蛋白质二硫键异构酶ERp57抑制剂的发现及其抗血栓活性研究

Thrombotic disease is a common critical event that causes life-threatening health condition in human. The current antiplalate drugs, such as aspirin and clopidogrel, effectively reduce the risk of thrombus formation to human, unfortunatley, no pharmacological responses exsit in some cases, and what's wores is that the drugs increase incidence of major hemorrhage and other unwanted side effects. There is a great need for the improvement of existing therapeutic agents and development of new ones for the prevention and treatment of thrombotic disease. New antithrombotic agents targeted novel proteins with improved efficacy and free of the side effects are necessary to be developed to address the current unmet needs. In the recent days, it is reported that the disulfide isomerase ERp57 is a potential target for the treatment antithrombotic therapy. The ideal inhibotor targeting ERp57 has yet to be developed. There are an increasing number of compounds in drug development trace their origins back to structure based drug design. In this project, we previously reported a novel compound, named ADTM, which was synthesized by modifying a heterodimer of Danshensu and chuanxiongqin, exhibited strong cardioprotective effects in vitro and in vivo. In addition, using chemical proteomic approach, we found that ADTM targets ERp57 and displays antiplatelet and antithrombotic effects. In the recent times, computor aided drug design strateries have been used successfully in drug design and protein inhibitor development processes. The current our new findings and relevant research literature provide important evidences for further development of ADTM analogues as a novel target-based anti-thrombotic agent. The crystal structure of human ERp57 ligand-binding domain bound to ADTM will be clarified. The high resolution insights into ERp57 and ADTM complex structure will enable the application of structure based virtual screening to develop better ERp57 chemical inhibitors for the treatment of thrombotic desease. A pharmacophore model for binding of ADTM to ERp57 will be derived. We proposed that the new ERp57 inhibitor with more inhibitory effects than ADTM will be found based upon the pharmacophore searching. Nest, the effects of the new coumpounds with effectively inhibitory effeficary to ERp57 on platelet aggregation and thrombotic formation will be evaluated. Finally, the novel small molelcules, highly effective inhibitors of ERp57 can be easy to synthesized, were found and were further evaluated to expore its possibility to be a promising lead candidate for the treatment of thrombotic desease.

血栓性疾病是威胁人类健康的常见病。然而，目前血栓性疾病的治疗药物都存在不同程度的毒副作用。针对新靶标研制抗血栓新药，是应对血栓性疾病的有效手段。最近文献报道了二硫键异构酶ERp57是抗血栓的潜在药靶，但目前尚无ERp57特异性抑制剂的报道。因此，发现可拮抗ERp57酶活性的先导化合物，阐明其抗血栓机制对抗血栓新药的研发尤为重要。本项目组前期报道了新型化合物ADTM具有心肌保护作用，还发现该化合物可与ERp57蛋白结合并抑制其酶活性，具有较强的抗血小板聚集和抗血栓活性。本课题在前期创新性结果的基础上，拟解析该化合物和ERp57蛋白复合物的晶体结构，综合应用计算虚拟筛选等手段，以期发现活性更高的ERp57抑制剂、评价其抗血小板聚集和抗血栓的活性并探讨其作用机制。本课题将确证ERp57这一抗血栓的新靶点、首获高活性的ERp57小分子抑制剂、并评价成为抗血栓药物先导化合物的可能性。

血栓性疾病是威胁人类健康的常见病。然而，目前血栓性疾病的治疗药物都存在不同程度的毒副作用。针对新型靶点研制抗血栓新药，是应对血栓性疾病的有效手段。最近文献报道了二硫键异构酶ERp57是抗血栓的潜在药物靶点，但目前尚无ERp57特异性抑制剂和该抑制剂与抗血小板聚集等相关的报道。本项目组发现具有心肌保护作用的丹参素衍生物ADTM，可与ERp57蛋白结合并抑制其酶活性，具有较强的抗血小板聚集和抗血栓活性。然而，ADTM对ERp57的IC50较大（IC50 > 100 μM），其抑制活性有一定的改善空间。以ERp57第二活性位点(C406-G407-H408-C409)为活性口袋，用计算机虚拟筛选技术与固化酶结合质谱鉴定与实验验证的方法，发现一系列ERp57的抑制剂，包括丹参素的有效成分迷迭香酸(IC50 = 109.06 ± 0.43 μM)、绿茶中的没食子儿茶素没食子酸酯（IC50 > 100 μM）、二氢丹参酮I（IC50 = 3.46±0.29 μM）、丹参酚酸A（IC50 = 5.960.42）、异丹参酚酸C（IC50 = 6.100.18）和丹参酚酸C（IC50 = 6.350.34）等，其中对酶活性抑制效果最强的是化合物二氢丹参酮I，其活性较ADTM提高了30被左右。迷迭香酸具有抗多种诱导剂诱导的血小板聚集的功能。其次，发现作用于ERp57的化合物中，ADTM对扩展的研究结果发现该化合物也具有抗帕金森综合症的功能，其作用机制可能和抗氧化和抗炎有关，同时也发现其有促血管新生和心脏保护的药理作用，为脑心统治理论的药物开发提供了科学依据。基于抑制ERp57蛋白活性会抑制血栓形成的理论和ADTM靶标ERp57具有抗血栓活性的实验基础，通过虚拟筛选和实验相结合的方法，发现了系列活性更强的具有的ERp57抑制剂。本课题为血小板聚集和血管新生不足等相关疾病的研究提供了新的药物研发思路，也为后续ERp57及其抑制剂的新功能的发现打下了坚实的基础。