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Application of small molecule compound in anti-African swine fever virus infection

A technology of African swine fever virus and compounds, applied in antiviral agents, medical preparations containing active ingredients, organic active ingredients, etc., can solve the problems of lower ASFV amplification efficiency, achieve the effect of inhibiting replication and high application value

Active Publication Date: 2020-10-20
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The dUTPase encoded by ASFV is called E165R protein, which plays a very important role in the efficient replication of ASFV in porcine macrophages. Studies have shown that knocking out E165R leads to a significant decrease in the amplification efficiency of ASFV in alveolar macrophages

Method used

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  • Application of small molecule compound in anti-African swine fever virus infection
  • Application of small molecule compound in anti-African swine fever virus infection
  • Application of small molecule compound in anti-African swine fever virus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Expression and purification of ASFV-E165R protein

[0032] The full-length DNA sequence of the E165R protein of the Pig / HLJ / 18 virus strain (GenBank accession number is CBW46796.1) was connected to the pET-21a vector through the restriction sites NdeI and XhoI. Wherein the 3' end of the ASFV-E165R protein coding region has added 6 histidine tags (His 6 -tag) coding sequence and translation stop codon. After the ASFV-E165R expression vector was constructed, the BL21 Escherichia coli competent cells were transformed. Pick a single clone and inoculate it into 50mL LB medium, and after 12 hours of shaking culture, transfer 20ml of the bacterial liquid into 2L of LB medium, and cultivate to OD at 37°C 600 = 0.6-0.8, add IPTG to a final concentration of 0.5 mM, and continue culturing at 16°C for 16 hours. After expression, the bacterial cells were collected, resuspended with protein buffer (20 mM Tris, 150 mM NaCl, pH 8.5), and crushed at low temperature and ...

Embodiment 2

[0033] Example 2: Virtual screening of small molecule inhibitors targeting the ASFV-E165R enzyme active center structure

[0034] The ASFV-E165R protein purified in Example 1 was mixed and co-crystallized with the substrate dUTP, and its high-resolution protein molecular structure was analyzed. As a result, the complex crystal structure of ASFV-E165R and the product dUMP was obtained. The complex structure is as follows: figure 2shown. The crystal structure analysis of the complex clarified the enzymatic activity center of E165R to exert hydrolytic activity, and then the anti-ASFV small molecule drug was obtained by virtual screening with the structure of the E165R enzymatic activity center as a target. Firstly, a drug library containing 41,384 small molecules was obtained from TargetMol, Selleck and MCE. The drug library went through initial processing, drug-like "five" principle screening, PAINS filtering, molecular docking, docking score analysis, molecular diversity analy...

Embodiment 3

[0038] Example 3: Virtual Screening Drugs Inhibit E165R Enzyme Activity and ASFV Replication

[0039] (1) Inhibition of E165R enzyme activity by 1,2,3,4,6-O-galloylglucose (Pentagalloylglucose)

[0040] The 52 small-molecule drugs screened in Example 2 were verified to confirm whether they had dUTPase inhibitory activity, and the production of PPi was detected by in vitro enzyme activity inhibition experiments to determine the ability of small-molecule drugs to inhibit ASFV E165R from hydrolyzing dUTP.

[0041] First draw the standard curve of pyrophosphoric acid (PPi): ① Dilute the 100×PPi stock solution at a ratio of 1:100 to 1×PPi working solution. ② Add 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 μl of standard PPi working solution (1 mM) to 10 EP tubes in sequence, make up to 800 μl with deionized water, and mix well. ③ Add 50 μl molybdic acid solution to each tube in turn, mix well; 100 μl sulfurous acid A solution, mix well; 50 μl mercapto solution, mix well. ④Let stand ...

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Abstract

The invention discloses application of a small molecule compound in anti-African swine fever virus ( ASFV) infection, and belongs to the technical field of medicine. According to the invention, an enzyme active center of E165R to exert hydrolytic action is defined by carrying out a crystal structure analysis of an E165R-dUMP complex, and then 1,2,3,4,6-O-Pentagalloylglucose capable of matching theE165R enzyme active center and significantly inhibiting E165R enzyme activity is obtained through in vitro screening by using the enzyme active center of E165R as a target. Further virus level experiments show that the 1,2,3,4,6-O-Pentagalloylglucose can significantly inhibit the replication of the ASFV in porcine alveolar macrophages, with high application value in clinical treatment or prevention of ASFV infection.

Description

technical field [0001] The invention relates to the application of small molecule compounds in anti-African swine fever virus infection, belonging to the technical field of medicine. Background technique [0002] African swine fever (ASF) is a highly fatal disease caused by African swine fever virus (ASFV) infection that seriously harms the pig industry and poses a serious threat to the world economy and food security. The ASF epidemic first broke out in Kenya in 1921, and then became popular in some countries and regions in Africa. In recent decades, ASFV has spread rapidly from Africa to Europe and Asia, and is showing a trend of further spread. As a type of enveloped double-stranded DNA virus, ASFV has a complex and unique multilayer membrane structure, which consists of core, nucleocapsid, inner membrane, capsid and outer membrane from inside to outside. The diameter of the enveloped ASFV virus particle is about 250nm, the genome length is 170-194kb, and it can encode ...

Claims

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Application Information

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IPC IPC(8): A61K31/7024A61P31/20
CPCA61K31/7024A61P31/20
Inventor 高福宋豪李长尧施一苏佳岐翁长江黄丽
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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