ETA-selective Antagonist Research Articles

Structural basis of antagonist selectivity in endothelin receptors

Endothelins and their receptors, ETA and ETB, play vital roles in maintaining vascular homeostasis. Therapeutically targeting endothelin receptors, particularly through ETA antagonists, has shown efficacy in treating pulmonary arterial hypertension (PAH) and other cardiovascular- and renal-related diseases. Here we present cryo-electron microscopy structures of ETA in complex with two PAH drugs, macitentan and ambrisentan, along with zibotentan, a selective ETA antagonist, respectively. Notably, a specialized anti-ETA antibody facilitated the structural elucidation. These structures, together with the active-state structures of ET-1-bound ETA and ETB, and the agonist BQ3020-bound ETB, in complex with Gq, unveil the molecular basis of agonist/antagonist binding modes in endothelin receptors. Key residues that confer antagonist selectivity to endothelin receptors were identified along with the activation mechanism of ETA. Furthermore, our results suggest that ECL2 in ETA can serve as an epitope for antibody-mediated receptor antagonism. Collectively, these insights establish a robust theoretical framework for the rational design of small-molecule drugs and antibodies with selective activity against endothelin receptors.

RAPID ENDOGENOUS PRODUCTION OF ENDOTHELIN-1 REVEALED BY AN ENDOTHELIN NEUTRALIZING ANTIBODY IN A SERIES OF EXPERIMENTAL AND MODELING STUDIES

Objective: The endothelin pathway is implicated in driving pathophysiological changes leading to pulmonary and systemic hypertension, chronic kidney disease and heart failure. Small molecule antagonists that selectively blocked endothelin A receptors (ETA) as well as non-selective ETA and endothelin B receptor (ETB) antagonists were successfully developed for the treatment of these vascular diseases. However, the treatment-related adverse events, such as fluid retention and edema, significantly limit the utility of this class of therapeutics, especially in the heart failure patients. Design and method: A monoclonal antibody that selectively neutralizes endothelin-1 (ET-1) may offer an attractive alternative therapeutic approach that remedies ETA-mediated vasoconstriction without impacting desirable ET-3/ETB mediated fluid regulation. A selective ET-1/2 neutralizing antibody, S69glC21 was discovered and optimized through protein engineering to 80 pM and 30 pM binding affinities to ET-1 and ET-2, respectively. Results: When administered into rats exogenously infused with ET-1, S69glC21 prevented blood pressure increase. In a hypoxia-induced pulmonary hypertension rat model, S69glC21 reduced pulmonary pressure. Compared to atrasentan, a selective small molecule ETA antagonist, S69glC21 had a distinct pharmacodynamic (PD) effect characterized by initial complete inhibition followed by a delayed rise of pulmonary pressure as the hypoxia condition persisted. To further dissect these findings, S69glC21 pharmacokinetics (PK) and circulating ET-1 target engagement (TE) analysis were performed. In the S69glC21 treated rats, antibody bound ET-1 was shown to accumulate relative to baseline plasma levels at all dose levels. In the 30 mpk group, the antibody bound ET-1 accumulated nearly 40,000-fold above the baseline plasma level of 2.25 ± 0.87 pg/mL indicating a rapid production rate of ET-1. Conclusions: Thus, PK/PD modeling reveals significant druggability challenges for the development of a highly effective, commercially viable ET-1 selective neutralizing antibody for the treatment of cardiovascular, pulmonary, and/or renal diseases.

POS-527 A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study of Atrasentan in Patients with IgA Nephropathy (The ALIGN Study)

IgA nephropathy (IgAN) is the most common primary glomerulonephritis. Up to 40% of patients with IgAN are at risk of progressing to end-stage kidney disease (ESKD); proteinuria is the strongest predictor of progression. Endothelin A receptor (ETA) activation promotes proteinuria, along with kidney inflammation and fibrosis. Atrasentan, a potent and selective ETA antagonist, has been studied in >5,000 patients in a global phase 3 outcome clinical trial in patients with diabetic kidney disease who were on a maximum tolerated dose of RAS inhibitor (RASi).

The acute pressure natriuresis response is suppressed by selective ETA receptor blockade.

