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Application of compound for mediating cbl to degrade P-TFEB and exciting TFEB in preparation of antitumor drugs

A technology of P-TFEB and antineoplastic drugs, applied in the field of medicine

Pending Publication Date: 2022-06-03
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many ways to induce TFEB agonism with drugs, but the method of activating TFEB by degrading phosphorylated TFEB by cbl and increasing TFEB / TFEB dimer has not been reported yet
And there is no report of inducing ferroptosis in lung cancer cells through this pathway.

Method used

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  • Application of compound for mediating cbl to degrade P-TFEB and exciting TFEB in preparation of antitumor drugs
  • Application of compound for mediating cbl to degrade P-TFEB and exciting TFEB in preparation of antitumor drugs
  • Application of compound for mediating cbl to degrade P-TFEB and exciting TFEB in preparation of antitumor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Ginkgo biflavonoids bind to TFEB protein

[0025] The TFEB protein and its truncates were covalently immobilized on the CM5 chip through amine groups, and the immobilization level was 6500RU. Ginkgo biflavonoids solutions of different concentrations (100 μM, 33.3 μM, 11.1 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM) flowed over the chip surface to collect interaction signals in real time. The entire system was performed in 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P2O, 2% DMSO buffer solution. GE Life Sciences Biacore S200 system was used for detection and data analysis. The results showed that Ginkgo biflavonoids had a binding effect with TFEB protein ( figure 1 ).

Embodiment 2

[0026] Example 2: Ginkgo biflavonoids induce P-TFEB to bind to cbl, and cbl is positively correlated with Ginkgo biflavonoids-induced P-TFEB degradation

[0027] A549 cells were cultured in 1640 medium containing 10% fetal bovine serum, penicillin 100U / ml, gentamicin 100ug / ml, and the incubator temperature was 37°C, containing 5% CO 2 . Using molecular cloning technology, the phosphorylation site S122 of TFEB was mutated to construct the phosphorylation site mutant plasmid pcDNA3.1-TFEB S122D , mimicking the persistent phosphorylation of TFEB.

[0028] Combining pcDNA3.1, pcDNA3.1-TFEB with pcDNA3.1-TFEB S122D They were transfected into A549 cells and given Ginkgo biflavonoids. Then cells were collected, lysed to extract protein, incubated with TFEB antibody and protein G-beads, washed protein G-beads with buffer 4 times, and dissolved the protein with urea. The peptides were then digested with trypsin and subjected to proteomic analysis by LC-MS / MS. The results show that...

Embodiment 3

[0029] Example 3: Ginkgo biflavonoids promote TFEB phosphorylation, dissociate from binding protein 14-3-3, and promote TFEB / TFEB dimer formation.

[0030] A549 cells were collected at different time (3, 6, 12, 24h) after administration of Ginkgo biflavonoids, the cells were lysed, the protein was extracted, and the changes of P-TFEB were detected by western blot. The level of TFEB ( image 3 A).

[0031]The decrease in the phosphorylation level of TFEB will lead to its separation from the binding protein 14-3-3, and three forms of dimers will be formed between free TFEB and P-TFEB, namely P-TFEB dimer and TFEB dimer. polymer, P-TFEB\TFEB heterodimer. Among them, the TFEB dimer can enter the nucleus to play the function of transcription factor, and the appearance of P-TFEB in the dimer will affect the activation of TFEB activity. Immunoprecipitation was used to observe the binding of TFEB to 14-3-3 after Ginkgo biflavonoids were administered to A549 cells at different time ...

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Abstract

The invention discloses an application of a compound for mediating cbl to degrade P-TFEB and exciting TFEB in preparation of antitumor drugs. And the compound capable of mediating cbl to degrade P-TFEB and exciting TFEB can be combined with TFEB protein. Under the compound action of mediating the cbl to degrade the P-TFEB and exciting the TFEB, the Cbl is combined with the P-TFEB, the P-TFEB is reduced, and the formation of a TFEB / TFEB homodimer is promoted. The TFEB is a key regulatory protein for mediating cbl to degrade P-TFEB and exciting a compound of the TFEB to resist lung cancer, and TFEB knock-down can reverse ferroptosis induced by biflavone of ginkgo biloba. The anti-tumor drug is screened on the basis of an anti-tumor pathway for promoting the combination of P-TFEB and cbl, reducing P-TFEB and increasing TFEB / TFEB homodimer to excite TFEB to induce ferroptosis on the basis of the targeted TFEB.

Description

Technical field [0001] The present invention relates to the field of medical technology, and in particular to the application of compounds that mediate cbl to degrade P-TFEB and stimulate TFEB in the preparation of anti-tumor drugs. Background technique [0002] Lung cancer is currently the leading cause of cancer death, accounting for nearly 20% of all cancer deaths. The treatments for lung cancer mainly include surgery, radiotherapy, chemotherapy, targeted and immunotherapy. Surgery and radiotherapy have certain limitations and should not be used alone. The most prominent problems with chemotherapy, targeted and immunotherapy are drug resistance and explosive progression, and the long-term clinical efficacy is still unsatisfactory. As recent clinical studies have shown, 10% of patients receiving immunotherapy experience explosive tumor progression. So far, there has been no significant breakthrough in the average five-year survival time of lung cancer patients. Therefo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/352A61K45/06A61P35/00
CPCA61K31/352A61K45/06A61P35/00
Inventor 楼剑书王浩洁胡碟赵丽萍胡君虎张君颖
Owner HANGZHOU NORMAL UNIVERSITY
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