Separable Binding Sites for the Natural Agonist Endothelin-1 and the Non-Peptide Antagonist Bosentan on Human Endothelin-A Receptors

Abstract

A three-dimensional model for the transmembrane domains of human endothelin-A receptor was built using structural information from bacteriorhodopsin and sequence alignment to other guanine-nucleotide-binding regulatory(G) protein-coupled receptors. Based on this model, 18 amino acids located at the inside of the receptor were mutated and analyzed for binding of the natural ligand endothelin-1 and bosentan, a recently described potent orally active endothelin antagonist [Clozel, M., Breu, V., Gray, G., Kalina, B., Löffler, B.-M., Burri, K., Cassal, J.-M., Hirth, G., Müller, M., Neidhart, W. & Ramuz, H. (1994) Pharmacological characterization of bosentan, a new potent orally active nonpeptide endothelin receptor antagonist, J. Pharmacol. Exp. Ther. 270, 228-235]. Mutation of Gly97, Lys140, Lys159, Gln165 and Phe315, located in transmembrane region 1, 2, 3, 3, and 6, respectively, caused reduced specific binding of 125I-labelled endothelin-1, despite an expression level similar to wild-type endothelin-A receptor. Mutation of Tyr263, Arg326 and Asp351 preserved endothelin-1 binding but caused reduced binding of bosentan. These amino acids, located on transmembrane regions 5, 6 and 7, respectively, are conserved among endothelin-A and endothelin-B receptors but not in other G-protein-coupled receptors. These observations demonstrate a dissociation of the binding site for the peptidic natural agonist endothelin-1 and the synthetic non-peptide antagonist bosentan. They provide the molecular basis for bosentan being a specific antagonist for both, endothelin-A as well as endothelin-B receptors and may in combination with studies on structure/activity relationship support the design of novel and more potent endothelin receptor antagonists.

Mutations in the Endothelin Receptor Type A Cause Mandibulofacial Dysostosis with Alopecia

“…4,5,22 EDN3 is expressed in the first PA in humans, 23 chick, 24 and mice ( Figure S5 and microarray data from the FaceBase Consortium, 25 EDN3. To limit our analysis to the effects resulting from EDNRA signaling, we also included BQ788, an EDNRBspecific antagonist.…”

Substrates, Inducers, Inhibitors and Structure-Activity Relationships of Human Cytochrome P450 2C9 and Implications in Drug Development

“…Bosentan, a specific and competitive endothelin receptor (ET A and ET B ) antagonist [660,661], is the first oral drug for the treatment of pulmonary arterial hypertension [662,663]. The absolute oral bioavailability of bosentan in healthy volunteers is 50% and is unaffected by food intake [664].…”

Endothelin-1 and -2: Two amino acids matter

“…Whether also some of the low molecular weight non-peptidergic ET-receptor antagonists are negative allosteric modulators rather than neutral competitive antagonists (De Mey et al, 2011) largely remains to be established. For bosentan, however, it has been shown that its binding site does not fully coincide with that of ET-1 on ET A -receptors (Breu et al, 1995) and ABT-627 was shown to promote internalization of ET A -receptors (Chiou et al, 2000); an effect that is not compatible with neutral competitive antagonism.…”