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3CLpro protease inhibitor and antiviral application thereof

A protease inhibitor and anti-virus technology, applied in the field of medicine, can solve the problems of high specificity and no discovery, and achieve the effect of strong binding affinity and strong anti-new coronavirus effect

Pending Publication Date: 2022-04-12
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no small molecule 3CLpro protease inhibitor with good curative effect and high specificity has been found

Method used

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  • 3CLpro protease inhibitor and antiviral application thereof
  • 3CLpro protease inhibitor and antiviral application thereof
  • 3CLpro protease inhibitor and antiviral application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Enzyme activity evaluation system confirms that acriflavine, proflavin sulfate, acriflavine acetate, and acriflavine succinate inhibit coronavirus 3CLpro protease activity

[0044] 1. Protein expression and purification of coronavirus 3CLpro protease;

[0045]For the detailed steps of expression and purification of coronavirus 3CLpro protease (NCBI ID 43740578), refer to the methods disclosed in the literature (DOI: 10.1126 / science.1085658). The gene sequence encoding amino acids 3264-3569 of coronavirus 3CLpro protease was cloned into the pGEX-6p-1 vector with N-terminal secretion signal peptide and C-terminal His tag. The expression plasmid was transformed into competent cells E.coli BL21(DE3) Condon Plus, spread evenly on a solid plate with ampicillin resistance, and cultured overnight at 37°C. A small amount of bacteria was first cultivated by using bacterial plaque, and then a large amount of bacteria was cultured. Before the culture, corresponding antibiotics wer...

Embodiment 2

[0054] Determine the drug that significantly inhibits the activity of 3CLpro protease by the above-mentioned examples, and then further detect its binding affinity with coronavirus 3CLpro protease, with the acetate of acriflavine, proflavin sulfate, acriflavine, acriflavine Take the succinate of pyriflavin as an example, specifically:

[0055] 1. Expression and purification of coronavirus 3CLpro protease

[0056] The method is the same as the expression and purification of the coronavirus 3CLpro protease in Example 1.

[0057] 2. Determination of the binding affinity of acriflavine, proflavin sulfate, acetate of acriflavine, succinate of acriflavine and coronavirus 3CLpro protease by microthermophoresis

[0058] Micro-thermophoresis (MST) is a technique based on fluorescence detection and thermophoresis for detecting protein-protein or protein-small molecule interactions. To further validate the results of the enzyme activity assay, the binding affinity between acriflavine, ...

Embodiment 3

[0064] Anti-2019-nCoV activity experiment of acriflavine, proflavin sulfate, acetate of acriflavine, and succinate of acriflavine.

[0065] 1. Determination of virus titer

[0066] MDCK cells were seeded in a 96-well culture plate and cultured to a monolayer for experiments. The culture solution containing 10% serum in the original cell culture plate was removed, and the virus was -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 and 10 -8 The dilution of the above-mentioned solution was added to the cell plate, and after adsorption at 35°C for 30 minutes, the cell maintenance solution was added, and a normal cell control was set up at the same time, and cultured at 35°C. Observe the lesions of the cells every day and record the results. Calculate TCID 50 .

[0067] 2. Determination of sample cytotoxicity

[0068] Select the virus-susceptible cell MDCK, add different concentrations of samples (μg / mL), culture for 7 days, observe the cytotoxicity every day, use the R-M f...

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Abstract

The invention discloses a 3CLpro protease inhibitor and antiviral application thereof, and belongs to the technical field of medicines. The structural general formula of the 3CLpro protease inhibitor is shown in the specification, wherein R is one or combination of more of unsubstituted C1-C4 alkyl, acid radical of organic acid or inorganic acid and organic acid or inorganic acid. The 3CLpro protease inhibitor is used for preparing antiviral drugs, especially anti-coronavirus drugs. Based on an enzymatic activity evaluation system and an MST binding force determination test, the 3CLpro protease inhibitor has relatively strong binding affinity with 3CLpro protease, can directly inhibit the enzymatic activity of the 3CLpro protease, shows a very strong effect on resisting viruses containing 3CLpro protease at a cellular level, and can be used for preparing drugs for resisting viruses containing 3CLpro protease.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a 3CLpro protease inhibitor and its antiviral application. Background technique [0002] 3CLpro protease is also called 3C-like protease (3CLpro), because the specificity of its cleavage site is very similar to that of picornavirus 3C protease (3C protease), and both are called 3CLpro protease. Viral RNA is initially translated into a polyprotein after entering human cells, and 3CLpro protease can cut 12 smaller proteins from the polyprotein, and these proteins will participate in the replication of viral RNA. Since 3CLprotease is an important protease, it plays a vital role in the process of inhibiting virus replication, and there is no homologous protein in the human body, so 3CLprotease is an ideal target for antiviral drug development. In addition, 3CLprotease It is highly conserved in β-coronavirus, and the screened 3CLpro protease inhibitor has a certain degre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/473A61P31/14
Inventor 陈丽霞李华邹鹏巫国谊
Owner SHENYANG PHARMA UNIVERSITY
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