Thrombin bivalent inhibitors

Bivalent inhibitors of thrombin have been synthesized to bind the anion-binding exosite and active (catalytic) site of thrombin simultaneously. By coupling the carboxy terminal fragment of hirudin to a tripeptide (D-Phe-Pro-Arg) by including a spacer molecule, both the anion exosite and the catalytic site are blocked. An example of such a molecule is Hirulog, which has 20 amino acids and has a Kj of 2 nM (61). Its ability to block the active site has been questioned, since thrombin has been shown to cleave the Arg-Pro bond of Hirulog slowly in vivo (58). In addition to hirudin and hirudin-like compounds, three other classes of site-directed thrombin inhibitors deserve mention. 

Bivalirudin, another bivalent inhibitor of thrombin, is administered intravenously, with a rapid onset and offset of action. 

Most of the peptide-based bivalent inhibitors were slowly cleaved by thrombin. Incorporation of a ketomethylene pseudo peptide bond (3-4) resulted in a noncleavable bivalent inhibitor that retained high thrombin affinity [24]. Decreased proteolysis in bivalent inhibitors increasingly nonpeptide in character continues to be observed. 

Increasingly nonpeptide substituents have been incorporated into the primary specificity pocket binding portion of the bivalent inhibitors. Higher affinity for thrombin was achieved by replacement of the (d-Phe)-Pro-Arg with either dansyl-Arg-(D-pipecolic acid) (3-17, [27]) or 4-tert-butylbenzenesulfonyl-Arg-(D-pipecolic acid) (3-18, [27]). While the arginine side chain of these and the (D-Phe)-Pro-Arg-containing inhibitors make similar interactions with the aspartic acid within the SI specificity pocket, the dansyl-Arg-(D-pipecolic acid) inhibitors bind in a nonsubstrate mode [27]. This initial result suggests that other nonpeptide thrombin inhibitors may be successfully incorporated into bivalent inhibitors. 

Bivalirudin, another bivalent inhibitor of thrombin, is administered intravenously, with a rapid onset and offset of action. The drug has a short half-life with clearance that is 20% renal and the remainder metabolic. Bivalirudin inhibits platelet activation and been FDA-approved for use in percutaneous coronary angioplasty. 

Maraganore J. M., Bourdon P Jablonskj J., Ramachandran K. L. Design and characterization of hirulogs A novel class of bivalent peptide inhibitors of thrombin. Biochemistry 1990 229, 7095-101. 

The direct thrombin inhibitors (DTIs) exert their anticoagulant effect by directly binding to the active site of thrombin, thereby inhibiting thrombin s downstream effects. This is in contrast to indirect thrombin inhibitors such as heparin and LMWH (see above), which act through antithrombin. Hirudin and bivalirudin are bivalent DTIs in that they bind at both the catalytic or active site of thrombin as well as at a substrate recognition site. Argatroban and melagatran are small molecules that bind only at the thrombin active site. 

IV. Bivalent Thrombin Inhibitors Directed at the Fibrinopeptide a Binding Pocket and the Fibrinogen Recognition Site... 

Table 2 Bivalent Thrombin Inhibitors Interacting with both Fibrinopeptide and Fibrinogen Recognition Binding Sites...

Tsuda Y, Cygler M, Gibbs BF, Pedyczak A, Fethiere J, Yue SY, Konishi Y. Design of potent bivalent thrombin inhibitors based on hirudin sequence incorporation of nonsubstrate-type active site inhibitors. Biochemistry 1994 33 14443-14451. 

Warkentin TE. Bivalent direct thrombin inhibitors hirudin and bivalirudin. Best Pract Res Clin Hematol 2004 17 105-125. 

Stteinmetzer T, Zhu BY, Konishi Y (1999) Potent bivalent thrombin inhibitors replacement of the scissile peptide bond at P(l)-P(l) with arginyl ketomethylene isosteres. J Med Chem 42 3109-3115... 

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