MDL-28050

Synthetic peptides based on the sequence of the negatively charged carboxyl tail of hirudin exhibit anticoagulant activity. Several antithrombin agents are being developed by chemical and structural optimization of these "hirupeptides". The present work demonstrates the design and use of novel spin-labeled and fluorescent-labeled C-terminal hirudin analogs to study the interactions of these antithrombin agents with thrombin in solution. Three labeled hirulabels were synthesized based upon the amino acid sequence of the antithrombin agent MDL 28050, X-NH-(CH2)7-CO-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha-D-Glu-OH, where X = anthraniloyl, 1,5-dansyl, or 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl. The modifications did not significantly alter the potency of these inhibitors which showed Ki values of 100 nM. Their interactions with human and bovine thrombin were studied by ESR and fluorescence techniques. The spin-labeled hirupeptide was able to discern subtle differences in binding to human versus bovine thrombin. The 8-aminooctanoic acid spacer arm placed the nitroxide moieties near the active site, near regions of the autolysis loops which differentiates between human alpha- and gamma-thrombin. It was also able to discern paramagnetic quenching and fluorescence energy transfer interactions, respectively, between covalently attached spin labels and fluorescent probes at the active site Ser 195 and the fluorophore on the hirupeptide.

In an effort to prepare crystals and determine the structure of alpha-thrombin complexed to a synthetic peptide inhibitor (MDL-28050) of the hirudin 54-65 COOH-terminal region, it was discovered that the crystals were not those of the complex but of gamma-thrombin. Gel electrophoresis studies revealed that autolytic degradation had occurred prior to crystallization. NH2-terminal sequence analysis of these autolytic fragments confirmed the gamma-thrombin product (cleavages at Arg75-Tyr76 and/or Arg77A-Asn78, and Lys149E-Gly150; chymotrypsinogen numbering) with a minor amount of another autolysis product, beta-thrombin (first two cleavages only). The final structure has an R-factor of 0.156 for 7.0-2.5-A data, and includes 186 water molecules. A comparison of gamma-thrombin with the thrombin structure in the alpha-thrombin-hirugen complex revealed that the two structures agreed well (r.m.s. delta = 0.39 A for main chain atoms). These structures possess uninhibited active sites where the disposition of the catalytic triad residues is nearly identical. The electron density in the vicinity of the gamma-thrombin cleavage regions is poor, and only becomes well-defined several residues prior to and after the actual cleavage sites. The extensive disorder evoked by beta-cleavage(s) in the Lys70-Glu80 loop region indicates that this part of the molecule is severely disrupted by autolysis and is the reason exosite functions are dramatically impaired in beta-and gamma-thrombin. Since autolysis did not lead to a major reorganization of the folded structure of alpha-thrombin, the likely structural features of the interaction of thrombin substrate with thrombin enzyme during beta-cleavage have been modeled by docking the exosite region of one molecule at the active site of another.