Hypertension is a major risk factor for cardiovascular disease. In a significant minority of people, it develops when salt intake is increased (salt-sensitivity). It is not clear whether this represents impaired vascular function or disruption to the relationship between blood pressure (BP) and renal salt-handling (pressure natriuresis, PN). Endothelin-1 (ET-1) regulates BP via ETA and ETB receptor subtypes. Blockade of ETA receptors reduces BP but promotes sodium retention by an unknown mechanism. ETB blockade increases both BP and sodium retention. We hypothesized that ETA blockade promotes sodium and water retention by suppressing PN. We also investigated whether suppression of PN might reflect off-target ETB blockade. Acute PN was induced by sequential arterial ligation in male Sprague Dawley rats. Intravenous atrasentan (ETA antagonist, 5 mg/kg) halved the normal increase in medullary perfusion and reduced sodium and water excretion by >60%. This was not due to off-target ETB blockade because intravenous A-192621 (ETB antagonist, 10 mg/kg) increased natriuresis by 50% without modifying medullary perfusion. In a separate experiment in salt-loaded rats monitored by radiotelemetry, oral atrasentan reduced systolic and diastolic BP by ∼10 mmHg, but additional oral A-192621 reversed these effects. Endogenous ETA stimulation has natriuretic effects mediated by renal vascular dilation while endogenous ETB stimulation in the kidney has antinatriuretic effects via renal tubular mechanisms. Pharmacological manipulation of vascular function with ET antagonists modifies the BP set-point, but even highly selective ETA antagonists attenuate PN, which may be associated with salt and water retention.

POS-378 SELECTIVE ETA ANTAGONIST ATRASENTAN, RAPIDLY REDUCES ALBUMINURIA AND DOWNREGULATES INTRA-RENAL PRO-INFLAMMATORY AND PRO-FIBROTIC TRANSCRIPTIONAL NETWORKS IN THE GDDY MOUSE MODEL OF SPONTANEOUS IGA NEPHROPATHY

Endothelin pathway activation has been observed in kidney biopsies from IgA nephropathy (IgAN) patients which may be an important driver of disease progression by promoting proteinuria along with kidney inflammation and fibrosis via ETA receptor activation. Atrasentan is a potent and selective ETA antagonist which has demonstrated rapid and sustained reductions in proteinuria, preservation of kidney function and improved kidney outcomes in diabetic kidney disease patients. However, the effects of atrasentan have not been previously investigated in IgAN. The objective of this study was to evaluate the effect of short-term treatment of varying doses of atrasentan in the gddY mouse model of spontaneous IgAN, with a focus on dynamic changes in the intra-renal transcriptional profile. 6-week-old male gddY mice were administered atrasentan (10, 20 or 30 mg/kg/day, n=3/group or 0 mg/kg/day, n=2/group) in drinking water for 5 days. Urine albumin to creatinine ratio (UACR) was measured at baseline and on Day 4 of treatment. On Day 5, a terminal plasma sample was collected for the measurement of atrasentan plasma concentrations and kidney cortex was flash-frozen for RNA-seq which was analyzed by pairwise differential gene expression using edgeR’s quasi-likelihood F-test (FDR adjusted p-value < 0.05) and gene pathway analysis (FDR adjusted p-value filter of < 0.001). At baseline, gddY mice had substantial albuminuria, with a mean UACR of 293.1 ± 16 mg/g. Atrasentan reduced UACR from baseline by 28 ± 44 % (p=ns), 62 ± 8 % (p=0.0498) and 63 ± 6 % (P=0.029) at 10, 20 and 30 mg/kg/day, respectively. Free terminal atrasentan plasma concentrations were 90 pM, 100 pM and 207 pM at the 10, 20 and 30 mg/kg/day doses respectively, providing free atrasentan concentrations 2.6-fold, 2.9-fold and 6.1-fold above the atrasentan ETA Ki (34 pM), respectively and in the same range as observed clinically at the optimized atrasentan dose of 0.75 mg. High quality RNA sequencing reads revealed dose-dependent effects of atrasentan on differential gene expression compared to untreated control ddY mice, including reductions in known ETA target genes ET-1 and ETA. Ingenuity Pathway Analysis to calculate enrichment scores showed that atrasentan produced prominent, dose-dependent downregulation of inflammatory, proliferative, and fibrotic gene networks. Atrasentan was observed to downregulate signaling pathways previously implicated in IgAN pathogenesis including the PDGF, NF-κB, IL-6 and TNF pathways. In the gddY mouse model of spontaneous IgAN, selectively blocking ETA with atrasentan leads to rapid reductions in albuminuria and results in intra-renal transcriptional downregulation of inflammatory and fibrotic signaling. The dynamic transcriptional changes in the kidney, including downregulation of known direct ETA receptor target genes, following only 5 days of treatment and prior to sustained long-term reductions in albuminuria and blood pressure that could mediate this benefit, is consistent with direct anti-inflammatory and antifibrotic effects of ETA blockade in IgAN. These results support further characterization of the effects of long-term treatment of atrasentan in the gddY mouse model and also support the therapeutic potential of atrasentan in IgAN patients to reduce proteinuria and kidney inflammation and fibrosis, key drivers of IgAN progression.

