cit25","doi-asserted-by":"publisher","first-page":"2315","DOI":"10.1016\/j.bmcl.2005.03.032","article-title":"Carbonic anhydrase inhibitors. Zonisamide\nis an effective inhibitor of the cytosolic isozyme II and mitochondrial\nisozyme V: solution and X-ray crystallographic studies","volume":"15","author":"De Simone","year":"2005","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"2026090302082371300_cit26","doi-asserted-by":"publisher","first-page":"10102","DOI":"10.1038\/s41598-017-10012-x","article-title":"Structural and\nFunctional View of Polypharmacology","volume":"7","author":"Moya-Garci\u0301a","year":"2017","journal-title":"Sci. Rep."},{"key":"2026090302082371300_cit27","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1080\/17460441.2020.1743676","article-title":"Exploring\nthe multiple binding modes of inhibitors to carbonic anhydrases for\nnovel drug discovery","volume":"15","author":"Supuran","year":"2020","journal-title":"Expert Opin. Drug Discovery"},{"key":"2026090302082371300_cit28","doi-asserted-by":"publisher","first-page":"239","DOI":"10.1016\/j.bpj.2018.11.3132","article-title":"Real-Time Insights into Biological\nEvents: In-Cell\nProcesses and Protein-Ligand Interactions","volume":"116","author":"Cerofolini","year":"2019","journal-title":"Biophys.\nJ."},{"key":"2026090302082371300_cit29","doi-asserted-by":"publisher","first-page":"9997","DOI":"10.1021\/acs.analchem.0c01677","article-title":"Real-Time Quantitative\nIn-Cell NMR: Ligand Binding and Protein Oxidation Monitored in Human\nCells Using Multivariate Curve Resolution","volume":"92","author":"Luchinat","year":"2020","journal-title":"Anal.\nChem."},{"key":"2026090302082371300_cit30","doi-asserted-by":"publisher","first-page":"12705","DOI":"10.1002\/chem.201301657","article-title":"19F NMR spectroscopy\nas a probe of cytoplasmic viscosity and weak protein interactions\nin living cells","volume":"19","author":"Ye","year":"2013","journal-title":"Chem. - Eur. J."},{"key":"2026090302082371300_cit31","doi-asserted-by":"publisher","first-page":"52","DOI":"10.1016\/j.ab.2015.11.015","article-title":"Fluorine nuclear magnetic\nresonance-based assay in living mammalian cells","volume":"495","author":"Veronesi","year":"2016","journal-title":"Anal. Biochem."},{"key":"2026090302082371300_cit32","doi-asserted-by":"publisher","first-page":"1101","DOI":"10.1038\/nprot.2016.061","article-title":"Characterization of\nproteins by in-cell\nNMR spectroscopy in cultured mammalian cells","volume":"11","author":"Barbieri","year":"2016","journal-title":"Nat. Protoc."},{"key":"2026090302082371300_cit33","doi-asserted-by":"publisher","first-page":"1243","DOI":"10.1107\/S0907444906029799","article-title":"A time- and cost-efficient system for high-level protein\nproduction in mammalian cells","volume":"62","author":"Aricescu","year":"2006","journal-title":"Acta Crystallogr.,\nSect. D: Biol. Crystallogr."},{"key":"2026090302082371300_cit34","doi-asserted-by":"publisher","first-page":"297","DOI":"10.1038\/nchembio.1202","article-title":"Atomic-resolution\nmonitoring of protein\nmaturation in live human cells by NMR","volume":"9","author":"Banci","year":"2013","journal-title":"Nat.\nChem. Biol."},{"key":"2026090302082371300_cit35","doi-asserted-by":"publisher","first-page":"661","DOI":"10.1007\/BF02192855","article-title":"Gradient-tailored excitation for\nsingle-quantum NMR spectroscopy of aqueous solutions","volume":"2","author":"Piotto","year":"1992","journal-title":"J. Biomol. NMR"},{"key":"2026090302082371300_cit36","doi-asserted-by":"publisher","first-page":"1126","DOI":"10.1107\/S0021889810030499","article-title":"Fityk: a general-purpose\npeak fitting program","volume":"43","author":"Wojdyr","year":"2010","journal-title":"J. Appl. Crystallogr."},{"key":"2026090302082371300_cit37","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1080\/10408340600970005","article-title":"Multivariate Curve Resolution (MCR) from 2000: Progress\nin Concepts and Applications","volume":"36","author":"de Juan","year":"2006","journal-title":"Crit. Rev. Anal.\nChem."},{"key":"2026090302082371300_cit38","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.chemolab.2014.10.003","article-title":"MCR-ALS GUI 2.0: New features and\napplications","volume":"140","author":"Jaumot","year":"2015","journal-title":"Chemom. Intell. Lab. Syst."},{"key":"2026090302082371300_cit39","doi-asserted-by":"publisher","first-page":"8094","DOI":"10.1021\/acs.jpcb.7b05679","article-title":"High-Resolution Solid-State\nNMR Characterization of\nLigand Binding to a Protein Immobilized in a Silica Matrix","volume":"121","author":"Cerofolini","year":"2017","journal-title":"J. Phys. Chem. B"},{"key":"2026090302082371300_cit40","doi-asserted-by":"publisher","first-page":"9646","DOI":"10.1074\/jbc.M609679200","article-title":"Tautomerism\nof histidine 64 associated\nwith proton transfer in catalysis of carbonic anhydrase","volume":"282","author":"Shimahara","year":"2007","journal-title":"J. Biol. Chem."},{"key":"2026090302082371300_cit41","doi-asserted-by":"publisher","first-page":"5758","DOI":"10.1002\/anie.201811714","article-title":"Assessment of a Large\nEnzyme-Drug Complex by Proton-Detected Solid-State NMR Spectroscopy\nwithout Deuteration","volume":"58","author":"Vasa","year":"2019","journal-title":"Angew. Chem., Int. Ed."}],"container-title":["ACS Chemical Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acschembio.0c00590","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/15\/10\/2792\/67151720\/acschembio.0c00590.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/15\/10\/2792\/67151720\/acschembio.0c00590.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T06:08:30Z","timestamp":1788415710000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acbcct\/article\/15\/10\/2792\/588795\/Intracellular-Binding-Unbinding-Kinetics-of"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,21]]},"references-count":41,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2020,9,21]]},"published-print":{"date-parts":[[2020,10,16]]}},"URL":"https:\/\/doi.org\/10.1021\/acschembio.0c00590","relation":{},"ISSN":["1554-8929","1554-8937"],"issn-type":[{"value":"1554-8929","type":"print"},{"value":"1554-8937","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,9,21]]}}}