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-NH (1), GYPGKF-NH (2), Ac-AYPGKF-NH (3), trans-cinnamoyl-AYPGKF-NH (4), YPGKF-NH (5), Ac-YPGKF-NH (6), trans-cinnamoyl-YPGKF-NH (7), and caffeoyl-YPGKF-NH (8). Peptide (1) is a selective PAR-4 agonist inducing platelet aggregation with an IC 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-NH. 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.

Unveiling and tackling guanidinium peptide coupling reagent side reactions towards the development of peptide-drug conjugates

“…Synthesis of compounds 5 and 6 (Scheme 1). 5 0 -O-Gemcitabine hemisuccinate (3) and [D-Lys] 6 -GnRH (4) were synthesized as before. 24 DIPEA (130 ml, 0.746 mmol) was added to a solution of 3 (84 mg, 0.149 mmol) and HATU (85 mg, 0.223 mmol) in 10 ml DMF under nitrogen.…”

“…S29 †) to get the side product 5 and the desired PDC 6. MS (ESI + ) m/z for (6) (Fig. S30-S32 †) and its purity was evaluated via analytical RP-HPLC ( Fig.…”

“…Synthesis of compounds 15-21 ( 6 -GnRH (Glp-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH 2 ; Fig. 3B and S35 †), Fmoc-HER2-BP1 (Fmoc-Tyr-Trp-Pro-Ser-Val-Thr-Leu-COOH; Fig.…”

“…Among the most classically utilized guanidinium reagents are HATU (N-[(dimethylamino)-1H-1,2,3-triazo [4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexauorophosphate-N-oxide) (1), and HBTU (N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexauorophosphate-Noxide) (2). [1][2][3][4][5][6] Based on their structure, HBTU and HATU were originally classied as O-uronium salts; later were revised to N-guanidinium salts (Fig. 1).…”

“…24 Aer the construction and HPLC purication of the relevant conjugate, we observed the coexistence of a side product, bearing a mass of the expected PDC plus 99 amu. A template peptide coupling reaction was conducted to monitor the exact location of this side product using all amino acids of [D-Lys] 6 -GnRH sequence.…”