PAR4 (proteinase-activated receptor-4)

The agonistic efficacy of AYPGKF-NH2 is considerably reduced by a factor of 25 by GYPGKF-NH2 [1]. 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 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-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.[1]

In guinea pigs, GYPGKF-NH2 (500 μM) does not trigger IAS strips to contract or relax. Activation of proteinase-activated receptor-1 (PAR1) and PAR2 stimulates contraction of the rat but relaxation of the guinea pig colon. The aim of the present study was to investigate PAR effects on internal anal sphincter (IAS) motility. We measured relaxation of isolated muscle strips from the guinea pig IAS caused by PAR agonists using isometric transducers. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the existence of PAR. In the IAS, thrombin and PAR1 peptide agonists TFLLR-NH2 and SFLLRN-NH2 evoked moderate to marked relaxation in a concentration-dependent manner. In addition, trypsin and PAR2 peptide agonists 2-furoyl-LIGRLO-NH2, SLIGRL-NH2 and SLIGKV-NH2 produced relaxation. In contrast, both PAR1 and PAR2 inactive control peptides did not elicit relaxation. Furthermore, the selective PAR1 antagonist vorapaxar and PAR2 antagonist GB 83 specifically inhibited thrombin and trypsin-induced relaxations, respectively. RT-PCR revealed the presence of PAR1 and PAR2 in the IAS. This indicates that PAR1 and PAR2 mediate the IAS relaxation. The relaxant responses of TFLLR-NH2 and trypsin were attenuated by N(omega)-Nitro-L-arginine (L-NNA), indicating involvement of NO. These responses were not affected by tetrodotoxin, implying that the PAR effects are not neurally mediated. On the other hand, PAR4 agonists GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2 did not cause relaxation or contraction, suggesting that PAR4 is not involved in the sphincter motility. Taken together, these results demonstrate that both PAR1 and PAR2 mediate relaxation of the guinea pig IAS through the NO pathway. PAR1 and PAR2 may regulate IAS tone and might be potential therapeutic targets for anal motility disorders. [2].

[1]. Molecular requirements involving the human platelet protease-activated receptor-4 mechanism of activation by peptide analogues of its tethered-ligand. Platelets. 2017 Mar 7:1-10. doi: 10.1080/09537104.2017.1282607. [Epub ahead of print].

[2]. Proteinase-activated receptor-1 (PAR1) and PAR2 mediate relaxation of guinea pig internal anal sphincter. Regul Pept. 2014 Feb 10;189:46-50.

C33H46N8O7

666.76774

666.349

245443-52-1

90471153

H-Gly-Tyr-Pro-Gly-Lys-Phe-NH2

GYPGKF or GYPGKF-NH2

White to off-white solid

1.914

8

9

18

48

1090

4

NC([C@@H](NC([C@@H](NC(CNC([C@@H]1CCCN1C([C@@H](NC(CN)=O)CC2=CC=C(O)C=C2)=O)=O)=O)CCCCN)=O)CC3=CC=CC=C3)=O

NAIYNSCJEHHFMV-FWEHEUNISA-N

InChI=1S/C33H46N8O7/c34-15-5-4-9-24(31(46)40-25(30(36)45)17-21-7-2-1-3-8-21)38-29(44)20-37-32(47)27-10-6-16-41(27)33(48)26(39-28(43)19-35)18-22-11-13-23(42)14-12-22/h1-3,7-8,11-14,24-27,42H,4-6,9-10,15-20,34-35H2,(H2,36,45)(H,37,47)(H,38,44)(H,39,43)(H,40,46)/t24-,25-,26-,27-/m0/s1

(2S)-1-[(2S)-2-[(2-aminoacetyl)amino]-3-(4-hydroxyphenyl)propanoyl]-N-[2-[[(2S)-6-amino-1-[[(2S)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-1-oxohexan-2-yl]amino]-2-oxoethyl]pyrrolidine-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~75 mM)

Solubility in Formulation 1: 100 mg/mL (149.98 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: PBS: ~100 mg/mL (150 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)