Method for determining receptor affinity of GLP-1 receptor agonist

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...

AI Technical Summary

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 calculated by the radioactivity intensity on the radioactive substance, but this method relies on isotope labeling technology and has certain harm to the human body

(2) The detection of the second messenger cAMP / cGMP in the GPCR signaling pathway, represented by enzyme-linked immunoassay-sorbent assay (ELISA) and homogeneous radioimmuno-scintillation approach assay, can realize the single-function analysis of a single target, However, the specificity is poor, and there may be endogenous interference of cells

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