STAT3 CCD结构域的新型抗肿瘤抑制剂的设计优化和功能研究

Signal transducer and activator of transcription 3(STAT3) signaling pathway is constitutively activated in human cancer cells. It is now established that the specificity in signaling are mediated by STAT3 Coiled-coil domain (CCD), which regulate and mediate the interaction with the phosphopeptide docking sites displayed by receptors, dimerization and subsequent DNA binding. The goal of this project is to find a Novel Inhibitor of the Coiled-coil Domain of STAT3 Signaling Pathway against Cancer and develop a useful theoretical foundation in drug design for targeting STAT3.After structure-based screening we detected the activity of these compounds by fluorescence polarization (FP). We also identified that K116 inhibits the growth of tumor cells harboring abnormal activation of STAT3, such as, MDA-MB-468，MDA-MB-231 and DU145. The binding affinity of K116 to CCD and WT of STAT3 are 3.2μM and 2.397μM. Our studies indicate that K116 binds to STAT3 Coiled-coil domain, and we also detect the binding affinity of inhibitors and mutants. Based on these data, we analysis the SAR for optimizing the inhibitors. Meanwhile, we plan to detect the phosphorylation level of 705 Tyr on STAT3, Src, STAT1, STAT5 and downstream targets of STAT3, such as Cyclin D1, C-myc and Bcl-xl in MDA-MB-468 cells in protein level. The suppression of pY705STAT3 nuclear accumulation and migration of MDA-MB-468 cell will also be testing. Finally, we expect the inhibitors induce MDA-MB-468 cell apoptosis and inhibit the growth of tumor in tumor xenografts mouse. Here we aimed to identify a series novel allosteric inhibitors targeted to Coiled-coil domain of STAT3.

信号传导与转录活化因子3（STAT3）在乳腺癌细胞中持续异常活化，信号传递过程中STAT3的Coiled-coil（CCD）结构域参与并调控STAT3的活化。因此我们旨在发现一类新型的针对CCD结构域的小分子抑制STAT3的活性达到抗肿瘤的效果。前期我们通过结构依赖的虚拟筛选和荧光偏振的方法进行了筛选，在体外分子水平，通过SPR技术检测小分子与CCD的亲和力是3.20 μM；细胞水平，我们发现小分子能够选择性的抑制含有持续异常活化STAT3的肿瘤细胞系的生长，在此基础上我们对小分子初步进行了优化改造，并探讨了构-效关系，为下一步优化提供依据，我们希望通过点突变方法明确小分子的结合位点，并在细胞和动物水平观测小分子对STAT3信号通路的抑制效果。通过上述实验我们希望开发针对STAT3 CCD的新型小分子抑制剂，优化其成为潜在的针对含有持续活化STAT3的人类肿瘤的抗肿瘤抑制剂。

信号传导与转录活化因子3（STAT3）在乳腺癌细胞中持续异常活化，信号传递过程中STAT3的Coiled-coil（CCD）结构域参与并调控STAT3的活化。Coiled-coil 结构域对 SH2 结构域的影响以及对整个 STAT3 蛋白的作用都说明 Coiled-coil 结构域可以作为药物设计和小分子化合物筛选的靶标，因此我们旨在发现一类新型的针对CCD结构域的小分子抑制STAT3的活性达到抗肿瘤的效果。我们通过筛选和分析构-效关系，确定了活性更好，选择性更高的小分子 K116，K134；在体外分子水平测定K116小分子与 STAT3 的结合亲和力为 3.22μM，确定STAT3 Coiled-coil 结构域为结合位点并明确Asp171, Gln202, and Met213是关键氨基酸残基。在细胞水平， K116 小分子对 STAT3 信号通路具有抑制转录活性，促进细胞凋亡，抑制乳腺癌细胞生长增殖，迁移，磷酸化STAT3在细胞核内聚集的作用，同时还降低细胞内STAT3的稳定性；在体内生理水平，抑制成瘤小鼠肿瘤生长，但不影响小鼠神经行为学表现，因此这些结论说明K116具有抑制STAT3信号通路的功能。这对于研究针对 STAT3新别构位点的小分子抑制剂作为临床抗肿瘤药物奠定重要理论依据。

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