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Application of FRAX597 compound in preparation of medicine for treating Ebola virus disease

A technology of Ebola virus and compounds, applied in the direction of antiviral agents, pharmaceutical formulations, and resistance to vector-borne diseases, etc., to achieve the effect of reducing replication

Active Publication Date: 2022-08-09
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current effective prevention and control strategy against Ebola virus is still a world problem that needs to be solved urgently

Method used

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  • Application of FRAX597 compound in preparation of medicine for treating Ebola virus disease
  • Application of FRAX597 compound in preparation of medicine for treating Ebola virus disease
  • Application of FRAX597 compound in preparation of medicine for treating Ebola virus disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] In this example, the interaction between PAK2 and VP35 was confirmed by means of co-immunoprecipitation. VP35 is an important structural protein during Ebola virus replication.

[0025] The specific experimental methods are as follows:

[0026] (1) Construction of GFP-VP35 expression vector. The construction method is: search the VP35 gene sequence on the database NCBI, use primer5 to design VP35 amplification primers; PCR amplify the target fragment, use EcoRI and XhoI at 37 °C for double digestion, and agarose gel electrophoresis. for recycling. T4 ligase was used to react with pEGFP-C1 vector at 16°C for more than 2 hours to transform DH5α competent cells, and then the bacterial solution was evenly spread on solid LB medium containing antibiotics corresponding to the vector. After culturing at 37°C for 12 hours, a single colony was picked and cultured in 5 mL of liquid LB medium containing antibiotics, and the plasmid was extracted and sent to Beijing Nuosai Gene ...

Embodiment 2

[0031]In this example, the Ebola minimal genome was used to detect the changes of Ebola virus replication in cells after FRAX597 compound treatment of cells inhibited the activity of PAK2 in cells. This example uses the Ebola minimal genome to simulate the life cycle of Ebola virus under biosafety secondary conditions. In this system, a 4-cistronic minigenome (minigenome, MG) is formed by linking the Renilla luciferin reporter gene (RLUC) to the genome of the virus lacking NP, VP35, and L. When MG was co-transfected with NP, VP35, L and T7 polymerases into 293 cells, the virus could replicate and produce virus particles containing MG (without self-replication ability). When the virus replicates, RLUC is also expressed. Therefore, virus replication can be evaluated by detecting the value of RLUC by using a dual-luciferase detection kit. That is, the higher the RLUC value, the higher the virus replication level.

[0032] The experimental method is as follows:

[0033] On day...

Embodiment 3

[0036] In this example, CCK8 was used to detect the cytotoxicity of FRAX597 compounds.

[0037] The experimental method is as follows:

[0038] (1) Cell culture: inoculate 5×10 cells per well in a 96-well plate 3 HEK-293 cells, 100 μL of culture medium, 24 hours later, the DMSO solution of FRAX597 compound prepared at a predetermined concentration (10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0.0001 μM) was added. 8 repetitions);

[0039] (2) After culturing at 37°C for 48 hours, add 10 μL of CCK-8 solution to the culture plate;

[0040] (3) After co-incubating for 1 h in the incubator, use a microplate reader to measure the absorbance at 450 nm.

[0041] (4) By calculating the experimental group / control group*100%, the cell survival rate of the corresponding well can be obtained. The experimental results are as follows image 3 shown.

[0042] Among them, when the concentration of FRAX597 in the medium solution was 1 μM and below 1 μM, the cell viability was >100%. This show...

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PUM

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Abstract

The invention relates to application of an FRAX597 compound in preparation of a medicine for preventing Ebola virus infection or treating Ebola virus diseases. Researches show that after cells are treated with the FRAX597 inhibitor compound of serine / threonine kinase PAK2 (Gene ID: 5062), replication of the Ebola virus in the cells is remarkably reduced, and proliferation of the Ebola virus is inhibited. CCK8 is used for detecting the cytotoxicity of the FRAX597, and it is proved that the FRAX597 shows extremely low or even negligible cytotoxicity under the condition that the concentration is controlled. Therefore, the FRAX597 can be used as an active ingredient for preparing a candidate medicine for treating and resisting the Ebola virus disease.

Description

technical field [0001] The invention relates to the technical field of drug development, in particular to the application of a FRAX597 compound in the preparation of a drug for preventing Ebola virus infection or treating Ebola virus disease. Background technique [0002] Ebola virus disease (EVD) is a severe zoonotic infectious disease caused by the Ebola virus (EBOV), which has occurred in Africa since its discovery in 1976. The Ebola virus genome sequence is 3'-end non-coding region-NP-VP35-VP40-GP-VP30-VP24-L-5'-end non-coding region, which can encode nucleoprotein NP, virion protein VP35, matrix protein VP40, Glycoprotein GP, ​​VP30, VP24, RNA-dependent RNA polymerase L, a total of 7 structural proteins. The mortality rate of Ebola virus disease is about 50%-90%. . At present, effective prevention and control strategies for Ebola virus are still a world problem that needs to be solved urgently. SUMMARY OF THE INVENTION [0003] (1) Technical problems to be solved ...

Claims

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Application Information

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IPC IPC(8): A61K31/519A61K45/00A61P31/14
CPCA61K31/519A61K45/00A61P31/14Y02A50/30
Inventor 刘海楠刘萱曹诚张迅柏宇
Owner ACADEMY OF MILITARY MEDICAL SCI
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