Molecular requirements involving the human platelet protease-activated receptor-4 mechanism of activation by peptide analogues of its tetheredligand

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), AcAYPGKF-NH2 (3), trans-cinnamoyl-AYPGKF-NH2 (4), YPGKF-NH2 (5), Ac-YPGKF-NH2 (6), transcinnamoyl-YPGKF-NH2 (7), and caffeoyl-YPGKF-NH2 (8). Peptide (1) is a selective PAR-4 agonist inducing platelet aggregation with an IC50 value 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-YPGKFNH2. 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 activit

Molecular Mechanisms Regulating the Platelet Thrombin Receptor PAR4

Proteinase activated receptor 4 (PAR4) is a G-protein-coupled receptor with an important role in the platelet response to vascular injury. In platelets, PAR4 activation, by thrombin-cleavage of its N-terminus and unmasking of a tethered ligand, leads to G-protein- and beta-arrestin-mediated intracellular signalling pathways which result in platelet activation, shape change, and ultimately, platelet aggregation. As an important platelet thrombin receptor, PAR4 is an interesting target for the development of anti-platelet therapeutics. However, molecular determinants of PAR4 activation, signalling, and signal regulation remain poorly understood. In this thesis, mechanisms of PAR4 activation and signalling were studied through determination of the molecular basis of agonist binding at the extracellular surface of the receptor that result in G-protein signalling and beta-arrestin recruitment and evaluation of the role of Helix-8 and C-terminal tail residues on effector interaction and signalling. In our first series of experiments, we evaluated the impact of single amino acid substitutions to the parental PAR4 agonist peptide, AYPGKF-NH2, in an effort to understand the chemical characteristics enabling activity at the PAR4 receptor. We identified key residue characteristics contributing to agonist peptide activation of PAR4. We further showed that different chemical modifications affect G-protein and beta-arrestin signalling through PAR4, providing leads for further development of biased ligands of PAR4. Additionally, using in silico receptor modelling and peptide docking, we identify a key residue in the ECL2 of the receptor that is required for receptor activation by either peptide or enzyme-revealed tethered ligand. Subsequent studies evaluated the role of the Helix-8 and C-terminal tail motifs in PAR4 and the related receptor, PAR2, on intracellular effector interaction and signalling. PAR4 was compared to PAR2 since several key Class A GPCR regulatory sites are missing in PAR4 and are retained in PAR2. These studies have revealed residues and structural features that are involved in PAR4 interaction with G-proteins and beta-arrestin. Importantly, we also identified key differences in residues that are important for signalling following enzyme- versus peptide-activation of PARs. Taken together, this body of work enhances our understanding of how PAR4 engages and is activated by agonists to promote signalling and cellular function

Molecular requirements involving the human platelet protease-activated receptor-4 mechanism of activation by peptide analogues of its tethered-ligand

<p>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.</p> <p>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.</p> <p>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, <i>in silico</i> 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.</p> <p>The following peptide analogues were synthesized: AYPGKF-NH₂ (1), GYPGKF-NH₂ (2), <i>Ac-</i>AYPGKF-NH₂ (3), <i>trans-cinnamoyl-</i>AYPGKF-NH₂ (4), YPGKF-NH₂ (5), <i>Ac-</i>YPGKF-NH₂ (6), <i>trans-cinnamoyl-</i>YPGKF-NH₂ (7), and <i>caffeoyl-</i>YPGKF-NH₂ (8). Peptide (1) is a selective PAR-4 agonist inducing platelet aggregation with an IC₅₀ value 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 <i>trans-cinnamoyl-</i>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 <i>trans-cinnamoyl-</i>YPGKF-NH₂.</p> <p>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.</p