Method and screening model for screening small-molecule inhibitor of main protease of new coronavirus

A small molecule inhibitor and main protease technology, applied in the field of medicine and biology, can solve the problems of long screening period, limited application, cumbersome operation, etc., and achieve the effect of sensitive detection, easy operation and low cost

Pending Publication Date: 2021-11-26
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Abstract
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  • Application Information

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Problems solved by technology

However, the above-mentioned screening methods generally have shortcomings such as high false positive rate, high screening cost, cumbersome operation, poor stability, and long screening cycle, which greatly limit their application in large-scale high-throughput screening (Qi Haiyan, et al. Chemistry, 2021)

Method used

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  • Method and screening model for screening small-molecule inhibitor of main protease of new coronavirus
  • Method and screening model for screening small-molecule inhibitor of main protease of new coronavirus
  • Method and screening model for screening small-molecule inhibitor of main protease of new coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Prokaryotic expression and isolation and purification of the new coronavirus Mpro recombinant protein

[0038] Using Escherichia coli prokaryotic expression technology, the codon-optimized Mpro gene ( figure 1) was connected to the pET-21a (+) expression vector to construct the recombinant plasmid Mpro-pET-21a. Then the recombinant plasmid was transformed into E. coli Rosetta (DE3) competent cells, and the recombinants were screened by ampicillin resistance. The recombinants were inoculated into 1 liter of LB liquid medium (containing 100 μg / mL ampicillin), cultured at 37° C. for 7 hours, added with 0.2 mM IPTG, and induced at 30° C. for 8 hours. After the cells are disrupted by ultrasonic method, the lysed supernatant is separated and purified by HisTrap affinity chromatography column. The purified Mpro has an apparent molecular weight of 34kDa, and only one formylmethionine remains at the amino terminal, and polyhistidine is fused to the carboxyl terminal....

Embodiment 2

[0045] Example 2. Biological Activity Identification of New Coronavirus Mpro Recombinant Protein

[0046] 1. Dilute 10 μM MCA (7-methoxycoumarin, 7-methoxycoumarin-4-acetic acid, MCA) in TBS solution (50mM Tris, 150mM NaCl pH8.0) to 5 concentrations by 2 times For gradient, the wells containing only TBS solution were used as negative control wells. The above MCA dilution was added to a 384-well plate at 50 μL / well, and the total amount of MCA in each well was 0, 31.25, 62.5, 125, 250, and 500 pmol, and the automatic gain mode was set to detect the relative fluorescence intensity with a multi-functional microplate reader Value (relative fluorescence unit, RFU). According to the total amount of MCA in each well and the ΔRFU value (ΔRFU=RFU MCA -RFU 0 ) to fit the regression equation, draw the MCA fluorescence intensity standard curve ( figure 2 B).

[0047] 2. Add 2mM MCA-Substrate (MCA-AVLQSGFR-Lys(Dnp)-Lys-NH 2 ) was diluted to 10 μM with TBS solution, and Mpro was adde...

Embodiment 3

[0050] Example 3. The hydrolysis of the new coronavirus Mpro recombinant protein to the FITC-S-Biotin substrate

[0051] Dilute 2mM FITC-S-Biotin to 60nM with fluorescence polarization reaction solution (10mM Tris, 50mM NaCl, 1mM DTTpH8.0), add 20μL / well into a 384-well plate, and then add 0, 25, 50, 100, 150, 175, 200, 300, 400, 500, 600, 700nM Mpro, 30μL per well, set 3 sets of duplicate wells for each group, incubate at room temperature for 20min, then add 300nM avidin reaction solution at 10μL / well, incubate at room temperature in the dark After 5 min, the mP value was detected with a multifunctional microplate reader.

[0052] The Mpro hydrolysis reaction curve shows that 200nM Mpro can fully hydrolyze the FITC-S-Biotin substrate into the product FITC-AVLQ( image 3 ).

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Abstract

The invention relates to a method and a screening model for screening a small-molecule inhibitor of main protease (Mpro) of a new coronavirus. The method and the screening model are based on the basis of a fluorescence polarization principle, a fluorescent probe FITC-Substrate-Biotin is used as a hydrolysis substrate of the new coronavirus Mpro, avidin is used for terminating the hydrolysis reaction of the new coronavirus Mpro, and a multifunctional microplate reader is used for detecting the millipolarization units (mP) of an experimental system. The active compound shows a high mP value in the screening model, and the inactive compound shows a low mP value.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and specifically relates to a method and a screening model for screening small molecule inhibitors of the main protease of the new coronavirus. Background technique [0002] The rapid development of new coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) vaccines, humanized antibodies and anti-new coronavirus drugs has become a major scientific problem facing the scientific community. Although significant progress has been made in the development of a new coronavirus vaccine, safe and effective broad-spectrum anti-new coronavirus drugs are also crucial for unvaccinated individuals or high-frequency gene mutations of the virus that lead to reduced vaccine protection (WangZ, et al. Nature, 2021). Although some candidate antiviral drugs have been used in clinical treatment, such as remdesivir, favipiravir, ribavirin, telbivudine, etc., they have shown limited or ineffectiv...

Claims

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Application Information

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IPC IPC(8): C12Q1/37C12N9/50C12N15/70C12N15/57
CPCC12Q1/37C12N9/506C12N15/70
Inventor 陈云雨司书毅张晶闫干干李东升李妍许艳妮陈明华刘超李顺旺王晨吟
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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