Slow Receptor Dissociation Kinetics Differentiate Macitentan from Other Endothelin Receptor Antagonists in Pulmonary Arterial Smooth Muscle Cells

Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH), a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC). The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP₁) assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with K_b values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt_1/2) compared to bosentan and ambrisentan (ROt_1/2∶17 minutes versus 70 seconds and 40 seconds, respectively). Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP₁ assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1) concentrations. However, prolongation of the ET-1 stimulation time beyond ROt_1/2 rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with significantly slower receptor dissociation kinetics than the currently approved ERAs. Slow dissociation caused insurmountable antagonism in functional PASMC-based assays and this could contribute to an enhanced pharmacological activity of macitentan in ET-1-dependent pathologies.     

Design,synthesis and evaluation of 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acid derivatives as ETA receptor selective antagonists using FRET assay

The endothelin axis and in particular the two receptor subtypes, ET_A and ET_B, are under investigation for the treatment of various diseases such as pulmonary arterial hypertension, fibrosis, renal failure and cancer. Previous work in our lab has shown that 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acid derivatives exhibit noteworthy endothelin receptor antagonist activity. A series of analogues with modifications centered around position 6 of the heterocyclic quinolone core and replacement of the aryl carboxylic acid group with an isosteric tetrazole ring was designed and synthesized to further optimize the structure activity relationship. The endothelin receptor antagonist activity was determined by in vitro Förster resonance energy transfer (FRET) using GeneBLAzer® assay technology. The most potent member of this series exhibited ET_A receptor antagonist activity in the subnanomolar range with an IC₅₀ value of 0.8 nM, and was 1000-fold selective for the ET_A receptor compared to the ET_B receptor. Its activity and selectivity profile resembles that of the most recently approved drug, macitentan.     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Evaluation of the Endothelin Receptor Antagonists Ambrisentan,Bosentan, Macitentan,and Sitaxsentan as Hepatobiliary Transporter Inhibitors and Substrates in Sandwich-Cultured Human Hepatocytes

Background

Inhibition of the transporter-mediated hepatobiliary elimination of bile salts is a putative mechanism for liver toxicity observed with some endothelin receptor antagonists (ERAs).

Methods

Sandwich-cultured human hepatocytes were used to study the hepatobiliary distribution and accumulation of exogenous taurocholate, ERAs and endogenous bile acids. The molecular mechanisms for findings in hepatocytes or clinical observations were further explored using either vesicular assays (efflux transporters) or transfected cell-lines (uptake transporters). Inhibition constants (IC₅₀) were measured for the human hepatobiliary transporters bile salt export pump (BSEP), sodium taurocholate cotransporting polypeptide (NTCP), multidrug resistance protein 2 (MRP2), P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3.

Results

The ERAs showed dose-dependent reductions in exogenous taurocholate cellular accumulation in human hepatocytes, with macitentan having the greatest effect. Consistent with their effects on bile acids, the ERAs inhibited bile transporters. IC₅₀ values for OATP1B1 and OATP1B3 ranged from 2 µM for macitentan to 47 µM for ambrisentan. Macitentan and bosentan also inhibited NTCP with IC₅₀ values of 10 and 36 µM, respectively. Similar to previously reported findings with sitaxsentan, BSEP inhibition was observed for bosentan and macitentan with IC₅₀ values of 42 and 12 µM, respectively. In contrast, ambrisentan showed little or no inhibition of these transporters. Other transporters tested were weakly inhibited by the ERAs. Accumulation in hepatocytes was also a factor in the effects on bile transport. Macitentan demonstrated the greatest accumulation in human hepatocytes (∼100x) followed by sitaxsentan (∼40x), bosentan (∼20x) and ambrisentan (∼2x).

Conclusions

Significant differences in the inhibition of hepatic transporters were observed between the evaluated ERAs in vitro. Macitentan had the highest level of cellular accumulation and caused the greatest effects on bile acid distribution in human hepatocytes followed by sitaxsentan and bosentan. Ambrisentan showed a low potential to affect bile acids.     

