Thrombin receptor-activating peptides (TRAPs): Investigation of bioactive conformations via structure-activity, spectroscopic, and computational studies

The thrombin receptor (PAR-1) is an unusual transmembrane G-protein coupled receptor in that it is activated by serine protease cleavage of its extrac ellular N-terminus to expose an agonist peptide ligand, which is tethered t o the receptor itself. Synthetic peptides containing the agonist motif, suc h as SFLLRN for human PAR-1, are capable of causing full receptor activatio n. We have probed the possible bioactive conformations of thrombin receptor -activating peptides (TRAPs) by systematic introduction of certain conforma tional perturbations, involving alpha-methyl, ester Psi(COO), and reduced-a mide Psi(CH2N) scans, into the minimum-essential agonist sequence (SFLLR) t o probe the importance of the backbone conformation and amide NH hydrogen b onding. We performed extensive conformational searches of representative pe ntapeptides to derive families of putative bioactive structures. In additio n, we employed H-1 NMR and circular dichroism (CD) to characterize the conf ormational disposition of certain pentapeptide analogues experimentally. Ac tivation of platelet aggregation by our pentapeptide analogues afforded a s tructure-function correlation for PAR-1 agonist activity. This correlation was assisted by PAR-1 receptor binding data, which gauged the affinity of p eptide ligands for the thrombin receptor independent of a functional cellul ar response derived from receptor activation (i.e. a pure molecular recogni tion event). Series of alanine-, proline-, and N-methyl-scan peptides were also evaluated for comparison. Along with the known structural features for PAR-1 agonist peptides, our work adds to the understanding of peptide topo graphy relative to platelet functional activity and PAR-1 binding. The abso lute requirement of a positively charged N-terminus for strong agonist acti vity was contradicted by the N-terminal hydroxyl peptide Psi(HO)S-FLLR-NH2. The amide nitrogen between residues 1 and 2 was found to be a determinant of receptor recognition and the carbonyl groups along the backbone may be i nvolved in hydrogen bonding with the receptor. Position 3 (P3) of TRAP-5 is known to tolerate a wide variety of side chains, but we also found that th e amide nitrogen at this position can be substituted by an oxygen, as in SF -Psi(COO)-LLR-NH2, without diminishing activity. However, this peptide bond is sensitive to conformational changes in that SFPLR-NH2 was active, where as SF-NMeL-LR-NH2 was not. Additionally, we found that position 3 does not tolerate rigid spacers, such as 3-aminocyclohexane-1-carboxylic acid and 2- aminocycloalkane-1-carboxylic acid, as analogues 1A, 1B, 2A, 2B, 3, 4, 5A a nd 5B lack agonist activity. On the basis of our results, we suggest that a n extended structure of the agonist peptide is principally responsible for receptor recognition (i.e. binding) and that hydrophobic contact may occur between the side chains of the second (Phe) and fourth (Leu) residues (i.e. P2-P4 interaction). (C) 1999 Elsevier Science Ltd. All rights reserved.