Distinct ETA Receptor Binding Mode of Macitentan As Determined by Site Directed Mutagenesis

John Gatfield,Oliver Nayler,Celia Mueller Grandjean,Martin H Bolli,Daniel Bur,James West

doi:10.1371/journal.pone.0107809

Abstract

The competitive endothelin receptor antagonists (ERA) bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min) occupancy half-lives at the ETA receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ETA receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ETA receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ETA receptor-antagonist interaction modes, we performed functional studies using ETA receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ETA receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that – in contrast to bosentan and ambrisentan - macitentan-ETA receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan's sustained target occupancy and insurmountable antagonism.

Highlights

Endothelins are vasoactive peptides mainly produced by endothelial cells, and by smooth muscle cells, fibroblasts and macrophages
Kinetic properties of macitentan and a diverse set of structural analogs Previous experiments in human primary pulmonary arterial smooth muscle cells (PASMC) revealed an almost 20-fold longer endothelin receptor subtype A (ETA) receptor occupancy half-life of macitentan compared to ambrisentan and bosentan
This longer ETA receptor occupancy translated into insurmountable antagonism of ET-1-induced ETA receptor signaling by macitentan, but not bosentan or ambrisentan [18]

Summary

Introduction

Endothelins (endothelin-1, endothelin-2 and endothelin-3) are vasoactive peptides mainly produced by endothelial cells, and by smooth muscle cells, fibroblasts and macrophages. ET-1 responses are mediated via activation of two homologous G protein-coupled receptor subtypes, endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) [2,3] Both receptor subtypes activate Gq protein-mediated pathways leading to phospholipase Cb and PKC activation and increased intracellular calcium concentrations [4]. In lung tissue of patients suffering from pulmonary arterial hypertension (PAH) ET-1 concentrations are elevated [5,6]. These increases in local ET-1 concentrations cause activation of endothelin receptors in pulmonary arterial smooth muscle cells (PASMC). Increased intracellular calcium levels promote cytoskeletal contraction and cell proliferation [4,5,7] and thereby mediate persistent constriction and remodeling of pulmonary arteries, two hallmarks of PAH pathology [8,9,10,11]

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