Cerebrospinal Fluid from Patients with Subarachnoid Haemorrhage and Vasospasm Enhances Endothelin Contraction in Rat Cerebral Arteries

Introduction

Previous studies have suggested that cerebrospinal fluid from patients with subarachnoid hemorrhage (SAH) leads to pronounced vasoconstriction in isolated arteries. We hypothesized that only cerebrospinal fluid from SAH patients with vasospasm would produce an enhanced contractile response to endothelin-1 in rat cerebral arteries, involving both endothelin ET_A and ET_B receptors.

Methods

Intact rat basilar arteries were incubated for 24 hours with cerebrospinal fluid from 1) SAH patients with vasospasm, 2) SAH patients without vasospasm, and 3) control patients. Arterial segments with and without endothelium were mounted in myographs and concentration-response curves for endothelin-1 were constructed in the absence and presence of selective and combined ET_A and ET_B receptor antagonists. Endothelin concentrations in culture medium and receptor expression were measured.

Results

Compared to the other groups, the following was observed in arteries exposed to cerebrospinal fluid from patients with vasospasm: 1) larger contractions at lower endothelin concentrations (p<0.05); 2) the increased endothelin contraction was absent in arteries without endothelium; 3) higher levels of endothelin secretion in the culture medium (p<0.05); 4) there was expression of ET_A receptors and new expression of ET_B receptors was apparent; 5) reduction in the enhanced response to endothelin after ET_B blockade in the low range and after ET_A blockade in the high range of endothelin concentrations; 6) after combined ET_A and ET_B blockade a complete inhibition of endothelin contraction was observed.

Conclusions

Our experimental findings showed that in intact rat basilar arteries exposed to cerebrospinal fluid from patients with vasospasm endothelin contraction was enhanced in an endothelium-dependent manner and was blocked by combined ET_A and ET_B receptor antagonism. Therefore we suggest that combined blockade of both receptors may play a role in counteracting vasospasm in patients with SAH.     

Discovery of a series of pyrrolidine-based endothelin receptor antagonists with enhanced ETA receptor selectivity

Endothelins, ET-1, ET-2, and ET-3 are potent vasoconstricting and mitogenic 21-amino acid bicyclic peptides, which exert their effects upon binding to the ET_A and ET_B receptors. The ET_A receptor mediates vasoconstriction and smooth muscle cell proliferation, and the ET_B receptor mediates different effects in different tissues, including nitric oxide release from endothelial cells, and vasoconstriction in certain vascular cell types. Selective antagonists of endothelin receptor subtypes may prove useful in determining the role of endothelin in various tissue types and disease states, and hence as therapeutic agents for such diseases. The pyrrolidine carboxylic acid A-127722 has been disclosed as a potent and ET_A-selective antagonist, and is currently undergoing clinical trials. In our efforts to find antagonists with altered selectivity (ET_A-selective, ET_B-selective, or nonselective), we investigated the SAR of the 2-substituent on the pyrrolidine. Compounds with alkyl groups at the 2-position possessed ET_A selectivity improved over A-127722 (1400-fold selective), with the best of these compounds showing nearly 19,000-fold selectivity.     

C-terminal modifications of an endothelin antagonist RES-701-1: Production of ETA/ETB dual selective analogs

RES-701-1 is an endothelin B receptor (ET_B) selective peptidic antagonist, which has a novel cyclic structure of microbial origin. Modification at the C-terminal free carboxyl group of RES-701-1 by a methyl ester results in an ET_A/ET_B dual selective analog, which showed relatively high affinity for ET_A receptor subtype, while retaining the affinity for ET_B. The carboxyl-group-deleted analog with tryptamine as the C-terminal residue also showed relatively weak affinity for ET_A; however, benzyl ester or amide analogs did not show remarkable affinity for ET_A. It is suggested that the binding mode of RES-701-1 and its analogs is different from those of known ligands for ET receptors.     

