Application of BRAF inhibitor to preparation of novel drugs for treating programmed necrotic diseases and screening method of BRAF inhibitor

A technique of programmed necrosis and screening methods, which is applied in the field of cytology and pharmacology, can solve the problems of difficult compound families, long screening cycle and verification experiments, and long research and development time, so as to achieve targeting and safety , reduce cost and failure risk, and avoid the effect of long investment time

Inactive Publication Date: 2019-04-05
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

[0005] Traditional screening of drugs based on natural products or small molecule compound libraries has two main defects. One is the long R&D time and high investment risk in the development and innovation of new drugs and therapeutic biological products; the other is the solubility and safety of the screened drugs. Urgent need for animal and clinical validation
At the same time, the drug screening and application of targeted necrosis inhibitors currently mainly have the following shortcomings, including: (1) The necrostatin family represented by Nec-1 is a small molecular compound, although it is easy to pass through the blood Brain barrier is a good tool for molecular mechanism research, but because its compound family is insoluble in water or electrolyte solutions, it is difficult to use it as a clinical drug for new drug development; (2) Nec-1 i

Method used

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  • Application of BRAF inhibitor to preparation of novel drugs for treating programmed necrotic diseases and screening method of BRAF inhibitor
  • Application of BRAF inhibitor to preparation of novel drugs for treating programmed necrotic diseases and screening method of BRAF inhibitor
  • Application of BRAF inhibitor to preparation of novel drugs for treating programmed necrotic diseases and screening method of BRAF inhibitor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Use the positive control TNF (TNFα group) and the antagonistic group (Nec-1 group) of Nec-1 anti-necrosis inhibitors to detect and determine the sensitivity of the cell line to different functional drugs in the FADD-KO cell line, each group of drugs After the L929-FADD-KO cells were treated for 24 hours, the cell survival rate was detected by the ATP level, the Control group was used as the standard, and the data analysis of the chemiluminescence instrument was shown in Table 1 and figure 1 As shown, the lethality of TNFα group (10ng / mL) to cells almost reached 50%, and Nec-1 group (30μM) could restore the cells to 90%.

[0058] Table 1 Cell survival rate of L929 FADD-KO cells treated with TNF, Nec-1, TNF+Nec-1

[0059]

Embodiment 2

[0061] The L929 cell proliferation model was used to screen the FDA drug library for necrosis. After more than a thousand FDA drugs (10 μM) were treated for 24 hours, if the cell survival rate of the three replicate wells treated with the same drug was higher than 50% ( value above 0.5), it is considered that the drug has a strong necrosis-inhibiting effect on L929-FADD-KO cells and can be used as a candidate drug.

[0062] The necrosis inhibitory function of the above candidate drugs was tested at different concentrations and different treatment times. Using the human cell line HT29 as a cell model, it was found that there was no difference in treatment at different times for 8 hours and 24 hours, and drug concentrations of 10uM and 30uM could be obtained. Very good results, with BRAF inhibitors working best, such as figure 2 shown.

[0063] The cells were used as control group, TNF necrosis group, TNF+BRAF inhibitor group, TNF+Nec-1 group for target verification, Nec-1 was...

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Abstract

The invention discloses application of a BRAF inhibitor to preparation of novel drugs for treating programmed necrotic diseases and a screening method of the BRAF inhibitor. The method is characterized by taking necrotic pathway sensitive cell line L929-FADD gene knockout cell strains as model cells of cell proliferation and necrosis, screening out small-molecule compounds influencing and inhibiting cell death from an FDA drug library, detecting different drug concentration of the small-molecule compounds obtained by screening, determining toxicity-effect concentration, and performing comprehensive evaluation from toxicity and recovery efficiency to obtain the BRAF inhibitor; and through inhibition of the BRAF inhibitor on cell necrosis, the BRAF inhibitor can be used for preparing novel drugs for treating the programmed necrotic diseases. The BRAF inhibitor has excellent necrosis inhibition effects on necrotic sensitive cell line L929-FADD knockout strains, human cell line HT29 and animal models; the water solubility of the BRAF inhibitor is better than that of Nec-1; the BRAF inhibitor is an FDA-approved clinical drug; the targeting property and the safety of the BRAF inhibitor are guaranteed; and the BRAF inhibitor is easy for clinical promotion.

Description

technical field [0001] The present invention relates to the field of cytology and pharmacology, in particular to the use of BRAF inhibitors in the preparation of novel drugs for treating programmed necrosis diseases, and also to the screening method for preparing new drugs for treating programmed necrosis diseases. Background technique [0002] The dynamic balance between orderly proliferation, differentiation and death of cells is the primary condition for maintaining the homeostasis of the internal environment. Programmed necrosis is an important way of cell death. It is a protein kinase through RIP3 and RIP1, and its interaction and phosphorylation affect necroptosis. Cell necrosis is a death process in which cells are subjected to strong physical, chemical or biological factors that cause disordered changes in cells. It is manifested as cell swelling, cell membrane rupture, cell content overflow, nuclear changes are slow, DNA degradation is insufficient, and local severe...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61P43/00C12Q1/02
CPCA61K45/00A61P43/00G01N33/5008
Inventor 王慧巴乾牟为
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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