ROCK Kinase has demonstrated good clinical efficacy, but possesses some limitations. For instance, the ability of the compound to potentiate the activity of the CaSR in both parathyroid cells and thyroidCcells can induce a marked reduction in serum Cao 2 sufficient to induce hypocalcemia despite a relative selectivity of phenylalkylamines for parathyroid CaSR. The development of a biased CaSR ligand selective for pathway leading to the inhibition of PTH release without inducing calcitonin release may potentially benefit dialysis patients by preventing hypocalcemia and promoting bone turnover and, in addition, permit use in patients with normocalcaemic hyperparathyroidism or early stage renal disease. Encouragingly, proof of concept of biased signaling among familyCGPCRs exists. Activation by caspase glutamate of the metabotropic glutamate mGluR1 receptor results in recruitment of both Gq and Gs protein coupled pathways, but this is biased toward Gq proteins in the presence ofGd3.
At the CaSR, an allosteric autoantibody isolated from a patient with acquired Dihydrofolate Reductase hypocalciuric hypercalcemia potentiated the effects of Cao 2 via Gq signaling and concomitantly suppressed the Cao 2 response elicited via Gi proteins. L Amino acids have also been reported to alter intracellular Ca2 oscillations differently to the effects of elevated Cao 2 by differential recruitment ofGq vs.G12 pathways. Finally, the first generation synthetic calcimimetic, NPS R467, promotes different kinetics of ERK1/2 phosphorylation, depending on the levels of Cao 2. To rationally study the phenomenon of stimulus bias and facilitate novel allosteric drug discovery, two important requirements must be addressed. The first is the need to investigate the signaling of the targetGPCRacross multiple pathways to increase the likelihood of capturing behaviors that arise from different receptor conformations. The second is the need to quantify the affinity of allosteric ligands for their target GPCR as well as the effects they mediate on the signaling of the orthosteric ligand, stimulus bias can manifest as alterations in either or both of these properties. Thus, the aim of the current study was to use an analytical approach to quantify the allosteric properties of the positive allosteric Streptozotocin modulators, cinacalcet and NPS R568, and the negative allosteric modulator, NPS 2143, across multiple intracellular signaling pathways mediated by activation of the CaSR.
The availability of a clinically effective allosteric agent provided an unprecedented opportunity to quantify potential signal bias by a clinically used small molecule modulator. Wepresent, for the first time, quantitative evidence for the generation of stimulus bias by both represents positive and negative allosteric modulators of theCaSRthat reflects the differential stabilization of distinct affinity states of the receptor or differential cooperativity between the modulator and Cao 2 in a signaling pathway dependent manner. Materials and Methods Materials Flp In human embryonic kidney 293 tetracycline regulated expression cells, DMEM, calcium free DMEM, blasticidin, zeocin, Lipofectamine 2000, tetracycline free fetal bovine serum, Fluo 4 AM, Alexa Fluor 568 conjugated phalloidin, and Hoechst 33342 were obtained from Invitrogen, whereas hygromycin B was from Roche.