title: Molecular requirements involving the human platelet protease-activated receptor-4 mechanism of activation by peptide analogues of its tethered-ligand.
abstract: Thrombin is the most potent agonist of human platelets and its effects are primarily mediated through the protease-activated receptors (PARs)-1 and -4. Although PAR-1 has higher affinity for thrombin than PAR-4, both receptors contribute to thrombin-mediated actions on platelets. Recently, a potent and selective PAR-1 antagonist (vorapaxar) was approved for clinical use in selected patients. In contrast, despite the fact that several PAR-4 antagonists have been developed, few of them have been tested in clinical trials. The aim of the present study was to elucidate the molecular requirements involving the PAR-4 mechanism of activation by peptide analogues of its tethered-ligand. Eight synthetic PAR-4 tethered-ligand peptide analogues were synthesized and studied for their agonistic/antagonistic potency and selectivity toward human washed platelet aggregation, using light transmittance aggregometry. In addition, in silico studies were conducted to describe the receptor-peptide interactions that are developed following PAR-4 exposure to the above analogues. To provide a first structure-activity relationship rationale on the bioactivity profiles recorded for the studied analogues, molecular docking was applied in a homology model of PAR-4, derived using the crystal structure of PAR-1. The following peptide analogues were synthesized: AYPGKF-NH2(1), GYPGKF-NH2(2), Ac-AYPGKF-NH2(3), trans-cinnamoyl-AYPGKF-NH2(4), YPGKF-NH2(5), Ac-YPGKF-NH2(6), trans-cinnamoyl-YPGKF-NH2(7), and caffeoyl-YPGKF-NH2(8). Peptide (1) is a selective PAR-4 agonist inducing platelet aggregation with an IC50value of 26.2 μM. Substitution of Ala-1 with Gly-1 resulted in peptide (2), which significantly reduces the agonistic potency of peptide (1) by 25-fold. Importantly, substitution of Ala-1 with trans-cinnamoyl-1 resulted in peptide (7), which completely abolishes the agonistic activity of peptide (1) and renders it with a potent antagonistic activity toward peptide (1)-induced platelet aggregation. All other peptides tested were inactive. Tyr-2, residue, along with its neighboring environment was a key determinant in the PAR-4 recognition mode. When the neighboring residues to Tyr-2 provided an optimum spatial ability for the ligand to enter into the binding site of the transmembrane receptor, a biological response was propagated. These results were compared with the predicted binding poses of small molecule antagonists of PAR-4, denoted as YD-3, ML-354, and BMS-986120. π-π stacking interaction with Tyr-183 appears to be critical and common for both small molecules antagonists and the peptide trans-cinnamoyl-YPGKF-NH2. Conclusively, the lipophilicity, size, and aromatic nature of the residue preceding Tyr-2 are determining factors on whether a human platelet PAR-4 tethered-ligand peptide analogue will exert an agonistic or antagonistic activity.
authors.affiliation: a Atherothrombosis Research Centre/Laboratory of Biochemistry, Department of Chemistry , University of Ioannina , Ioannina , Greece.
authors.forename: I C
authors.initials: IC
authors.lastname: Moschonas
authors.affiliation: b Sector of Organic Chemistry and Biochemistry, Department of Chemistry , University of Ioannina , Ioannina , Greece.|c Department of Chemistry , National and Kapodistrian University of Athens, Panepistimiopolis Zografou , Athens , Greece.
authors.forename: T F
authors.identifier: http://orcid.org/0000-0001-6977-9946
authors.initials: TF
authors.lastname: Kellici
authors.affiliation: c Department of Chemistry , National and Kapodistrian University of Athens, Panepistimiopolis Zografou , Athens , Greece.|d Department of Chemistr y, York College and the Graduate Center of the City University of New York , Jamaica , NY , USA.
authors.forename: T
authors.identifier: http://orcid.org/0000-0001-5309-992X
authors.initials: T
authors.lastname: Mavromoustakos
authors.affiliation: b Sector of Organic Chemistry and Biochemistry, Department of Chemistry , University of Ioannina , Ioannina , Greece.
authors.forename: P
authors.initials: P
authors.lastname: Stathopoulos
authors.affiliation: b Sector of Organic Chemistry and Biochemistry, Department of Chemistry , University of Ioannina , Ioannina , Greece.
authors.forename: V
authors.initials: V
authors.lastname: Tsikaris
authors.affiliation: e Laboratory of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy , University of Patras , Patras , Greece.
authors.forename: V
authors.initials: V
authors.lastname: Magafa
authors.affiliation: b Sector of Organic Chemistry and Biochemistry, Department of Chemistry , University of Ioannina , Ioannina , Greece.
authors.forename: A G
authors.identifier: http://orcid.org/0000-0001-6391-0288
authors.initials: AG
authors.lastname: Tzakos
authors.affiliation: a Atherothrombosis Research Centre/Laboratory of Biochemistry, Department of Chemistry , University of Ioannina , Ioannina , Greece.
authors.forename: A D
authors.initials: AD
authors.lastname: Tselepis
mesh_terms: D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D018179:Receptors, Thrombin / Q000378:metabolism*; D013917:Thrombin
chemical_list: D008024:Ligands; D018179:Receptors, Thrombin; D013917:Thrombin; C112908:protease-activated receptor 4
keywords: Peptides; platelets; protease-activated receptors; thrombin; vorapaxar