Smirnovienė Joana, Smirnovas Vytautas, Matulis Daumantas
Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio al. 7, LT-10257 Vilnius, Lithuania.
Anal Biochem. 2017 Apr 1;522:61-72. doi: 10.1016/j.ab.2017.01.022. Epub 2017 Jan 31.
The K of carbonic anhydrase (CA) inhibitors is often determined by the stopped- flow CO hydration assay, the method that directly follows the inhibition of CA enzymatic activity. However, the assay has limitations, such as largely unknown concentration of CO and the inability to determine the K below several nM. The widely used direct binding assay, isothermal titration calorimetry, also does not determine the K below several nM. In contrast, the thermal shift assay can accurately determine picomolar affinities. New equations estimating CO concentration were developed for the determination of k and K 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. 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 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.
碳酸酐酶（CA）抑制剂的解离常数（K）通常通过停流CO水合测定法来确定，该方法可直接监测CA酶活性的抑制情况。然而，该测定法存在局限性，比如CO浓度大多未知，且无法测定低于几纳摩尔的K值。广泛使用的直接结合测定法——等温滴定量热法，也无法测定低于几纳摩尔的K值。相比之下，热位移测定法能够准确测定皮摩尔亲和力。为了测定CA I和CA II的k值和K值，开发了估算CO浓度的新方程。使用希尔方程和莫里森方程分析抑制剂剂量反应曲线，结果表明只有莫里森模型适用于测定紧密结合抑制剂的K值。对十种抑制剂与七种CA同工型之间相互作用的测量显示了这三种技术的局限性和优势。抑制剂6的K值为50皮摩尔，对人CA IX具有高度选择性，CA IX在健康人体内几乎不存在，但在多种癌症中高度过表达。抑制和结合技术的结合对于精确测定CA高亲和力抑制剂相互作用以及未来的药物设计是必要的。
