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Application of dicoumarol in preparation of HBx protein stability inhibitor

A dicoumarin and inhibitor technology, which is applied in the direction of antiviral agents, medical preparations containing active ingredients, active ingredients of heterocyclic compounds, etc., can solve the problem of lack of treatment methods for hepatitis B, reduce stability and inhibit replication Effect

Pending Publication Date: 2020-09-18
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a use of dicoumarin in the preparation of HBx protein stability inhibitors, to solve the problem of lack of effective treatment means for hepatitis B in the prior art

Method used

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  • Application of dicoumarol in preparation of HBx protein stability inhibitor
  • Application of dicoumarol in preparation of HBx protein stability inhibitor
  • Application of dicoumarol in preparation of HBx protein stability inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1 Cell Culture and Transfection

[0107] HepG2-NTCP cell line was cultured in DMEM medium containing 10% fetal bovine serum and 2.5 μg / mL puromycin; PHH cells were cultured in HM medium; Huh-7 cell line was cultured in DMEM medium containing 10% fetal bovine serum medium. All cells were in 5% CO 2 , Routine culture in a 37°C incubator. The plasmid was transfected according to the instructions of Lipofectamin 3000TM (Invitrogen).

Embodiment 2

[0108] Example 2 HiBiT cleavage assay screened out that Dicoumarol can significantly inhibit the expression of HBx

[0109] In order to screen drugs targeting HBx inhibition, we constructed a drug screening model targeting HBx based on Promega's HiBiT cleavage detection system. First, the polypeptide HiBiT containing 11 amino acid residues is fused with HBx to obtain the HiBiT-HBx expression plasmid, which is transferred into HepG2 and Huh-7 cells, and then treated with candidate drugs;

[0110] The cells after drug treatment were taken out, washed twice with PBS, and operated in strict accordance with the instructions of the kit and protected from light. configuration HiBiT Lytic Reagent: Calculate the required total amount based on 100 μL per well, taking the amount of 1 well as an example, add 100 μL in sequence to a sterile EP tube HiBiT Lytic Buffer, 1 μL HiBiT LyticLgBiT Protein (1:100) and 2μL HiBiT Lytic Substrate (1:100), mix thoroughly; add 100μL to each we...

Embodiment 3

[0112] Example 3 MTT assay detects the cytotoxicity of Dicoumarol

[0113] In order to detect the cytotoxicity of the NQO1 inhibitor Dicoumarol, we first seeded different cells (including HepG2-NTCP, HepAD38, HepG2.2.15, HepG2, Huh-7 and PLC / PRF / 5) in 96-well plates, using different concentrations Dicoumarol (DIC) treated cells for 72 hours, specific operations:

[0114] Dissolve DIC with 0.13N NaOH to prepare a 40mM stock solution. will be 1.5×10 4 Huh-7 cells or HepG2-NTCP cells were inoculated in 96-well plates. After 24 hours, the DIC stock solution was diluted with growth medium to obtain 0 μM, 1.925 μM, 3.90 μM, 7.8125 μM, 15.625 μM, 31.25 μM μM, 62.5 μM, 125 μM, 250 μM, 500 μM, and 1000 μM DIC culture medium; and the no-drug treatment group was used as a control, and the pure medium was used as a blank. Each concentration was replicated in three wells, and 100 μl of the above solution was added to each well. After 72h, add 10ul of MTT reagent, incubate at 37°C for 4h...

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Abstract

The invention provides an application of dicoumarol in preparation of an HBx protein stability inhibitor. Experiments find that the HBx protein stability inhibitor, especially dicoumarol, can effectively reduce the stability of HBx, so as to inhibit the replication of HBV virus, achieve the purpose of treatment of hepatitis B, provide a new path for treatment of hepatitis B, and a high positive significance for the health of human beings is achieved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the use of dicoumarin in the preparation of HBx protein stability inhibitors. Background technique [0002] Hepatitis B Virus (HBV) infection is an important public health problem that seriously threatens human health. According to WHO reports, about 2 billion people in the world have been infected with HBV, of which 350 million people are chronic HBV infected people. About 1 million people die from liver failure, cirrhosis and primary hepatocellular carcinoma caused by HBV infection. In recent years, with the development of medical technology and the use of liver cancer vaccines, my country has made remarkable achievements in the prevention of HBV infection and the transformation of patients with chronic HBV infection to end-stage liver diseases such as liver cirrhosis and liver cancer. The number of infected people is still as high as 78 million, and HBV infection is still a public h...

Claims

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Application Information

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IPC IPC(8): A61K31/37A61K45/00A61P1/16A61P31/20
CPCA61K31/37A61K45/00A61P1/16A61P31/20
Inventor 陈娟黄爱龙程胜桃
Owner CHONGQING MEDICAL UNIVERSITY
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