e competition between an increase in physiological ligand concentration and the drug at the target binding site. In case of strongly fluctuating ligand concentrations at the target, which can be caused by local pulsatile secretion combined with rapid degradation, sustained target blockade can lead to enhanced maximal efficacy of an inhibitor. Many autocrine and paracrine GPCR ligands display high temporarily restricted local concentrations, and sustained receptor blockade/insurmountable antagonism seems ideally suited to enhance maximal antagonistic efficacy in such systems. Well known examples of GPCR antagonists dealing with such fluctuating para/autocrine ligands are histamine H1 antagonists for the blockade of allergic responses induced by mast-cell degranulation and P2Y12 antagonists for the blockade of platelet aggregation induced 
by platelet-derived ADP. Another class of well-studied GPCR antagonists targeting an at least partially autocrine/paracrine system are the angiotensin II receptor blockers for the treatment of hypertension and heart disease. The slow-offset ARBs candesartan and olmesartan show increased maximal efficacy compared to the fast-offset antagonist losartan, which has a 1525-fold shorter ROt1/2. It is thus not surprising that kinetic investigations are becoming a widespread practice in modern drug discovery and may either use biochemical assays to characterize compoundtarget complexes or to increase functional relevance employ functional assays. In the present study, using second messenger assays in human primary PASMC, we characterized the inhibitory potency, kinetics and mode of antagonism of macitentan, which recently concluded testing in a Phase III, long-term, event-driven, morbidity/mortality clinical trial in patients suffering from PAH. PASMC are known target cells for successful PAH therapies and endothelin-mediated effects on PASMCs were shown to contribute to PAH pathology. Two ERAs, ambrisentan and bosentan, are currently approved PAH therapies and therefore the molecular receptor inhibition properties of macitentan were compared with those of ambrisentan and bosentan. The ET-1-induced calcium signaling in human primary PASMC was largely ETA driven, which was shown by using endothelin receptor subtype specific reference antagonists. The observed functional contribution of the ETA receptor subtype in comparison to the ETB receptor subtype was in agreement with our quantitative assessments of subtype specific mRNA expression and in agreement with the previously published reports on ETA/ ETB expression ratios in human PASMCs. In this diseaserelevant primary cell system macitentan, but not ambrisentan and bosentan displayed slow receptor dissociation causing insurmountable antagonism in ET-1-induced IP1 accumulation and ET-1induced sustained elevation of intracellular calcium levels when ET-1 stimulation times were limited. Allowing for full equilibration of agonist and antagonist revealed the competitive nature of the antagonism exerted by macitentan. Our findings using second messenger assays in human primary PASMC confirm the competitive antagonism of macitentan previously shown in rat aortic ring contraction assays. Since the development of vessel contraction by MedChemExpress PBTZ 169 exogenously added ET-1 is a slow process, contraction assays with exogenously added ET-1 do not capture the insurmountability of macitentan which occurs with short ET-1 stimulation times only. Macitentan and ambrisentan had the same antagoni
