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Structure-based design of novel thrombin inhibitors
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Elizabeth A. Girnys
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The serine protease thrombin is the main clotting enzyme in the hemostatic system, in addition to being an effective platelet activator. Thrombin cleaves soluble fibrinogen into insoluble fibrin, to form the protein portion of blood clots. Thrombin also has two substrates that are G protein-coupled receptors (GPCRs), protease-activated receptors 1 and 4 (PAR1 and PAR4), which, upon thrombin activation, are very effective platelet activators. The activated platelets can then crosslink with fibrin, resulting in a blood clot. Thus, the development of a direct thrombin inhibitor offers an approach for the treatment of acute coronary syndromes through a two-prong modulation of the hemostatic system: blocking thrombus formation by inhibiting clotting, as well as inhibition of platelet aggregation. Previous studies have shown that the naturally occurring pentapeptide Arg-Pro-Pro-Gly-Phe inhibits the function of thrombin by interacting with its active site in a retro-binding fashion, as well as binding to PAR1 to prevent cleavage by thrombin. Structure-activity relationship studies led to the development of a lead compound FM 19, and an x-ray structure of FM 19 in the active site of thrombin has recently been determined (Figure 1).
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Figure 1 : (A) Crystal structure of FM 19 (colored yellow) in the active site of thrombin; aromatic and hydrophobic residues in the binding pocket are colored orange, hydrophilic residues are colored cyan. (B) Structure of FM 19. (C) Interaction of FM19 (colored grey with D-Arg colored yellow) with proximal residues in the active site of thrombin (colored cyan).
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The crystal structure has suggested several places for modification to improve potency, and our initial focus has been on replacements to the D-Arg residue. The side chain of this residue adopts a strained eclipsed conformation, rather than a lower energy extended conformation. Making the side chain shorter or adding conformational restriction to lock in this particular conformation could potentially improve potency by reducing the energetic penalty associated with the binding conformation. Additionally, this eclipsed conformation places the guanidino group in close proximity to the N-terminal amine. At physiological pH, both of these groups would be positively charged, resulting in an unfavorable electrostatic interaction. Since the guanidino group makes polar interactions with D189, as well as the backbone oxygen atoms of G218 and A190, it seems that a positively charged group on the side chain is necessary for activity. In contrast, the N-terminal amine of FM 19 has no significant interactions. So, elimination of the N-terminal amine should eliminate the unfavorable electrostatic interaction and potentially improve potency. Finally, replacement of the guanidino group with another positively charged group has also been explored.
These studies have resulted in four compounds thus far which show significant thrombin inhibition potency over FM 19. Two of these compounds are shown in Figure 2.
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Figure 2: Two compounds, BG 5 (left) and BG 6 (right), which show approximately 7-fold improvement in potency over FM 19.
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Additional modifications, including changes to other residues as well as modifications to improve bioavalability, are underway to potentially develop an orally administered direct thrombin inhibitor; a therapeutic alternative currently lacking in the U.S.
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Related Publications
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Girnys EA, Sobczyk-Kojiro K, Mosberg HI.
Structure-based design of residue 1 analogs of the direct thrombin inhibitor pentapeptide FM 19.
Chem Biol Drug Des, 75(1): 35-39 (2010)
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Nieman MT, Burke F, Warnock M, Zhou Y, Sweigart J, Chen A, Ricketts D, Lucchesi BR, Chen Z, Di Cera E, Hilfinger J, Kim JS, Mosberg HI, Schmaier AH.
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