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Active ingredient high throughput screen method based on target protein and selection

A screening method and active ingredient technology, applied in the direction of measuring devices, material separation, analysis of materials, etc., can solve problems such as lack of structural information and achieve high-affinity effects

Active Publication Date: 2011-12-14
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The problem to be solved by the present invention is to establish a method based on target protein affinity selection that can be applied to both monomeric compounds or compound groups with known structures and complex natural product groups (such as traditional Chinese medicine extracts) that lack structural information. High-throughput screening methods for active ingredients

Method used

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  • Active ingredient high throughput screen method based on target protein and selection
  • Active ingredient high throughput screen method based on target protein and selection
  • Active ingredient high throughput screen method based on target protein and selection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Drug screening for Alzheimer's disease-related targets:

[0046] The components with high affinity to acetylcholinesterase (Acetylcholinesterase, AChE) were screened among monomeric compounds composed of 27 steroidal alkaloids.

[0047] The monomer compounds to be screened are composed of 27 steroid alkaloids, which are pyrene A, pyrebene B, pyrebene C, pyrebene D, pyrebene E, pyrebene F, fritillin , Sibeisu, Sibeisu glycoside, Pubekone base, Pubekone glycoside, N-norpubekone base, Ibekine glycoside A, Zhebeijiasu, Zhebeijiasu N-oxide, Zhejiang Puqiedinone, Chuanbei ketone, puqiedine-7-01, puqiedinone, puqiedine, puqietinedinone, veratridine, ebeienine, ebeiedinone, ebeiedine, solasodine, isoverticine. It is time-consuming, laborious and inefficient to screen them one by one for their activity. process. The possible active ingredients with high affinity with acetylcholinesterase can be quickly found out by adopting the screening method of the present invention.

[00...

Embodiment 2

[0054] Drug screening for Alzheimer's disease-related targets:

[0055] The components with high affinity to butyrylcholinesterase (Butyrylcholinesterase, BChE) were screened among monomeric compounds composed of 27 steroidal alkaloids.

[0056] The composition of the screened monomer compound is the same as in Example 1.

[0057] Preparation of BChE: Butyrylcholinesterase freeze-dried powder 1200U (Butyrylcholinesterase from equine serum, Sigma), fully dissolved with 3ml ammonium acetate buffer (10mM, pH7.5), prepared to 400U / ml, put in -20℃ refrigerator For middle equipment.

[0058] Preparation of steroidal alkaloid solution: 27 steroidal alkaloids were dissolved in methanol, the concentration of each steroidal alkaloid was 50 μM, and then diluted with ammonium acetate buffer (10 mM, pH7.5) to a concentration of 4 μM.

[0059] Screening: 30 μl of BChE solution (400 U / ml) and 10 μl of steroidal alkaloid solution (4 μM) were evenly mixed, and allowed to stand at 25° C. for ...

Embodiment 3

[0063] Drug screening for cardiovascular disease-related targets

[0064] The components with high affinity for angiotensin converting enzyme (Angiotensin Converting Enzyme, ACE, sigma) were screened among the monomeric compounds composed of 17 fritillaria steroidal alkaloids.

[0065] The screened monomeric compounds are composed of 17 steroidal alkaloids, which are Pubeisuin E, Pubeisuin A, Pubeisuin B, Pubeisuin C, Pubeisuin D, puqiedine, Pubeisuanine, Pubeisu Bequinone glycoside, plebetin, ebeidinone, ebeidine, zhebeijiasu, zhebeijingsu, sibeisu, sibeisu glycoside, fritillary glycoside, ibetinine glycoside A.

[0066] Preparation of ACE: Angiotensin Converting Enzyme lyophilized powder 6U (Angiotensin Converting Enzyme from rabbitlung, Sigma), fully dissolved with 200ul ammonium acetate buffer (10mM, pH7.5), prepared to 30U / ml, placed in a -20°C refrigerator For middle equipment.

[0067] Preparation of steroidal alkaloid solution: 17 Fritillaria steroidal alkaloids were...

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Abstract

The invention relates to the medical field, in particular to the high throughput screening field, and relates to a high throughput screening method of active ingredients or active ingredient groups based on selective affinity of target protein; the invention leads the target protein and the compound to be detected to incubate in buffer solution, the obtained mixed solution is sampled at a molecular exclusion chromatographic column or an online solid extracting column, the buffer solution is eluted, the elution is dissociated, the dissociation solution is separated by the online solid extracting column, and finally guided into a high performance liquid phase mass spectrum system to carry out analysis, carry out the structure confirmation of compounds and qualitative analysis and calculate the binding ratio of the active ingredients to the target protein so as to evaluate the activity. The screening method of the invention improves the efficiency of screening the active ingredients (groups) from the compound groups largely and is a high throughput drug-screening method with relatively simple operation and lower cost.

Description

technical field [0001] The invention relates to the field of high-throughput screening, in particular to a high-throughput screening method for active ingredients or active ingredient groups based on target protein affinity selection, which is a high-affinity screening method for target proteins using multidimensional chromatography-mass spectrometry Active ingredients, especially groups of active ingredients. Background technique [0002] In the process of innovative drug research, the discovery of biologically active lead compounds (Lead Compounds) is the basis of all research work. At present, the screening of lead compounds mostly adopts screening models at the level of animals, organs (tissues) and cells. Although some effective drugs have been screened from them, this method has high screening costs, requires a large amount of compound samples, and is time-consuming and laborious. The cycle is long, so it is not suitable for the screening of natural products with low ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/00G01N27/64
Inventor 李萍周建良安婧婧李会军
Owner CHINA PHARM UNIV
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