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 ET(A) 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 
ET(A) 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 ET(A) 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 ET(A) 
receptor-antagonist interaction modes, we performed functional studies using 
ET(A) receptor variants harboring amino acid point mutations in the presumed ERA 
interaction site. Three ET(A) 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-ET(A) 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.