Combined hydroxyurea and ETA receptor blockade reduces renal injury in the humanized sickle cell mouse.

The objective of this study is to determine if ambrisentan (ETA selective antagonist) and hydroxyurea (HU) treatment has a synergistic effect on renal injury in sickle cell nephropathy when compared to HU treatment alone. The premise of the study is based on recent studies showing that endothelin-1 (ET-1) contributes to the pathophysiology of nephropathy in sickle cell disease (SCD) and that ETA receptor blockade improves renal function and protects against renal injury. Hydroxyurea (HU) is commonly prescribed for the treatment of SCD and has been shown to reduce renal injury in patients with SCD. Male 12-week-old humanized sickle mice (HbSS) and their genetic controls (HbAA) were treated with vehicle, HU, ambrisentan, or HU with ambrisentan for 2weeks and renal structure and function were assessed. Vehicle treated HbSS mice exhibited significant proteinuria compared to vehicle treated HbAA mice. HbSS mice also displayed significantly elevated plasma ET-1 concentrations and decreased urine osmolality compared to HbAA controls. Proteinuria was significantly lower in both HU and ambrisentan treated animals compared to vehicle treated HbSS mice; however, there was no additional improvement in HbSS mice treated with combined ambrisentan and HU. The combination of HU and ambrisentan resulted in significantly lower KIM-1 excretion, glomerular injury, and interstitial inflammation than HU alone. These findings indicate that HU and ETA receptor blockade produce similar reductions in renal injury in the humanized sickle mouse suggesting that both treatments may converge on the same mechanistic pathway.

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

BackgroundArthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient’s lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation.MethodsThe articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors.ResultsPlasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat).ConclusionThese results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

Endothelin ETA Receptor Blockade, by Activating ETB Receptors, Increases Vascular Permeability and Induces Exaggerated Fluid Retention.

Endothelin (ET) receptor antagonists have been associated with fluid retention. It has been suggested that, of the two endothelin receptor subtypes, ETB receptors should not be blocked, because of their involvement in natriuresis and diuresis. Surprisingly, clinical data suggest that ETA-selective antagonists pose a greater risk of fluid overload than dual antagonists. The purpose of this study was to evaluate the contribution of each endothelin receptor to fluid retention and vascular permeability in rats. Sitaxentan and ambrisentan as ETA-selective antagonists and bosentan and macitentan as dual antagonists were used as representatives of each class, respectively. ETA-selective antagonism caused a dose-dependent hematocrit/hemoglobin decrease that was prevented by ETB-selective receptor antagonism. ETA-selective antagonism led to a significant blood pressure reduction, plasma volume expansion, and a greater increase in vascular permeability than dual antagonism. Isolated vessel experiments showed that ETA-selective antagonism increased vascular permeability via ETB receptor overstimulation. Acutely, ETA-selective but not dual antagonism activated sympathetic activity and increased plasma arginine vasopressin and aldosterone concentrations. The hematocrit/hemoglobin decrease induced by ETA-selective antagonism was reduced in Brattleboro rats and in Wistar rats treated with an arginine vasopressin receptor antagonist. Finally, the decrease in hematocrit/hemoglobin was larger in the venous than in the arterial side, suggesting fluid redistribution. In conclusion, by activating ETB receptors, endothelin receptor antagonists (particularly ETA-selective antagonists) favor edema formation by causing: 1) fluid retention resulting from arginine vasopressin and aldosterone activation secondary to vasodilation, and 2) increased vascular permeability. Plasma volume redistribution may explain the clinical observation of a hematocrit/hemoglobin decrease even in the absence of signs of fluid retention.

