DEVELOPMENT OF POTENT THROMBIN RECEPTOR ANTAGONIST PEPTIDES

A peptide-based structure-activity study is reported leading to the di scovery of novel potent thrombin receptor antagonists. Systematic subs titution of nonproteogenic amino acids for the second and third residu es of the human thrombin receptor ''tethered ligand'' sequence (SFLLR) led to a series of agonists with enhanced potency. The most potent pe ntapeptide agonist identified was Ser-p-fluoroPhe-p-guanidinoPhe-Leu-A rg-NH2, 9 (EC(50) similar to 0.04 mu M for stimulation of human platel et aggregation, similar to 10-fold more potent than the natural pentap eptide). Systematic substitution of the NH2-terminal Ser in 9 with neu tral hydrophobic NH2-acyl groups led to partial agonists and eventuall y antagonists with unprecedented potency (greater than 1000-fold incre ase over the previously reported antagonist ropionyl-Phe-Cha-Cha-Arg-L ys-Pro-Asn-Asp-Lys-NH2). In the series of NH2-acyl tetrapeptide antago nists, cinnamoyl-p-fluoroPhe-p-guanidino-Phe-Leu-Arg-NH2, 41 (BMS-1975 25), was identified as the tightest binding (IC50 similar to 8 nM) and most potent with an IC50 similar to 0.20 mu M for inhibition of SFLLR NP-NH2-stimulated platelet aggregation. Systematic single substitution s in 41 indicated that, in addition to the NH2-terminal acyl group, th e side chains at the second and third positions were also responsible for important and specific receptor interactions. The p-fluoroPhe and p-guanidinoPhe residues in the second and third positions of 41 were o bserved to be optimal in both the agonist and antagonist series. In th e case of antagonists, however, an appropriately positioned positively charged group (i.e., protonated base) at the third residue was requir ed. In contrast, such a substitution was not required for potent agoni st activity. An even more potent antagonist resulted when 41 was exten ded at the C-terminus by a single Arg residue giving rise to analog 90 (BMS-200261) which had an IC50 similar to 20 nM for inhibition of SFL LRNP-NH2-stimulated platelet aggregation. When the C-terminal Arg of 9 0 was replaced by an Orn-(N-delta-propionyl) residue, the resulting an tagonist 91 (BMS-200661) was suitable for use in radioligand binding a ssays (K-d = 10-30 nM). Antagonist activity observed for selected comp ounds was verified through secondary assays in that these analogs prev ented SFLLRNP-NH2-stimulated GTPase activity in platelet membranes and Ca2+ mobilization in cultured human smooth muscle cells and mouse fib roblasts. Furthermore, this inhibition occurred at concentrations that had no effect on thrombin catalytic activity indicating a specific ac tivity attributable to receptor binding and not enzyme inhibition.