c-Src的SH3结构域作为低出血风险的抗血栓新靶点的研究

经典的抗血栓药物αIIbβ3拮抗剂因同步影响止血和血栓形成，因此难以使用足够的剂量以达成充分的抗血栓作用，而解决这一问题的策略是靶向信号转导通路。我们前期发现，c-Src的SH3结构域在结合整合素β3并转导外向内信号中起关键作用，是潜在的影响止血功能较小的抗血栓靶点。在解析SH3/β3相互作用晶体结构的基础上，经计算机模拟寻找并筛选出能靶向SH3的DCDBS84。本项目拟探讨其：一、与SH3相互作用的靶位特异性；二、靶向SH3后能否解离β3/c-Src相互作用；三、对整合素αIIbβ3信号转导及其介导的血小板功能的影响；四、对c-Src的激酶功能有无直接影响；五、在活体动物层面验证对止血和血栓形成的作用。此外，通过建立敲入SH3靶点失功能型c-Src的模式动物，检测模拟靶向c-Src/SH3的长期和全面体内效应。本研究将为新型抗血栓药物的研发打下坚实基础。

Owing to the indiscriminately inhibitory effects on hemostasis and thrombosis, the typical αIIbβ3 antagonists tend not to be used at aggressive dosages to avoid the fatal bleeding adverse effects. A consensus has been reached that targeting signaling pathways instead of blocking receptors may solve this antithrombotic-bleeding dilemma. We have previously established that the c-Src SH3 domain critically contributes to its association with integrin β3 transducing outside-in signals and thereby has potential to serve as a novel target exerting an inhibitory effect preferentially on thrombosis rather than on hemostasis. Based on our data from the crystallographic determination of the SH3/β3 interaction, we obtained a large number of synthetic small molecules via in silico screening and selected one of them, named DCDBS84, characterized by targeting c-Src/SH3. In the proposed project we plan to assay DCDBS84 for 1. the specific definition of its targeting site within the SH3 domain, 2. its capability to separate the c-Src/β3 interaction by targrting SH3, 3. its effects on integrin αIIbβ3 signaling-mediated platelet functions, 4. whether it is per se able to influence the kinase activity of c-Src directly, 5. its in vivo effects on thrombosis and hemostasis. In addition, to assess the long-term and systemic in vivo effects of targeting the c-Src SH3, an animal model will be established by knock-in of the c-Src harboring the loss-of-function mutations in the SH3 domain. By implementing this project we may lay a solid foundation for the development of the new generation of antithrombotics.