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Medicine for inducing autophagy and treating disease caused by wrong unfolded protein aggregation, and filtration method thereof

A misfolded protein and autophagy technology, which is applied in the field of drugs and their screening for treating diseases caused by misfolded protein aggregation by inducing autophagy

Inactive Publication Date: 2008-07-23
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only the mechanism of starvation-induced autophagy is relatively clear

Method used

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  • Medicine for inducing autophagy and treating disease caused by wrong unfolded protein aggregation, and filtration method thereof
  • Medicine for inducing autophagy and treating disease caused by wrong unfolded protein aggregation, and filtration method thereof
  • Medicine for inducing autophagy and treating disease caused by wrong unfolded protein aggregation, and filtration method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Compounds increase the expression and aggregation of LC3-GFP

[0038] experiment method:

[0039] Microtubule-associated protein light chain 3 (LC3) is a mammalian protein homologue of yeast autophagy protein ATG8 (Aut7 / Apg8), localized on the surface of preautophagic vacuoles and autophagic vacuoles, and is a widely used autophagosome membrane Labeling (Mizushima N. Int J Biochem Cell Biol 2004; 36: 2491-502). The present invention uses a high-content microscope to analyze and count the changes in the fluorescence intensity and distribution of the green fluorescent (GFP)-labeled autophagosome marker LC3 before and after the action of the compound

[0040]Specific method: The fusion protein LC3-GFP was transferred into H4 cells, and the H4-LC3 cell line was constructed and screened as a screening platform for high-content screening of 480 compounds with known biological activity (ICCB known bioactive library, BIOMOL). Method: The compound was diluted and dis...

Embodiment 2

[0047] Example 2 Compounds increase or do not affect intracellular PI(3)P levels

[0048] experiment method:

[0049] Type III PI3K vps34 is a multifunctional protein that, on the one hand, catalyzes the phosphorylation of PI to generate PI(3)P, which is critical for endocytosis and autophagosomal membrane trafficking (14). At the same time, the complex formed by Vps34 / beclin1 is involved in the regulation of autophagy initiation signal (15). Therefore, PtdIns(3)P levels should at least not decrease too much during autophagy. The FYVE domain is a protein domain consisting of ~70 amino acid residues and containing a zinc finger protein structure, which can specifically bind to PI(3)P(16). Usually, PI(3)P recruits proteins containing the FYVE domain to bind to Organelle membrane, involved in processes such as protein transport. Therefore, fluorescently labeled FYVE domains can be used to detect intracellular PtI(3)P levels and localization (17). We transferred the fusion pro...

Embodiment 3

[0056] Example 3 Eight kinds of compounds increase the degradation of long-lived proteins

[0057] experiment method

[0058] The essence of the autophagy process is a protein degradation process, which mainly mediates the degradation of organelles and intracellular long-lived proteins (5). Therefore, detecting whether a compound promotes the degradation of long-lived proteins in cells is an important evidence that the compound induces autophagy.

[0059] Specific method: the compound was diluted and dissolved in DMSO to prepare different concentrations, and each concentration was repeated three times; H4 cells were planted in a 96-well plate at an appropriate density, and treated with the compound for 2, 4, and 24 hours respectively. The following experimental groups were set up: blank control (DMSO treatment), positive control (inducer rapamycin treatment), and experimental group (compound treatment). One day before the experiment, the cells were planted in a 12-well plate...

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Abstract

The invention relates to a drug with autophagia inductivity for treating the diseases caused by the accumulation of misfolded proteins and a screening method thereof. Quantitative analysis is carried out to the changes of the fluorescence marked LC3 in the cells of the known medicine combination and to the changes of the fluorescence marked FYVE in the cells to obtain the candidate compound of an autophagia inductive agent; and then longevous protein degradation and polyQ degradation is carried out to finally determine the drug with the autophagia inductivity for treating the diseases caused by the accumulation of misfolded proteins. The method of the invention is a simple and high effective compound screening method which can affect autophagia.

Description

technical field [0001] The invention relates to a drug and a drug screening method capable of inducing autophagy to treat diseases caused by misfolded protein aggregation factors. Background technique [0002] Autophagy is a lysosome-dependent degradation process of intracellular proteins or damaged organs. (Klionsky, D.J. & Emr, S.D.Science, 2000, 290, 1717-21.) In this process, after the double-membrane autophagosome coats the cellular content that needs to be degraded, it fuses with the lysosome to perform the degradation function, Recycling of amino acids is achieved. This degradation / recycling system is highly conserved in evolution and is critical in processes such as development, growth, aging, disease, and death. The autophagy-lysosome pathway and the ubiquitin-proteasome pathway have become the two main degradation systems in eukaryotic cells, but unlike the proteasome pathway, autophagy mainly degrades long-lived proteins, protein aggregates and organelles. Ther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/445A61K31/343A61K31/4523A61K31/437A61P43/00
CPCA61K31/445A61P25/00A61P25/16A61P25/28A61P35/00A61P43/00C07D211/44C07D211/82C07D401/12C07D491/22
Inventor 张立红于佳潘鹤龄马大为袁钧瑛
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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