Design of Bifunctional Inhibitors for Enzymes in Coagulation-Fibrinolytic System

1. 体内双功能凝血酶抑制剂的评价及针对血栓的多功能凝血酶抑制剂的进一步研究：在大鼠体内评价了强效双功能凝血酶抑制剂对He-Ne激光诱导血栓形成的抗血栓作用，并与其他类型的凝血酶抑制剂进行了比较。双功能抑制剂的抗血栓活性高于水蛭素和水蛭素，与典型的活性位点定向凝血酶抑制剂阿加曲班相当。这一结果促使我们设计了一种针对血栓的多功能凝血酶抑制剂。这种抑制剂不仅更有效，而且副作用更小。将Arg-Gly-Asp基序引入双功能抑制剂分子的连接部分，并检测了体外抗凝血酶活性。双功能纤溶酶抑制剂的设计：我们设计了双功能纤溶酶抑制剂，它由活性位点阻断序列t- amcha - tir - eaca - nh_2 （IC_<50>=4.6 × 10^<-4>M）、赖氨酸结合位点阻断片段Lys （IC_<50>=5.0 × 10^<-2>M）和连接这些抑制剂片段的连接体组成。在合成的肽中，P-1抑制纤溶蛋白，在酶解实验中Ki值为1.9x10^<-6>M。t-AMCHA（一种LBS结合抑制剂）的加入使P-1的抑制活性降低到Ki值为1.4 × 10^<-5>M。这表明P-1与纤溶酶的LBS结合，是一种双功能的纤溶酶抑制剂。血浆钾激肽（PK）抑制剂的设计：合成的PK抑制剂PKSI-527由三部分组成。每个部分都被类似物取代，试图提高PKSI-527的效力和选择性。在PKSI-527的范围内，我们发现了具有高选择性且IC_<50>值的抑制PK的肽，该化合物可作为活性位点阻断片段与双功能PK抑制剂结合。

1. Evaluation of bifunctional thrombin inhibitors in vivo and further investigation of multifunctional thrombin inhibitors targeting thrombus : The antithrombotic effect of potent bifunctional thrombin inhibitors on He-Ne laser-induced thrombosis was evaluated in rat and compared with other types of thrombin inhibitors. The anthithrombotic activity of bifunctional inhibitors was higher than that of hirudin and hirulogs and comparable to that of argatroban, typical active site directed thrombin inhibitor. This result made us to design a multifunctional thrombin inhibitor targeting thrombus. Such a inhibitor would be not only more effective but also less side effects. A motif of Arg-Gly-Asp was introduced to the linker part on the molecule of bifunctional inhibitor and anthithrombin activities in vitro were examined.2. Design of bifunctional plasmin inhibitor : We design bifunctional plasmin inhibitor, which consists of an active site blocking sequence, t-AMCHA-Tyr-EACA-NH_2 (IC_<50>=4.6x10^<-4>M) , alysine binding sites (LBS) blocking moiety, Lys (IC_<50>=5.0x10^<-2>M) , and a linker, connecting those inhibitor moieties. Among the peptides synthesized, P-1 inhibited plasmin with a Ki value of 1.9x10^<-6>M in the amidolytic assay. The addition of t-AMCHA,a LBS binding inhibitor, reduced inhibitory activity of P-1 to a Ki value of 1.4x10^<-5>M.This result suggests that P-1 binds to the LBS of plasmin and acts as a bifunctional plasmin inhibitor.3. Design of plasma kallikrein (PK) inhibitor : The synthetic PK inhibitor, PKSI-527 consists of three parts. Each part was replaced by analogues in an attempt to improve the potency and the selectivity of PKSI-527. We found the peptide that inhibited PK with a high selectivity and an IC_<50> value in the same range of PKSI-527, This compound could be incorporated to bifunctional PK inhibitor as a active site blocking segment.