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Method for accurately and rapidly identifying drug target of active compound obtained by phenotypic screening

A compound and active technology, applied in the field of medicine, can solve problems such as unknown targets, and achieve the effect of acceleration speed and precision

Active Publication Date: 2014-12-24
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In our research, we unexpectedly found that this compound analog can block the lipid accumulation of 3T3-L1 in Caenorhabditis elegans and mouse preadipocytes, but its target is unknown

Method used

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  • Method for accurately and rapidly identifying drug target of active compound obtained by phenotypic screening
  • Method for accurately and rapidly identifying drug target of active compound obtained by phenotypic screening
  • Method for accurately and rapidly identifying drug target of active compound obtained by phenotypic screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. AA005 and its structural analogs can specifically inhibit nematode body fat accumulation without toxicity

[0050] Synchronized L1-stage N2 nematodes were reared on NGM plates with OP50 bacteria for 28 hours, and the nematodes were evenly divided into six-well plates, and the nematodes were treated with 4 μM AA005, A1, A2, and A3 compounds for 14 hours, and the control group did not add drugs , and appropriate OP50 bacteria feeding. Each group of nematodes after drug treatment were placed on the NGM plate containing OP50 bacteria to recover for 16 hours, and an equal amount of nematodes were collected for staining with oil red O, and oil red O was extracted for quantification, and the effect of AA005 and its derivatives on the lipid accumulation of nematodes was observed . Observation under the microscope ( figure 1 C) Compared with the control group, the lipids in nematodes treated with compounds AA005 and A3 were significantly reduced, while the lipids of...

Embodiment 2

[0051] Example 2. AA005 and its structural analogues can specifically inhibit the accumulation of triglycerides in preadipocyte 3T3-L1

[0052] 3T3-L1 cells were continuously grown to complete contact inhibition for two days, and MDI triple drug was added to DMEM high-glucose medium containing 10% fetal bovine serum to induce differentiation, and 300nM AA005 and structural analogues A1, A2, and A3 were added to the cells in the treatment group , after oil red O staining of cells in the control group and the four compound-treated groups induced to differentiate for 8 days, the macroscopic and microscopic photographs were taken ( image 3 A), oil red O dye in cells was extracted and quantified by spectrophotometer. The results suggested that compared with the control group, the oil red O staining in the AA005 treatment group and the A3 treatment group cells was reduced, and the inhibitory effect of AA005 was the most significant, while the oil red O staining in the A1 and A2 tre...

Embodiment 3

[0053] Example 3. Chemical proteomics fishing for interacting proteins of AA005

[0054] In order to obtain AA005-interacting proteins by affinity chromatography, we connected Biotin outside its active center to construct biotin-labeled AA005 (Biotin-AA005)( Figure 4 A). The modified small molecule still maintained the biological activity of AA005 in inhibiting lipid accumulation in 3T3-L1 cells, and could significantly inhibit the formation of intracellular lipid droplets ( Figure 4 B). We also analyzed the localization of Biotin-AA005 in 3T3-L1 cells by immunofluorescence staining experiment of Mito-tracker labeled mitochondria and FITC-labeled streptavidin co-staining, the results showed that Biotin-AA005 was almost all localized in mitochondria ( Figure 4 C). Accordingly, we speculate that the interacting protein of AA005 is likely to be located in mitochondria. Next, the mitochondria, nucleus and cytoplasm components of 3T3-L1 cells were separated and purified by d...

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Abstract

The invention discloses a method for accurately and rapidly identifying a drug target of an active compound obtained by phenotypic screening. The seamless joint of compound phenotypic screening and target identification is established by a chemical proteomics-driven caenorhabditis elegans feeding RNAi technology; the active compound is an active compound with a corresponding phenotype in the elegans; the phenotype includes but is not limited to lipid dysbolism, cell apoptosis, autophagy, senility, neurodegenerative disease and anti-infection. The problem that at present, false positive and false negative are easily produced when the proteomics method is purely used for analyzing the interacting protein of drug can be solved, and the aim of eliminating the false and retaining the true can be realized; furthermore, the method is simple and convenient in operation and rapid and accurate in screening, and provides a feasible scheme for the analysis of the large-flux interacting protein of the drug; a more perfect chemical proteomics technology system is formed, and the possibility of breaking through the choke point of phenotypic screening protein target identification is provided.

Description

technical field [0001] The present invention relates to the field of medicine, and more specifically, relates to a method for accurately and rapidly identifying drug targets of active compounds obtained by phenotypic screening. Background technique [0002] Drug discovery strategies mainly include "phenotype-based screening" and "target-based screening". These two types of drug screening have successively dominated drug development for the past hundred years. The former focuses on compound-induced effects or phenotypes in cells, tissues, or whole organisms, while the latter analyzes the effects of compounds on purified target proteins in vitro. Among them, target-based drug screening has dominated in the past 30 years. However, the statistical analysis of new drugs approved by the FDA from 1999 to 2008 shows that although target-based screening has an advantage in the approval of follower tracking drugs, there are only 17 discoveries of first-in-class original new drugs. ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 王立顺韩冰黄黎莹
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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