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Antidiabetic medicament screening method using Nur77-LKB1 interaction as target point

A nur77-lkb1, anti-diabetic technology, applied in biochemical equipment and methods, testing pharmaceutical preparations, biological testing, etc., can solve the problem of low phosphorylation activity of AMPKα in the liver, and achieve intuitive effect, stable system, and drug screening mechanism clear effect

Inactive Publication Date: 2013-11-20
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Liver-specific knockout of LKB1 mice exhibit hyperglycemia symptoms, and their livers have very low phosphorylation activity of AMPKα (Shaw RJ, et al., Science, 2005, 310, 1642-1646)

Method used

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  • Antidiabetic medicament screening method using Nur77-LKB1 interaction as target point
  • Antidiabetic medicament screening method using Nur77-LKB1 interaction as target point
  • Antidiabetic medicament screening method using Nur77-LKB1 interaction as target point

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Compounds that inhibit the Nur77-LKB1 interaction were screened by co-immunoprecipitation and Western blotting.

[0059] The test method is as follows:

[0060] 1) Cell transfection and drug treatment:

[0061] The cells were inoculated on a 6-em-diameter culture dish, transfected 24 hours later, and the cells were replaced with fresh culture medium before transfection. The transfection steps are: take a 1.5mL centrifuge tube, add 180μL of water, the corresponding plasmid, 2.5M CaCl 2 20 μL, HBS 200 μL, mix gently, let stand at room temperature for 15-30 minutes, then slowly add dropwise to the culture medium, shake the culture plate gently to make the distribution even.

[0062] After 36 hours of transfection, the cell culture medium was replaced with serum-free DMEM (Dulbecco's Modified Eagle's Medium), and the corresponding drugs were added. In the control group, the medium was changed at the same time, and an equal volume of drug solvent dimethyl sulfoxide (DMSO)...

Embodiment 2

[0087] In the further screening method, Western blotting was used to detect the effect of the compound to be screened in Example 1 on the phosphorylation level of AMPKα in normal mouse liver cells AML12.

[0088] The test method is as follows:

[0089] 1) Cell Lysis

[0090] Cells were seeded on a culture plate, and after 24 hours of culture, treated with drugs for an appropriate time, discarded the culture medium, washed once with PBS buffer, and then added PMSF and Cocktail containing 1mM (add just before use, add 1μL for every 100μL lysate) lysisbuffer cell lysate (50mM HEPES (pH 7.4), 100mM NaCl, 10% glycerol, 1% Triton-X-100, 1.5mM MgCl 2 , 25mM NaF) 400μL, sonicate on ice, centrifuge at 13000rpm at 4°C for 30min, collect the supernatant, measure the protein concentration and perform western blotting (method refer to Example 1).

[0091] 2) Determination of protein concentration

[0092] a. Add 200 μL Bradford protein assay solution to each well of the 96-well plate, a...

Embodiment 3

[0101] The method of measuring blood glucose by blood sampling from tail vein of mice was used to determine the hypoglycemic ability of TAPA1 on type II diabetes mouse model db / db mice.

[0102] The test method is as follows:

[0103] Take 16 hyperglycemic db / db mice (male, 8 weeks old) and randomly divide them into the experimental group (TAPA1 administration group; first dissolve TAPA1 in DMSO to make 1M mother solution, then take the required amount and 3 times the volume The emulsifier Tween-80 is mixed evenly, after standing for 15min, slowly add normal saline, flick to make the TAPA1-DMSO-Tween-80 mixture slowly dissolve in the normal saline to form an aqueous solution) and the control group (solvent group: use DMSO Replace TAPA1-DMSO solution, the configuration method is the same as above). Administered by intraperitoneal injection, the dose of TAPA1 was 50mg / kg, the mice in the solvent group were injected with the same volume of solvent as the experimental group, once...

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Abstract

The invention relates to an antidiabetic medicament screening method using Nur77-LKB1 interaction as a target point and provides a protein-protein interaction target point, a detecting method of the interaction, application of the interaction target point and a method for screening an antidiabetic medicament using Nur77-LKB1 interaction as the target point. The protein-protein interaction target point is the interaction between an orphan receptor Nur77 and the upstream kinase LKB1 of adenosine monophosphate-activated protein kinase (AMPK). The detecting method of Nur77-LKB1 interaction is selected from a cell molecular biology detecting method or a chemical biology detecting method. The invention provides application of Nur77-LKB1 interaction used as the target point in screening the antidiabetic medicament. The screening method comprises the following steps of: enabling a candidate compound to be in contact with a substance entity in the detecting method of Nur77-LKB1 interaction; and observing the influence of the candidate compound on a result in the Nur77-LKB1 interaction detecting method.

Description

technical field [0001] The invention relates to the screening of anti-diabetic drugs, in particular to a method for screening anti-diabetic drugs with Nur77-LKB1 protein interaction as the target. Background technique [0002] In recent years, the global incidence of diabetes has increased rapidly, and diabetes has become the third chronic disease that seriously threatens human health after cancer and cardiovascular disease. At present, there are nearly 180 million diabetic patients in the world. According to the World Health Organization, by 2025, the number of adults with diabetes in the world will increase to 300 million, while the number of diabetics in China will reach 40 million. Diabetes will still be a very serious problem in China in the next 50 years. Diabetes can cause a variety of serious chronic complications, such as diabetic nephropathy, retinopathy, diabetic foot, myocardial infarction, etc. These complications are the main cause of death in diabetic patien...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/15C12Q1/02
Inventor 吴乔占艳艳陈航姿田敏陈艳郑忠辉
Owner XIAMEN UNIV
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