Endothelin Receptor Ligands Research Articles

Synthesis and Molecular Modelling Studies of New 1,3-Diaryl-5-Oxo-Proline Derivatives as Endothelin Receptor Ligands.

The synthesis of seventeen new 1,3-diaryl-5-oxo-proline derivatives as endothelin receptor (ETR) ligands is described. The structural configuration of the new molecules was determined by analyzing selected signals in proton NMR spectra. In vitro binding assays of the human ETA and ETB receptors allowed us to identify compound 31h as a selective ETAR ligand. The molecular docking of the selected compounds and the ETA antagonist atrasentan in the ETAR homology model provided insight into the structural elements required for the affinity and the selectivity of the ETAR subtype.

Nucleoligands-repurposing G Protein–coupled Receptor Ligands to Modulate Nuclear-localized G Protein–coupled Receptors in the Cardiovascular System

There is significant evidence that internal pools of G protein-coupled receptors (GPCRs) exist and may be affected by both endogenous signaling molecules and hydrophobic pharmaceutical ligands, once assumed to only affect cell surface versions of these receptors. Here, we discuss evidence that the biology of nuclear GPCRs in particular is complex, rich, and highly interactive with GPCR signaling from the cell surface. Caging existing GPCR ligands may be an excellent means of further stratifying the phenotypic effects of known pharmacophores such as β-adrenergic, angiotensin II, and type B endothelin receptor ligands in the cardiovascular system. We describe some synthetic strategies we have used to design ligands to go from in cellulo to in vivo experiments. We also consider how surface and intracellular GPCR signaling might be integrated and ways to dissect this. If they could be selectively targeted, nuclear GPCRs and their associated nucleoligands would represent a completely novel area for exploration by Pharma.

Creating a Soluble Binder to Endothelin-1 Based on the Natural Ligand Binding Domains of the Endothelin-1 (G-Protein-Coupled) Receptor

Since its discovery in 1989, there has been extensive research on endothelin (ET)-1 physiology, as well as pathology. Accordingly, there is considerable research on the discovery of therapeutics based around ET-1, amongst which current treatment options include endothelin receptor antagonists. These target the ET-1 receptors, which are G-protein–coupled receptors (GPCRs). We have effectively developed a soluble form of a GPCR that binds to ligands, by constructing a fusion polypeptide of different endothelin receptor ligand binding domains. Phage experiments identified strong binders to ET-1. We then constructed Fc-fusions of the top binders and further binding assays revealed a KD of 21.2 nM for the Fc-ETtr1 construct and KD of 77.3 nM for the Fc-ETtr2 construct. These constructs are soluble and have the ability to bind and potentially sequester elevated ET-1 levels that are prevalent in different diseases. These results provide a novel approach to targeting GPCR–binding ligands, and thereby to contribute to a very important class of therapeutics.

Creating a Soluble Binder to Endothelin-1 Based on the Natural Ligand Binding Domains of the Endothelin-1 (G-Protein-Coupled) Receptor

Since its discovery in 1989, there has been extensive research on endothelin (ET)-1 physiology, as well as pathology. Accordingly, there is considerable research on the discovery of therapeutics based around ET-1, amongst which current treatment options include endothelin receptor antagonists. These target the ET-1 receptors, which are G-protein–coupled receptors (GPCRs). We have effectively developed a soluble form of a GPCR that binds to ligands, by constructing a fusion polypeptide of different endothelin receptor ligand binding domains. Phage experiments identified strong binders to ET-1. We then constructed Fc-fusions of the top binders and further binding assays revealed a KD of 21.2 nM for the Fc-ETtr1 construct and KD of 77.3 nM for the Fc-ETtr2 construct. These constructs are soluble and have the ability to bind and potentially sequester elevated ET-1 levels that are prevalent in different diseases. These results provide a novel approach to targeting GPCR–binding ligands, and thereby to contribute to a very important class of therapeutics.

Synthesis and endothelin receptors binding affinity of new 1,3,5- substituted pyrrole-2-carboxylic acid derivatives.

The interest of researchers for ligands of the endothelin receptors ETA and ETB is due to their extensive therapeutic potential. In particular, receptor antagonists are useful in a number of diseases such as pulmonary hypertension, acute myocardial infarction, congestive heart failure, renal failure, and atherosclerosis. In the context of our research program aimed to the development of new endothelin receptor ligands, in this paper we describe the synthesis and structure- activity relationships of a new series of 1,3,5-substituted pyrrole-2-carboxylic acid derivatives 27-40 possessing the structural features for ET receptors binding. New synthesized compounds were tested on ETA and ETB receptors stably expressed in CHO cells and some of them showed interesting affinity and selectivity towards ETA receptors.