PET-compatible endothelin receptor radioligands: Synthesis and first in vitro and in vivo studies

The expression and function of endothelin (ET) receptors is abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. In this study, we prepared two [¹⁸F]-fluorinated derivatives of the non-peptide ET_A receptor antagonist PD 156707 and evaluated their ET receptor binding potencies. Ex vivo as well as in vivo biodistribution studies in mice were performed, as well as the metabolism of the radiotracer, which was examined by metabolite analysis in mice and rats. All tested derivatives of PD 156707 exhibited potent in vitro pharmacological characteristics with K_i values comparable to that of the lead compound. The biodistribution studies showed a high accumulation of the tracer in bile and intestine. In vivo we were able to show that the visualization of the heart as a major target organ with high ET_AR expression is possible.     

C-terminal modifications of an endothelin antagonist RES-701-1: Production of ETA/ETB dual selective analogs

Summary RES-701-1 is an endothelin B receptor (ET_B) selective peptidic antagonist, which has a novel cyclic structure of microbial origin. Modification at the C-terminal free carboxyl group of RES-701-1 by a methyl ester results in an ET_A/ET_B dual selective analog, which showed relatively high affinity for ET_A receptor subtype, while retaining the affinity for ET_B. The carboxyl-group-deleted analog with tryptamine as the C-terminal residue also showed relatively weak affinity for ET_A; however, benzyl ester or amide analogs did not show remarkable affinity for ET_A. It is suggested that the binding mode of RES-701-1 and its analogs is different from those of known ligands for ET receptors.     

An endothelin type A receptor-expressing cell to characterize endothelin-1 binding and screen antagonist

Endothelin-1 (ET-1) induces contraction of vascular smooth muscle through binding to endothelin type A receptor (ET_AR). COS-7 cells stably expressing high levels of the ET_AR were established (designated COS-7(ET_AR)). The COS-7(ET_AR) cell bound [¹²⁵I]ET-1 with a K_d of 932 ± 161 pM and a B_max of 74 ± 13 fmol/2 × 10⁵ cells. [¹²⁵I]ET-1 binding was inhibited by ET-1 and the ET_AR antagonist BQ-610, but not by the endothelin type B receptor (ET_BR) antagonist BQ-788. In clones expressing two ET_AR mutants containing D46N or R53Q substitutions in the first extracellular domain of the receptor, [¹²⁵I]ET-1 binding activity was dramatically reduced. This suggests that these single amino acid substitutions alter the three-dimensional structure of the ligand-binding domain of the ET_AR. Using COS-7(ET_AR) cell, we showed that Ca²⁺ or Mg²⁺ was essential for ET-1 binding to the ET_AR and that ET-1 treatment induced postreceptor signaling, that is, intracellular accumulation of cyclic AMP (cAMP) and Ca²⁺ mobilization. The COS-7(ET_AR) established in this study will be a useful tool for screening ET-1 antagonists for treating hypertension.     

Improvement in cardiac function of diabetic rats by bosentan is not associated with changes in the activation of PKC isoforms

We previously demonstrated that chronic treatment with the mixed endothelin A and B (ET_A and ET_B) receptor blocker bosentan improved isolated working heart function in streptozotocin (STZ) diabetic rats. Endothelin-1 (ET-1) peptide levels, ET-1 mRNA and ET_A and ET_B receptor mRNA were all increased in diabetic hearts, but were unaffected by bosentan treatment, indicating that the beneficial effects of bosentan on heart appear to be on downstream effectors of ET-1 and ET receptors rather than the ET-1 system itself. Stimulation of ET-1 receptors leads to increased activation of protein kinase C (PKC), which is associated with PKC translocation from the cytosol to the membrane. Persistent activation of specific PKC isoforms has been proposed to contribute to diabetic cardiomyopathy. The purpose of this study was to determine whether chronic treatment with bosentan influences the activation of PKC isoforms in hearts from diabetic rats. Male Wistar rats were divided into four groups: control, bosentan-treated control, diabetic, and bosentan-treated diabetic. Diabetes was induced by the intravenous injection of 60 mg/kg streptozotocin. One week later, treatment with bosentan (100 mg/kg/day) by oral gavage was begun and continued for 10 weeks. The heart was then removed, homogenized, separated into soluble (cytosolic) and particulate (membrane) fractions and PKC isoform content in each fraction was determined by Western blotting. PKC α, β2, δ, ε and ζ were all detected in hearts from both control and diabetic rats. However, no change in the levels or distribution between the soluble and particulate fractions of any of these isoforms could be detected in chronic diabetic hearts compared to control, whether untreated or treated with bosentan. These observations indicate that bosentan does not improve cardiac performance in STZ diabetic rats by affecting the activation of PKC isoforms.     

