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Application of oridonin and/or prodrug thereof in preparation of medicine for inhibiting SARS-CoV-2

A technology of oridonin and sars-cov-2, which is applied in the application field of medicine to achieve the effect of reducing activity and inhibiting RNA replication

Inactive Publication Date: 2021-12-31
SUN YAT SEN UNIV SHENZHEN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, Rubescensine A also has strong anti-tumor activity, and has a significant inhibitory effect on the proliferation of more than 20 human cancer cell lines, such as common esophageal cancer, lung cancer and liver cancer, etc., as disclosed in patent CN201210011120.2 The therapeutic effect of oridonin on liver cancer, lung adenocarcinoma and pancreatic cancer, but there is no relevant research on the effect of oridonin on new coronavirus pneumonia

Method used

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  • Application of oridonin and/or prodrug thereof in preparation of medicine for inhibiting SARS-CoV-2
  • Application of oridonin and/or prodrug thereof in preparation of medicine for inhibiting SARS-CoV-2
  • Application of oridonin and/or prodrug thereof in preparation of medicine for inhibiting SARS-CoV-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Construction and protein expression and purification of Mpro protease fusion plasmid in Example 1 SARS-CoV-2

[0038] 1. Codon optimization:

[0039] Select the Mpro protease gene in SARS-CoV-2, with a full length of 918bp; analyze its gene sequence according to the general genetic code table and the codon preference of E. The codons preferred by Bacillus replace the codons with different preferences in the Mpro protease gene, and the codon-optimized Mpro protease gene sequence is designed and chemically synthesized. The coding sequence is shown in SEQ ID NO.1.

[0040] 2. Primer design:

[0041] Design primers F1 and R1 to amplify the codon-optimized Mpro protease gene, design primers F2 and R2 to amplify pET-20b: primer F1 is shown in SEQ ID NO.2, primer F2 is shown in SEQ ID NO.3, R1 is the reverse complementary sequence of F2, and R2 is the reverse complementary sequence of F1.

[0042] Primer sequence Primer F1 (SEQ ID NO.2) 5′-GATATACATATGAGC...

Embodiment 2

[0058] Example 2 Effect of Rubescensine A on Fluorescent Thermal Shift of Mpro Protease in SARS-CoV-2

[0059] Mpro protease was diluted to a final concentration of 0.2 mg / mL with reaction buffer (20 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM DTT), and 100 μM oridonin and 4×SYPRO orange fluorescent dye (purchased (from Sigma-Aldrich Company), the final reaction volume was 20 μL, the mixture was incubated at 25°C for 10min, and then heated from 25°C to 95°C at a heating rate of 1°C / min; only 0.2mg / mL of Mpro protease and 4× The mixture of SYPRO orange fluorescent dye was the negative control group. Three replicate wells were set up for each experiment, and the experiment was repeated 3 times. Use the StepOnePlus real-time PCR instrument (purchased from Life Technologies) to record the fluorescence intensities of the two groups, and explore the effect of oridonin on the Mpro protease fluorescence heat in SARS-CoV-2 by fluorescence thermal shift analysis (Fluorescence-based Thermal sh...

Embodiment 3

[0060] Example 3 Oridonin A inhibits Mpro protease in SARS-CoV-2 in vitro

[0061] (1) Mpro protease substrate design:

[0062]Known Mpro protease hydrolysis substrate peptide sequence SEQ ID NO.4: -KTSAVLQSGFRKME-(-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu-), The Edans fluorescent group was added to the N-terminal of the peptide chain, and the Dabcyl fluorescent quenching group was added to the C-terminal of the peptide chain to finally synthesize Edans-KTSAVLQSGFRKME-Dabcyl (synthesized by Jill Biochemical (Shanghai) Co., Ltd.).

[0063] (2) Detection of the inhibitory effect of oridonin on Mpro protease by fluorescence resonance energy transfer method:

[0064] Gradient concentrations of oridonin (0μM, 0.12μM, 0.47μM, 1.87μM, 3.75μM, 7.5μM, 15μM, 60μM) were added to the above enzyme activity experiment, and reaction buffer (20mM Tris-HCl pH8.0 , 150mM NaCl, 1mM DTT) to dilute Mpro protease to a final concentration of 0.5μM, and at the same time add 50μM of t...

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Abstract

The invention provides application of oridonin and / or a prodrug thereof in preparation of a medicine for inhibiting SARS-CoV-2. The oridonin can target Mpro protease in the SARS-CoV-2 and significantly inhibit the enzyme activity, effectively inhibits the RNA replication of the SARS-CoV-2 and reduces the activity of the SARS-CoV-2, it is indicated that the small molecule oridonin can be used for treating or preventing the novel coronavirus pneumonia infected by the SARS-CoV-2 and an efficient, safe and novel natural medicine source without toxic and side effects is provided for treatment of novel coronavirus pneumonia.

Description

technical field [0001] The invention belongs to the field of antiviral drugs. More specifically, it relates to the application of oridonin and / or its prodrug in the preparation of drugs for inhibiting SARS-CoV-2. Background technique [0002] Novel coronavirus disease 2019 (COVID-19) refers to the pneumonia caused by the infection of 2019 novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2). Therefore, the research and development of anti-new coronavirus pneumonia drugs is very critical to curb the development of the virus, control the epidemic of the virus, and avoid the ravages of COVID-19. [0003] At present, the drugs for the treatment of new coronary pneumonia are mainly antiviral drugs, and there is currently no specific anti-SARS-CoV-2 virus drug, and α-interferon, ribavirin, lopinavir, ritonavir, and phosphoric acid are commonly used in clinical practice. Chloroquine, Arbidol and other chemical drugs are used for antiviral treatment. Ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/352A61K36/53A61P31/14A61P11/00
CPCA61K31/352A61K36/53A61P31/14A61P11/00
Inventor 孙力涛钟柏森杜珊
Owner SUN YAT SEN UNIV SHENZHEN
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