A Comparison of 3D Conformations of Endothelin-1 Analogs to Find the Pharmacophore Model Required for Endothelin Receptor Ligand Activity

Endothelin receptors (ETRs) are GPCRs whose activation results in the increase of intracellular calcium levels, and are ultimately involved in vasoconstriction and vasodilation. Because control of ETR activity has important medical implications, it is beneficial to rapidly discover effective synthetic ETR ligands by virtual drug screening methods. Despite its biological importance, little is known about ETR structure, since there are no reported X-ray structures to date. However, structural information is available for Endothelin-1, the natural ligand to ETR and the most potent vasoconstrictor yet identified. Thus, our work takes the ligand-based approach instead of receptor-based approach to ETR ligand pharmacophore discovery. Two well-studied ETR ligands, BQ-123 and bosentan, were chosen to be reference compounds in addition to Endothelin-1, and 30 known synthetic ligands were defined as the sample active compounds set. Conformers of the sample set were prepared by genetic algorithm-based approach using OpenBabel, while conformers of the reference set were prepared by molecular dynamics, using myPresto/cosgene. For molecular superimpositions, we built and used a program called 'BMSIP,' which is an algorithmic extension of the ROCS program. While it performs rigid-body superimpositions using a volume overlap score function based on spherical Gaussian descriptions of atomic shape, BMSIP also introduces a scores-weighting matrix to bias atom-pairings by atom-type correspondence. Superimposition of the sample set against the reference set was performed to obtain a density map that shows the frequency of atom overlaps between the superimposed compounds and reference compounds. This map can indicate the essential chemical features (the pharmacophore) required for interaction with ETRs. The results suggest that nonpolar groups alone, in particular aromatic groups, are important for ETR binding affinity and provide a good initial guess for directed ETR ligand design.

Non-Invasive Approaches to Visualize the Endothelin Axis In Vivo Using State-of-the-Art Molecular Imaging Modalities

The endothelin axis plays a major role in cardiovascular diseases and a number of human cancers. This review summarizes the work that has been published in the past ten years using labeled endothelin receptor ligands for the visualization of endothelin receptor expression in vivo.

Synthesis and Binding Properties of New Endothelin Receptor Ligands

Synthesis and Binding Properties of New Endothelin Receptor Ligands

Novel (E)‐α‐[(1H‐Indol‐3‐yl)methylene]benzeneacetic Acids as Endothelin Receptor Ligands.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Novel ( E)-α-[(1 H-indol-3-yl)methylene]benzeneacetic acids as endothelin receptor ligands

Endothelin-1 (ET-1), a peptide of 21 amino acid residues, is the most potent vasoconstrictor substance known and now it is understood to be one of a family of three mammalian vasoactive peptides that also includes ET-2 and ET-3. The endothelins (ETs) affect multiple organ systems and seem to be involved in the pathogenesis of many diseases such as hypertension, pulmonary hypertension, atherosclerosis, apoptosis inhibition and angiogenesis. The ETs exert their effects via activation of two distinct G-protein coupled receptor subtypes termed ET A and ET B. To date a number of ET receptor ligands with good affinity and selectivity is known, nevertheless these compounds belong only to few chemical classes. The aim of this work was the identification of a “hit compound” with novel chemical structure, endowed with reasonable ET affinity and selectivity. Accordingly, a new class of ( E)-α-[(1 H-indol-3-yl)methylene]benzeneacetic acid derivatives ( 1– 23) was synthesized for evaluation of their binding profiles.

Effect of endothelin-1 on astrocytic protein content.

The astrocytic endothelin (ET) receptors, ET(A) and ET(B), modulate calcium signaling and the astrocytic gap junctional network. The nonselective ET receptor ligand ET-1 inhibits gap junction permeability, an effect that can be blocked by tolbutamide. This mechanism may play a role in pathophysiological conditions such as ischemic stroke, characterized by elevated tissue ET-1 levels and hypertrophic-appearing reactive astrocytes. Therefore, the effect of ET-1 on cellular protein content was investigated in confluent once-passaged rat astrocyte cultures under serum-free conditions, by the Lowry method. Gap junction permeability was determined by the dye transfer technique. ET-1 prevented the decrease in astrocytic protein content observed in controls. The effect of ET-1 on cellular protein content was most pronounced in cultures seeded at high density, but it was attenuated in ET(B)-deficient (sl/sl) astrocytes. This effect could be blocked by the nonselective ET antagonist LU 302872 (10 micro M), as well as by the protein synthesis inhibitor cycloheximide (10 micro M). This increase in astrocytic protein content was inhibited by the ATP-sensitive K(+) channel blocker tolbutamide, which also antagonized the ET-1-induced reduction of gap junction permeability and reversed the morphological changes observed in astrocytes upon ET-1 treatment. Cytosine arabinoside (10 micro M), a DNA synthesis blocker, inhibited the ET-1-induced BrdU uptake without affecting the ET-1-induced increase in astrocytic protein content. To conclude, ET-1 induces an increase in astrocytic protein content as well as changes in astrocyte morphology in vitro. This hypertrophic response involves uncoupling of the astrocytic gap junctional network and is not dependent on DNA synthesis.