Endothelin-1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention.

Haemorrhoids is a common anorectal condition affecting millions worldwide. We have studied the effect of endothelin-1 (ET-1) and the role of endothelin ETA and ETB receptors in haemorrhoid tissue. Protein expression of ET-1, ETA and ETB receptors were compared between haemorrhoids and normal rectal submucosa using Western blot analysis, with the localization of proteins determined by autoradiography and immunohistochemistry. Effects of ET-1 and sarafotoxin 6a on human colonic and rectal arteries and veins was assessed by wire myography and the involvement of receptor subtypes established by selective antagonists. Dense binding of [125 I]-ET-1 to haemorrhoidal sections was reduced by selective receptor antagonists. A higher density of ETB than ETA receptors was found in haemorrhoidal, than in control rectal tissue and confirmed by Western blot analysis. ETA and ETB receptors were localized to smooth muscle of haemorrhoidal arteries and veins, with ETB receptors on the endothelium. Human colonic and rectal arteries and veins were similarly sensitive to ET-1 and affected by the ETA selective antagonist, but sarafotoxin S6a-induced contractions were more pronounced in veins and antagonized by a selective ETB receptor antagonist. ETA and ETB receptors are present in human haemorrhoids with ETB receptors predominating. ETA receptors are activated by ET-1 to mediate a contraction in arteries and veins, but the latter are selectively activated by sarafotoxin S6a - a response that involves ETB receptors at low concentrations. Selective ETB agonists may have therapeutic potential to reduce congestion of the haemorrhoidal venous sinusoids.

Endothelin.

The endothelins comprise three structurally similar 21-amino acid peptides. Endothelin-1 and -2 activate two G-protein coupled receptors, ETA and ETB, with equal affinity, whereas endothelin-3 has a lower affinity for the ETA subtype. Genes encoding the peptides are present only among vertebrates. The ligand-receptor signaling pathway is a vertebrate innovation and may reflect the evolution of endothelin-1 as the most potent vasoconstrictor in the human cardiovascular system with remarkably long lasting action. Highly selective peptide ETA and ETB antagonists and ETB agonists together with radiolabeled analogs have accurately delineated endothelin pharmacology in humans and animal models, although surprisingly no ETA agonist has been discovered. ET antagonists (bosentan, ambrisentan) have revolutionized the treatment of pulmonary arterial hypertension, with the next generation of antagonists exhibiting improved efficacy (macitentan). Clinical trials continue to explore new applications, particularly in renal failure and for reducing proteinuria in diabetic nephropathy. Translational studies suggest a potential benefit of ETB agonists in chemotherapy and neuroprotection. However, demonstrating clinical efficacy of combined inhibitors of the endothelin converting enzyme and neutral endopeptidase has proved elusive. Over 28 genetic modifications have been made to the ET system in mice through global or cell-specific knockouts, knock ins, or alterations in gene expression of endothelin ligands or their target receptors. These studies have identified key roles for the endothelin isoforms and new therapeutic targets in development, fluid-electrolyte homeostasis, and cardiovascular and neuronal function. For the future, novel pharmacological strategies are emerging via small molecule epigenetic modulators, biologicals such as ETB monoclonal antibodies and the potential of signaling pathway biased agonists and antagonists.

Endothelin-1 contributes to the progression of renal injury in sickle cell disease via reactive oxygen species.

Endothelin-1 (ET-1) is increased in patients with sickle cell disease and may contribute to the development of sickle cell nephropathy. The current study was designed to determine whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of sickle cell disease. Adult, humanized HbSS (homozygous for sickle Hb) mice had increased ET-1 mRNA expression in both the cortex and the glomeruli compared with mice heterozygous for sickle and Hb A (HbAS controls). In the renal cortex, ETA receptor mRNA expression was also elevated in HbSS (sickle) mice although ETB receptor mRNA expression was unchanged. Ligand binding assays confirmed that sickle mice had increased ETA receptors in the renal vascular tissue when compared with control mice. In response to PKC stimulation, reactive oxygen species production by isolated glomeruli from HbSS sickle mice was increased compared with that from HbSA controls, an effect that was prevented by 1 week in vivo treatment with the selective ETA antagonist, ABT-627. Protein and nephrin excretion were both elevated in sickle mice, effects that were also significantly attenuated by ABT-627. Finally, ETA receptor antagonism caused a significant reduction in mRNA expression of NADPH oxidase subunits, which may contribute to nephropathy in sickle cell disease. These data support a novel role for ET-1 in the progression of sickle nephropathy, specifically via the ETA receptor, and suggest a potential role for ETA receptor antagonism in a treatment strategy.

