Application of 3, 4-broken cycloartane type triterpenoid compound in preparation of autophagy inhibitors and antitumor drugs

A cycloaltin-type, anti-tumor drug technology, applied in the field of medicine, achieves the effects of diversified dosage forms and administration methods, broad clinical application prospects, and increased sensitivity

Active Publication Date: 2020-08-04
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Schisandra chinensis contains a large number of 3,4-broken ring Altin-type triterpenoids, and there is no report on their autophagy inhibition

Method used

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  • Application of 3, 4-broken cycloartane type triterpenoid compound in preparation of autophagy inhibitors and antitumor drugs
  • Application of 3, 4-broken cycloartane type triterpenoid compound in preparation of autophagy inhibitors and antitumor drugs
  • Application of 3, 4-broken cycloartane type triterpenoid compound in preparation of autophagy inhibitors and antitumor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The 3,4-cleaved cycloaltin-type triterpenoid black schisandralic acid inhibits ubiquitination of the autophagy receptor p62.

[0055] (1) Detection of p62 ubiquitination in vitro: ATP, HA-Ub, E1 ubiquitin activating enzyme UbE1, E2 ubiquitin conjugating enzyme UbcH5b, E3 ubiquitin ligase HECTD3 and its substrate were sequentially added to the reaction buffer. For p62 (with His tag), black schisandraic acid of different concentrations (5 μM, 10 μM, 20 μM) was added, DMSO was added as a positive control, and ATP was not added as a negative control, and reacted at 37°C for 40 minutes. Afterwards, it was incubated overnight with a nickel column, and after sufficient elution, the ubiquitination of p62 was detected by Western Blot.

[0056] The result is as Figure 1A As shown, compared with the negative control and positive control, 20 μM black schisandraic acid significantly inhibited the ubiquitination of p62.

[0057] (2) Detection of p62 ubiquitination in cells: p62-Fla...

Embodiment 2

[0060] The 3,4-cleaved cycloaltin-type triterpenoid black schisandralic acid inhibits lapatinib-induced autophagy.

[0061] (1) Inoculate MDA-MB-468 cells at a density of 8000 cells / well in a 24-well culture plate with glass slides placed in advance. After the cells are completely adhered to the wall, transfect the GFP-LC3 plasmid, and add lapatidine 24 hours later. Ni (10 μM), black schisandrin (20 μM) and the combination of the two (10 μM lapatinib and 20 μM black schisandrin) were treated for 4 hours (DMSO was also used as a control group), and fixed with 4% paraformaldehyde for 1 hour , soaked slides in PBS for 3 times, then sealed with DAPI-containing blocking solution, and finally observed GFP-LC3 fluorescent spots under a fluorescence microscope, and quantitatively calculated the number of GFP-LC3 fluorescent spots.

[0062] See the experimental results Figure 2A with 2B , it can be seen from the figure that the number of fluorescent spots in the lapatinib treatment ...

Embodiment 3

[0066] The 3,4-cleaved cycloaltin-type triterpenoid black schisandralic acid inhibits cisplatin-induced autophagy.

[0067] (1) Inoculate MDA-MB-468 cells at a density of 8000 cells / well in a 24-well culture plate with glass slides placed in advance. After the cells are completely adhered to the wall, transfect the GFP-LC3 plasmid, and add black schisandraic acid respectively after 16 hours (20μM), cisplatin (5μM) and the combination of the two (20μM black schisandralic acid and 5μM cisplatin) for 4 hours (DMSO was also used as a control group), fixed with 4% paraformaldehyde for 1 hour, and soaked in PBS The slides were sealed three times with DAPI-containing blocking solution, and finally the fluorescent spots of GFP-LC3 were observed under a fluorescence microscope, and the number of fluorescent spots of GFP-LC3 was quantitatively calculated.

[0068] See the experimental results Figure 3A with 3B , the results showed that the number of fluorescent spots in the cisplatin...

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Abstract

The invention discloses new use of a 3, 4-broken cycloartane type triterpenoid compound. The compound is nigranoic acid or kadsuric acid, and is used in preparation of autophagy inhibitors and preparation of drugs for increasing the treatmnet sensitivity of anti-tumor drugs. The compound can be used for inhibiting ubiquitination modification of an autophagy receptor p62 and prevent activation of ap62 autophagy receptor function, and can inhibit the protective autophagy of tumor cells induced by anti-tumor drugs. The invention also provides an anti-tumor pharmaceutical composition, which comprises the 3, 4-broken cycloartane triterpenoid compound and an anti-tumor drug capable of easily causing tumor cell protective autophagy, can inhibit tumor cell growth and promote tumor cell death, andcan be used for treating cancers such as breast cancer and the like.

Description

technical field [0001] The invention belongs to the technical field of medicine; in particular, it relates to the application of a 3,4-broken cycloaltin-type triterpenoid compound in the preparation of autophagy inhibitors and antitumor drugs. Background technique [0002] Autophagy is a highly conserved catabolic process in cells, which is mainly divided into four stages: the formation of autophagic vesicle precursors, the formation and extension of autophagosomal membranes, the fusion of lysosomes and the degradation in the vesicle. Degrades damaged proteins or organelles to maintain cellular homeostasis. According to the different ways of transporting intracellular substrates to the lysosome cavity, mammalian cell autophagy can be divided into three main ways: macroautophagy, microautophagy and molecular chaperone-mediated autophagy, but the most widely studied one is Macroautophagy. Autophagy can help tumor cells cope with oxidative stress and DNA damage caused by adve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/194A61P35/00
CPCA61K31/194A61P35/00
Inventor 陈策实普诺·白玛丹增孙汉董黄茂波周元飞
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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