The Endothelin ETA Receptor Exists in the Caudal Solitary Tract Nucleus of the Rat Brain

1. The receptor autoradiographic method done on the rat lower brain stem and cerebellum plus ¹²⁵I-endothelin-1, BQ-123, an antagonist for the endothelin ET_A receptor, and sarafotoxin S6c, an agonist for the ET_B receptor, revealed minute amounts of the ET_A receptor coexisting with the ET_B receptor in the caudal solitary tract nucleus of the rat lower brain stem.2. The ET_B receptor is present predominantly in other parts of the lower brain stem.3. Knowledge of the heterogeneous distribution of the central endothelin receptor subtypes aids in understanding the neurophysiology of endothelins.     

Endothelin Receptors in Rat Pituitary Gland

1. We used the quantitative receptor autoradiographic method plus ¹²⁵I-endothelin-1 (¹²⁵I-ET-1), BQ-123, a specific antagonist for the endothelin ET_A receptor, and sarafotoxin S6c, a selective agonist for the ET_B receptor to investigate the ET receptor in the rat pituitary gland.2. The method revealed that the BQ-123-sensitive ET_A receptor was present predominantly in the anterior lobe and Rathke's pouch.3. The posterior lobe contained BQ-123-sensitive ET_A and sarafotoxin S6c-sensitive ET_B receptors, in almost the same proportion. There was no significant ¹²⁵I-ET-1 binding to the intermediate lobe.4. Knowledge of the heterogeneous distribution of ET receptor subtypes in the pituitary gland supplies information that will be pertinent to physiological investigations of the gland.     

Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB

The molecular basis for recognition of peptide ligands endothelin‐1, ‐2 and ‐3 in endothelin receptors is poorly understood. Especially the origin of ligand selectivity for ET_A or ET_B is not clearly resolved. We derived sequence‐structure‐function relationships of peptides and receptors from mutational data and homology modeling. Our major findings are the dissection of peptide ligands into four epitopes and the delineation of four complementary structural portions on receptor side explaining ligand recognition in both endothelin receptor subtypes. In addition, structural determinants for ligand selectivity could be described. As a result, we could improve the selectivity of BQ3020 about 10‐fold by a single amino acid substitution, validating our hypothesis for ligand selectivity caused by different entrances to the receptors' transmembrane binding sites. A narrow tunnel shape in ET_A is restrictive for a selected group of peptide ligands' N‐termini, whereas a broad funnel‐shaped entrance in ET_B accepts a variety of different shapes and properties of ligands. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Desensitization and Internalization of Endothelin Receptor A: IMPACT OF G PROTEIN-COUPLED RECEPTOR KINASE 2 (GRK2)-MEDIATED PHOSPHORYLATION*

