Method for determining receptor affinity of GLP-1 receptor agonist

A receptor agonist, GLP-1 technology, applied in the field of medicine, can solve the problems of relying on isotope labeling technology, limited application, poor specificity, etc.

Inactive Publication Date: 2015-08-19
CHINA PHARM UNIV
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Problems solved by technology

However, GLP-1 is easily degraded by dipeptidyl peptidase IV (DPP-IV) in the human body, and its half-life in vivo is less than 5 minutes, which largely limits its clinical application.
Commonly used assay methods include the following three categories: (1) Based on the principle of radioisotope receptor ligand binding, isotope-labeled drug molecules and prototype molecules act together on cells with high expression of GLP-1R, and detect the most total binding to cells. The affinity of the drug can be cal

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  • Method for determining receptor affinity of GLP-1 receptor agonist
  • Method for determining receptor affinity of GLP-1 receptor agonist
  • Method for determining receptor affinity of GLP-1 receptor agonist

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Embodiment

[0038] Determination of Receptor Affinity of 5 Different Kinds of GLP-1R Agonists Using This Method

[0039] The five GLP-1R agonists are: Exendin-4 and four kinds of polyethylene glycol site-modified Exendin-4 analogues with different molecular weights. By adding a cysteine ​​to the C-terminus of Exendin-4 to the Exendin-4 analogue (Ex4C for short), use monomethoxypolyethylene glycol-maleimide with molecular weights of 5kDa, 10kDa, 20kDa and 40kDa, respectively. The amine (mPEG-MAL) reacted with Ex4C and purified to obtain 4 kinds of modified products, namely mPEG 5k -Ex4C, mPEG 10k -Ex4C, mPEG 20k -Ex4C and mPEG 40k -Ex4C. The assay steps for the receptor affinity of the above five GLP-1R agonists are as follows:

[0040] (1) Take out the frozen CHO-GLP-1R-CRE-Luc from the liquid nitrogen tank + Cells, after the cells were quickly thawed and recovered, transferred to a culture flask containing 5mL of cell growth medium, at 37°C, 5% CO 2 cultured in an incubator. When...

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Abstract

The invention provides a method for determining the receptor affinity of a GLP-1 receptor agonist. The method is based on a tool cell line, namely CHO-GLP-1R-CRE-Luc<+>, when a sample to be determined is combined with GLP-1 receptor on the surface of a cell, the receptor-mediated signal cascade reaction can be activated, cAMP-response element (CRE) is activated specifically, the expression of the luciferase reporter gene is promoted, the quantity of luciferase in cytosol is detected for drawing and fitting to obtain a dose-effect curve of acting of the sample and the GLP-1 receptor; and the half effective concentration (EC50) is calculated. By inspecting and optimizing all influence factors in the determining process, the method for determining the receptor affinity of the GLP-1 receptor agonist is finally established. The method has the advantages of being high in specificity, precision and accuracy, good in durability and convenient to operate, and the like. The method can be used for determining the receptor affinity of the GLP-1 receptor agonist, and thus such type of drug can be fast evaluated and screened.

Description

technical field [0001] The invention belongs to the technical field of medicine and relates to a method for measuring receptor affinity, in particular to a method for measuring receptor affinity of a GLP-1 receptor agonist. Background technique [0002] Incretin refers to the general term for a class of polypeptides that can cause blood sugar-lowering effects secreted by the human body after eating. Insulin secretion caused by incretin accounts for about 50% to 70% of its total secretion. In recent years, incretin drugs represented by glucagon-like peptide-1 (GLP-1) have received extensive attention in the field of diabetes treatment. After GLP-1 binds to its receptor, it can activate the G protein coupled with it, increase the concentration of intracellular cAMP and activate protein kinase A (PKA), thereby up-regulating the transcription and translation of insulin gene in β cells, thereby increasing insulin secretion And enhance the sensitivity of glucose stimulation sign...

Claims

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Application Information

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IPC IPC(8): C12Q1/66C12Q1/02
Inventor 姚文兵田浤郭林峰高向东胡晓静
Owner CHINA PHARM UNIV
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