Positive and Negative Allosteric Modulators of the Ca2+-sensing Receptor Interact within Overlapping but Not Identical Binding Sites in the Transmembrane Domain (original) (raw)
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F1000 - Post-publication peer review of the biomedical literature, 2016
BACKGROUND AND PURPOSE Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. EXPERIMENTAL APPROACH We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca 2+ i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. KEY RESULTS Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca 2+ i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G 670 E). CONCLUSIONS AND IMPLICATIONS The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses. Abbreviations Ca 2+ o, extracellular calcium; Ca 2+ i, intracellular calcium; CaS receptors, calcium-sensing receptors; IP1, inositol 1-phosphate; Mg 2+ o, extracellular magnesium; pERK1/2, phosphorylated ERK1/2; PTH, parathyroid hormone
Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics
British journal of pharmacology, 2015
Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca(2+) i ) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca(2+) i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-26534...
British Journal of Pharmacology, 2019
Background and Purpose: Negative allosteric modulators (NAMs) that target the calcium-sensing receptor (CaS receptor) were originally developed for the treatment of osteoporosis by stimulating the release of endogenous parathyroid hormone, but failed in human clinical trials. Several chemically and structurally distinct NAM scaffolds have been described, but it is not known how these different scaffolds interact with the CaS receptor to inhibit receptor signalling in response to agonists. Experimental Approach: In the present study, we used a mutagenesis approach combined with analytical pharmacology and computational modelling to probe the binding sites of four distinct NAM scaffolds.
Characterization of Highly Efficacious Allosteric Agonists of the Human Calcium-Sensing Receptor
Journal of Pharmacology and Experimental Therapeutics, 2011
We discovered structurally novel human calcium-sensing receptor (CaSR) allosteric agonists, and compared their pharmacology to phenylalkylamine calcimimetics. AC-265347 activated CaSR signaling in cellular proliferation and phosphatidyl inositol (PI) hydrolysis assays with potencies of 30 and 10 nM, respectively. (S)-AC-265347, the (S) enantiomer of AC-265347, was approximately 10 to 20-fold more potent than (R)-AC-265347. The phenylalkylamines cinacalcet and calindol had similar activity to AC-265347 in cellular proliferation assays, but less activity in PI assays. All compounds had reduced activity when extracellular Ca 2+ was removed indicating they cooperate with Ca 2+ to activate CaSRs, and all activated CaSR isoforms with the N-terminal extracellular domain deleted, indicating they interact with the transmembrane (TM) domains. In both cases AC-265347 and therefore (S)-AC-265347 were significantly more efficacious than the phenylalkylamines. Mutations E837A 7.39 and I841A 7.43 strongly reduced phenylalkylamine-induced signaling, but not AC-265347 or (S)-AC-265347 induced signaling, suggesting different modes of binding. AC-265347 and (S)-AC-265347 stimulated significantly greater responses than cinacalcet or calindol at each of four lossof-function human polymorphic CaSR variants. AC-265347 did not inhibit the CYP2D6 cytochrome p450 isozyme, unlike cinacalcet which is a potent CYP2D6 inhibitor. In rats, AC-265347, (S)-AC-265347 and (R)-AC-265347 each reduced serum parathyroid hormone (PTH) with a rank order potency correlated to their in vitro potencies. AC-265347 and (S)-AC-265347 also reduced plasma ionizable calcium ([Ca 2+ ] o). AC-265347 was orally active, and its plasma concentrations correlated well with its effects on This article has not been copyedited and formatted. The final version may differ from this version.
Journal of Biological Chemistry, 2005
The extracellular calcium-sensing human Ca 2؉ receptor (hCaR), 2 a member of the family-3 G-protein-coupled receptors (GPCR) possesses a large amino-terminal extracellular ligand-binding domain (ECD) in addition to a seven-transmembrane helical domain (7TMD) characteristic of all GPCRs. Two calcimimetic allosteric modulators, NPS R-568 and Calindol ((R)-2-{1-(1-naphthyl)ethylaminom-ethyl}indole), that bind the 7TMD of the hCaR have been reported to potentiate Ca 2؉ activation without independently activating the wild type receptor. Because agonists activate rhodopsinlike family-1 GPCRs by binding within the 7TMD, we examined the ability of Calindol, a novel chemically distinct calcimimetic, to activate a Ca 2؉ receptor construct (T903-Rhoc) in which the ECD and carboxyl-terminal tail have been deleted to produce a rhodopsinlike 7TMD. Here we report that although Calindol has little or no agonist activity in the absence of extracellular Ca 2؉ for the ECDcontaining wild type or carboxyl-terminal deleted receptors, it acts as a strong agonist of the T903-Rhoc. In addition, Ca 2؉ alone displays little or no agonist activity for the hCaR 7TMD, but potentiates the activation by Calindol. We confirm that the activation of T903-Rhoc by Calindol is truly Ca 2؉ independent using in vitro reconstitution with purified G q . These findings demonstrate distinct allosteric linkages between Ca 2؉ site(s) in the ECD and 7TMD and the 7TMD site(s) for calcimimetics. VOLUME 280 • NUMBER 44 • NOVEMBER 4, 2005 by guest on September 7, 2016 http://www.jbc.org/ Downloaded from
Towards a structural understanding of allosteric drugs at the human calcium-sensing receptor
Cell research, 2016
Drugs that allosterically target the human calcium-sensing receptor (CaSR) have substantial therapeutic potential, but are currently limited. Given the absence of high-resolution structures of the CaSR, we combined mutagenesis with a novel analytical approach and molecular modeling to develop an "enriched" picture of structure-function requirements for interaction between Ca(2+)o and allosteric modulators within the CaSR's 7 transmembrane (7TM) domain. An extended cavity that accommodates multiple binding sites for structurally diverse ligands was identified. Phenylalkylamines bind to a site that overlaps with a putative Ca(2+)o-binding site and extends towards an extracellular vestibule. In contrast, the structurally and pharmacologically distinct AC-265347 binds deeper within the 7TM domains. Furthermore, distinct amino acid networks were found to mediate cooperativity by different modulators. These findings may facilitate the rational design of allosteric modulators...
