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Examination of Two Independent Kinetic Assays for Determining the Inhibition of Carbonic Anhydrases I and II: Structure–Activity Comparison of Sulfamates and Sulfamides
Author(s) -
Shank Richard P,
McComsey David F,
SmithSwintosky Virginia L,
Maryanoff Bruce E
Publication year - 2006
Publication title -
chemical biology and drug design
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.59
H-Index - 77
eISSN - 1747-0285
pISSN - 1747-0277
DOI - 10.1111/j.1747-0285.2006.00423.x
Subject(s) - sulfamide , carbonic anhydrase , chemistry , enzyme , carbonic anhydrase ii , selectivity , isozyme , enzyme kinetics , carbonic anhydrase i , stereochemistry , biochemistry , active site , organic chemistry , catalysis
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).