Smoothened Pathway receptor properties, stimulus bias manifests as a pathway dependent alteration in modulator KB, and/or values. Application of our analytical model of allosteric modulation to the Cao 2 C/R curve potency shifts mediated by each of the modulators across all three different functional assays enabled the estimation of affinity and cooperativity values describing these interactions. From this analysis, two important conclusions were drawn. First, both the Cai 2 mobilization and pERK1/2 data sets were fitted best by models in which the affinities of the modulators for the allosteric site did not change between signaling pathways, the KB estimates varied from approximately 186 nM for cinacalcet to 575 nM for NPS R568. In contrast, the PM ruffling data were consistently characterized by significantly higher affinity values for the modulators, which varied from approximately 0.4 nMfor NPS R568 to 15 nM for NPS 2143. This finding suggests that PM ruffling is mediated by a state of the CaSRthat displays Adrenergic Receptors significantly higher affinity for the phenylalkylamines than the state of the receptor mediating Cai 2 mobilization or pERK1/2.
Second, evidence of bias engendered by the allosteric AUY922 modulators was also noted at the level of the signaling pathway, with significantly greater degrees of positive or negative cooperativity being manifested in Cai 2 mobilization compared with pERK1/2. Discussion The current study has identified stimulus bias in the actions of both positive and negative allosteric modulators of the CaSR, including the clinical agent cinacalcet. Withregard to Cai 2 mobilization and pERK1/2, the stimulus bias was manifested as a preference toward allosteric modulation ofCai 2mobilization rather than pERK1/2, as previously described for allosteric modulation by L amino acids. With regard to the PM ruffling pathway, however, the bias was manifested through a higher affinity of the modulators toward the conformational state of the receptor that mediates that response. Mechanistically, these findings reflect two sides of the same coin, biased signaling results from the stabilization by ligands of unique receptor active Daunorubicin states that are further stabilized/ selected by the intracellular effectors with which they interact.
The detection of a higher affinity state governing a given response suggests that the ligand stabilization mechanism is effectively compartmentalized and primarily driving the bias, whereas the detection of pathway biased modulation for a common affinity state indicates that the observed bias is primarily driven by the states selected by intracellular coupling partners. Because the ultimate link between intracellular signaling preference and therapeutic outcome is not known in most instances, our findings mortality highlight the need to incorporate bias determinations into the study and characterization of small molecule modulators of the CaSR and, more broadly, to all GPCRs. Although the ability of phenylalkylamines to act as positive or negative allosteric modulators of the CaSR is well known, only one prior study has attempted to rationalize these effects in terms of an allosteric receptor model, and our study is the first to apply this approach to the quantification of the actions of cinacalcet and NPS 2143.