Characterization of a new endothelin receptor ligand by in vitro assays.

Following the discovery of endothelin (), functional characterization of the three endothelin (ET) isoforms (ET-1, ET-2 and ET-3) predicted that two mammalian receptor subtypes are present: the ETA receptor that is selective for ET-1, and the ETB receptor that has equal affinity for the three isoforms (). The existence of the two distinct high-affinity ET receptor subtypes has been confirmed by cloning. Unique cDNAs that code for ETA and ETB belonging to the G-protein linked heptahelical receptor superfamily are identified in human, bovine and rat tissues (, , , ). While pharmacological studies suggest that there may be more ET receptor subtypes (), no additional homologous mammalian cDNAs have been identified.

Functional Cross-talk of Ca2+-Mobilizing Endothelin Receptors in C6 Glioma Cells

There are conflicting results concerning the receptor subtype(s) involved in calcium-mediated endothelin signaling in the glial cells. In order to elucidate the role of endothelin A and B receptors in these processes, we have studied the effect of a complex spectrum of endothelin receptor ligands on intracellular calcium concentration changes in proliferating and differentiated C6 rat glioma cells. Cell differentiation was induced by dibutyryl-cAMP and assessed by the glial fibrillar acidic protein content. Intracellular calcium changes were measured in cell suspensions using fluorescent probe Fura-2. The specific endothelin B receptor agonists sarafotoxin S6c and IRL-1620 did not influence the intracellular calcium concentration. However, calcium changes induced by endothelin-1 and especially by endothelin-3 after the pretreatment of cells with one of these endothelin B receptor specific agonists were significantly enhanced even above the values attained by the highest effective endothelin concentrations alone. Such endothelin B-receptor ligand-induced sensitization of calcium signaling was not observed in differentiated C6 cells. Moreover, endothelin-induced calcium oscillations in differentiated C6 cells were less inhibited by BQ-123 and BQ-788 than in their proliferating counterparts. In conclusion, the specific activation of endothelin B receptor in C6 rat glioma cells does not affect intracellular calcium per se, but probably does so through interaction with the endothelin A receptor. The pattern and/or functional parameters of endothelin receptors in C6 rat glioma cells are modified by cell differentiation.

Endothelin ET B receptors show different binding profiles in intact cells and cell membrane preparations

We examined the affinity of endothelin-1, endothelin-3 and four endothelin receptor ligands, BQ788 ( cis-2,6-dimethylpiperidinocarbonyl- γ-methyl-Leu- d-Trp(1-CO 2CH 3- d-Nle-ONa), SB-209670 ((+)-(1S,2R,3S)-3-(2-carboxymethoxy-4-methoxyphenyl)-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylic acid), IRL-1620 (succinyl-[Glu 9,Ala 11,15]endothelin-1(8-21)), and L-749329 (3′,4′-methylenedioxy-1-(2-propyl-4-carboxyphenoxy)- N-(4-isopropyl-phenylsulfonyl)-benzene acetamide), for endothelin ET B receptors in human and rat heart cells. The affinities of these ligands showed good correlation between both types of living cells and between their membrane preparations ( r=0.861, P<0.001), but less significant correlation between each of the living cells and its respective membrane preparation ( r=0.569, 0.02< P<0.05). These results suggest that there is no species difference in the affinities of these ligands and that destruction of the intact cell membrane structure may lead to changes in binding properties of the endothelin ET B receptor.

Endothelin antagonists: search for surrogates of methylendioxyphenyl by means of a Kohonen neural network.

The methylendioxyphenyl group, present in a number of potent endothelin receptor antagonists, could have undesirable metabolic interactions with cytochrome P450 in vivo. Using a self-organizing neural network we analysed the features of molecular electrostatic potentials of several endothelin receptor ligands. A library of small "fragments and functional groups" together with their corresponding Kohonen maps was generated. By means of this Kohonen map library we discovered the benzothiadiazole group as a surrogate for methylendioxyphenyl.

