基于蛋白质构效关系研究新型抗凝防栓药物作用机制

A novel recombinant hirudin, r-RGD-hirudin, was designed in the previous research. The r-RGD-hirudin is a single-chain; carbohydrate-free polypeptide containing 65-66 amino acids with a molecular weight of 7,000 Da. It has a trisul?de-linked core structure, forming a compact NH2-terminal domain and a long COOH-terminal extended domain. Tripeptide RGD sequence was fused into the given domain of native hirudin (wt-hirudin), and the r-RGD-hirudin was available. It inhibits activity of thrombin and aggregation of platelet..The crystal structure of hirudin-thrombin complex revealed abundant polar and polar interactions between these two molecules, which may account for the tight af?nity and speci?city of hirudin binding. Adhesion of platelets to vessel walls, their activation, and aggregation are key events in blood coagulation. On the activated platelets, the glycoprotein IIb/IIIa (GP IIb/IIIa) functions as a receptor for ?brinogen to mediate platelet aggregation and cross-linking. The capability of GP IIb/IIIa to bind to ?brinogen and other adhesive proteins is due to its ability to recognize the Arg-Gly-Asp (RGD) motif within their sequences. Peptides containing the RGD motif or RGD itself competitively inhibit the binding of ?brinogen to GP IIb/IIIa on the platelets, thus inhibiting platelet aggregation. So, it showed that wt-hirudin containing RGD motif ef?ciently prevents platelet aggregation which is induced by ADP..R-RGD-hirudin is a novel anti-coagulant for injection in vein. We hope it can be transformed to oral delivery. So, the r-RGD-hirudin needs to be redesigned based on its structural and functional studies. The most important structural alteration of r-RGD-hirudin is to reduce its molecular weight. NMR will be used to study the interaction between r-RGD-hirudin and thrombin or platelet. Here, the 15N or 15N, 13C-labeled r-RGD-hirudin was expressed in Pichia pastoris. The protein was subsequently purified to yield sufficient quantities for NMR experiments. .Then, a new anti-thrombin molecular will be designed through the computer modeling. Its Bio-activity will be characterized by the means of Bioassay and animal model.

课题组研制了一种新型抗凝防栓药物-重组双功能水蛭素(RGD-Hirudin)，具有抗凝血酶和抗血小板聚集双重功能，静脉注射用药后，凝血时间延长，有效防止血栓形成。初步的蛋白质化学结构研究发现RGD-Hirudin分子C端与凝血酶非活性底物识别位点结合，抗凝活性中心在RGD-Hirudin分子N端，RGD三肽融合在环形结构域，深入研究RGD-Hirudin的抗凝作用机制，改变分子结构，降低分子量，使之成为模拟短肽，可以用于制备口服或皮下注射制剂。本项目综合运用多维核磁共振技术、分子动力学研究技术和生物信息学，与抗凝防栓药物作用机制研究相结合，基于RGD-Hirudin的构效关系，研究RGD-Hirudin与凝血酶和血小板的相互作用位点和作用机理，利用计算机模拟，设计具有双重活性，分子量更小的模拟短肽，保持有效的抗凝血酶和抗血小板聚集的活性，通过体外试验和动物模型试验药物功能。

重组双功能水蛭素，具有抑制凝血酶和抗血小板聚集双重活性，是新型抗凝防栓药物。双功能水蛭素是蛋白质类药物，分子量大，临床应用受限。进一步研究其精细分子结构及其与凝血酶相互作用位点，指导设计新型低分子量抗凝活性多肽是本课题的研究内容。项目组首先制备了13C/15N双标记的双功能水蛭素，标记率＞98%。利用 15N/13C 双标记的 RGD-Hirudin，作异核多维 NMR 试验，并进行谱峰归属。利用化学位移扰动实验，在 15N/13C 均匀标记的 RGD-Hirudin 重水/水溶液中滴加凝血酶，记录异核单量子相关谱，跟踪由于相互作用引起氨基酸残基化学位移的变化，研究 RGD-Hirudin-凝血酶相关的键合位残基，发现了与凝血酶的结合区域。根据核磁共振实验的结果和计算机模拟的结果，对 RGD-Hirudin 的C 末端作了一系列突变，并对突变体作性质研究，利用 BIAcore 生物分子相互作用分析仪研究 RGD-hirudin 及其突变体与凝血酶之间的相互作用。基于上述研究，以其序列为基础，保留其功能氨基酸残基，借助分子模拟软件设计出两条 RGD-hirudin多肽类似物，理论分子量分别为3924.6730 Da(35肽) 和4023.7275 Da(36肽)。本课题综合运用多维核磁共振技术、酶反应动力学研究技术和生物信息学，1.研究双功能水蛭素(1-66)分子结构及其与凝血酶的作用位点和作用机制；2.利用计算机辅助设计，提出分子改造的方案；3.设计并制备具有抑制凝血酶活性的低分子量多肽，通过体外试验和动物模型研究其抗凝防栓的功能。