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Functional assay for agonist activation of receptors

A technology of receptor agonists and receptors, which is applied in the direction of material analysis, analytical materials, biological material analysis, etc. by observing the influence on chemical indicators, and can solve problems such as limitations in analytical throughput

Inactive Publication Date: 2006-05-17
PFIZER PRODS ETAT DE CONNECTICUT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Analysis of these signals has been used to determine the role of these receptor ligands; however, these assays remain limited in terms of throughput

Method used

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  • Functional assay for agonist activation of receptors
  • Functional assay for agonist activation of receptors
  • Functional assay for agonist activation of receptors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0026] Use FLIPR 384 HEK cell D3 protocol

[0027] Description of the cell line

[0028] The HEK293-Gα15.D3 cell line stably expressing Gα15 was obtained by transfecting D3 cDNA into the HEK293 cell line expressing G-α. The expression of the D3 receptor is maintained in the presence of screening factors such as puromycin and blasticidin. This cell line has poor attachment to typical tissue culture flasks. To obtain a strong attachment phenotype, the cells were grown in Matrigel (Becton Dickinson, diluted 1:200 with serum-free DMEM)-coated culture flasks.

[0029] Load the cells according to the following steps:

[0030] (a) 20,000 cells were plated in a 384-well plate at 50 μl / well, or 60,000 cells per well in a 96-well plate, and the multi-well plate was coated with poly-D-lysine. Return the plate to the 37°C incubator.

[0031] (b) After 16-24 hours, the growth medium is removed, and then the growth medium is replaced with a serum-free medium in the presence of calcium-sen...

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Abstract

The invention provides a novel high throughput functional assay for certain agonist-activated receptors, including Alpha 1A , Alpha 2A, H1, 5HT1A, SHT2A, D2 and D3 receptors. The assay method of the invention uses an elevated temperature and a cell line that stably expresses both the receptor and the promiscuous G protein G1S wherein agonist-induced intracellular Ca<2+> release was monitored by a Fluorometric Imaging Plate Reader (FLIPR). The magnitude of the agonist-induced response was dramatically enhanced by performing the assay at an elevated temperature, rather than at room temperature. The novel assay of the invention is useful for selecting compounds which are effective in the treatment of disorders related to the activation of certain neuroreceptors.

Description

Background of the invention [0001] The present invention relates to a new high-throughput functional analysis of agonist-activated receptors. [0002] The new analysis of the present invention can be used to screen compounds that are effective in the treatment of disorders related to activation of neuroreceptors, including α2A, H1, 5HT1A, 5HT2A, D2, and D3 receptors. Molecules that regulate the function of these receptors are potential therapeutic drugs for many mental illnesses. Although ligand binding analysis has been used for the discovery of these molecules, functional analysis that measures receptor ligands can often provide more information. The development of high-throughput functional assays for the activation of these receptors is still problematic. [0003] The analytical method of the present invention uses elevated temperature and Fluorometric Imaging PlateReader (FLIPR 384 ) Is used to measure agonist activation of these receptors. For example, the present invention ...

Claims

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Application Information

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IPC IPC(8): G01N33/50C12N15/09C12Q1/02G01N21/78G01N33/15G01N33/566G01N33/68G01N33/94
CPCG01N33/5008G01N33/502G01N33/5058G01N33/6872G01N33/9413G01N33/942G01N33/9433G01N2333/726G01N2800/52G01N33/50
Inventor 王国风阿尔卡·V·施里克汉德
Owner PFIZER PRODS ETAT DE CONNECTICUT
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