{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T09:28:08Z","timestamp":1773048488553,"version":"3.50.1"},"reference-count":45,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2021,12,23]],"date-time":"2021-12-23T00:00:00Z","timestamp":1640217600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004504","name":"Lietuvos Mokslo Taryba","doi-asserted-by":"publisher","award":["S-SEN-20-10"],"award-info":[{"award-number":["S-SEN-20-10"]}],"id":[{"id":"10.13039\/501100004504","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Among the twelve catalytically active carbonic anhydrase isozymes present in the human body, the CAIX is highly overexpressed in various solid tumors. The enzyme acidifies the tumor microenvironment enabling invasion and metastatic processes. Therefore, many attempts have been made to design chemical compounds that would exhibit high affinity and selective binding to CAIX over the remaining eleven catalytically active CA isozymes to limit undesired side effects. It has been postulated that such drugs may have anticancer properties and could be used in tumor treatment. Here we have designed a series of compounds, methyl 5-sulfamoyl-benzoates, which bear a primary sulfonamide group, a well-known marker of CA inhibitors, and determined their affinities for all twelve CA isozymes. Variations of substituents on the benzenesulfonamide ring led to compound 4b, which exhibited an extremely high observed binding affinity to CAIX; the Kd was 0.12 nM. The intrinsic dissociation constant, where the binding-linked protonation reactions have been subtracted, reached 0.08 pM. The compound also exhibited more than 100-fold selectivity over the remaining CA isozymes. The X-ray crystallographic structure of compound 3b bound to CAIX showed the structural position, while several structures of compounds bound to other CA isozymes showed structural reasons for compound selectivity towards CAIX. Since this series of compounds possess physicochemical properties suitable for drugs, they may be developed for anticancer therapeutic purposes.<\/jats:p>","DOI":"10.3390\/ijms23010130","type":"journal-article","created":{"date-parts":[[2021,12,23]],"date-time":"2021-12-23T10:16:18Z","timestamp":1640254578000},"page":"130","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Methyl 2-Halo-4-Substituted-5-Sulfamoyl-Benzoates as High Affinity and Selective Inhibitors of Carbonic Anhydrase IX"],"prefix":"10.3390","volume":"23","author":[{"given":"Audrius","family":"Zak\u0161auskas","sequence":"first","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Edita","family":"\u010capkauskait\u0117","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vaida","family":"Paketuryt\u0117-Latv\u0117","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexey","family":"Smirnov","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Janis","family":"Leitans","sequence":"additional","affiliation":[{"name":"Latvian Biomedical Research and Study Centre, Ratsupites 1 k-1, LV-1067 Riga, Latvia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andris","family":"Kazaks","sequence":"additional","affiliation":[{"name":"Latvian Biomedical Research and Study Centre, Ratsupites 1 k-1, LV-1067 Riga, Latvia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2873-0139","authenticated-orcid":false,"given":"Elviss","family":"Dvinskis","sequence":"additional","affiliation":[{"name":"Latvian Biomedical Research and Study Centre, Ratsupites 1 k-1, LV-1067 Riga, Latvia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laimonas","family":"Stan\u010daitis","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aurelija","family":"Mickevi\u010di\u016bt\u0117","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jelena","family":"Jachno","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linas","family":"Jezep\u010dikas","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vaida","family":"Linkuvien\u0117","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2715-067X","authenticated-orcid":false,"given":"Andrius","family":"Sakalauskas","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena","family":"Manakova","sequence":"additional","affiliation":[{"name":"Department of Protein\u2014DNA Interactions, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Saulius","family":"Gra\u017eulis","sequence":"additional","affiliation":[{"name":"Department of Protein\u2014DNA Interactions, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jurgita","family":"Matulien\u0117","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kaspars","family":"Tars","sequence":"additional","affiliation":[{"name":"Latvian Biomedical Research and Study Centre, Ratsupites 1 k-1, LV-1067 Riga, Latvia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6178-6276","authenticated-orcid":false,"given":"Daumantas","family":"Matulis","sequence":"additional","affiliation":[{"name":"Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saul\u0117tekio al. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,12,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Dodgson, S.J., Tashian, R.E., Gros, G., and Carter, N.D. (1991). The Carbonic Anhydrases, Springer.","DOI":"10.1007\/978-1-4899-0750-9"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Chegwidden, W.R., Carter, N.D., and Edwards, Y.H. (2000). The Carbonic Anhydrases, Birkh\u00e4user.","DOI":"10.1007\/978-3-0348-8446-4"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1002\/ijc.2910380406","article-title":"Monoclonal antibody G 250 recognizes a determinant present in renal-cell carcinoma and absent from normal kidney","volume":"38","author":"Oosterwijk","year":"1986","journal-title":"Int. J. Cancer"},{"key":"ref_4","first-page":"2877","article-title":"Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment","volume":"9","author":"Pastorek","year":"1994","journal-title":"Oncogene"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/S0002-9440(10)65569-1","article-title":"Immunohistochemical Study of Colorectal Tumors for Expression of a Novel Transmembrane Carbonic Anhydrase, MN\/CA IX, with Potential Value as a Marker of Cell Proliferation","volume":"153","author":"Saarnio","year":"1998","journal-title":"Am. J. Pathol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1021\/cr050262p","article-title":"Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein\u2212Ligand Binding","volume":"108","author":"Krishnamurthy","year":"2008","journal-title":"Chem. Rev."},{"key":"ref_7","first-page":"9419521","article-title":"Crystallography and Its Impact on Carbonic Anhydrase Research","volume":"2018","author":"Lomelino","year":"2018","journal-title":"Int. J. Med. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/j.ejmech.2018.06.059","article-title":"Design of two-tail compounds with rotationally fixed benzenesulfonamide ring as inhibitors of carbonic anhydrases","volume":"156","author":"Smirnov","year":"2018","journal-title":"Eur. J. Med. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"111825","DOI":"10.1016\/j.ejmech.2019.111825","article-title":"Halogenated and di-substituted benzenesulfonamides as selective inhibitors of carbonic anhydrase isoforms","volume":"185","author":"Smirnov","year":"2020","journal-title":"Eur. J. Med. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1513","DOI":"10.1016\/j.bpj.2020.08.037","article-title":"Isoform-Selective Enzyme Inhibitors by Exploring Pocket Size According to the Lock-and-Key Principle","volume":"119","author":"Kairys","year":"2020","journal-title":"Biophys. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1985","DOI":"10.1002\/cmdc.200900386","article-title":"Thermodynamic Optimisation in Drug Discovery: A Case Study using Carbonic Anhydrase Inhibitors","volume":"4","author":"Scott","year":"2009","journal-title":"ChemMedChem"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1016\/j.ejmech.2012.02.050","article-title":"Design of [(2-pyrimidinylthio)acetyl]benzenesulfonamides as inhibitors of human carbonic anhydrases","volume":"51","author":"Manakova","year":"2012","journal-title":"Eur. J. Med. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"6937","DOI":"10.1016\/j.bmc.2013.09.029","article-title":"Benzenesulfonamides with pyrimidine moiety as inhibitors of human carbonic anhydrases I, II, VI, VII, XII, and XIII","volume":"21","author":"Smirnov","year":"2013","journal-title":"Bioorg. Med. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Singh, S., Lomelino, C., Mboge, M., Frost, S., and McKenna, R. (2018). Cancer Drug Development of Carbonic Anhydrase Inhibitors beyond the Active Site. Molecules, 23.","DOI":"10.3390\/molecules23051045"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"14726","DOI":"10.1021\/ja3068098","article-title":"Neutron Diffraction of Acetazolamide-Bound Human Carbonic Anhydrase II Reveals Atomic Details of Drug Binding","volume":"134","author":"Fisher","year":"2012","journal-title":"J. Am. Chem. Soc."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Matulis, D. (2019). Observed Versus Intrinsic Thermodynamics of Inhibitor Binding to Carbonic Anhydrases. Carbonic Anhydrase as Drug Target: Thermodynamics and Structure of Inhibitor Binding, Springer International Publishing.","DOI":"10.1007\/978-3-030-12780-0"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"9435","DOI":"10.1021\/jm501003k","article-title":"Discovery and characterization of novel selective inhibitors of carbonic anhydrase IX","volume":"57","author":"Smirnov","year":"2014","journal-title":"J. Med. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1322","DOI":"10.1021\/bi802035f","article-title":"Design of a carbonic anhydrase IX active-site mimic to screen inhibitors for possible anticancer properties","volume":"48","author":"Genis","year":"2009","journal-title":"Biochemistry"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"7210","DOI":"10.1016\/j.bmc.2013.08.033","article-title":"Structural study of interaction between brinzolamide and dorzolamide inhibition of human carbonic anhydrases","volume":"21","author":"Pinard","year":"2013","journal-title":"Bioorg. Med. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5360","DOI":"10.1002\/slct.201700531","article-title":"Combinatorial Design of Isoform-Selective N-Alkylated Benzimidazole-Based Inhibitors of Carbonic Anhydrases","volume":"2","author":"Smirnov","year":"2017","journal-title":"ChemistrySelect"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e4412","DOI":"10.7717\/peerj.4412","article-title":"Crystal structure correlations with the intrinsic thermodynamics of human carbonic anhydrase inhibitor binding","volume":"6","author":"Smirnov","year":"2018","journal-title":"PeerJ"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/S0301-4622(01)00209-5","article-title":"Thermodynamics of the hydrophobic effect. III. Condensation and aggregation of alkanes, alcohols, and alkylamines","volume":"93","author":"Matulis","year":"2001","journal-title":"Biophys. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3222","DOI":"10.1529\/biophysj.108.134973","article-title":"A Quantitative Model of Thermal Stabilization and Destabilization of Proteins by Ligands","volume":"95","author":"Cimmperman","year":"2008","journal-title":"Biophys. J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"9004","DOI":"10.1021\/acs.jmedchem.5b01343","article-title":"Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX","volume":"58","author":"Leitans","year":"2015","journal-title":"J. Med. