G protein-coupled endothelin receptors and their natural and therapeutic ligands

From the discovery of endothelin ligand…


Studying the role of acetylcholine, Furchgott and Zawadzki came across an anomaly in one of their experiments. They knew that the latter substance had a vasodilatory effect thanks to its action on the muscarinic transmembrane receptors involved in the parasympathetic nervous system. But the action of acetylcholine present in their preparation was in no way comparable to the known effects.


Going back in time, they realized that they had made the mistake of scratching the animal's epithelial cell layer that lines the interior of the vessel walls during the preparation of the tested sample.


They then hypothesized that the binding of acetylcholine to its transmembrane receptor triggers the release of a substance from the endothelial cells. This factor would then have an activity on muscle tone that would be uncoupled from the nervous pathway (1). Years later, Furchgott, Ignarro, and Murad, independently of each other, demonstrated that this substance was nitric oxide, which allowed them to be awarded the Nobel Prize in Physiology or Medicine.


In 1985, Hickey and his team were also working on the vasoconstriction of coronary arteries following their treatment with various compounds derived from the endothelium. But as trypsin treatment inhibited isometric tension in the muscles of treated animals, they concluded that this time they were dealing with a substance totally different from nitric oxide. A new compound of polypeptide nature seemed to have a significant impact in the regulation of vascular smooth muscle contractility (2).


It was finally in 1988, from the supernatant of a culture of porcine aortic endothelial cells, that Yanagisawa's team isolated a peptide of 21 amino acids with a powerful vasoconstrictive effect. This new factor, never previously described, will henceforth be called endothelin (3).


…to that of G protein-coupled ET-A and ET-B transmembrane receptors


Yanagisawa's team endeavored to extend their work to humans, succeeded in obtaining a human endothelin cDNA from a placenta cDNA library and confirmed the existence of analog human peptide (4). While searching for the existence of molecules close to human endothelin, they eventually stumbled upon three distinct human genes for putative endothelin precursors (5).


It turns out that the three genes, from respectively chromosome 6, 1, and 20, encode closely related but different peptides, making these three isoforms…a small family! Endothelin was then renamed endothelin-1 or ET-1, while the new peptides were logically called endothelin-2 (ET-2) and endothelin-3 (ET-3).


The three endothelins are single 21-residue peptide backbone chains containing two intrachain disulphide bridges that divide the  structures into two rings at fixed positions (residues 1 and 15, and 3 and 11), from which a flexible hydrophobic carboxyl-terminal tail extends with a terminal tryptophan residue.


Compared with endothelin-1, endothelin-2 has two different amino acids, endothelin-3 has six amino acid substitutions at residues 2, 4, 5, 6, 7 and 14, and the sarafotoxins, from the venom of Atractaspis engaddensis snake, are very homologous (6).


Endothelin precursors are cleaved by two proteases to produce mature, active endothelins. Preproendothelin (PPET) are cleaved at dibasic sites by furin-like endopeptidases to form biologically inactive intermediates, called big endothelin (big ET). Big ETs are then cleaved at the Trp-Val bond of big ET-1 and big ET-2 or at the Trp-Ile bond of big ET-3. This last step is performed by membrane zinc metalloproteases of the neprilysin superfamily, called endothelin-converting enzymes.


ET-1 is produced by endothelial cells (major source), epithelial cells, macrophages, fibroblasts, cardiac myocytes and neurons. ET-2 is expressed by intestinal epithelial cells and ET-3 by neurons, tubular epithelial cells of the kidney and the intestine.


In mammals, two G protein-coupled endothelin receptors (ETAR and ETBR) have been identified and cloned (7,8). Like all GPCR members, ET-A and ET-B are receptors with seven hydrophobic transmembrane domains of 427 and 416 amino acids respectively, but with only 59% sequence similarity.


Each receptor activates the same G-proteins, but with different responses depending on the cell type, such as activation of phospholipase C, increase of intracellular calcium and induction of early genes. The ET-A receptor has nanomolar affinities for the ET-1 and ET-2 peptides and a lower affinity for ET-3. In contrast, the ET-B receptor has the same nanomolar affinity for all three peptides.


Review of the clinical relevance of endothelin receptors and therapeutic development of agonist and antagonist drugs to ETAR and ETBR

Endothelin impact on blood vessels and related therapeutic strategies


Given the natural role of endothelin as a potent vasoconstrictor, the first clinical studies were logically directed towards diseases related to the vascular system, such as atherosclerosis, hypertension, myocardial infarction, vasospasm but also nephropathies.