Endothelin receptor A (ET_A), a G protein-coupled receptor, mediates endothelin signaling, which is regulated by GRK2. Three Ser and seven Thr residues recently proven to be phosphoacceptor sites are located in the C-terminal extremity (CTE) of the receptor following its palmitoylation site. We created various phosphorylation-deficient ET_A mutants. The phospholipase C activity of mutant receptors in HEK-293 cells was analyzed during continuous endothelin stimulation to investigate the impact of phosphorylation sites on ET_A desensitization. Total deletion of phosphoacceptor sites in the CTE affected proper receptor regulation. However, proximal and distal phosphoacceptor sites both turned out to be sufficient to induce WT-like desensitization. Overexpression of the Gα_q coupling-deficient mutant GRK2-D110A suppressed ET_A-WT signaling but failed to decrease phospholipase C activity mediated by the phosphorylation-deficient mutant ET_A-6PD. In contrast, GRK2-WT acted on both receptors, whereas the kinase-inactive mutant GRK2-D110A/K220R failed to inhibit signaling of ET_A-WT and ET_A-6PD. This demonstrates that ET_A desensitization involves at least two autonomous GRK2-mediated components: 1) a phosphorylation-independent signal decrease mediated by blocking of Gα_q and 2) a mechanism involving phosphorylation of Ser and Thr residues in the CTE of the receptor in a redundant fashion, able to incorporate either proximal or distal phosphoacceptor sites. High level transfection of GRK2 variants influenced signaling of ET_A-WT and ET_A-6PD and hints at an additional phosphorylation-independent regulatory mechanism. Furthermore, internalization of mRuby-tagged receptors was observed with ET_A-WT and the phosphorylation-deficient mutant ET_A-14PD (lacking 14 phosphoacceptor sites) and turned out to be based on a phosphorylation-independent mechanism.     

Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells

Background  

Endothelin-1 (ET-1) is a potent vasoactive peptide, which induces vasoconstriction and proliferation in vascular smooth muscle cells (VSMCs) through activation of endothelin type A (ET_A) and type B (ET_B) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigate the ET_A and ET_B receptor intracellular signaling in human VSMCs and used phosphorylation (activation) of ERK1/2 as a functional signal molecule for endothelin receptor activity.     

Selective and non-selective et antagonists reveal an ETA/ETB receptor mediated ET-1-induced antinociceptive effect in PAG area of mice

The injection of endothelin-1 (ET-1) (2 pmol) into the dorsolateral periaqueductal gray area (PAG) of mice produces antinociceptive effect as underscored by increases in the latency time for the reaction to a hot plate. Pretreatment of the PAG area with bosentan (10 nmol) (a mixed ET_A/ET_B receptor antagonist), FR 139317 (5 nmol) (ET_A receptor selective antagonist) or BQ-788 (5 nmol) (ET_B receptor selective antagonist) greatly reduced the antinociceptive effect induced by ET-1. Therefore, ET-1 induces antinociceptive effects via both ET_A/ET_B receptors. In addition, since ET-antagonists lowered per se the control reaction time of the mice when administered alone to the PAG area, we would suggest that endogenous ET-1 acting within the PAG area contributes to the suppression of pain.     

Number and brightness analysis to study spatio-temporal distribution of the angiotensin II AT1 and the endothelin-1 ETA receptors: Influence of ligand binding

The angiotensin II AT₁ and the endothelin 1 ET_A receptors play a crucial role in the pathogenesis of cardiovascular diseases like hypertension, heart failure, stroke, pulmonary hypertension, and cardiac hypertrophy. Both receptors are members of the rhodopsion-like superfamily of G protein-coupled receptors which can exist as monomers, dimers, and higher order aggregates.Recently, oligomerization of these two receptors have been described by several biophysical methods based mainly on luminescence and fluorescence energy transfer. Since this oligomerization can occur either spontaneously or it can be induced by ligand-binding, the aim of this work was to address whether the oligomerization of these receptors occurs upon ligand-binding. For this purpose the Number and Brightness analysis, a method that allows the identification, localization, and quantification of protein aggregates in the plasma membrane of a single cell, was used. An advantage of this method is that it is not limited to certain dyes specially required for Fluorescence Resonance Energy Transfer measurements.Our results showed that stably transfected angiotensin II AT₁ receptors and transiently transfected endothelin 1 ET_A receptors, were found as monomeric, dimeric, and tetrameric receptor aggregates. Interestingly, the binding of antihypertensive agents like losartan and BQ123, earlier suggested to be inverse agonists, significantly increased the proportion of monomers and reduced the occurrence of dimers on the cell membrane; while the kown endothelin 1 ET_A antagonist sitaxentan did not influence the aggregation state of these receptors.