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

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.

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide.

A novel liposomal formulation was developed for the encapsulation of the oligopeptide leuprolide (GlpHisTrpSerTyr-D-LeuLeuArgProNHEt), a potent analogue of gonadotropin releasing hormone used in the treatment of advanced prostate cancer, endometriosis and precocious puberty. Leuprolide was synthesized using solid phase methodology on a {3-[(ethyl-Fmoc-amino)-methyl]-1-indol-1-yl}-acetyl AM resin and Fmoc/tBu chemistry. The new liposomal formulation, called 'liposomes in liposomes' is composed of egg phosphatidylcholine:dipalmitoylphosphatidylglycerol in a molar ratio of 98.91:1.09 (internal liposomes) and egg phosphatidylcholine:dipalmitoylphosphatidylglycerol:cholesterol in a molar ratio of 68.71:0.76:30.53 (external liposomes). It offers high encapsulation efficiency (73.8% for leuprolide); it can provide new delivery characteristics and it may have possible advantages in future applications regarding the encapsulation and delivery of bioactive peptides to target tissues. Furthermore, the physicochemical characteristics (size distribution and zeta-potential) of the liposomal formulations and the thermal effects on leuprolide in model lipidic bilayers composed of dipalmitoylphosphatidylcholine were studied using differential scanning calorimetry. Finally, the dynamic effects of leuprolide in an egg phosphatidylcholine/cholesterol system were examined using solid state 13C MAS NMR spectroscopy.

Angiotensin II analogues with sulphur-containing side-chains in position 5. A structure-activity relationship study.

Four sets of angiotensin II (AngII) analogues with position 5 modifications, two agonist series with either Asp or Sar in position 1 and L-Phe in position 8, and two antagonist series with again Asp or Sar in position 1 and Leu in position 8 were synthesized. Modifications in positions 5 were introduced successively: Ile, Nle, Met, S-ethyl Cys, S-n-propyl-Cys, S-n-butyl Cys, S-t-butyl Cys and S-benzyl Cys in all four series. The study was undertaken in order to investigate the 5-position residue of AngII by replacing the hydrophobic side-chain by another containing an electrophilic moiety. The analogues were synthesised by solid phase synthesis using the Boc/Bzl or Fmoc/But strategy. All analogues were evaluated by their binding properties to the AT1 receptor on bovine adrenocortical membranes (bAT1). The results indicate that AngII analogues bind, irrespective of their agonistic or antagonistic nature or of their position 1 modification, in a similar manner and that position 5 modifications without beta-branching behave in an additive manner towards their affinity.

Insertion of an electronegative sulfur atom in the side chain of position 5 of angiotensin II: changes in the tachyphylactic properties of the peptide.

Angiotensin II (AII) analogs bearing n-Leu, Met or S-substituted groups for cysteine at position 5 were studied regarding their agonistic and tachyphylactic properties. It was shown that these analogs lowered the relative affinity towards the AT1 receptor as determined by contractile responses, which could be due to the removal of the beta-branching residue at position 5. Insertion of a sulfur atom in a different position away from the attached backbone carbon atom presented no significant difference in EC50 values for these analogs. Interestingly, the S-bearing analogs at position 5 were full agonists but the tachyphylactic property was lost, in contrast to [n-Leu5]AII, which still induced reduction of the contractile responses. Nevertheless after replacing the Asp with Sar in position 1 (Sar1) tachyphylaxis was again established. It is concluded that the insertion of Met or an S-substituted cysteine into the side chain at position 5 of AII may promote interactions with its receptor due to the slight electronegative character of the sulfur atom and changes in the restricted conformational freedom of the Ile5 residue in the AII molecule. This was overcome by Sar1, probably through interactions due to its fully protonated N-terminal amino group and favoring the conformation responsible for the tachyphylaxis phenomenon.

Spectral analysis of a series of partially protected and deprotected tetrapeptides, analogues of AS-I phytotoxin.