Discovery of phenoxybutanoic acid derivatives as potent endothelin antagonists with antihypertensive activity

A series of phenoxybutanoic acid derivatives were synthesized and tested for their antagonistic activity on the contraction of the rat thoracic aortic ring induced by endothelin-1. Preliminary screening results showed that 6e and 6g with benzoheterocycles demonstrated significant antagonistic activities when compared to the reference compound BQ123. The results from additional assays for the binding affinity and selectivity for endothelin receptors showed that 6e was a selective ETA antagonist with a nanomolar IC50. Moreover, 6e was effective in relieving hypoxia-induced pulmonary arterial hypertension and right ventricular weight ratio. Therefore, 6e may have potential for further development as a therapeutic agent for the treatment of cardiovascular diseases.

High salt diet increases glomerular ROS formation through an ET‐1/ETA dependent mechanism (1134.9)

Endothelin‐1 (ET‐1) stimulates the production of reactive oxygen species (ROS) in pulmonary artery smooth muscle cells and also activates NADPH oxidase in isolated aortic rings. The connection between ROS production provides mechanistic insight into possible deleterious effects of ET‐1 dysregulation during pathological processes such as hypoxia. ET‐1 production is increased by a high salt diet (HSD), so we sought to test the effects of HSD on the capacity of glomeruli to produce ROS. C57BL6/J mice were placed on a normal or HSD and then treated with or without selective ETA antagonist ABT‐627 for one week (5 mg/kg/d p.o.). Untreated animals on a HSD had significantly increased levels of glomerular ROS production when stimulated by phorbol (PMA) mediated PKC activation compared to glomeruli from normal salt controls (77.5±7.8 v. 23.7±7.3 thousand ALU/mg protein, p&lt;0.01, n=5‐6). However, HSD animals treated with ABT‐627 had a blunted increase in glomerular ROS production (44.3±11.5 thousand ALU/mg protein, n=6). To further test the etiology of ROS production, glomeruli were incubated with 100 µM apocynin, a NADPH oxidase inhibitor, which completely prevented PMA stimulation of ROS to sub‐control levels in normal and HSD treated animals (10.5±2.75 and 13.02±4.3 thousand ALU/mg protein, n=5‐6). These data provide evidence for ET‐1/ETA mediated glomerular injury via ROS production during increased dietary salt intake.Grant Funding Source: Supported by the National Institutes of Health

12 Pharmacology of Human ETA and ETB Receptor Signalling VIA G-Protein and Beta-Arrestin Pathways

The endothelin (ET) system is altered in cardiovascular diseases and ET receptor antagonists are licenced for treatment of pulmonary arterial hypertension (PAH). Endothelins act via two G-protein-coupled receptors (GPCRs), ETA and ETB. However, it is recognised that GPCRs may also activate signalling pathways in a G-protein-independent manner via beta-arrestin. Ligand-specific pathway modulation (biassed agonism/antagonism) may have therapeutic application and therefore we have investigated the potential for pathway bias of ET agonists and antagonists. Concentration-response curves were constructed to ET agonists using ETA and ETB beta-arrestin assays. ET receptor antagonists were tested for their ability to block ET-1 responses in each assay and agonist-dependence of the ETA selective antagonist BQ123 was investigated. These data were compared to results from binding experiments in human heart (that expresses both subtypes) and to ETA-mediated vasoconstrictor experiments in human saphenous vein. The relative potency of ET peptides in the ETA and ETB beta-arrestin assays was as expected. Interestingly, for ETA, compared to ET-1, all other agonists tested were partial agonists. Differences from the known pharmacology of antagonists were also revealed in the beta-arrestin assays. Specifically, BQ123 behaved as a negative allosteric modulator in the ETA assay, exhibiting agonist-dependent affinities. Bosentan was a potent ETA-selective antagonist in the beta-arrestin assay in contrast to its non-selective profile determined in human heart. The apparent ETA beta-arrestin bias of bosentan may contribute to its clinical effectiveness in PAH but further investigation of a role for functional selectivity and biassed signalling via the ET receptors in cardiovascular disease is required.

