靶向成纤维生长因子受体4(FGFR4)选择性抑制剂的发现及其抗肿瘤机理研究

Fibroblast growth factor receptors are important tyrosine kinases that regulate numerous capital cellular processes, such as proliferation, cell growth, differentiation and migration. Dysregulation of the FGF/FGFR signaling pathway has been associated with multiple developmental disorders and cancers. Among four FGFRs family members, FGFR4 is least well understood but playing a significant role in many cancers. Overexpression of FGFR4 has been described in various malignancies. FGFR4 differs from FGFR1-3 in several aspects which shares the least homology with the other FGFR members. Above all, unlike FGFR1-3, FGFR4 has not been linked to skeletal disorders. Deletion of FGFR4 does not lead to an embryonic lethal phenotype. These findings suggest the possibility that its inhibition in cancer therapy might not cause grave adverse effects, and hence highlights the clinical value in developing FGFR4 inhibitors. To date, there is no specific inhibitor for FGFR4. Most of the reported FGFR inhibitors are more potent against FGFR1-3 than FGFR4. A more selective inhibitor is in great need to be discovered so as to get a better understanding the role played by FGFR4 signaling in both physiological and pathological processes, and to enable exploration of FGFR4 as a therapeutic target in cancer. In our previous studies, the crystal structures of FGFR4-ligand (data from the collaborating lab) were analyzed. This structural information illustrates FGFR4 kinase conformational state is DFG-out conformation. We perform library screening and hit finding to find flavonoids which can provide a more appropriate space skeleton. The chromenone moiety of the ?avonoid can fit into ATP-binding domain and the allosteric pocket of FGFR4. About 20 flavonoid derivatives are thereby designed and synthesized, which show selective inhibition effect on FGFR4 in a series of biological tests. In this project, we aim to develop potent and selective inhibitor of FGFR4 based on flavonoid skeleton. A variety of flavonoid derivatives will be designed and synthesized. To verify their selective and inhibition to FGFR4, we choose MKN45 and SGC7901 cells for FGFR4 siRNA assays to investigate the knockdown of FGFR4 expression. In addition, we perform a series of functional assays in the several cell lines, including proliferation assays, migration assays, apoptosis detection and immunoprecipitation. On the basis of their in vitro profile, 2-3 compunds are selected and evaluated in a gastric cancer model based on the human gastric cancer cell line SGC7901 which overexpresses FGFR4. Protein crystallization of the protein?ligand complex and X-ray diffraction experiments are performed to provide insight and understanding. As a research tool, flavonoid derivatives will be invaluable for mechanistic studies to delineate the precise role of FGFR4 in the pathogenesis of such diseases so that their clinical applications as drug targets can be justified.

FGFR受体酪氨酸激酶家族已成为肿瘤治疗的重要靶点。在该家族中，FGFR4的功能和结构与FGFR1-3有较大的差异，且FGFR4的敲除不会导致早期胚胎死亡，抑制FGFR4副作用较小。但目前的抑制剂对FGFR4的抑制活性和选择性都较差。本课题组前期通过对天然活性化合物库的药效团筛选，发现黄酮骨架化合物具有选择性抑制FGFR4的作用，并初步分析了其结合机制，为FGFR4选择性抑制剂提供了新的先导结构和方向。本项目中，我们以黄酮骨架为母核，分别进行基于受体和配体的FGFR4选择性抑制剂设计，发现选择性和活性更好的抑制剂，并通过多种肿瘤细胞株，测定它们通过抑制FGFR4对下游信号通路、细胞存活、凋亡及迁移的影响；表征其体内抗肿瘤活性，明确其作用靶点，并通过蛋白-抑制共结晶明确抑制剂与FGFR4的结合模式和位点。本项目将为以FGFR4为靶标的抗肿瘤药物研究提供新的作用位点和新型选择性高效抑制剂。

成纤维细胞生长因子受体4（FGFR4）通过与FGF1、FGF19结合激活PI3K-AKT、RAS-MAPK等信号通路，从而参与调节许多重要生物学过程。FGFR4的过表达和突变会导致下游信号通路的持续激活，与多种恶性肿瘤的发生发展密切相关。FGFR4基因异常与胃癌发生有密切关系，但目前的抑制剂对FGFR4的抑制活性和选择性都较差。本课题组分析FGFR4蛋白晶体结构，构建了虚拟筛选模型，从Spec商业库中筛选后，购买83个化合物，并用SPR测试其激酶活性，结果得到7个活性较好先导化合物，其中两个化合物呈Type-1/2型构象，这种构型的抑制剂在FGFR家族中首次报道。进一步对先导物进行结构改造，合成50多个化合物。体外活性筛选得到有IC50小于100nM的三个化合物chen23、chen27和chen35。然后用FGFR4高表达的胃癌细胞（MKN45和SGC7901）验证了化合物剂量相关的抑制细胞生长，测定它们通过抑制FGFR4对下游信号通路、细胞存活、凋亡及迁移的影响。通过体外裸鼠模型，将抑制剂chen35连续给药 28d，表现出了较好的抑瘤率和肿瘤生长抑制作用，抑瘤率为77.6%。本项目将为以FGFR4为靶标的抗肿瘤药物研究提供新的作用位点和新型抑制剂。