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"7413","DOI":"10.1016\/j.bmc.2010.09.011","article-title":"4-[N-(Substituted 4-pyrimidinyl)amino]benzenesulfonamides as inhibitors of carbonic anhydrase isozymes I, II, VII, and XIII","volume":"18","author":"Golovenko","year":"2010","journal-title":"Bioorg. Med. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1431","DOI":"10.1016\/j.bmc.2012.10.016","article-title":"Characterization of human carbonic anhydrase XII stability and inhibitor binding","volume":"21","author":"Matulis","year":"2013","journal-title":"Bioorg. Med. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1177\/108705710100600609","article-title":"High-density miniaturized thermal shift assays as a general strategy for drug discovery","volume":"6","author":"Pantoliano","year":"2001","journal-title":"J. Biomol. Screen"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1007\/s00249-021-01537-1","article-title":"Standard operating procedure for fluorescent thermal shift assay (FTSA) for determination of protein-ligand binding and protein stability","volume":"50","author":"Kazlauskas","year":"2021","journal-title":"Eur. Biophys. J."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1002\/(SICI)1097-0282(199905)49:6<451::AID-BIP3>3.0.CO;2-6","article-title":"1-Anilino-8-naphthalene sulfonate as a protein conformational tightening agent","volume":"49","author":"Matulis","year":"1999","journal-title":"Biopolymers"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"863","DOI":"10.3109\/14756360903571685","article-title":"Inhibition and binding studies of carbonic anhydrase isozymes I, II and IX with benzimidazo[1,2-c][1,2,3]thiadiazole-7-sulphonamides","volume":"25","author":"Hilvo","year":"2010","journal-title":"J. Enzyme Inhib. Med. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1002\/jmr.2550","article-title":"Applications of isothermal titration calorimetry\u2014The research and technical developments from 2011 to 2015: Review of Isothermal Titration Calorimetry from 2011 to 2015","volume":"29","author":"Falconer","year":"2016","journal-title":"J. Mol. Recognit."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1039\/C5NP00094G","article-title":"Application of isothermal titration calorimetry as a tool to study natural product interactions","volume":"33","author":"Callies","year":"2016","journal-title":"Nat. Prod. Rep."},{"key":"ref_33","first-page":"1","article-title":"Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design","volume":"51","author":"Manakova","year":"2018","journal-title":"Q. Rev. Biophys."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"17889","DOI":"10.1073\/pnas.1114107108","article-title":"Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase","volume":"108","author":"Snyder","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1107\/S0907444909047337","article-title":"XDS","volume":"66","author":"Kabsch","year":"2010","journal-title":"Acta Crystallogr. Sect. D"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1107\/S0907444910048675","article-title":"iMOSFLM: A new graphical interface for diffraction-image processing with MOSFLM","volume":"67","author":"Battye","year":"2011","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1107\/S0907444905036693","article-title":"Scaling and assta quality","volume":"62","author":"Evans","year":"2006","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_38","unstructured":"Bruker (2012). SAINT (Version 8.18c). Bruker Advanced X-ray Solutions, Bruker AXS."},{"key":"ref_39","unstructured":"Bruker (2012). SADABS (Version 2.11). Bruker Advanced X-ray Solutions, Bruker AXS."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1107\/S0907444909042589","article-title":"Molecular replacement with MOLREP","volume":"66","author":"Vagin","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1107\/S0907444911001314","article-title":"REFMAC5 for the refinement of macromolecular crystal structures","volume":"67","author":"Murshudov","year":"2011","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1107\/S0907444910007493","article-title":"Features and development of \u0131t Coot","volume":"66","author":"Emsley","year":"2010","journal-title":"Acta Crystallogr. Sect. D"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1186\/1758-2946-4-17","article-title":"Avogadro: An advanced semantic chemical editor, visualization, and analysis platform","volume":"4","author":"Hanwell","year":"2012","journal-title":"J. Cheminform."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1107\/S090744491200251X","article-title":"JLigand: A graphical tool for the CCP4 template-restraint library","volume":"68","author":"Lebedev","year":"2012","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2184","DOI":"10.1107\/S0907444904023510","article-title":"REFMAC5 dictionary: Organization of prior chemical knowledge and guidelines for its use","volume":"60","author":"Vagin","year":"2004","journal-title":"Acta Crystallogr. D Biol. Crystallogr."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/1\/130\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:51:57Z","timestamp":1760169117000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/1\/130"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,12,23]]},"references-count":45,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,1]]}},"alternative-id":["ijms23010130"],"URL":"https:\/\/doi.org\/10.3390\/ijms23010130","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,12,23]]}}}