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Biological Activity
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Description
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NPS 2143 is a novel potent and selective antagonist of Ca(2+) receptor with IC50 of 43 nM in HEK 293 cells. |
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Targets
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Ca(2+) receptor |
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IC50 |
43 nM [1] |
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In Vitro
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NPS 2143 blocks increases in cytoplasmic Ca2+ concentrations with IC50 of 43 nM elicited by activating the Ca2+ receptor in HEK 293 cells expressing the human Ca2+ receptor. [1] NPS 2143 stimulates parathyroid hormone (PTH) secretion from bovine parathyroid cells with EC50 of 41 nM. Moreover, NPS 214 also blocks the inhibitory effects of calcimimetic NPS R-467 on PTH secretion from bovine parathyroid cells and the inhibitory effects of extracellular Ca2+ on isoproterenol-stimulated increases in cyclic AMP formation. [1] In HEK-293 cells transiently expressing hCaSRs, NPS 2143 significantly suppresses the kokumi taste by effectively inhibiting the activity of both GSH (data not shown) and γ-Glu-Val-Gly. [3] A recent study shows that NPS 2143 treatment suppresses low molecular weight fractions of azuki hydrolysate-induced cholecystokinin (CCK) secretion in CaSR-transfected HEK 293 cells. [4] |
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In Vivo
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In rats, NPS 2143 results in a rapid 4- to 5-fold increase in plasma PTH levels and also a transient increase in plasma Ca2+ levels. [1] In normotensive rats, NPS 2143 administration (1 mg/kg) by i.v. markedly increases mean arterial blood pressure (MAP) in the presence of parathyroid glands. [2] |
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Clinical Trials
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Features
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Protocol
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Kinase Assay
[1]
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| Assays for Assessing Potency and Selectivity of Compounds on Ca2+ Receptor |
This clonal cell line, referred to as HEK 293 4.0-7 cells, are used in a high-throughput screening format to detect agonists and allosteric activators of the Ca2+ receptor. Changes in the concentration of cytoplasmic Ca2+([Ca2+]i) provide a quantitative and functional assessment of Ca2+receptor activity in these cells and the results using this assay parallel those obtained using a homologous expression system of bovine parathyroid cells. On-line continuous measurements of fluorescence in fluo-3- or fura-2-loaded HEK 293 4.0-7 cells are obtained using a custom-built spectrofluorimeter or a fluorescence imaging plate reader instrument. NPS 2143 is incubated with cells for 1 minute before increasing the concentration of extracellular Ca2+ from 1.0 mM to 1.75 mM. NPS 2143 is tested individually at a concentration of 100 μg/ml (20 μM–80 μM) and those causing more than a 40% inhibition of the control response are considered to be biologically active. To determine the potencies (IC50) of NPS 2143 with biological activity, concentration-response curves are obtained and then, as an initial assessment of selectivity, the effects of NPS 2143 on [Ca2+]i evoked by other G protein-coupled receptors are examined at a concentration several times their IC50. Wild-type HEK 293 cells (and HEK 293 4.0-7 cells) express receptors for thrombin, bradykinin, and ATP, which couple to the mobilization of intracellular Ca2+. These responses can be studied to quickly assess any nonselective action of compounds on G protein-coupled receptors. Additional assays for selectivity include HEK 293 cells engineered to express receptors most homologous in sequence and topology to the Ca2+ receptor. These include native or chimeric receptors for various metabotropic glutamate and γ-aminobutyric acid type B receptors (GABABRs). Chimeric receptors are created using partial sequences of metabotropic glutamate receptors and Ca2+ receptors, engineered to couple to activation of phospholipase C and release of intracellular Ca2+ in HEK 293 cells. NPS 2143 lacking pan-activity are then subjected to structural modifications and their potencies and selectivities monitored using these HEK 293 4.0-7 cell assays in an iterative process. |
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Animal Study
[1]
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| Animal Models |
Chronic indwelling catheters are implanted in the inferior vena cava and in the abdominal aorta of male Sprague-Dawley rats. |
| Formulation |
NPS 2143 is dissolved in 20% aqueous solution of 2-hydroxypropyl-β-cyclodextrin. |
| Doses |
≤0.1 μmol/kg · min |
| Administration |
Administered via i.v. |
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References |
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[1] Nemeth EF, et al. J Pharmacol Exp Ther. 2001, 299(1), 323-331.
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[2] Rybczynska A, et al. J Endocrinol. 2006, 191(1), 189-195.
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[3] Ohsu T, et al. J Biol Chem. 2010, 285(2), 1016-1022.
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[4] Nakajima S, et al. Mol Nutr Food Res. 2012, 56(5), 753-760.
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