c-Src的SH3结构域作为低出血风险的抗血栓新靶点的研究

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.

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。

抗血小板是治疗心脑血管血栓性疾病的关键，经典抗血小板药物同步影响血栓形成和止血功能，因此难以发挥充分的抗血栓作用。鉴于Src SH3在结合整合素β3及外向内信号转导中起关键作用，通过结构推理及将Src SH3 RT loop（介导非经典结合）及n-Src loop（介导经典结合）的关键氨基酸进行突变，发现β3倾向于结合在RT loop, 尤其以E97为主，这是本项目发现的新型抗血栓靶点。为验证该靶点，构建了SrcE97A转基因小鼠，发现其β3/Src相互作用明显减弱，FeCl3诱导的颈动脉血栓形成能力明显受到抑制；而在剪尾实验中，其出血时间与野生型小鼠无差异。基于此，根据SH3/β3相互作用晶体结构解析，综合考虑β3胞内段以及Src SH3的RT loop的信息，经计算机虚拟筛选及血小板伸展实验，筛选到DCDBS84为候选小分子。应用表面等离子共振、核磁共振、氢氘交换质谱以及突变分析，证实了DCDBS84特异靶向上述新型抗血栓靶点，并能解离β3/Src相互作用。血小板功能实验表明，DCDBS84抑制血小板不可逆聚集、在固相纤维蛋白原的粘附和伸展以及纤维蛋白凝块回缩等与血小板外向内信号转导相关的功能，而不影响关联血小板内向外信号转导的可溶性纤维蛋白原结合能力。在流体状态下，DCDBS84抑制代表狭窄动脉的5000s-1剪切力下的血栓形成，但在代表正常微小动脉的1500s-1剪切力下的影响较小。小鼠体内实验表明DCDBS84显著抑制FeCl3诱导的颈动脉血栓形成和激光诱导的提睾肌小动脉血栓形成，而对正常止血功能几乎没有影响。本研究发现了Src SH3 RT loop以E97为核心的新型抗血栓靶点，并且筛选到了特异靶向该靶点的小分子化合物DCDBS84，证实了DCDBS84在维持正常止血功能条件下显著抑制血栓形成。研究成果有助于阐明对血栓形成与止血的区别调控机制（即非经典的整合素β3结合Src/外向内信号转导/高剪切力下血栓增长）和开发更为安全有效的新型抗血栓药物。