A series of six tetrapeptides, analogues of AS-I phytotoxin, pathogenic to sunflower, have been synthesized either in solution and/or by solid phase methods and have been tested for phytotoxic activity in various plants and cytotoxic activity in three cancer cell lines. These peptides were identified as model compounds by fast atom bombardment (FAB), plasma desorption (PD), electrospray ionization (ESI) mass spectrometry and by 1H, 1H-1H, 13C and 1H-13C NMR. The data presented show that in protected tetrapeptides the molecular ion was easily identified whereas some difficulties appeared with the fully deprotected peptides. NMR spectra are given.

Substance P C-terminal octapeptide analogues augment tumor necrosis factor-alpha release by human blood monocytes and macrophages.

We have investigated the effects of the substance P C-terminal octapeptide analogues [Pro4, Glu (OBzl)11] SP4-11, [Hyp4, Glu(OBzl)11] SP4-11, [cHyp4, Glu(OBzl)11] SP4-11 and [kPro4, Glu(OBzl)11] SP4-11 on the constitutive and/or lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF-alpha) in both freshly isolated human blood monocytes (FIBM) and monocyte-derived macrophages (MDM). The cells were treated with substance P and the substance P analogues at various concentrations (10-14 to 10-6 M) in the presence or absence of LPS and culture supernatants were analyzed for TNF-alpha as measured by an enzyme immunosorbent assay (ELISA). Monocytes and macrophages treated with the substance P analogues alone increased TNF-alpha secretion at a magnitude similar to the effect of entire undecapeptide substance P. The stimulatory effects of the substance P analogues on TNF-alpha secretion are inhibited by substance P antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-9-Leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the substance P receptor), indicating that these effects are specific and substance P receptor-mediated. Treatment of monocytes and macrophages with the substance P analogues in combination with LPS, however, showed no synergistic interaction in upregulation of TNF-alpha. These data indicate that the biological effect of substance P on TNF-alpha production by human monocytes and macrophages depends mainly on the sequence of the C-terminal region of the molecule.

Chimeric peptides: a new approach to enhancing the immunogenicity of peptides with low MHC class I affinity: application in antiviral vaccination.

Recruitment of the CTL repertoire specific for subdominant epitopes that have a low MHC class I-binding affinity could be the way to achieve an efficient protective immunity against spontaneous tumors and viruses with high mutation rate. However, we have reported recently that subdominant peptides of influenza A Puerto Rico/8/34 (flu PR8) nucleoprotein (NP) with low Db affinity are only partially able to protect mice against lethal influenza infection. This seems to be due to their inability to recruit the specific CTL repertoire, and suggests that subdominant peptides could be used for vaccination only if they become highly immunogenic. In this work, we describe an approach that allows enhancement of the immunogenicity of every low affinity peptide presented by the Db molecule. It consists in producing chimeric peptides composed by amino acids from a high Db affinity peptide (NP366) in positions that interact with the MHC, and amino acids from low Db affinity nonimmunogenic influenza NP-derived peptides (NP17, NP97, NP330, and NP469) in positions that are exposed to the TCR. All chimeric peptides tested exhibited a high Db affinity and efficiently recruited the CTL repertoire specific for the corresponding low Db affinity peptide. Furthermore, vaccination with chimeric peptides that corresponded to subdominant NP17 and NP97 peptides induced a very potent anti-flu PR8 protective immunity.

Nuclear magnetic resonance studies of thyrotropin-releasing hormone (TRH) and analogues incorporating D-histidine and 4-hydroxy-L-proline.

NMR studies have been used to examine conformational effects in thyrotropin-releasing hormone (TRH), the epimer incorporating D-His, and their analogues where trans- and cis-4-hydroxy-L-proline replace L-proline (Pro). In all six compounds the observed overall conformation of the major conformer around the Pro-His amide bond, and the observed increase of the cis/trans ratio between the conformers when L-His is replaced by D-His, can be accommodated by assuming that a ten-membered ring is formed by hydrogen bonding between the N-H of the Pro carboxamide function and the N pi-atom of the His imidazole nucleus.