Design of Bifunctional Inhibitors for Enzymes in Coagulation-Fibrinolytic System

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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.