All luciferase results were normalized to Renilla activity from the co-transfected pRL-TK plasmid

nds after ET-1 addition and lasted for about 3 min. It was followed by a second, prolonged but less pronounced increase in cytosolic calcium, which lasted for more than 25 min. Both phases showed a concentrationdependent, saturable behavior. Macitentan, ambrisentan and bosentan were investigated in Schild experiments with 120-min pre-incubation times and the effects on the two phases of calcium increase were analyzed independently. The first response phase was quantified by using the fluorescence peak height within the first 3 minutes, and the second phase was quantified by calculating the area under the curve between 3 minutes and 23 minutes of observation. Due to the fast signal development of the first calcium response, all three compounds MG 516 displayed a certain degree of insurmountable antagonism as expected, although macitentan was the most pronounced. The extent of insurmountability was further illustrated by displaying the ERA inhibition curves at a fixed ET-1 concentration. For ambrisentan and bosentan these curves were biphasic and reached an intermediate plateau of antagonistic efficacy with the residual unblocked signal being descriptive of the proportion of receptor that is subject to surmountable antagonism. In fact, bosentan showed a surmountable mode of antagonism for,50% of the ET-1-induced signal and ambrisentan showed this surmountable mode for,30% of the ET-1-induced signal. Macitentan showed no surmountable behavior. Therefore, based 6 Receptor Dissociation Kinetics of Macitentan on these observations it can be estimated that within the first 30 seconds, bosentan had dissociated from,50% of the receptors and ambrisentan from,30% of receptors. These considerations yield a very short ROt1/2 of,1 minute for ambrisentan and bosentan. Fig. 7C shows the antagonistic effects of the three compounds on the second sustained phase of calcium elevation. Macitentan displayed an insurmountable mode of antagonism while the other two compounds showed surmountable antagonism. Using the Cheng-Prusoff equation, the Kb values were calculated for the first and second phase. Once again, macitentan and ambrisentan were almost equipotent and,10-fold more potent than bosentan. It is interesting to note that ambrisentan and bosentan treatment did not only lack inhibitory capacity on sustained calcium release at high ET-1 concentrations, but their presence reproducibly increased the maximal efficacy of ET-1 in this readout. Discussion Relevance of Drug-target Binding Kinetics The pharmacological activity of a drug depends on target affinity and on pharmacokinetic variables such as free fraction and plasma half-life and on physicochemical properties that influence the speed and degree of compound distribution into the target tissue. All of these factors are now well established and are part of any medicinal chemistry compound optimization program. However, there is increasing evidence that the kinetic behavior of the drugtarget complex also influences the clinical activity of a compound. Prolonged target engagement is common among effective inhibitors and in fact, of the new molecular entities approved by the FDA between 2001 and 2004, only 18% of the orthosteric inhibitors or antagonists displayed a mechanism of inhibition following purely mass action competition, whereas the majority of inhibitors were capable of sustained target blockade. Thus, sustained target blockade by slow dissociation is an effective strategy to avoid or at least delay th