计算机辅助设计HER2/neu酪氨酸激酶小分子抑制剂及其生物活性研究

Discovery of HER2/neu Tyrosine Kinase Inhibitor through Computer- Aided Drug Design Approach and its Biological Activity

目的：用计算机辅助设计法寻找 HER2/neu受体酪氨酸激酶小分子抑制剂。方法：用 MODERLAR软件模拟出 HER2/neu和 EGFR受体酪氨酸激酶的三维 (three- dimensional,3D)空间结构；根据 HER2/neu酪氨酸激酶 ATP结合区的空间结构，搜索数据库，找出候选化合物；用 Western blot方法检测化合物对 HER2/neu受体酪氨酸激酶磷酸化的影响； MTT法检测细胞增殖抑制作用。结果：比较 HER2/neu和 EGFR（靶蛋白）与胰岛素受体酪氨酸激酶、 FGFR1受体酪氨酸激酶和 Src酪氨酸激酶（模板蛋白）的氨基酸序列发现有 35％～ 41％的相同和 52％～ 55％的相似，用 MODELLER程序模拟出 HER2/neu和 EGFR激酶区域的 3D结构。根据 HER2/neu受体酪氨酸激酶结构模型， 3D数据库的搜索，获得潜在 HER2/neu酪氨酸激酶抑制剂化合物，经筛选、优化发现 ST2325对 HER2/neu磷酸化有明显的抑制作用，其 IC50值为 6.6μ mol/L，且抑制作用是可逆的，对 EGFR受体磷酸化没有抑制作用。 ST2325对 HER2/neu受体表达没有影响。 ST2325对 HER2/neu过表达的肿瘤细胞 MDA- MB- 453 m1的抑制作用更强，而对 EGFR过表达的肿瘤细胞 MDA- MB- 468抑制作用相对较弱，其 IC50值分别为 29.05μ mol/L和 60.40μ mol/L。结论：基于 HER2/neu的结构设计，筛选?

Objective： To discover HER2/neu tyrosine kinase inhibitor through computer- aided drug design approach. Methods： The three- dimensional(3D) structure of HER2/neu and EGFR tyrosine kinase domain was modeled using MODERLAR software. To search database and pick up candidate compounds based 3D structure of HER2/neu tyrosine kinase domain. Inhibition of HER2/neu tyrosine kinase phosphorylation by the compounds was detected using Western blot analysis. Inhibition of cell proliferation was determined by MTT assay. Results： The amino acid identity and similarity between HER2/neu and EGFR kinase domain, insulin receptor kinase domain, FGFR1 kinase domain, Src kinase domain were compared. The identity was 35％－ 41％ and the similarity was 52％－ 55％ . The 3D structure of HER2/neu and EGFR was obtained by MODERLAR software. Through searching, screening and optimization, the authors found that ST2325 had significantly inhibitory effect on HER2/neu tyrosine kinase phosphorylation with IC50 of 6.6μ mol/L. The inhibition of HER2/neu phosphorylation was selective and reversible. ST2325 inhibited cell proliferation of HER2/neu- overexpressing MDA- MB- 453m1 preferentially compared with EGFR- overexpressing MDA- MB- 468, The IC50 values were 29.05 and 60.4μ mol/L. Conclusion： ST2325 which was discovered through structure- based approach had notable inhibitory effect on HER2/neu tyrosine kinase.