NPS-2143 (SB 262470A) is a novel potent and selective antagonist of Ca(2+) receptor.

Ca2+ receptor:43 nM

NPS 2143 stimulates the secretion of parathyroid hormone (PTH) from bovine parathyroid cells with an effective concentration (EC50) of 41 nM. Moreover, NPS 2143 inhibits the suppressive effect of the calcimimetic NPS R-467 on PTH secretion from bovine parathyroid cells and the inhibition of cAMP formation stimulated by isoproterenol in the presence of extracellular Ca2+. In HEK 293 cells expressing the human Ca2+ receptor, NPS 2143 blocks the increase in cytosolic Ca2+ concentration induced by receptor activation, with an inhibitory concentration (IC50) of 43 nM. Furthermore, in HEK-293 cells transiently expressing hCaSRs, NPS 2143 significantly inhibits the umami taste perception by effectively suppressing GSH (data not shown) and γ-Glu-Val-Gly activity.

In normotensive rats, intravenous administration of NPS 2143 (1 mg/kg) significantly increased mean arterial pressure (MAP) in the presence of the parathyroid gland. Additionally, NPS 2143 induced a rapid 4-5 fold increase in plasma parathyroid hormone (PTH) levels and an instantaneous rise in plasma calcium (Ca2+) levels.

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+]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.