Binding characterization of [ [formula omitted]]S-0139, an antagonist of the endothelin ET A receptor subtype

S-0139 (27-O-3-[2-(3-carboxy-acryloylamino)-5-hydroxyphenyl]-acryloyloxy myricerone, sodium salt) is a highly specific nonpeptide endothelin ET A receptor antagonist. The binding of [ 3H ]S-0139 was compared to that of [ 125I ]endothelin-1 to characterize the binding of the antagonist in porcine aortic smooth muscle membranes. Scatchard analysis revealed a single class of [ 3H ]S-0139 binding sites with a K d value of 0.61±0.10 nM and a B max of 0.72±0.16 pmol/mg protein. These sites were saturable and reversible. [ 125I ]Endothelin-1 also showed binding with high affinity ( K d=0.12±0.02 nM) to a homogenous population of binding sites, whose B max (0.71±0.20 pmol/mg protein) was almost the same as that for [ 3H ]S-0139. In both cases, the binding could be displaced by known endothelin receptor ligands and their IC 50 values in each case showed a very close correlation ( r=0.986). The potency of seven endothelin receptor antagonists to displace [ 3H ]S-0139 binding also correlated highly to the potency for inhibiting the endothelin-1-induced increase in cytosolic Ca 2+ concentration ( r=0.949). Myriceric acid A showed a more potent functional activity than expected from its binding affinity, but this seemed to result from the different assay conditions, such as incubation time. Together, the results suggest that S-0139 labels only endothelin ET A receptor binding sites in porcine aortic smooth muscle.

Characterization of three non-peptide endothelin receptor ligands using human cloned ETA and ETB receptors.

1. A number of putative endothelin (ET) receptor ligands were synthesized with a view to assessing their relative affinity for human recombinant ET receptors. 2. Human (h) and endothelin ETA and ETB receptor open reading frames were cloned by reverse transcription-polymerase chain reaction into the mammalian expression vector pcDNA1 and stable cell lines were created by transfection of Chinese hamster ovary cells. 3. Scatchard analyses of saturation isotherms for the specific binding of [125I]-endothelin-1 ([125I]-ET-1) to membranes, prepared from Chinese hamster ovary cells transfected with hETA or hETB receptors, yielded values for equilibrium dissociation constants (Kd) of 20.5 +/- 1.8 pM and 25.5 +/- 5.5 pM, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogeneous, non-interacting receptor populations. 4. Pharmacological characterization of the transfected hETA and hETB receptors was undertaken by measuring the relative abilities of ETA and ETB receptor-selective peptide ligands to inhibit binding of [125I]ET-1. For interaction with hETA receptors, the relative order of potency was ET-1 > ET-3 = FR139317 = BQ123 >[Ala1,3,11,15]-ET-1 = sarafotoxin S6c (S6c). In contrast, the relative order of potency, at hETB receptors, was ET-1 = ET-3 = [Ala1,3,11,15]-ET-1 = S6c >> FR139317 = BQ123. 5. The novel non-peptide ligands, Ro 46-2005, SB 209670 and BMS 182874, were found to inhibit [125I]-ET-1 binding to human recombinant ETA and ETB receptors. At hETA receptors, the calculated pIC50 values were 6.7 (Ro 46-2005), 8.7 (SB 209670) and 5.8 (BMS 182874), while at hETB receptors, the corresponding pIC50 values were 6.8, 7.5 and <5, respectively.6. In conclusion, we have characterized the pharmacology of human cloned ETA and ETB receptors and used these in membrane binding assays to determine the affinity and selectivity of three structurally diverse non-peptide ET receptor ligands. SB 209670 is, to date, the highest affinity non-peptide ligand to be described for ET receptors. As such, it may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of endothelins.

Endothelin eceptor Ligands. Multiple D-Amino acid replacement net approach

All possible single and multiple L- to D- amino acid replacements of a potent hexapeptide endothelin receptor ligand were synthesized and tested. While most of these 64 analogues were inactive on the ETR A receptor, three showed submicromolar activity. Interestingly, two of these contain 5D-amino acids and may be stable to proteolysis.

Endothelin receptor ligands. replacement net approach to SAR determination of potent hexapeptides

We determined the SAR of the potent hexapeptide endothelin ligand Ac-Dphe-Orn-Asp-Ile-Ile-Trp-OH 1 ( 1) through the systematic replacement of each residue with 50 amino acid substitutes. Multipin peptide synthesis methods allowed us to rapidly synthesize and then screen all 300 analogues.