F1000 - Post-publication peer review of the biomedical literature, 2016
Drugs that allosterically target the human calcium-sensing receptor (CaSR) have substantial therapeutic potential, but are currently limited. Given the absence of high-resolution structures of the CaSR, we combined mutagenesis with a novel analytical approach and molecular modeling to develop an "enriched" picture of structure-function requirements for interaction between Ca 2+ o and allosteric modulators within the CaSR's 7 transmembrane (7TM) domain. An extended cavity that accommodates multiple binding sites for structurally diverse ligands was identified. Phenylalkylamines bind to a site that overlaps with a putative Ca 2+ o-binding site and extends towards an extracellular vestibule. In contrast, the structurally and pharmacologically distinct AC-265347 binds deeper within the 7TM domains. Furthermore, distinct amino acid networks were found to mediate cooperativity by different modulators. These findings may facilitate the rational design of allosteric modulators with distinct and potentially pathway-biased pharmacological effects.
Behind the curtain: cellular mechanisms for allosteric modulation of calcium-sensing receptors
British Journal of Pharmacology, 2012
Calcium-sensing receptors (CaSR) are integral to regulation of systemic Ca 2+ homeostasis. Altered expression levels or mutations in CaSR cause Ca 2+ handling diseases. CaSR is regulated by both endogenous allosteric modulators and allosteric drugs, including the first Food and Drug Administration-approved allosteric agonist, Cinacalcet HCl (Sensipar®). Recent studies suggest that allosteric modulators not only alter function of plasma membrane-localized CaSR, but regulate CaSR stability at the endoplasmic reticulum. This brief review summarizes our current understanding of the role of membrane-permeant allosteric agonists in cotranslational stabilization of CaSR, and highlights additional, indirect, signalling-dependent role(s) for membrane-impermeant allosteric drugs. Overall, these studies suggest that allosteric drugs act at multiple cellular organelles to control receptor abundance and hence function, and that drug hydrophobicity can bias the relative contributions of plasma membrane and intracellular organelles to CaSR abundance and signalling.
Endocrinology, 2013
Cinacalcet is predominantly used to treat secondary hyperparathyroidism due to end-stage renal failure, but, more recently, its potential clinical efficacy in treating patients with loss-of-function mutations in the calcium-sensing receptor (CaSR) has been recognized. Many clinically relevant CaSR mutations are located in the heptahelical membrane spanning and extracellular loop regions of the receptor, where allosteric modulators are predicted to bind. The aim of the present study was to investigate the impact of such mutations on the pharmacoregulation of the CaSR by the positive and negative allosteric modulators, cinacalcet and NPS-2143, respectively. Both cinacalcet and NPS-2143 effectively rescued mutants whose cell surface expression was substantially impaired, suggesting that both classes of drug can stabilize a receptor conformation that is trafficked more effectively to the cell surface. In addition, functional impairments in almost all mutant CaSRs were rescued by either ...
The Journal of Clinical Endocrinology & Metabolism, 2013
Objective: Activating mutations in the calcium-sensing receptor (CASR) gene cause autosomal dominant hypoparathyroidism, and heterozygous inactivating CASR mutations cause familial hypocalciuric hypercalcemia. Recently, there has been a focus on the use of allosteric modulators to restore the functional activity of mutant CASRs. In this study, the effect of allosteric modulators NPS R-568 and NPS 2143 on CASR mutants was studied in vitro. Methods: DNA sequence analysis of the CASR gene was undertaken in autosomal dominant hypoparathyroidism and familial hypocalciuric hypercalcemia Japanese patients, and the functional consequences for the Gi-MAPK pathway and cell surface expression of CASR were determined. Furthermore, we studied the effect of NPS R-568 and NPS 2143 on the signal transduction activity and cell surface expression of each mutant CASR. Results: We identified 3 activating mutations (S122C, P569H, and I839T) and 2 inactivating mutations (A110T and R172G) in patients. The...