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Screening method for epidermal growth factor receptor tyrosine kinase inhibitor and inhibitors identified using same

A technology of epidermal growth factor and tyrosine kinase, which is applied in the screening of compounds, biochemical equipment and methods, and the determination/inspection of microorganisms, etc. It can solve the problems of multiple proteins and unsuitable mass screening, so as to reduce time and cost, The effect of significantly inhibiting the effect

Inactive Publication Date: 2012-02-01
HOSEO UNIV ACADEMIC COOP FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method not only requires a large amount of protein, but also has a non-specific reaction, and there is a problem that it is not suitable for mass screening.

Method used

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  • Screening method for epidermal growth factor receptor tyrosine kinase inhibitor and inhibitors identified using same
  • Screening method for epidermal growth factor receptor tyrosine kinase inhibitor and inhibitors identified using same
  • Screening method for epidermal growth factor receptor tyrosine kinase inhibitor and inhibitors identified using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Pharmacophore search and docking simulation of compounds

[0052] figure 2 It is a schematic diagram for explaining an example of the process of performing pharmacophore search and docking simulation when performing the screening method according to the present invention, using a chemical computing group (Chemical Computing Group) drug for virtual screening Develop a visual simulation (Molecular Operating Environment, MOE) program. The virtual screening method uses both pharmacophore search and molecular docking simulation.

[0053] First of all, a basic pharmacophore model similar to the structure of IRESSA and TARCEVA, which are well known as EGFR tyrosine kinase inhibitors, was produced, and 7,000 compounds with the same properties as above were selected from the 40,000 library compounds of IBS Basic Model Approximation of Pharmacophore Compounds.

[0054] Then, using these 7000 compounds as objects, a docking simulation was performed on the X-Ray st...

Embodiment 2

[0058] Embodiment 2: The phosphorylation reaction experiment of the substrate caused by EGFR kinase

[0059] First, in this example, in order to understand the stability of the EGFR kinase substrate bound on the chip, the degree of reaction between the above-mentioned EGFR kinase substrate and EGFR kinase was confirmed.

[0060] Protein chip using ProteoChip TM (Proteogen, Inc., Seoul, Korea), at ProteoChip TM (Proteogen, Inc., Seoul, South Korea) in the microarray (CM-1000; Proteogen, Inc., Seoul, South Korea) the EGFR kinase substrates were spotted at different concentrations to form EGFR kinase substrate microarrays (microarray). The EGFR kinase substrate used PLC-gamma1 purchased from Calbiochem, dissolved the above substrate in a phosphate-buffered saline (PBS) containing 30% glycerol, and applied it to the ProteoChip TM and then fixed overnight at 4°C in a humid environment. Then, the protein chip immobilized with the EGFR kinase substrate (PLC-gamma1) was blocked ...

Embodiment 3

[0067] Example 3: A large number of high-speed screening out of EGFR kinase inhibitors from a single compound library

[0068] First, the EGFR kinase substrate (PLC-gamma1) was attached to the protein chip by the same method as in Example 2 above, and the concentration of the substrate at this time was 20 μg per 1 mm of the chip. Then, on the protein chip attached with the above substrate, with ATP / Mg 2+ The mixture and EGFR tyrosine kinase were used as a positive control 1mM tyrosine phosphorylation inhibitor 51 (Tyrphostin51) and 300 library compounds selected in the above-mentioned Example 1 were differentiated according to different compounds and carried out at a concentration of 50mM respectively. reaction. Other process is identical with embodiment 2, after reaction finishes, the result that chip is carried out fluorescence intensity analysis is as follows: Figure 4 shown.

[0069] Figure 4 According to the present invention, PLC-gamma1 is used as a substrate to ...

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Abstract

The present invention relates to a fast screening method for identifying large quantities of new substances which can effectively inhibit the activity of EGFR tyrosine kinase, and comprises attaching an EGFR tyrosine substrate, which is activated by reaction with EGFR tyrosine kinase, to a protein chip and reacting it with a compound pool consisting of EGFR tyrosine kinase, ATP, Mg2+ and a single candidate library, to provide superior and rapid screening for tyrosine kinase inhibitors.

Description

technical field [0001] The present invention relates to epidermal growth factor receptor (Epidermal Growth Factor Receptor: EGFR), in particular, relates to a screening method capable of rapidly screening out a large number of inhibitors (inhibitors) that inhibit the activity of the above-mentioned EGFR tyrosine kinase (tyrosine kinase), and more Specifically, it relates to a method for ultra-high-speed screening of a large number of substances that inhibit EGFR tyrosine kinase activity using a protein chip, and an inhibitor screened by the method. Background technique [0002] Epidermal Growth Factor Receptor (hereinafter referred to as "EGFR") is a membrane-bound protein with a molecular weight of 170 kilodaltons (kDa), which is expressed on the surface of epithelial cells. EGFR belongs to the growth factor receptor family of protein tyrosine kinases as a class of cell cycle regulating molecules. EGFR is activated when the extracellular domain binds its own ligand (EGF or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573
CPCG01N2500/00G01N2333/71G01N33/566C12Q1/485G01N33/74
Inventor 康仁哲金應允崔荣晋
Owner HOSEO UNIV ACADEMIC COOP FOUND
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