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Application of PLpro protein inhibitor in medicine for treating or preventing novel coronavirus infection

A new type of coronavirus technology, applied in antiviral agents, drug combinations, pharmaceutical formulations, etc., can solve problems such as high risk of side effects

Pending Publication Date: 2022-07-05
PEKING UNIV SHENZHEN GRADUATE SCHOOL +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Common side effects include bone marrow suppression, liver damage, and oral inflammation, and people with thiopurine methyltransferase deficiency have a higher risk of side effects

Method used

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  • Application of PLpro protein inhibitor in medicine for treating or preventing novel coronavirus infection
  • Application of PLpro protein inhibitor in medicine for treating or preventing novel coronavirus infection
  • Application of PLpro protein inhibitor in medicine for treating or preventing novel coronavirus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Induced expression and purification of PLpro protein

[0053] The PLpro prokaryotic expression plasmid (see SEQ ID NO.1 for the expressed PLpro protein amino acid sequence) was transformed into BL21 (DE3) E. coli competent cells, and grown in LB medium at 37°C until OD600 was 0.6-0.8 Time. Protein expression was induced by the addition of 0.5 mM IPTG and 1 mM zinc chloride (ZnCl 2 ), and then E. coli was grown overnight at 18°C. After collection by centrifugation, the cell pellet was resuspended with 50 mM Tris-HCl, 150 mM NaCl, 10 mM Imidazole, 2 mM DTT, and pH=8.5 buffer, disrupted by sonication, and centrifuged at 18,000 rpm to take the supernatant. Purified on a His-TRAPTM column and finally eluted with buffer (50 mM Tris-HCl, 150 mM NaCl, 250 mM Imidazole, 1 mM DTT, pH 7.4) and further purified on a gel chromatography column (Superdex 20016 / 60, GE) , the SEC buffer was 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, pH 7.4. The purified PLpro protein was conc...

Embodiment 2

[0054] Example 2: In vitro assay to measure the activity of thioguanine (6-TG) in inhibiting PLpro protein (IC50)

[0055]The reaction system was 50 μL, wherein the concentration of PLpro was 30 nM, the concentration of substrate Ub-AMC (Boston Biochem) was 250 nM, and the reaction buffer was 50 mM Hepes (pH=7.5), 0.01% TrixtonX-100, 0.1 mg / ml BSA and 2 mM DTT. A gradient concentration (100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 μM) of inhibitor thioguanine (6-TG) was added, and then the fluorescence emission intensity was measured using a microplate reader (excitation: 340 nm; emission: 430 nm) ), the activity of thioguanine (6-TG) to inhibit PLpro enzyme (IC50) was calculated according to the fluorescence intensity, and the inhibition curve was fitted using GraphPad Prism. For fitting results, see figure 2 .

Embodiment 3

[0056] Example 3: P3 laboratory tested the inhibitory activity of thioguanine (6-TG) on the new coronavirus

[0057] (1) Compound cytotoxicity test:

[0058] Will 1x10 4 Vero cells (from: VERO cells from the Cell Resource Center, Shanghai Academy of Biological Sciences, Chinese Academy of Sciences) were seeded in a 96-well plate, set up three duplicate wells, grown at 37 degrees for 20-24 hours, removed the medium, and added 100 μL containing different concentrations. Compounds (100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0 μM) were grown in new medium for 72 hours, with DMSO as a control, using MTS / CCK8 reagent (the above reagents were purchased from: Beijing Soleibao Technology Co., Ltd., Cat. No. CK04-500T), cell viability was measured, and compound cytotoxicity (CC50) was calculated.

[0059] (2) Compound virus inhibition activity test:

[0060] Will 1x10 4 Vero cells were seeded in 96-well plates, three replicate wells were set up, and they were grown at 37°C for 20-24 hour...

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Abstract

The invention relates to application of a PLpro protein inhibitor to preparation of a medicine for treating or preventing novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection related diseases, and provides a pharmaceutical composition which contains the PLpro protein inhibitor and is used for preparing the medicine composition for treating or preventing the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection related diseases.

Description

technical field [0001] The present invention relates to the use of PLpro protein inhibitors in the preparation of medicines for treating or preventing new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-related diseases, and provides a PLpro inhibitor for preparing new drugs for treating or preventing new types of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Pharmaceutical compositions for diseases associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Background technique [0002] Coronaviruses are a class of positive-strand RNA viruses that can pose significant health risks. Coronaviruses are genetically divided into four main virus genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. The first two genera mainly infect mammals, while the latter two mainly infect birds. Before the discovery of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7076A61P11/00A61P31/14
CPCA61P31/14A61K31/7076A61P11/00
Inventor 黄昊张国良汤金乐刘淑燕付子阳刘明
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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