Thrombosis-related disorders have driven the interest on thrombin inhibitors as potential antithrombotic drugs. By employing in silico structure-based design, our group designed novel hexapeptides, pentapeptides and tetrapeptides as a new class of compounds that may be used as additives in the treatment of acute coronary diseases (ACD). The newly-designed peptides competitively inhibited the alpha-thrombin induced cleavage of a chromogenic substrate (S2238), at 18-0.8 uM. Herein, we describe the biochemical and structural characterization of some of these non-covalent direct thrombin inhibitors (DTI) derived from the common sequence D-Phe-Pro-D-Arg-P1'-CONH2. The three-dimensional structures of three complexes of human alpha-thrombin with the three lead peptidic inhibitors (with L-isoleucine (p3), L-cysteine (p4) or D-threonine (p6) at the P1' position) is reported by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of thrombin. The cleavable bond adopts an unfavorable geometry for nucleophilic attack by the serine side chain (3.69�, 2.85� and 2.96� distance between the Ser-195 OG and the arginine carbonyl carbon for p3, p4 and p6, respectively), making these compounds stable to proteolysis. The binding affinity for each inhibitor correlates well with the extent of interactions established by the residue at position P1'. In addition, we report the specific binding in solution between thrombin and other lead tetrapeptides from the D-Phe-Pro-D-Arg-P1'-CONH2 class (P1'=Gly and P1'=Ala) using the 2D-transferred NOESY method.

2D-transferred NOESY NMR and X-ray diffraction analysis of D-Phe-Pro-D-Arg-derived direct thrombin inhibitors

245th National Meeting (2013)

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