Stimulus bias at the calcium sensing receptor

The calcium sensing receptor (CaSR), a Family C G protein-coupled receptor (GPCR), is an attractive drug target due to its critical role in systemic mineral homeostasis. The CaSR is activated by a plethora of orthosteric ligands, and the action of these ligands can be modulated by an increasing number of allosteric ligands. Most notably, the positive allosteric modulator of the CaSR, cinacalcet (Sensipar®/Mimpara®), was the first allosteric compound for any GPCR to reach the market, currently approved for the treatment of secondary hyperparathyroidism (HPT) and primary HPT in cases of parathyroid cancer. Study of the CaSR is complicated by two unique features: chronic exposure to many endogenous ligands and that activation by its primary orthosteric ligand, Ca2+, occurs over a very narrow concentration range. These two features of the CaSR present a challenge to both the initial optimisation of assays to study CaSR signalling, and also for the interpretation of agonism and allosteric modulation of the CaSR. Furthermore, study of the CaSR is hampered by a paucity of selective ligands. Therefore, several allosteric ligands of the CaSR were synthesised and an allosteric receptor model was modified to investigate the effect of ambient agonist concentrations on concentration-response (C/R) curves. GPCRs do not exist solely in either an inactive or active state, but rather a multitude of conformational states may be stabilised by different ligands (stimulus) giving rise to preferential association (bias) that particular intracellular coupling partners. The detection of such stimulus-bias at a GPCR requires that the receptor has multiple ligands and is promiscuously coupled; requirements met by the CaSR. Quantification of stimulus-bias by allosteric ligands (that is, the allosteric effects on orthosteric ligand affinity and cooperativity) at the CaSR in our HEK293-TREx c-myc-CaSR cell line was made possible by investigation across multiple signalling pathways (intracellular Ca2+i (Ca2+i) mobilisation, ERK1/2 phosphorylation, plasma membrane (PM) ruffling and cAMP accumulation assays) and application of an allosteric receptor model. The evidence herein establishes the CaSR as a prime example of a GPCR for which allosteric ligands can bias the signalling pathways activated by orthosteric ligands. The current thesis has presented four novel findings. First, several examples of allosteric ligands engendering stimulus-bias at the CaSR have been uncovered, including that of a prescribed drug, cinacalcet. A second finding was the identification of a non-interconvertable subset of CaSR with a higher affinity for NPS-R568, cinacalcet & NPS-2143 as evident in the PM ruffling assay. Third, mathematical modelling provided a parsimonious explanation for the experimentally observed changes in CaSR C/R curve shape: changes reflect modulation of the ambient, as well as added, agonist concentrations. Fourth, none of the allosteric modulators tested altered the signalling of Ca2+o to inhibit forskolin-induced cAMP production or to recruit β-arrestin1, β-arrestin2 or GRK2, likely due to weak coupling. Our findings of stimulus-bias at the CaSR imply that further delineation of the desired and undesired CaSR pathways for a given condition, coupled with further discovery and development of biased compounds, has the potential to sculpt therapeutics with greater selectivity and improved patient outcomes.