title: Carbonic anhydrase inhibitors: cloning, characterization, and inhibition studies of the cytosolic isozyme III with sulfonamides.
abstract: The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(-1) and k(cat)/K(M) of 2.5 x 10(5) M(-1) s(-1), being a slower catalyst for the physiological reaction as compared to the genetically related cytosolic isoforms hCA I and II. An inhibition study with a library of sulfonamides and one sulfamate, some which are clinically used compounds, is reported. hCA III is less prone to be inhibited by these compounds as compared to hCA I and II for which many low nanomolar inhibitors were detected earlier. The best hCA III inhibitors were prontosil, sulpiride, indisulam, benzolamide, aminobenzolamide, and 4-amino-6-chloro-benzene-1,3-disulfonamide which showed K(I)s in the range of 2.3-18.1 microM. Clinically used compounds such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, topiramate, zonisamide, celecoxib, and valdecoxib were less effective hCA III inhibitors, with affinities in the range of 154-2200 microM. This is the first study in which low micromolar hCA III inhibitors are reported.
authors.affiliation: Department of Gastroenterology and Hepatology, Kochi Medical School, Nankoku, Kochi 783-8505, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Nishimori
authors.forename: Tomoko
authors.initials: T
authors.lastname: Minakuchi
authors.forename: Saburo
authors.initials: S
authors.lastname: Onishi
authors.forename: Daniela
authors.initials: D
authors.lastname: Vullo
authors.forename: Alessandro
authors.initials: A
authors.lastname: Cecchi
authors.forename: Andrea
authors.initials: A
authors.lastname: Scozzafava
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
mesh_terms: D000595:Amino Acid Sequence; D024403:Carbonic Anhydrase III / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000302:isolation & purification; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology*; D013451:Sulfonic Acids / Q000737:chemistry / Q000494:pharmacology