Picomolar inhibitors of carbonic anhydrase: Importance of inhibition and binding assays

Abstract

The K_i of carbonic anhydrase (CA) inhibitors is often determined by the stopped- flow CO₂ hydration assay, the method that directly follows the inhibition of CA enzymatic activity. However, the assay has limitations, such as largely unknown concentration of CO₂ and the inability to determine the K_i below several nM. The widely used direct binding assay, isothermal titration calorimetry, also does not determine the K_d below several nM. In contrast, the thermal shift assay can accurately determine picomolar affinities. New equations estimating CO₂ concentration were developed for the determination of k_cat and K_M of CA I and CA II. The inhibitor dose-response curves were analyzed using Hill and Morrison equations demonstrating that only the Morrison model is applicable for the determination of tight-binding inhibitor K_i. The measurements of interactions between ten inhibitors and seven CA isoforms showed the limitations and advantages of all three techniques. Inhibitor 6 exhibited the K_d of 50 pM and was highly selective towards human CA IX, an isoform which is nearly absent in healthy human, but highly overexpressed in numerous cancers. Combination of inhibition and binding techniques was necessary for precise determination of CA-high-affinity inhibitor interactions and future drug design.

Keywords: Carbonic anhydrase; Differential scanning fluorimetry; Fluorescent thermal shift assay; Isothermal titration calorimetry; Stopped-flow CO(2) hydration assay; Tight-binding inhibitors.