Regulation of tyrosine hydroxylase by ETA receptors in the olfactory bulb of DOCA‐salt hypertensive rats

We recently showed that olfactory bulb (OB) exposure to endothelins (ETs) differentially modulate tyrosine hydroxylase (TH) activity and expression in normotensive rats. In the present study we evaluated the regulation of TH by ETs in hypertension. BQ610 (selective ETA antagonist) (20uM/ul/min) was intracerebroventriculary applied to normotensive and DOCA‐Salt hypertensive rats. Blood pressure and heart rate were monitored by 60 min and then animals sacrificed to obtain OB where TH activity (radioenzymatic method) and expression of total enzyme and phosphorylated forms (western blot) were assessed. Following BQ610 administration blood pressure and heart rate were reduced in hypertensive rats but no changes were observed in control animals (control, salt or DOCA only). Hypertensive rats exhibited an increase in TH activity as well as in total TH and its phosphorylated forms (pSer19, pSer31 and pSer40) that were diminished to control levels by BQ610 administration. These findings support an interaction between ETs and TH in the OB of DOCA‐salt hypertensive rats likely related with the onset and/or development of hypertension in this animal model (ANPCyT, CONICET, UBA).

Comparison of human ETA and ETB receptor signalling via G-protein and β-arrestin pathways

AimsTo determine the pharmacology of ETA- and ETB-mediated β-arrestin recruitment and compare this to established human pharmacology of these receptors to identify evidence for endothelin receptor biased signalling and pathway specific blockade by antagonists. Main methodsThe ability of ET-1, ET-2, ET-3, sarafotoxin 6b and sarafotoxin 6c to activate ETA and ETB-mediated β-arrestin recruitment was determined in CHO-K1 cells. Affinities were obtained for ETA selective (BQ123, sitaxentan, ambrisentan), ETB selective (BQ788) and mixed (bosentan) antagonists using ET-1 and compared to affinities obtained in competition experiments in human heart and by Schild analysis in human saphenous vein. Agonist dependence of affinities was compared for BQ123 and BQ788 in the ETA and ETB β-arrestin assays respectively. Key findingsFor β-arrestin recruitment, order of potency was as expected for the ETA (ET-1≥ET-2>>ET-3) and ETB (ET-1=ET-2=ET-3) receptors. However, at the ETA receptor sarafotoxin 6b and ET-3 were partial agonists. Antagonism of ET peptides by selective and mixed antagonists appeared non-competitive. BQ123, but not BQ788, exhibited agonist-dependent affinities. Bosentan was significantly more effective an inhibitor of β-arrestin recruitment mediated by ETA compared to the ETB receptor. In the ETA vasoconstrictor assay, ET-1, ET-2 and S6b were equipotent, full agonists and antagonists tested behaved in a competitive manner, although affinities were lower than predicted from the competition binding experiments in left ventricle. SignificanceThese data suggest that the pharmacology of ETA and ETB receptors linked to G-protein- and β-arrestin mediated responses was different and bosentan appeared to show bias, preferentially blocking ETA mediated β-arrestin recruitment.

Fluorescence Correlation Spectroscopy in Drug Discovery: Study of Alexa532-Endothelin 1 Binding to the Endothelin ETAReceptor to Describe the Pharmacological Profile of Natural Products

Fluorescence correlation spectroscopy and the newly synthesized Alexa532-ET1 were used to study the dynamics of the endothelin ETA receptor-ligand complex alone and under the influence of a semisynthetic selective antagonist and a fungal extract on living A10 cells. Dose-dependent increase of inositol phosphate production was seen for Alexa532-ET1, and its binding was reduced to 8% by the selective endothelin ETA antagonist BQ-123, confirming the specific binding of Alexa532-ET1 to the endothelin ETA receptor. Two different lateral mobilities of the receptor-ligand complexes within the cell membrane were found allowing the discrimination of different states for this complex. BQ-123 showed a strong binding affinity to the “inactive” receptor state characterized by the slow diffusion time constant. A similar effect was observed for the fungal extract, which completely displaced Alexa532-ET1 from its binding to the “inactive” receptor state. These findings suggest that both BQ-123 and the fungal extract act as inverse agonists.

