NPS-2143  (Synonyms: SB-262470A)

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.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Rats: On the day of study, the rats are infused intravenously (0.1 mL/kg·min) for 120 min with NPS-2143 (0.1 μmol/kg·min) or vehicle, a 20% aqueous solution of 2-hydroxypropyl-β-cyclodextrin. Blood samples (0.5 mL) are collected before and at various times after the start of the infusion for measurements of plasma levels of PTH and Ca²⁺. To prevent excessive blood volume loss during the course of the experiment, for each blood sample the erythrocyte pellet is resuspended in an equal volume of normal rat plasma and reinjected. Plasma levels of Ca²⁺ are measured immediately after collection using a model 634 ionized calcium analyzer. PTH levels are measured using the Immutopics rat PTH(1-34) immunoradiometric assay kit.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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