Development of the 4th generation calcium antagonists based on molecular revealing the binding sites in calcium channels

本课题旨在利用光亲和标记技术揭示钙拮抗剂在心肌钙通道中的结合位点，并将其应用于新药的设计和合成。新的调查结果如下。(1)我们确定了心脏通道中的二氢吡啶位点，其与骨骼肌对应物相同，但一些氨基酸被取代。这种取代可以解释为主导两种钙通道结合亲和力的差异。(2)苯并硫氮杂卓结合位点，确定在第5和第6段重复IV的光亲和标记使用叠氮丁酰克仑硫卓与一个相当小的光探针。最近国外研究者的报道认为，重复序列III和IV中的两个标记位点是一个错误的结论，这是由于使用了大体积的长侧链光探针。(3)我们揭示了一种新型钙拮抗剂1，4-苯并噻嗪结构I的结合位点，它与苯并硫氮杂卓2同源，但所鉴定的位点不同。结果可以解释两种药物的药理学差异。(4)X射线晶体学分析表明，化合物1和2的三维结构不对称。这表明，我们将设计和合成改进的钙拮抗剂，衍生自2作为先导化合物。

In this project, we aim to reveal binding sites for calcium antagonists in cardiac calcium channels by photoaffinity labeling and the results are to be applied to design and synthesis of new drugs. New findings are as follows. (1) We identified the dihydropyridine sites in cardiac channel, which were identical to the skeletal muscle counterpart but some amino acids were substituted. The substitution can be interpreted to dominate the difference of binding affinity in two calcium channels. (2) Benzothiazepine binding sites were identified in segment 5 and 6 in repeat IV by photoaffinity labeling using azidobutyryl clentiazem with a rather small-sized photoprobe. The recent report by foreign investigaters where two labeled sites in repeat III and IV must be a wrong conclusion resulted from using a bulky photoprobe with long side chain. (3) We revealed the binding site of a new typed calcium antagonist of 1, 4-benzothiazine structure I which is homologous to benzothiazepine 2 but the identified sites were different. The results can explain the pharmacological difference between two drugs. (4) Three dimentional structures of 1 and 2 are not ovelapped by x-ray crystallographic analysis. It suggests that we will design and synthesis of improved calcium antagonists which are derived from 2 as a lead compound.