Endothelin receptor antagonist activity of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy)acetamide hydrochloride (PABSA) in rat aortic smooth muscle cells and isolated rat thoracic aorta.

Antagonist activities of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy) acetamide hydrochloride (CAS 188710-94-3, PABSA), a novel endothelin (ET) receptor antagonist, for ETA and ETB receptors were evaluated using rat aortic smooth muscle A7r5 cells and isolated rat thoracic aorta. PABSA concentration-dependently inhibited the ET-1-induced increase in intracellular calcium concentration ([Ca2+]i) mediated via ETA receptors in A7r5 cells with an IC50 of 0.17 nmol/l. PABSA antagonized the ETA receptor-mediated contraction induced by ET-1 in endothelium-denuded rat aorta with a Kb of 0.74 nmol/l. The potency of PABSA in inhibiting ETA receptor-mediated vasocontraction was approximately 40- and 100-fold greater than those of BQ-123, a selective ETA antagonist, and bosentan, a mixed ETA/ETB receptor antagonist, respectively. ETB receptor-mediated endothelium-dependent vasorelaxation induced by ET-3 in the aorta was also antagonized by PABSA, with a Kb of 9.8 nmol/l. In contrast, PABSA affected neither the vasocontraction induced by KCl or norepinephrine nor the vasorelaxation induced by acetylcholine or prostaglandin I2 in the aorta. These results suggest that PABSA is a highly potent and selective ETA receptor antagonist.

Endothelin-1 promotes cytoplasmic accumulation of RIP140 through a ETA–PLCβ–PKCε pathway

The physiological signal activating cytoplasmic accumulation of nuclear receptor interacting protein 140 (RIP140) in adipocytes was unclear. We uncover that endothelin-1 (ET-1) promotes cytoplasmic accumulation of RIP140 in 3T3-L1 adipocytes. We determine ET-1’s signal transduction pathway in adipocytes, which is by activating ETA receptor-PLCβ-nuclear PKCε. Blocking this pathway in 3T3-L1 adipocyte cultures, by treating cells with an ETA antagonist, inhibiting PLCβ, or silencing PKCε, reduces ET-1-stimulated cytoplasmic accumulation of RIP140. In a HFD-fed obese mouse model, administration of a selective ETA antagonist, ambrisentan, effectively dampens cytoplasmic accumulation of RIP140 in the epididymal adipose tissue and reduces HFD-caused adipocyte dysfunctions. Importantly, ambrisentan improves blood glucose control and reduces the severity of hepatic steatosis in HFD-fed mice. This study reports a physiological signal that stimulates nuclear export of RIP140 in adipocytes and provides evidence for a strategy using selective ETA antagonist to treat obesity-induced insulin resistance and, possibly, other metabolic disorders.

Contractile Effects of Endothelins on Isolated Human Ureter

The aim of our study was to investigate mechanism of action of endothelins 1, 2 and 3 on spontaneous activity, tone and intraluminal pressure of human ureter. Both longitudinal tension and intraluminal pressure were recorded from the isolated segments of proximal human ureter. Endothelins 1, 2 and 3 (5.35x10(-11) M - 5.05x10(-8) M) produced concentration-dependent tonic contraction and sustained increase in intraluminal pressure of isolated preparations of human ureter. Endothelins 1 and 3 produced also concentration-dependent inhibition of spontaneous, phasic contractions of the isolated preparations. Selective antagonist of ET(A) receptors BQ123 and selective antagonist of ET(B) receptors BQ788 produced significant inhibition of endothelin-1-induced tonic contraction (pA(2)=8.80 and 6.55, respectively) and increase in intraluminal pressure (pA(2)=8.68 and 7.02, respectively), while they did not affect endothelin-1-induced inhibition of spontaneous activity. Endothelin 1 produces increase in tone and intraluminal pressure of isolated human ureter acting on both ET(A) and ET(B) receptors, the first one being functionally more important. Only endothelins 1 and 3 inhibit spontaneous, phasic activity of human ureter, but this effect was not blocked by selective antagonists of ET(A) and ET(B) receptors.