Structure Basis Of RGD-Hirudin And Development Of New Direct Thrombin Inhibitors

Cardiovascular disease is a leading cause of death both at home and abroad, and is a frequently-occurring disease in middle-aged and elderly people. Common clinical acute myocardial infarction, cerebral thrombosis and disseminated intravascular coagulation (DIC) are almost arterial thrombosis, which is the main reason of causing death or disability. Pathologic coagulation plays an integral role in the development, propagation, and intervention of cardiovascular disease. Blood coagulation abnormality is the central segment of the occurrence, spread and intervention in this type of disease.Thrombotic diseases need early intervention and treatment before the acute outbreaks of the disease. Thrombolytic therapy after acute outbreaks of the disease requires long-term medication to prevent blood clots formed again.The two classic anticoagulants, heparin and vitamin K antagonists, though having served humanity for nearly a century. However, both of them have some distinct disadvantages, inconvenient and nonspecific. Thrombin plays a central role in the generation of a thrombus, and makes for a very attractive target in medical intervention of pathologic thrombosis.Hirudin, which belongs to direct thrombin inhibitors, is the most potent and specific thrombin inhibitor currently known. RGD-hirudin is a novel bi-functional molecule that contains the Arg-Gly-Asp (RGD) adhesion site recognition sequence (Chinese invention patent, ZL01105798.X). The abilities of this protein to inhibit thrombin and the aggregation of platelets were confirmed in our previous study. However, as a protein drug, the intravenous injection administration makes it narrow therapeutic window, and limits its clinical use range.Recently, several bivalent and univalent DTIs have been successfully marketed. Yet significant limitations to use of current DTIs are their specific therapeutic window and certain side effects. Consequently, there was a large unmet need (as well as a large potential market) for developing new DTIs. The main purpose of this study is to study the structure and function of the RGD-hirudin, find the key sites in the interaction with thrombin, transform the structure of RGD-hirudin, design and develop small molecules having anti-thrombin activity for orally or subcutaneously using.There are four parts of the thesis as the following:1. We describe the successful expression of15N/13C-labeled and unlabeled RGD-hirudin in yeast, Pichia pastoris (GS115). In total, amount of15N/13C-labeled and unlabeled RGD-hirudin were generated through high density fermentation, the bioactivity, purity and molecular weight were determined, and we obtained a sufficient amount of purified and uniformly labeled RGD-hirudin for structure-function studies by NMR.2. One-(1D1H), Two-(2D1H-15N HSQC) and three-dimensional (3D CBCA(CO)NH,3D CBCANH,3D HNCO) double and triple resonance NMR techniques have been successfully applied to obtain most backbone1H15N,13C and13CO assignments of RGD-Hirudin, and NMR titration experiments of15N/13C-labeled RGD-hirudin and thrombin have also been conducted. We successfully determined the key amino acid sites of RGD-hirudin.3. We used homology modeling method to simulate the full length of the structure of the RGD-hirudin, and RGD-hirudin-thrombin complex model structure was obtained by molecular docking method, combined with NMR chemical shift perturbation experiment results, we analysised the interaction beteen RGD-hirudinin and thrombin. In order to verify the accuracy of the model, we determined the amino acid residues of RGD-hirudin which forming hydrogen bond with thrombin, and furthermore, these amino acid residues were mutated to alanine, we obtained six RGD-hirudin mutant protein, the activity of these mutants were measured, after that, we used surface plasmon resonance experiment (SPR) to determine the affinity of RGD-hirudin and these mutants with thrombin. Finally, we identified the C-terminal and N-terminal of the RGD-hirudin interact with the exosite I and activity site of thrombin, respectively.4. Based on the study of the structure and function of RGD-hirudin all above, we designed two peptide analogs of RGD-hirudin by molecular simulation software, two peptides were expressed by Pichia pastoris, and after the determination of purity and molecular weight, anti-thombin activity was also tested.Finally, we got two short peptides with anticoagulant activity, which molecular weight were3925.5274Da (35amino acids) and4024.5459Da (36amino acids), respectively.