title: Examination of two independent kinetic assays for determining the inhibition of carbonic anhydrases I and II: structure-activity comparison of sulfamates and sulfamides.
abstract: Enzyme inhibition assays often require deviations from physiological conditions. For carbonic anhydrases, procedures involving native CO(2) and non-native substrates have been used. We compared a native and a non-native substrate in the context of inhibition of human carbonic anhydrases I and II by examining various sulfamate and sulfamide compounds in two kinetic assays: hydration of CO(2) and hydrolysis of 4-nitrophenylacetate. For carbonic anhydrase II, the two assays consistently generated similar K(i) values, with the relative difference between the assays never exceeding 2.5-fold. However, for carbonic anhydrase I there was more variability between the two assays, with K(i) values for three compounds differing by more than 2.5-fold, up to eightfold. In the CO(2) hydration assay, some sulfamates and sulfamides exhibited mixed kinetics or partial inhibition. Our results indicate that K(i) or K(d) values from carbonic anhydrase assays involving non-native substrates should be confirmed by assays that use CO(2) (or HCO), to establish pharmacological relevance. From structure-activity comparisons, the sulfamate is more effective than the sulfamide in inhibiting carbonic anhydrase I and II, but the sulfamate does not confer selectivity. In contrast, the sulfonamide confers selectivity for carbonic anhydrase I (10- to 30-fold). Selectivity for carbonic anhydrase II occurred with the substituted fructose moiety, especially the d-enantiomer (>100-fold).
authors.affiliation: Research & Early Development, Johnson & Johnson Pharmaceutical Research & Development, Spring House, PA 19477-0776, USA.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Shank
authors.forename: David F
authors.initials: DF
authors.lastname: McComsey
authors.forename: Virginia L
authors.initials: VL
authors.lastname: Smith-Swintosky
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Maryanoff
mesh_terms: D024401:Carbonic Anhydrase I / Q000037:antagonists & inhibitors*; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors*; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D007700:Kinetics; D013329:Structure-Activity Relationship; D013451:Sulfonic Acids / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D013451:Sulfonic Acids; C005741:sulfamic acid; D024401:Carbonic Anhydrase I; D024402:Carbonic Anhydrase II