针对P-selectin外吐和GPIIb/IIIa识别双靶的抗血栓剂的设计、合成、活性和机理研究

Of the diseases, thrombosis has been known as a leading cause of morbidity and mortality around the world. There are two kinds of thrombi, which formed via two different pathways, in the patients. One is triggered by the exocytosis of P-selectin stored in the α-granule of the platelet. The other results from the eversion and the allosterism of GPIIb/IIIa. So far, all the known anti-thrombotic agents target either one or the other of the two pathways. Based on the mechanisms of both pathways, a strategy of designing novel anti-thrombotic agents that simultaneously inhibit the exocytosis of P-selectin and the allosterism of GPIIb/IIIa was established. Accordingly, a novel compound, N-(tetrahydroisoqunoline-3-carbonyl)-TARGD(F)F, was designed, of which the tetrahydroisoqunoline was for the binding at the active site pocket of P-selectin, the dipeptide TA was to form a transmembrane moiety and RGD(F)F was to occupy the binding sites of fibrinogen on GPIIb/IIIa. Preliminary experimental results prove that the lead compound N-(tetrahydroisoqunoline-3-carbonyl)-TARGD(F)F was capable of simultaneously inhibiting the exocytosis of P-selectin and the allosterism of GPIIb/IIIa, and consequently inhibiting the in vivo formation of thrombi. Further, molecular modelling studies was performed on its various analogs to optimize the lead compound. The modelling results show that the substitution of T with L or P, and the substitution of F with S or V will significantly improve the inhibiting activities of the lead compound. In this proposal, 8 optimized analogs of the lead compound will be synthesized, tested for their activities of inhibiting the exocytosis of P-selectin, inhibiting the allosterism of GPIIb/IIIa, inhibiting the aggregation of platelets, and inhibiting the in vivo formation of thrombsis. The preliminary results of the lead compound strongly support the feasibility and success of the following studies. Granting us the financial support is to ensure the discovery of novel effective anti-thrombotic agents and to benefit human beings suffer from thrombosis.

血栓可致心梗和脑梗等严重后果。患者体内的血栓是血小板活化诱发的两种机理形成的血栓混合体。一种是血小板膜内α-颗粒外吐P-选择素造成的血栓，一种是糖蛋白IIb/IIIa外化和变构造成的血栓。已知的药物设计通常只针对其中一种血栓。基于血栓对两种机理的依赖性和分子建模技术,申请人设计四氢异喹啉-3-甲酰基-TARGD(F)F同时阻断了两种机理及血栓形成。借助计算机模拟预测，它的T换为L或P，F换为S或V组成的8种新化合物更优秀。本项目拟合成这8种新化合物、研究这8种新化合物对P-选择素和糖蛋白IIb/IIIa的抑制活性、评价这8种新化合物的抗血小板聚集和抗血栓活性、研究这8种新化合物的作用机理。在科学层面，该研究会带来创新并揭露血小板膜内α-颗粒外吐P-选择素与糖蛋白IIb/IIIa外化和变构的关联性，深化对血栓病理的认识。在临床层面，该研究会为抗血栓新药研究发现可进入临床前研究的优秀化合物。

按照原计划，本项目圆满完成了本项目的计算机模拟的分子对接、新型四氢异喹啉-3-甲酰基-TARGD(F)F的对P-选择素和糖蛋白IIb/IIIa的抑制活性/抗血小板聚集活性/抗血栓活性和揭示血小板膜内α-颗粒外吐P-选择素与糖蛋白IIb/IIIa外化和变构的关联性三个要点的全部研究内容。在国际杂志上发表了10篇标注了本项目号的学术论文，总IF值为36.31。其中本项目负责人署名第一作者的论文3篇，1篇被邀请为封面论文，总IF值为9.956。负责人署名的论文中，1篇为美国化学含官方网站的周新闻论文，3篇被邀请为封面论文。2015年4月至11月在Dove出版社发表的3篇论文已经被读5549次。.本项目通过计算机辅助P-选择素晶体结构优化，获得了更加接近实际的分子对接活性口袋；采用更加接近实际的P-选择素的活性口袋，本项目将冬凌草水提物的5种核苷、21种酚酸和4种二萜与之对接，确认了主成分迷迭香酸是冬凌草水提物的抗血栓关键组分与P-选择素是作用靶点；发现了下调P-选择素和糖蛋白IIb/IIIa二者水平可获优秀抗血栓效果，并认定Cu2+-RGDFRGDS的抗血栓双重作用靶点；完善了糖蛋白IIb/IIIa检测方法，确认了Poly-α,β-aspartyl-Arg-Gly-Asp-Phe 对糖蛋白IIb/IIIa的靶向性作用；用计算机辅助的能量优化配合MS和NMR研究纳米结构形成，阐明了YW3-56的折叠构像、环五缔合、纳米结构及与PAD4的结合模式；用计算机辅助的能量优化配合MS和NMR研究，阐明了aspirin-Arg-Gly-Asp-Val在水中可形成三缔合体，24个三缔合体聚集成直径5nm的纳米球；用计算机辅助方法成功设计具有纳米性质的全新抗肿瘤化合物THPDTPI，并配合MS和NMR研究，阐明THPDTPI在水中形成π-π堆积二缔合体，304个二缔合体形成直径8.5nm的纳米球。基于计算机模拟的分子对接和3D-QSAR研究进行抗炎药物分子设计。这些研究结果，为合理设计针对P-选择素和糖蛋白IIb/IIIa的双靶点的抗血栓剂奠定了基础。完成本项目不仅使本项目负责人晋升为副教授，而且使本项目负责人凝练了可持续发展的研究方向。