18 research outputs found
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
Electronic sculpting of ligand-GPCR subtype selectivity:the case of angiotensin II
GPCR subtypes possess distinct functional
and pharmacological profiles,
and thus development of subtype-selective ligands has immense therapeutic
potential. This is especially the case for the angiotensin receptor
subtypes AT1R and AT2R, where a functional negative control has been
described and AT2R activation highlighted as an important cancer drug
target. We describe a strategy to fine-tune ligand selectivity for
the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl
interactions. Through this strategy an AT2R high affinity (<i>K</i><sub>i</sub> = 3 nM) agonist analogue that exerted 18,000-fold
higher selectivity for AT2R versus AT1R was obtained. We show that
this compound is a negative regulator of AT1R signaling since it is
able to inhibit MCF-7 breast carcinoma cellular proliferation in the
low nanomolar range
Design of New Liposome Formulation and Encapsulation of the Synthetic Anticancer Drug Leuprolide Acetate and Study of its Thermal Effects on Membrane Bilayers.
. Design of a New Liposomal Formulation and Encapsulation of the Synthetic Anticancer Drug Leuprolide Acetate. Study of its Thermal Effects on Membrane Bilayers.
Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling
The nonapeptide Leuprorelin, one of the LHRH agonists, was studied by means of 2D nuclear magnetic resonance spectroscopy and molecular modeling. NOESY spectra in aqueous/deuterated methanol solution (50% H2O/CD3OD) at low temperature (268 K) revealed short-range nOe connectivities (i, i+1), characteristic of flexibility of the molecule. The HN-HN sequential connectivities observed provide evidence that the sequence has the propensity to form a bend involving residues 5 and 6 and the N-terminal segment. The a-proton chemical shifts compared to random coil and additional data from the amide proton temperature coefficients support this assumption. One long-range nOe cross peak between H2α-HN Eth is indicative of proximity between C- and N-termini. © 2002 Kluwer Academic Publishers
αCGRP, another amyloidogenic member of the CGRP family
The Calcitonin-gene related peptide (CGRP) family is a group of peptide hormones, which consists of IAPP, calcitonin, adrenomedullin, intermedin, αCGRP and βCGRP. IAPP and calcitonin have been extensively associated with the formation of amyloid fibrils, causing Type 2 Diabetes and Medullary Thyroid Carcinoma, respectively. In contrast, the potential amyloidogenic properties of αCGRP still remain unexplored, although experimental trials have indicated its presence in deposits, associated with the aforementioned disorders. Therefore, in this work, we investigated the amyloidogenic profile of αCGRP, a 37-residue-long peptide hormone, utilizing both biophysical experimental techniques and Molecular Dynamics simulations. These efforts unravel a novel amyloidogenic member of the CGRP family and provide insights into the mechanism underlying the αCGRP polymerization. © 2018 Elsevier Inc
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 (All) 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 AT(1) 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-Leu(5)]All, which still induced reduction of the contractile responses. Nevertheless after replacing the Asp with Sar in position 1 (Sar(1)) 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 All 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 lle(5) residue in the All molecule. This was overcome by Sar(1), probably through interactions due to its fully protonated N-terminal amino group and favoring the conformation responsible for the tachyphylaxis phenomenon.Universidade Federal de São Paulo, Dept Biophys, Escola Paulista Med, BR-04041990 São Paulo, SP, BrazilUniv Patras, Dept Chem, Organ Chem Lab, Patras 26001, GreeceUniversidade Federal de São Paulo, Dept Biophys, Escola Paulista Med, BR-04041990 São Paulo, SP, BrazilWeb of Scienc
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 ζ-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. Copyright © 2005 European Peptide Society and John Wiley &amp; Sons, Ltd
Exploring new scaffolds for angiotensin II receptor antagonism
Nowadays, AT1receptor (AT1R) antagonists (ARBs) constitute the one of the most prevalent classes of antihypertensive drugs that modulate the renin-angiotensin system (RAS). Their main uses include also treatment of diabetic nephropathy (kidney damage due to diabetes) and congestive heart failure. Towards this direction, our study has been focused on the discovery of novel agents bearing different scaffolds which may evolve as a new class of AT1receptor antagonists. To fulfill this aim, a combination of computational approaches and biological assays were implemented. Particularly, a pharmacophore model was established and served as a 3D search query to screen the ChEMBL15 database. The reliability and accuracy of virtual screening results were improved by using molecular docking studies. In total, 4 compounds with completely diverse chemical scaffolds from potential ARBs, were picked and tested for their binding affinity to AT1receptor. Results revealed high nanomolar to micromolar affinity (IC50) for all the compounds. Especially, compound 4 exhibited a binding affinity of 199 nM. Molecular dynamics simulations were utilized in an effort to provide a molecular basis of their binding to AT1R in accordance to their biological activities. © 2016 Elsevier Lt
Structural studies and cytotoxicity assays of “aggregation-prone” IAPP8–16 and its non-amyloidogenic variants suggest its important role in fibrillogenesis and cytotoxicity of human amylin
Amyloid deposits to the islets of Langerhans are responsible for the gradual loss of pancreatic β-cells leading to type II diabetes mellitus. Human mature islet amyloid polypeptide (hIAPP), a 37-residue pancreatic hormone, has been identified as the primary component of amyloid fibrils forming these deposits. Several individual segments along the entire sequence length of hIAPP have been nominated as regions with increased amyloidogenic potential, such as regions 8–20, 20–29, and 30–37. A smaller fragment of the 8–20 region, spanning residues 8–16 of hIAPP has been associated with the formation of early transient α-helical dimers that promote fibrillogenesis and also as a core part of hIAPP amyloid fibrils. Utilizing our aggregation propensity prediction tools AmylPred and AmylPred2, we have identified the high aggregation propensity of the 8–16 segment of hIAPP. A peptide analog corresponding to this segment was chemically synthesized and its amyloidogenic properties were validated using electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, and polarized microscopy. Additionally, two peptides introducing point mutations L12R and L12P, respectively, to the 8–16 segment, were chemically synthesized. Both mutations disrupt the α-helical properties of the 8–16 region and lower its amyloidogenic potential, which was confirmed experimentally. Finally, cytotoxicity assays indicate that the 8–16 segment of hIAPP shows enhanced cytotoxicity, which is relieved by the L12R mutation but not by the L12P mutation. Our results indicate that the chameleon properties and the high aggregation propensity of the 8–16 region may significantly contribute to the formation of amyloid fibrils and the overall cytotoxic effect of hIAPP. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 196–205, 2015. © 2015 Wiley Periodicals, Inc