In atherosclerosis, ET-1 activates ET-A receptors present on macrophages, smooth muscle cells and fibroblasts. ET-1 synthesis is stimulated by oxidized LDL (low density lipoproteins) in endothelial cells, macrophages and smooth muscle cells of coronary arteries. Co-expression of ECE-1 and ET-1, correlated with the evolution of atherosclerotic plaques, has been observed in human arteries. Peripheral artery disease (PAD) is a major complication of atherosclerosis when blockages in the arteries to leg reduce blood flow and one of the resulting problems is termed intermittent claudication (9).


As early as 1999, Schriffin’s team has established the link between endothelin and hypertension. In some moderately hypertensive patients, an increase in mRNA encoding PPET-1 in the endothelium of resistance arteries has been observed (10).


Different therapeutic small molecules have been developed including:


Selective ETA receptor antagonists

  • Ambrisentan, respectively marketed by Gilead under Letairis name and by GSK under Volibris brand,
  • Sitaxentan, marketed by Pfizer under Thelin brand but withdrawn from the market following two cases of fatal liver damage,
  • Clazosentan from Idorsia Pharmaceuticals, currently in Phase 3
  • Atrasentan, initially developed by Abbott and then licensed to Chinook Therapeutics,
  • Avosentan from Speedel (now Novartis), terminated due to safety reasons like severe heart failure and deaths,
  • Edonantan (BMS 207940), from Bristol Myers Squibb, discontinued
  • TMB-003 (Sitaxsentan) from Timber for the treatment of sclerotic skin diseases in preclinical stage

Selective ETB receptor antagonists but without any clinical outcomes

  • BQ-788, from MelCure now in bankruptcy 

Dual antagonists which affect both endothelin A and B receptors

  • Macitentan developed by Actelion (now J&J) and approved under brand name Opsumit
  • Bosentan, developed by Actelion (now J&J) and approved under brand name Tracleer
  • Aprocitentan, from Idorsia Pharmaceuticals, currently in Phase 3
  • Tezosentan, developed by Actelion (now J&J)  and discontinued.

Dual antagonists which affect both endothelin A angiotensin II subtype 1 (AT1) receptors

  • Sparsentan, from Travere Therapeutics, currently in Phase 3

In 1990, Kondoh et al were the first to prove, despite the known difficulties of specifically targeting a GPCR with antibodies, that it was possible to isolate effector monoclonals against endothelin receptors (11).


Following this success, Zhang and his team developed getagozumab, an antagonistic therapeutic antibody against ETA  for the treatment of pulmonary arterial hypertension, developed by Gmax Biopharm under GMA301 (12).


But it seems that endothelin receptors could also prove to be targets of interest in cancer immunotherapy approaches...


Endothelin role in cancer progression: current status of small molecules and monoclonal antibodies to GPCR endothelin receptors


Tumor angiogenesis requires angiogenic factors, such as VEGF (vascular endothelial growth factor), produced by cancer cells to affect host tissue. ET-1 and its receptors are negative modulators of the angiogenic response in colon cancer progression by repressing fibroblast differentiation and inducing ET-B receptors. Other investigations have also have put their finger on the role of the endothelin axis in mitogenesis, apoptosis inhibition and invasiveness (13).


Different therapeutic small molecules have been developed including:


Selective ETA receptor antagonists

  • Zibotentan, developed by AstraZeneca but with no significant effect in prostate or lung cancer
  • Atrasentan (Xinlay), initially developed by Abbott and then licensed to Chinook Therapeutics, but with no significant effect in renal cell carcinoma
  • YM-598, from Astellas Pharma but discontinued 

Selective ETB receptor antagonists

  • SPI1620, from Spectrum Pharmaceuticals, but whose the study did not meet efficacy end point

Dual antagonists which affect both endothelin A and B receptors

  • Bosentan, developed by Actelion (now J&J)
  • Macitentan developed by Actelion (now J&J).

Given the lack of promising results, other companies have attempted to develop antibody approaches:


Selective ETA receptor antagonists

  • Rendomab A, from CEA (14)
  • AG8, developed by Ju et al (15)

Selective ETB receptor antagonists

  • Rendomab B from CEA (16,17)

Dual antagonists which affect both endothelin A and CD3 receptors

  • GMA202 from Gmax Biopharm for the ovarian cancer patients currently in IND enabling stage.

