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Application of Berbamine Dihydrochloride in Preparation of Ebola Virus Inhibitor

A technology of berbamine dihydrochloride and berbamine hydrochloride, which is applied in the field of application of berbamine dihydrochloride in the preparation of Ebola virus inhibitors, and can solve the problems of no specific therapeutic drugs and vaccines

Active Publication Date: 2020-07-03
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Currently, symptomatic and supportive treatments are mainly used for viral hemorrhagic fever, and there are no specific therapeutic drugs and vaccines that have been systematically and clinically proven effective

Method used

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  • Application of Berbamine Dihydrochloride in Preparation of Ebola Virus Inhibitor
  • Application of Berbamine Dihydrochloride in Preparation of Ebola Virus Inhibitor
  • Application of Berbamine Dihydrochloride in Preparation of Ebola Virus Inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. EBOV entry inhibitor screening model verifies that berbamine dihydrochloride can specifically inhibit EBOV activity.

[0047] Using cell-level recombinant virus technology, the GP of Zaire-EBOV was co-expressed with the HIV core plasmid (pNL4-3.Luc) to prepare recombinant viruses, and the high-throughput screening model of EBOV entry inhibitors targeting GP protein was used to evaluate the efficacy of compounds Antiviral activity. The specific steps are:

[0048] 293T cells were cultured, and after the cells filled the culture flask, the old medium was discarded and digested with a digestive solution containing 0.25% trypsin and 0.02% EDTA. When the cells become round, discard the digestion solution, immediately add high-sugar DMEM medium (GIBICO) containing 10% FBS (purchased from GIBCO), and gently blow the bottom of the bottle with a straw to completely separate the cells from the bottom of the bottle and disperse them into single cells. cell suspension....

Embodiment 2

[0055] Example 2. The antiviral activity of berbamine dihydrochloride has nothing to do with its cytotoxicity.

[0056] In order to exclude non-specific differences due to compound toxicity, the effect of berbamine dihydrochloride on the growth of 293T cells was evaluated using cell counting kit-8 (cellCounting Kit-8, CCK-8).

[0057] The CCK-8 kit is a kit for detecting cell proliferation, cell survival and cytotoxicity. It is a kit based on WST-8 (water-soluble tetrazolium salt, chemical name: 2-(2-methoxy-4-nitrophenyl )-3-(4-nitrophenyl)-5-(2,4-disulfobenzene)-2H-tetrazolium monosodium salt) is a widely used rapid and high-sensitivity detection kit, which is an alternative method for the MTT method, The water-soluble tetrazolium salt-WST-8 is used in the kit. In the presence of electron coupling reagents, it can be reduced by some dehydrogenases in the mitochondria to generate orange-yellow formazan. The more and faster the cell proliferation, the darker the color; the gr...

Embodiment 4

[0064] Example 4. Through the drug action time point experiment, it was determined that berbamine dihydrochloride acts on the entry stage of the virus.

[0065] The specific inhibitory effect of berbamine dihydrochloride on EBOV-Zaire GP / HIV-luc pseudoviruses suggests that they may act as EBVO entry inhibitors. In order to verify this point, the action stage of berbamine dihydrochloride in the virus infection cycle was studied through the Time of addition (TOA) experiment. The specific steps are:

[0066] One day before infection, 293T cells were divided into 6×10 4 Each cell / well was inoculated into a 96-well plate, and 50 μL of the EBOV-Zaire GP / HIV-luc of Example 1 were added to infect the cells. Berbamine dihydrochloride (dissolved in DMSO, in culture medium The content (final concentration) is 1×10 -5 mol L -1 ), with the EBOV entry inhibitor tetrandrine (tetrandrine, TET) (dissolved in DMSO, the content in the medium was 1×10 -7 mol L -1 ), the non-nucleoside reve...

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Abstract

The invention discloses the application of berbamine dihydrochloride in the preparation of Ebola virus inhibitors. The present invention takes the envelope glycoprotein (EBOV-GPcl) in the active state of Ebola virus as the target, and obtains an antiviral active compound with the ability to bind to EBOV-GPcl through structure-based virtual screening. The compound is small dihydrochloride beramine. Berbamine dihydrochloride can specifically inhibit the entry of Ebola recombinant virus by binding to the target protein EBOV-GPcl, and achieve the effect of resisting Ebola virus infection. The half-maximal effect concentration (EC50) of berbamine dihydrochloride against EBOV was 0.49 μM, indicating that berbamine dihydrochloride had a strong inhibitory effect on EBOV.

Description

technical field [0001] The invention relates to the application of berbamine dihydrochloride, in particular to the application of berbamine dihydrochloride in the preparation of Ebola virus inhibitors. Background technique [0002] Viral hemorrhagic fever is a group of natural foci diseases caused by viruses, with fever, hemorrhage and shock as the main clinical features. Such diseases are widely distributed in the world, with severe clinical manifestations and high fatality rate. At present, more than ten kinds have been found in the world. Common viral hemorrhagic fevers include Ebola hemorrhagic fever, Marburg hemorrhagic fever, Lassa fever, Crimea-Congo hemorrhagic fever, Rift Valley fever, dengue hemorrhagic fever, yellow fever and smallpox. [0003] Ebola hemorrhagic fever (Ebola virus disease) is an acute hemorrhagic infectious disease caused by Ebola virus (EBOV) of the Filoviridae family, with a mortality rate of up to 90%, and is the deadliest disease in humans O...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/472A61P31/14
CPCA61P31/14A61K31/472
Inventor 岑山李泉洁衣岽戎施一王寒周金明
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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