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Application of alkyl pyridine compound to preparation of medicine for inducing cell autophagy and method for inducing cell autophagy

A technology of alkylpyridines and compounds, applied in the field of inducing autophagy, can solve the problems of large side effects and high working concentration, and achieve the effect of low working concentration and good specificity

Active Publication Date: 2017-05-31
蒋培都
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In summary, the use of drugs to enhance autophagy is beneficial to the treatment of tumors, infections, aging, metabolic diseases, autoimmune diseases, and neurodegenerative diseases. However, the working concentration of many existing autophagy inducers high and highly nonspecific
For example, the autophagy inducer rapamycin (rapamycin), which is widely used in cell biology experiments, has an immunosuppressive effect 10 times stronger than cyclosporine, a commonly used immunosuppressant in clinical practice, and it is easy to produce large side effects, which limits its clinical application as an autophagy inducer

Method used

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  • Application of alkyl pyridine compound to preparation of medicine for inducing cell autophagy and method for inducing cell autophagy
  • Application of alkyl pyridine compound to preparation of medicine for inducing cell autophagy and method for inducing cell autophagy
  • Application of alkyl pyridine compound to preparation of medicine for inducing cell autophagy and method for inducing cell autophagy

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Effect test

Embodiment 1

[0045] Induction of autophagy by CPC was detected by observing the distribution of LC3 protein (autophagosome marker) under a fluorescence microscope

[0046] 1. Main experimental materials and reagents: cetylpyridinium chloride (CPC, purchased from Sigma-Aldrich), wherein the structural formula of cetylpyridinium chloride (CPC) is as in formula II (in formula I, n = 0, m = 15, X - for chloride ion):

[0047]

[0048] The above-mentioned CPC was prepared into a 10mM concentrated solution with ultrapure water, stored at -20°C after aliquoting, and diluted with cell culture medium to the required concentrations of 0.1μM, 1μM and 10μM respectively when used. mTOR( m echanistic T target O f R apamycin) inhibitor Torin1 (purchased from Tocris Bioscience) was prepared into 1 mM concentrated solution with dimethyl sulfoxide (DMSO), stored at -20°C after aliquoting, and diluted to 250 nM or other required concentrations with cell culture medium when used. Human cervical cance...

Embodiment 2

[0052] Induction of autophagy by CPC was detected by observation of cellular ultrastructure, including autophagosomes, under a transmission electron microscope

[0053] 1. Experimental method: the cells were digested, centrifuged and resuspended in the corresponding culture medium, planted on collagen-coated plastic coverslips (collagen-coated plastic coverslips) specially used for electron microscopy at a suitable density, and placed at 37°C. 5%CO 2 After culturing in the incubator for 24 h, the original culture medium was removed, and a freshly prepared culture medium containing 0.1 μM, 1 μM and 10 μM of MPC or 250 nM Torin1 was added to continue culturing for 2 h. Then, the coverslips with cells were rinsed twice with 0.1M PBS (pH7.4), and fixed with 2.5% glutaraldehyde for 2 hours. Glutaraldehyde was blotted dry, washed twice with 0.1M PBS, and post-fixed with 1.5% osmium tetroxide for 2h. Then dehydrated and embedded with Epon812 epoxy resin. Final trimming and ultrath...

Embodiment 3

[0056] The effect of CPC treatment on autophagy and mTORC1 pathway-related protein content changes and phosphorylation was monitored by Western Blotting to detect the induction of CPC on autophagy

[0057] 1. Main experimental materials and reagents: anti-LC3 rabbit polyclonal antibody was purchased from MBL (catalog number: PM036); anti-p62 rabbit polyclonal antibody was purchased from MBL (catalog number: PM045); anti-phospho-p70S6Kinase (Thr389) rabbit polyclonal The antibody was purchased from Cell Signaling Technology (catalog number: 9234); the anti-p70S6Kinase rabbit polyclonal antibody was purchased from Cell Signaling Technology (catalog number: 9202); the anti-4E-BP1 rabbit polyclonal antibody was purchased from Cell Signaling Technology (catalog number: 9452) ; Anti-β-actin mouse monoclonal antibody was purchased from Sigma-Aldrich (catalog number: A2228).

[0058] 2. Experimental method: cells were digested, centrifuged and resuspended in the corresponding culture ...

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Abstract

The invention discloses application of an alkyl pyridine compound to preparation of a cell autophagy inducer or application of an alkyl pyridine compound to preparation of a medicine for treating diseases capable of being benefit from autophagy induction and a method for inducing cell autophagy, and belongs to the field of biological medicines. The compound induces cell autophagy mainly by restraining the activity of mTORC1. When the compound serves as an autophagy inducer or an effective constituent of the medicine for treating diseases capable of being benefit from autophagy induction, the compound is low in working concentration and high in specificity, and has a wide application prospect.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of alkylpyridine compounds in the preparation of cell autophagy inducers or drugs for the treatment of diseases that can benefit from autophagy induction and the method for inducing cell autophagy. Background technique [0002] Autophagy refers to the process in which some intracellular substances such as proteins and other biological macromolecules and organelles that need to be degraded are encapsulated and finally transported to the lysosome for degradation. The amino acids and other small molecular substances produced by autophagic degradation Can be reused or generated energy. This is a tightly regulated, evolutionarily highly conserved process that plays an important role in cell growth, development, and homeostasis, helping to maintain the balance between the synthesis, degradation, and subsequent recycling of cellular products. So far, at least three forms of a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/4425A61P25/28A61P25/14A61P25/16A61P9/00A61P1/00A61P21/00A61P19/02A61P29/00A61P35/00A61P39/06
CPCA61K31/4425
Inventor 蒋培都
Owner 蒋培都
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