INTERACTIONS OF HIRUDIN-BASED INHIBITOR WITH THROMBIN - CRITICAL ROLE OF THE ILE(H59) SIDE CHAIN OF THE INHIBITOR

摘要

Hirudin is the most potent and specific thrombin inhibitor from medicinal leech with a K-i value of 2.2 x 10(-14) M. It consists of an active site inhibitor segment, hirudin(1-48), a fibrinogen-recognition exosite inhibitor segment, hirudin(55-65), and linker, hirudin(49-54), connecting these inhibitor segments. The role of the side chain of the hirudin 59th residue, Ile, is studied by using a series of synthetic bivalent thrombin inhibitors, which mimic the binding mode of hirudin. The synthetic inhibitors based on the hirudin sequence have a general sequence of Ac-(D-Phe)-Pro-Arg-Pro-(4-aminobutyric acid)-(7-aminoheptanoic acid)-Asp-Phe-Glu-Glu-Xaa-Pro-Glu-Glu-Tyr-Leu-Gln-OH, in which the 59th residue, Xaa, is substituted by various natural and unnatural L-amino acids. For example, substitution of Ile(H59) by Val, which is equivalent to removing the delta-methyl group of Ile(H59), reduces the affinity of the inhibitor 5.7-fold (Delta Delta G(o) = 1.0 kcal/mol) to a K-i value of 4.7 nM compared to that (K-i = 0.82 nM) of the corresponding inhibitor with Ile(H59). Removal of the entire side chain of Ile(H59), i.e., a substitution of Ile(H59) by Gly, reduces the affinity of the inhibitor 6300-fold, revealing the critical role of the Ile(H59) side chain in the inhibitor binding, Theoretical free energy calculation successfully reproduces the binding free energy of most of the analogs. It suggests that intra- and intermolecular van der Waals interactions of delta-CH3, gamma-CH3, and gamma-CH2 of Ile(H59) play the major role in the binding affinity. Further search of possible methyl group(s) which may be incorporated in the side chain of the 59th residue results in the substitution of Ile(H59) with tert-butylalanine, improving the inhibitor affinity 2.1-fold (K-i = 0.39 nM). [References: 35]