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Cell screening model of label-free muscarinic receptor M4

A muscarinic receptor, label-free technology, applied in the field of cell screening, can solve the problems of cumbersome operation, long experimental cycle, low throughput, etc., and achieve pathway integration research with simple operation, short experimental cycle, and high spatiotemporal resolution Effect

Pending Publication Date: 2020-06-30
TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have certain limitations, such as the traditional radioligand-receptor binding assay requires washing and filtration, long experimental cycle and low throughput, and this technique cannot distinguish receptor agonists and antagonists; the rest The current GPCR detection method is mainly aimed at the activation of a certain signaling pathway, and often does not consider the activation of multiple pathways. It often requires fluorescent protein labeling or additional indicators, which makes the operation cumbersome, and the addition of these indicators will also cause cells. Certain damage, affecting the reliability of screening results

Method used

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  • Cell screening model of label-free muscarinic receptor M4
  • Cell screening model of label-free muscarinic receptor M4
  • Cell screening model of label-free muscarinic receptor M4

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: DMR characteristic signal spectrum of the agonist carbachol on HEK-293-M4 cells

[0032] Human embryonic kidney cells HEK-293-M4 cells were derived from the cell bank independently constructed by the laboratory. The inverted microscope was purchased from OLYMPUS, and carbacholine and scopolamine were purchased from TCI and Aladdin, respectively. The cell culture plate is an Epic optical biosensing 384 microwell plate, purchased from Corning, and the detection platform is the third generation of Corning Imager, the detected signal is the wavelength shift caused by cell dynamic mass reset (DMR).

[0033] HEK-293-M4 cells in the logarithmic growth phase were seeded in a cell-compatible 384 microwell plate, the medium used was DMEM (#SH30022.01B, Thermo), the inoculation volume of each well was 40 μL, and each well was inoculated The number of cells is 2.0 x 10 4Firstly, place the inoculated cell plate in a cell incubator and culture it for 20-22 hours until t...

Embodiment 2

[0034] Example 2: DMR characteristic signal spectrum of antagonist scopolamine on HEK-293-M4 cells

[0035] HEK-293-M4 cells in the logarithmic growth phase were seeded in a cell-compatible 384 microwell plate, the medium used was DMEM (#SH30022.01B, Thermo), the inoculation volume of each well was 40 μL, and each well was inoculated The number of cells is 2.0 x 10 4 Firstly, place the inoculated cell plate in a cell incubator and culture it for 20-22 hours until the cell confluency reaches about 95%, and conduct an activity test. Replace the cell culture solution in the microwell plate with Hank's balanced salt solution (containing 20mM HEPES), and add a volume of 30 μL to each well. After adding, place in Equilibrate on the imager for 1h; re-scan the baseline for 2min, add different concentrations of scopolamine into the microwell plate, the volume of each well is 10μL, the concentration is 5000nM, 2500nM, 1250nM, 625nM, 312.5nM, 156.25nM, 78.125nM, 39.06 nM, 19.53nM, 9.7...

Embodiment 3

[0036] Example 3: Desensitized DMR characteristic signal spectrum of HEK-293-M4 cells

[0037] HEK-293-M4 cells in the logarithmic growth phase were seeded in a cell-compatible 384 microwell plate, the medium used was DMEM (#SH30022.01B, Thermo), the inoculation volume of each well was 40 μL, and each well was inoculated The number of cells is 2.0 x 10 4 Firstly, place the inoculated cell plate in a cell incubator and culture it for 20-22 hours until the cell confluency reaches about 95%, and conduct an activity test. Replace the cell culture solution in the microwell plate with Hank's balanced salt solution (containing 20mM HEPES), and add a volume of 30 μL to each well. After adding, place in Equilibrate on the imager for 1 hour; add different concentrations of carbachol to the microwell plate to pretreat HEK-293-M4 cells for 1 hour, the volume of each well is 10 μL, and the concentration is 5000nM, 2500nM, 1250nM, 625nM, 312.5nM, 156.25nM , 78.125nM, 39.06nM, 19.53nM, 9....

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Abstract

The present invention provides a cell screening model of a label-free muscarinic receptor M4. A method for screening agonists and antagonists of the M4 receptor is established based on a label-free cell integration pharmacology technology and a use of a cell line stably expressing the M4. The method can also be used to study modulators affecting downstream pathways of the M4 receptor. The constructed M4 cell screening model does not require fluorescent labeling and additional addition of indicators during a detection process, has characteristics of target-pathway integration response, no damage to cells, reliable detection results, high sensitivity, high screening throughput, easy operation, etc., and is used to find the agonists, antagonists and pathway regulators of the M4 receptor fromnatural product libraries, metabolite libraries and combined chemical libraries, and screen drugs for diseases of M4 receptor-participated Alzheimer's disease, Parkinson's disease, schizophrenia, etc.

Description

technical field [0001] The invention relates to the field of cell screening, in particular to a cell screening model of unmarked muscarinic receptor M4. Background technique [0002] G protein-coupled receptor (GPCR) is the most important class of membrane receptors in cell signal transduction, and it is also one of the most concerned drug targets in the development of small molecule drugs, with about 34% of Modern drugs directly target this family of receptors. Muscarinic receptor M4, also known as the cholinergic receptor, muscarinic 4 (CHRM4), is a protein encoded by the CHRM4 gene in humans. It is a G protein-coupled receptor, and the receptor reduces the level of cAMP in cells by coupling with Gi / o protein. Mainly distributed in the central nervous system, the activation of M4 receptors inhibits the release of acetylcholine in the striatum, so M4 receptors are used as inhibitory autoreceptors of acetylcholine; M4 receptors affect the extrapyramidal by regulating neuro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12Q1/02
CPCC07K14/705G01N33/502G01N33/5041C12N2510/00C12N2503/02G01N2500/10
Inventor 梁鑫淼王纪霞王志伟薛珍珍于广璞单彩龙
Owner TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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