靶向WT及V550M突变体的新型FGFR4抑制剂及抗肝癌活性研究

Hepatocellular carcinoma (HCC) is a common malignancy with high morbidity and mortality in China. FGFR4, a new member of the fibroblast growth factor receptor family, which plays sasignificant role in the angiogenesis, transplantation, division and proliferation of HCC, while the FGFR4 inhibitor can markedly block one step of above signals thereby prevent the HCC. The majority of reported small-molecule FGFR4 inhibitors for which in vitro pan-isoform selectivity data are available display much reduced potency toward FGFR4 compared to FGFR1-3 and other kinases. The lack of kinome selectivity invariably results in toxicity related to off-target activity and theV550M mutation in FGFR4 kinase often causes drug resistance to the inhibtiors. In our previous study, we found through computer-assisted analysis that the DFG-out domain and Cys-552 residue in ATP-binding pocket of FGFR4, are different from FGFR1-3 and other kinases, meanwhile the mutation of the so-called “gatekeeper” residue Met-550 displayed the strong “steric effect” with the rest of the core unit of Ponatinib. In this project, based on this finding and the special amino acid sequences of ATP-binding pocket, we plan to design and synthesize a novel series of potent and selective new generation inhibitors target FGFR4WT and FGFR4V550M. Meanwhile, these inhibitors will be chosen to test the inhibition and selectivity between FGFR4 （WT and V550M） and other RTKs, investigating the anti-tumor activity in vitro and in vivo,confirming the ligand binding mode and the sites of the FGFR4 protein through the crystallization of protein-ligand complexes. This project will provide new sites of action and selective and efficient inhibitors targeting FGFR4 in HCC.

肝癌是我国高发恶性肿瘤，FGFR家族中新成员FGFR4与肝癌的关系较为密切，其小分子抑制剂可有效治疗肝癌。目前的FGFR4抑制剂如Ponatinib等，普遍存在V550M继发性耐药、激酶选择性弱、成药性差等缺陷。我们前期发现FGFR4的“DFG-out”结构域及铰链区Cys-552残基均相异于其他激酶，且Ponatinib的母核与突变后的Met-550残基因存在“位阻效应”而导致耐药。本项目中，我们拟基于Ponatinib的耐药机制及在FGFR4（WT和V550M）结合口袋中的作用模式差异，针对受体结合域特有的空间结构和残基序列，设计并合成一类可双重作用于FGFR4WT和FGFR4V550M的新型选择性抑制剂。测试抑制剂的激酶抑制和选择性，表征体内外抗肿瘤活性，进而通过蛋白-药物共结晶确证活性化合物与受体的结合模式和位点。本项目将为FGFR4抑制剂类抗肝癌药物提供新的先导化合物和研究思路。

肝癌是我国高发恶性肿瘤，数据显示我国每年大约有19万人死于肝癌。成纤维生长因子受体（Fibroblast growth factor receptors，FGFR）家族中新成员FGFR4与肝癌的关系较为密切，其小分子抑制剂可有效治疗肝癌。目前的FGFR4抑制剂如Ponatinib等，临床试验中普遍存在V550M或V550L突变导致的继发性耐药、激酶选择性弱、成药性差等缺陷。本项目中，我们基于现有抑制剂的耐药机制及其在FGFR4（WT和V550M）结合口袋中的作用模式差异，利用计算机辅助药物设计平台及经典药物设计理念，设计并合成了2,4-二氨基嘧啶和苯并吲唑类两个系列共计70余新型小分子化合物，通过体外激酶筛选实验得到9f、9i和F-30共计三个结构新颖的抑制FGFR4或FGFR4V550L的高效选择性抑制剂。Western Blot、MTT、Hoechst染色和流式细胞技术等多种实验方法评价了三个活性抑制剂的体外抗肝癌活性。实验结果证实上述活性化合物不仅有效抑制FGFR4高表达的HUH7等肝癌细胞的增殖、阻滞细胞周期，而且可以有效抑制抑FGFR及其下游增殖相关的ERK、AKT蛋白的磷酸化，促进肿瘤凋亡。裸小鼠荷HUH7体内肿瘤模型评价了三个活性化合物的体内抗肿瘤活性，这些化合物均能有效抑制肿瘤生长，且不影响小鼠的体重。计算机辅助分子对接结果证实，活性化合物F-30的与FGFR4蛋白可形成共价结合，有效延长药物的作用时间和作用效果，高分辨质谱技术进一步确证了该结合模式。本课题的系列研究为FGFR4抑制剂的构效关系研究增加了新的内容，为肝癌靶向治疗研究提供了新的先导化合物和候选药物。