title: Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX.
abstract: Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regarding the expression and crystallization of the enzyme. Currently, only one baculovirus-produced CA IX structure in complex with a nonspecific CA inhibitor, acetazolamide, is available in Protein Data Bank. We have developed an efficient system for the production of the catalytic domain of CA IX in methylotrophic yeast Pichia pastoris. The produced protein can be easily crystallized in the presence of inhibitors, as we have demonstrated for several 2-thiophene-sulfonamide compounds. We have also observed significant differences in the binding mode of chemically identical compounds to CA IX and CA II, which can be further exploited in the design of CA IX-specific inhibitors.
authors.affiliation: Biomedical Research and Study Center , Ratsupites 1, LV-1067, Riga, Latvia.
authors.forename: Janis
authors.initials: J
authors.lastname: Leitans
authors.affiliation: Biomedical Research and Study Center , Ratsupites 1, LV-1067, Riga, Latvia.
authors.forename: Andris
authors.initials: A
authors.lastname: Kazaks
authors.affiliation: Latvian Institute of Organic Synthesis , Aizkraukles 21, LV-1006, Riga, Latvia.
authors.forename: Agnese
authors.initials: A
authors.lastname: Balode
authors.affiliation: Latvian Institute of Organic Synthesis , Aizkraukles 21, LV-1006, Riga, Latvia.
authors.forename: Jekaterina
authors.initials: J
authors.lastname: Ivanova
authors.affiliation: Latvian Institute of Organic Synthesis , Aizkraukles 21, LV-1006, Riga, Latvia.
authors.forename: Raivis
authors.initials: R
authors.lastname: Zalubovskis
authors.affiliation: NEUROFARBA Department, Section of Pharmaceutical Chemistry, Università degli Studi di Firenze , Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.|Laboratorio di Chimica Bioinorganica, Polo Scientifico, Università degli Studi di Firenze , Rm. 188, Via dellaLastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.affiliation: Biomedical Research and Study Center , Ratsupites 1, LV-1067, Riga, Latvia.|Faculty of Biology, Department of Molecular Biology, University of Latvia , Jelgavas 1, LV-1004, Riga, Latvia.
authors.forename: Kaspars
authors.initials: K
authors.lastname: Tars
mesh_terms: D000086:Acetazolamide / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000494:pharmacology; D016367:Baculoviridae / Q000378:metabolism; D000071231:Carbonic Anhydrase IX; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000096:biosynthesis / Q000737:chemistry*; D003001:Cloning, Molecular; D003460:Crystallization; D030562:Databases, Protein; D006801:Humans; D007527:Isoenzymes; D008958:Models, Molecular; D009369:Neoplasms / Q000201:enzymology*; D010843:Pichia / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D002257:Carbonic Anhydrase Inhibitors; D007527:Isoenzymes; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D000086:Acetazolamide