基于受体结构的新型氮杂吲哚-2-酮类FGFR1抑制剂的设计、合成及抗肿瘤活性研究

The singaling pathway induced by Fibroblast growth factor receptor (FGFR1) plays sasignificant role in tumor is relative to the tumor angiogenesis, transplantation, division and proliferation, while the FGFR1 inhibitor can markedly block one step of above signals thereby prevent the tumor. There are several FGFR1 inhibitors have currently used in clinical trials for cancer treatment, most inhibitors bind to the middle and outer of relatively conserved ATP-binding domain in RTKs and lack kinase selectivity, which gives rise to a variety of side and toxic effects. we found through computer-assisted analysis that the ATP-binding sites in currently available inhibitors account for only a fraction of the ATP-binding pocket in the FGFR1 protein and are localized in the middle and outside within the cavity of the whole ATP-binding pocket, it is different from other RTKs. As one of FGFR1 inhibitors, compound SU5402 displayed the significantly selective inhibitory effect. In our project, based on this finding and the amino acid sequence inside of ATP-binding pocket, we plan to design and synthesize a series of potent azaindolin-2-one FGFR1 inhibitors and further explore the structure-activity relationship on 3 position of the core which never be explored before. Meanwhile, several inhibitors will be choosed to test the inhibition on FGFR1 and other RTKs, investigating the anti-tumor activity in vitro and in vivo, confirmation of the target by siRNA technology, supplemented by CAD docking techconique and ATP competition experiments conclusive evidence of the binding mode and the sites of the FGFR1. The project will FGFR1 as targets of anticancer drugs to provide new sites of action and selective and efficient inhibitor.

成纤维生长因子受体(FGFR) 介导的信号转导途径参与肿瘤细胞形成和发展过程，而FGFR抑制剂能阻断信号传导的某个环节，达到抑制肿瘤发生、发展的目的。临床已有部分FGFR抑制剂应用于肿瘤治疗，如吲哚-2-酮类化合物SU5402等，但它们主要结合于受体口袋的中部或外侧，缺乏对激酶的选择性，显示出多种毒副作用。我们前期发现 FGFR1 结合口袋内部存在特异性的氨基酸序列和结构，相异于其他激酶。本项目中，我们基于SU5402在FGFR1结合口袋的作用模式，针对结合域内部特有的空间和氨基酸序列设计并合成高效氮杂吲哚-2-酮类FGFR1选择性抑制剂；测试该类抑制剂对FGFR1和其他激酶的抑制，表征体内外抗肿瘤活性，通过siRNA技术明确其作用靶点，并辅以分子对接技术和ATP竞争实验确证与FGFR1的结合模式和位点。本项目将为以FGFR1为靶标的抗肿瘤药物研究提供新的作用位点和新型选择型高效抑制剂。

成纤维细胞生长因子受体（FGFR）广泛高表达于乳腺癌、胃癌等多种肿瘤细胞中，已被认为是抗癌药物开发的重要靶点。开发FGFR小分子抑制剂类抗肿瘤药物有着良好的应用前景。然而，经典的FGFR竞争性抑制剂，如SU5402等都普遍存在特异性靶向性差、副作用大和容易产生耐药性的缺点。我们前期在SU5402与FGFR1的蛋白-小分子药物晶体结构中研究发现：SU5402主要结合于FGFR1受体口袋的中部或外侧，缺乏对激酶的选择性，从而显示出多种毒副作用。此外，FGFR1受体结合口袋内部存在特异性的氨基酸序列和结构，相异于其他激酶。本课题中，我们以FGFR1为靶标，以化合物SU5402为先导化合物，利用基于靶点蛋白三维结构的计算机辅助药物设计和经典药物设计理论，设计并合成了一系列吲哚-2-酮及双芳基-1，4-戊二烯-3-酮类似物，通过激酶筛选实验得到多个结构新颖的FGFR1抑制剂。Western Blot、 MTT、Hoechst染色和流式细胞技术等多种实验方法评价了活性抑制剂的体外抗肿瘤活性。其中，化合物A114和A117不仅有效抑制FGFR1高表达的非小细胞肺癌H460细胞的增殖和阻滞H460细胞的细胞周期，而且可以通过诱导凋亡蛋白Caspas3的表达，促进肿瘤凋亡。裸小鼠荷H460体内肿瘤模型评价了A114和A11的体内抗肿瘤活性，两个化合物均能有效抑制肿瘤生长，且不影响受试体的体重。肿瘤组织的Western Blot和免疫组化实验显示，上述化合物均能有效地抑制了FGFR1及其下游增殖相关的ERK、AKT蛋白的磷酸化，并下调Bcl-2、COX-2及Cyclin D1等多种促肿瘤相关蛋白的表达。本课题为FGFR1抑制剂的构效关系研究增加了新的内容，为肺癌靶向治疗研究提供了新的先导化合物。