Discovery and structure–activity relationship studies of a novel and specific peptide motif, pro-X-X-X-asp-X, as a platelet fibrinogen receptor antagonist Original Research Article

A novel hexapeptide, H-Pro-Ser-Nva-Gly-Asp-Trp-OH 6, a specific antagonist of platelet fibrinogen receptor (GpIIb/IIIa), was discovered in a structure–activity relationship (SAR) study where the role of the N-terminal Pro moiety of an RGD-containing peptide, H-Pro-Ser-Arg-Gly-Asp-Trp-OH 1, which is a potent but not specific antagonist toward GpIIb/IIIa integrin, was investigated. This novel peptide 6 exhibits very high activity as a human platelet aggregation inhibitor (IC50 0.59 μM, human PRP/collagen) as well as marked specificity for GpIIb/IIIa. A series of substitutions at the third position (Nva residue) in this hexapeptide, focused on the conformational rigidity, led to compounds which are superior to the original novel peptide 6 with regard to anti-platelet activity. The peptides, H-Pro-Ser-Hyp-Gly-Asp-Trp-OH 17 and H-Pro-Ser-ΔPro-Gly-Asp-Trp-OH 18 with the 5-membered ring structure, which restricted the conformation of the peptide backbone at the third position, inhibited the aggregation of human platelets at submicromolar concentrations (IC50 0.39 and 0.30 μM, respectively). Further structure–activity relationship studies at each position of the peptide sequence suggest a novel motif sequence, Pro-X1-X2-X3-Asp-X4, for specific GpIIb/IIIa integrin recognition, in which the N-terminal free Pro residue and the Asp residue at the fifth position are essential to the activity. This motif sequence is summarized as follows: (1) a small amino acid such as Ser, Ala or Gly is preferable at X1 position; (2) X2 may be any amino acid, preferably a bulky amino acid such as Tle or a cyclic amino acid such as Pro; (3) X3 must be a small amino acid such as Gly; and (4) X4 is preferably an amino acid with an aromatic side chain.