| Résumé | Hirudin from medicinal leech is the most potent and specific thrombin inhibitor from medicinal leech with a Ki value of 2.2 × 10⁻¹⁴ M. It consists of an active site blocking moiety, hirudin¹⁻⁴⁸, a fibrinogen-recognition exo-site binding moiety, hirudin⁵⁵⁻⁶⁵, and a lilnker, hirudin⁴⁹⁻⁵⁴, connecting these inhibitor moieties. Synthetic inhibitors were designed based on the C-terminal portion of hirudin. The bulky active site blocking moiety, hirudin¹⁻⁴⁸, was replaced by small nonsubstrate-type active site ibhibitors of thrombin, e.q., dansyl-Arg-(D-pipecolic acid). The linker moiety was replaced by ω-amino acids of (12-aminododecanoic acid)-(4-aminobutyric acid), and hirudin⁵⁵⁻⁶⁵ was used as a fibrinogen-recognition exo-site binding moiety in most of the inhibitors. The crystal structure of the inhibitor in complex with human α-thrombin showed that dansyl, Arg, and D-pipecolic acid of the active site blocking moiety occupy S3, S1, and S2 subsites of thrombin, respectively, and were therefore designated as P3, P1, and P2 residues. The use of dansyl-Arg-(D-pipecolic acid) improved the affinity (Ki) of the inihibitor 10-100-fold (down to 1.70 × 10⁻¹¹ M) comparted to that of the similar compounds having D-Phe-Pro-Arg as their substrate-type inhibitor moiety (Ki = 10⁻⁹-10⁻¹º M). The linker connected to P2 residue eliminated teh scissle peptide bond. The inhibitor was also stable against human plasma proteases. Further inhibitor design revealed that the toxic dansyl group could be replaced by 4-tert-butylbenzenesulfonyl group and 1- or 2-naphthalenesulfonyl group for in vivo studies. In addition, the replacement of hirudin⁵⁵⁻⁶⁵ with [Tyr⁵⁶,-Pro⁵⁸,Ala⁶³,Cha⁶⁴,D-Glu⁶⁵]hirudin⁵⁵⁻⁶⁵ improved the affinity of the inhibitors (Ki = 2.0 × 10⁻¹³ M). |
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