基于ERα/VEGFR双靶点抗乳腺癌药物设计、生物活性评价及作用机制研究

Some SERMs such as Tamoxifen have been used in the treatment of ER(+)-breast cancer and are associated with an increased incidence of endometrial carcinoma due to the selectivity deficiency. On account of this fact, a series of compounds targeting ERα were designed and synthesized by our research group at the previous study. We happened to find that some compounds including WXH520 not only bound to ERα well but also inhibited the activity of VEGFR obviously. WXH520 exhibited anti-proliferative activity against ER(+)-breast cancer and ER(+)-endometrial carcinoma cell lines, and had effects of antiangiogenesis. It has currently been known that ligand-induced ERα signaling pathway cross-talks with RTKs' signaling pathway of some Gowth Factors when regulating the proliferation and differentiation of breast cancer cells and endometrial carcinoma cells. In this research project, novel compounds will be creatively designed by using WXH520 as lead compound and ERα/VEGFR as dual targets with the combination of CADD strategy. These compounds are likely to have good anti-breast cancer activity and satisfactory selectivity with their structural profiles and characterization of both SERMs and VEGFR inhibitors. A cis-tetrahydroisoquinoline ring is orient-specifically formed with catalyst, and then an isoquinolinone scaffold is produced through dehydrogenation. The target compounds will be obtained by introducing different aminoalkylphenyl side chains into the scaffold under the guide of diversity-oriented synthesis. The bioactivity of these novel compounds are evaluated by the experiments of ERα binding affinity, VEGFR inhibition and antitumor effects in vitro and in vivo, and the lead compounds with dual targets will be found out. The molecular mechanism will be explored, including expression fluctuation of some key proteins and genes related to ERα/VEGFR signaling pathways and their interaction induced by lead compounds, and the function difference between lead compound and Tamoxifen. It is predictable that the breakthrough in our research will be crucial for the development of novel anti-ER(+)-breast cancer drugs which target ERα/VEGFR and overcome the drawback of Tamoxifen after this research project is fulfilled.

针对他莫昔芬等SERMs药物在治疗ER阳性乳腺癌时，易引发子宫内膜癌的缺陷，本项目前期以ERα为靶点设计的SERMs化合物WXH520同时能抑制VEGFR活性，对乳腺癌、子宫内膜癌的细胞增殖和血管生成有抑制作用。近年来已知配体诱导的ERα与生长因子受体信号通路在调控乳腺癌和子宫内膜癌细胞增殖与分化时串话关联。本项目以WXH520为先导结构，结合CADD手段，首次基于ERα/VEGFR双靶点，设计兼具SERMs和VEGFR抑制剂结构特征的新型化合物。采用选择性催化法构建顺式取代的四氢异喹啉环，多样性导向合成法在异喹啉酮母核中引入定向设计的基团，构建目标化合物。通过ERα亲和性、VEGFR抑制力、体内外抑瘤实验评价生物活性，筛选活性先导物，研究其作用于双靶点信号通路后关键蛋白和基因的表达变化以及通路间的协调关联，并与他莫昔芬比较，探索作用机制，为研发能克服他莫昔芬缺陷的抗乳腺癌新药奠定基础。

针对他莫昔芬等选择性雌激素受体调节剂（SERMs）在治疗雌激素受体（ER）阳性乳腺癌时，易引发子宫内膜癌的缺陷，本项目前期以ERα为靶点设计了一系列SERMs化合物，发现WXH520在抑制ERα基因转录的同时，还能抑制VEGFR活性，对乳腺癌细胞和子宫内膜癌细胞的增殖都有抑制作用。根据文献报道：配体诱导的ERα与生长因子受体信号通路在调控乳腺癌和子宫内膜癌细胞增殖与分化时有串话关联。.在此背景下，本项目开展了以下研究工作并取得了重要结果：（1）以WXH520为先导结构，结合CADD手段，基于ERα/VEGFR双靶点，设计了兼具SERMs和VEGFR抑制剂结构特征的新型化合物；（2）合成了6个系列共249个化合物，均未见文献报道。其中芳基吲哚异喹啉酮衍生物的合成采用了选择性催化法构建顺式取代的四氢异喹啉环、多样性导向合成法在异喹啉酮母核中引入定向设计基团的策略。（3）对目标化合物进行了ERα亲和性和抑制活性、VEGFR抑制力的测试，以及对ERα(+)乳腺癌细胞株及子宫内膜癌细胞株的抑制活性测试，部分化合物活性突出。比如化合物21c，其对ERα表现出较好的亲和力，对ERα和VEGFR-2的IC50值分别为7.2μM和0.099μM，对MCF-7、MDA-MB-231和Ishikawa细胞株的IC50值分别为1.2μM、0.5μM和8.2μM。（4）探索了化合物21c通过ERα/VEGFR双靶点信号通路抑制ER（+）乳腺癌、并且抑制子宫内膜癌的作用机制，发现其能剂量依赖性地抑制VEGFR-2下游Raf/MAPK/ERK信号通路的关键蛋白Raf-1和ERK1/2的表达，从而产生ERα/VEGFR双靶点抑制乳腺癌的协同作用；由于Raf/MAPK/ERK信号通路与他莫昔芬引发子宫内膜癌相关，因此，ERα/VEGFR双靶点抗乳腺癌药物集SERMs和VEGFR抑制剂的优势于一体，有望成为能克服他莫昔芬缺陷的新型抗肿瘤药物。

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