Examination of Two Independent Kinetic Assays for Determining the Inhibition of Carbonic Anhydrases I and II: Structure-Activity Comparison of Sulfamates and Sulfamides

Enzyme inhibition assays often require deviations from physiological conditions. For carbonic anhydrases, procedures involving native CO2 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 CO2 and hydrolysis of 4‐nitrophenylacetate. For carbonic anhydrase II, the two assays consistently generated similar Ki 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 Ki values for three compounds differing by more than 2.5‐fold, up to eightfold. In the CO2 hydration assay, some sulfamates and sulfamides exhibited mixed kinetics or partial inhibition. Our results indicate that Ki or Kd values from carbonic anhydrase assays involving non‐native substrates should be confirmed by assays that use CO2 (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).