The questions now are whether these antibodies will do better than the previous small molecules, but more importantly whether the endothelin pathway will remain a target of choice in cancer treatment or will be gradually abandoned in favor of other GPCRs.


The question remains open.


(1) Furchgott, Robert F.; Zawadzki, John V. (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. , 288(5789), 373–376. doi:10.1038/288373a0

(2) Hickey KA, Rubanyi G, Paul RJ, Highsmith RF. Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am J Physiol. 1985 May;248(5 Pt 1):C550-6. doi: 10.1152/ajpcell.1985.248.5.C550. PMID: 3993773.

(3) Yanagisawa, Masashi; Kurihara, Hiroki; Kimura, Sadao; Tomobe, Yoko; Kobayashi, Mieko; Mitsui, Youji; Yazaki, Yoshio; Goto, Katsutoshi; Masaki, Tomoh (1988). A novel potent vasoconstrictor peptide produced by vascular endothelial cells. , 332(6163), 411–415. doi:10.1038/332411a0 

(4) Inoue A, Yanagisawa M, Takuwa Y, Mitsui Y, Kobayashi M, Masaki T. The human preproendothelin-1 gene. Complete nucleotide sequence and regulation of expression. J Biol Chem. 1989 Sep 5;264(25):14954-9. PMID: 2670930.

(5) Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2863-7. doi: 10.1073/pnas.86.8.2863. PMID: 2649896; PMCID: PMC287019.

(6) Kloog, Y; Ambar, I; Sokolovsky, M; Kochva, E; Wollberg, Z; Bdolah, A (1988). Sarafotoxin, a novel vasoconstrictor peptide: phosphoinositide hydrolysis in rat heart and brain. Science, 242(4876), 268–270. doi:10.1126/science.2845579 

(7) Arai, Hiroshi; Hori, Seiji; Aramori, Ichiro; Ohkubo, Hiroaki; Nakanishi, Shigetada (1990). Cloning and expression of a cDNA encoding an endothelin receptor. , 348(6303), 730–732. doi:10.1038/348730a0 

(8) Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 1990 Dec 20-27;348(6303):732-5. doi: 10.1038/348732a0. PMID: 2175397.

(9) JIANGLIN FAN; HIROYUKI UNOKI; SATOSHI IWASA; TERUO WATANABE (2000). Role of Endothelin-1 in Atherosclerosis. , 902(none), 84–94. doi:10.1111/j.1749 6632.2000.tb06303.x 

(10) Schiffrin, E. L. (1999). Role of Endothelin-1 in Hypertension. Hypertension, 34(4), 876–881. doi:10.1161/01.HYP.34.4.876 

(11) Motohiro Kondoh; Hitoshi Miyazaki; Hirotoshi Watanabe; Takeshi Shibata; Masashi Yanagisawa; Tomoh Masaki; Kazuo Murakami (1990). Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization. , 172(2), 0–510. doi:10.1016/0006-291x(90)90701-n 

(12) Zhang C, Wang X, Zhang H, Yao C, Pan H, Guo Y, Fan K, Jing S. Therapeutic Monoclonal Antibody Antagonizing Endothelin Receptor A for Pulmonary Arterial Hypertension. J Pharmacol Exp Ther. 2019 Jul;370(1):54-61. doi: 10.1124/jpet.118.252700. Epub 2019 Apr 16. PMID: 30992315.

(13) Nelson J, Bagnato A, Battistini B, Nisen P. The endothelin axis: emerging role in cancer. Nat Rev Cancer. 2003 Feb;3(2):110-6. doi: 10.1038/nrc990. PMID: 12563310.

(14) https://patents.google.com/patent/WO2019155151A2/en

(15) Ju, MS., Ahn, HM., Han, SG. et al. A human antibody against human endothelin receptor type A that exhibits antitumor potency. Exp Mol Med 53, 1437–1448 (2021). https://doi.org/10.1038/s12276-021-00678-9

(16) Borrull A, Allard B, Wijkhuisen A, Herbet A, Lamourette P, Birouk W, Leiber D, Tanfin Z, Ducancel F, Boquet D, et al. Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration. MAbs. 2016;8:1371–85. https://doi.org/10.1080/19420862.2016.1208865.

(17) Allard B, Wijkhuisen A, Borrull A, Deshayes F, Priam F, Lamourette P, Ducancel F, Boquet D, Couraud JY. Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor. MAbs. 2013;5:56–69. https://doi.org/10.4161/mabs.22696.