title: Intrinsic binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI.
abstract: Carbonic anhydrase (CA) VI is a potential drug target for cariogenesis and cancer of the salivary gland. It is the only secreted human CA isozyme which is found in saliva and milk. Here, CA VI was expressed in bacterial and mammalian cell cultures and directly affinity-purified from human saliva. The binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to the native and recombinant CA VI from these three sources was compared. Interaction between the enzyme and inhibitors was determined by fluorescent thermal shift assay and isothermal titration calorimetry. The observed dissociation constants were the same within the error margin for all three CA VI preparations. The intrinsic binding parameters for the compounds were obtained by determining and dissecting the binding-linked protonation reactions. Intrinsic thermodynamic parameters of binding arrange the compounds in a buffer- and pH-independent manner. Intrinsic binding constants of nonfluorinated compounds were significantly stronger than those of fluorinated benzenesulfonamides. An opposite result was determined for the observed binding constants. The increase in observed affinity of the fluorinated compounds was due to the fluorine effect on diminishing the pKa of the compounds but not due to direct recognition of the protein. The temperature-stability profiles for recombinant and native CA VI were compared and showed that CA VI is more stable in slightly acidic than neutral conditions.
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Lithuania.
authors.forename: Justina
authors.initials: J
authors.lastname: Kazokaitė
authors.forename: Goda
authors.initials: G
authors.lastname: Milinavičiūtė
authors.forename: Joana
authors.initials: J
authors.lastname: Smirnovienė
authors.forename: Jurgita
authors.initials: J
authors.lastname: Matulienė
authors.forename: Daumantas
authors.initials: D
authors.lastname: Matulis
mesh_terms: D002151:Calorimetry / Q000379:methods; D002257:Carbonic Anhydrase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D000096926:Benzenesulfonamides
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D002256:Carbonic Anhydrases; D011994:Recombinant Proteins; D013449:Sulfonamides; D000096926:Benzenesulfonamides; C477051:carbonic anhydrase VI
keywords: ThermoFluor®; carbonic anhydrase VI; fluorescent thermal shift assay; intrinsic dissociation constants; isothermal titration calorimetry; salivary protein