Picomolar inhibitors of carbonic anhydrase: Importance of inhibition and binding assays.
The Kiof carbonic anhydrase (CA) inhibitors is often determined by the stopped- flow CO2hydration assay, the method that directly follows the inhibition of CA enzymatic activity. However, the assay has limitations, such as largely unknown concentration of CO2and the inability to determine the Kibelow several nM. The widely used direct binding assay, isothermal titration calorimetry, also does not determine the Kdbelow several nM. In contrast, the thermal shift assay can accurately determine picomolar affinities. New equations estimating CO2concentration were developed for the determination of kcatand KMof 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 Ki. The measurements of interactions between ten inhibitors and seven CA isoforms showed the limitations and advantages of all three techniques. Inhibitor 6 exhibited the Kdof 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.
Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio al. 7, LT-10257 Vilnius, Lithuania. Electronic address: gylyte@ibt.lt.
Joana
J
Smirnovienė
Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio al. 7, LT-10257 Vilnius, Lithuania. Electronic address: vytautas@smirnovas.info.
Vytautas
V
Smirnovas
Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio al. 7, LT-10257 Vilnius, Lithuania. Electronic address: daumantas.matulis@bti.vu.lt.
Daumantas
D
Matulis
D002245:Carbon Dioxide / Q000737:chemistry; D024401:Carbonic Anhydrase I / Q000737:chemistry*; D024402:Carbonic Anhydrase II / Q000737:chemistry*; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry*; D003124:Colorimetry / Q000379:methods; D006801:Humans
D002257:Carbonic Anhydrase Inhibitors; D002245:Carbon Dioxide; D024401:Carbonic Anhydrase I; D024402:Carbonic Anhydrase II
Carbonic anhydrase; Differential scanning fluorimetry; Fluorescent thermal shift assay; Isothermal titration calorimetry; Stopped-flow CO(2) hydration assay; Tight-binding inhibitors