CN114146072A

CN114146072A - New application of thymoquinone as coronavirus broad-spectrum inhibitor

Info

Publication number: CN114146072A
Application number: CN202010915362.9A
Authority: CN
Inventors: 徐欢; 严欢; 翟丽丽; 周美玲; 葛琳; 刘艳玲; 苏国庆; 王鑫; 尹福涛; 魏敬双; 朱秀良; 高健
Original assignee: North China Pharmaceutical New Drug R&d Co ltd; Wuhan University WHU
Current assignee: North China Pharmaceutical New Drug R&d Co ltd; Wuhan University WHU
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2022-03-08

Abstract

The invention discloses a new application of thymoquinone, which is used as a broad-spectrum inhibitor of coronavirus for treating 2019 novel coronavirus, Severe Acute Respiratory Syndrome (SARS) and common cold caused by human coronavirus NL-63(HCoV-NL 63). Thymoquinone can bind to the coronavirus receptor ACE2, affecting the virus-binding receptor, thereby blocking the virus from entering cells. The thymoquinone is used as an old medicine for new use, has no problem in safety, and therefore can be clinically applied.

Description

New application of thymoquinone as coronavirus broad-spectrum inhibitor

Technical Field

The invention belongs to the field of medicines, relates to a new application of thymoquinone, and particularly relates to an application of thymoquinone in treatment of coronavirus infection.

Background

Highly pathogenic coronavirus infection has become a public health concern over the last decade. Severe acute respiratory syndrome (SARS, 2002-2004), middle east respiratory syndrome (MERS, 2012-to-date), 2019-nCoV (COVID-19), each of which has a tremendous impact on human health and economic development.

Because there is no effective drug and vaccine, the research and development of new medicine of new coronavirus is significant, in the war without smoke, several scientific research institutions and pharmaceutical companies are dedicated to research and develop new coronavirus vaccine and therapeutic drug, so the invention has significant social and economic significance.

Thymoquinone, is a natural product extract used in the adjuvant treatment of upper respiratory tract infections.

Thymoquinone is a natural product isolated from nigella sativa. Thymoquinone has antioxidant, antiinflammatory, anticancer, antitumor activity and hepatoprotective effects. Nigella sativa Linn, having the name of Black Cummin, belonging to the genus Nigella of the family Ranunculaceae, is mainly distributed in the Mediterranean region, and also grows in Saudiabar, North Africa and Asian parts, and is distributed in Yunnan China, Xinjiang, etc. The nigella sativa has been used as a medicinal and edible plant in folk over 2000 years. The dried seeds can be used as auxiliary materials in bread and baked food, and can also be used in sauce for increasing flavor. As a medicine, it is often used for the adjuvant treatment of upper respiratory tract infections. Modern researches show that the nigella sativa seeds have the effects of dilating bronchus, resisting bacteria, stabilizing blood pressure and regulating bile. Record of Chinese herbage, the black grass has major functions: replenishing qi and nourishing the heart; dispel wind and relieve cough. Major palpitations; insomnia; asthenia; common cold due to wind-cold; and (5) cough.

Italic (980-. Nigella sativa seed oil containing thymoquinone has a preventive effect on depressive behavior, and can also reduce anxiety by increasing the level of serotonin (a neurotransmitter, a natural mood stabilizer) in the brain, thereby increasing mental energy level and mood level. The black grass seeds also have application potential in the aspect of relieving sleep. Long-term studies have found that regular ingestion of nigella sativa oil can help to eliminate sleep disorders, provide better sleep and complete sleep cycles. The underlying mechanism of the effect of nigella sativa seed oil in sleep may be due to its ability to enhance acetylcholine in the brain during the sleep cycle, as research results indicate that acetylcholine levels rise during sleep.

Disclosure of Invention

The purpose of the present invention is to provide an effective inhibitor for a coronavirus including a 2019 novel coronavirus (COVID-19), which can prevent and treat a coronavirus infection including a 2019 novel coronavirus. The invention provides a new application of thymoquinone, which is an effective inhibitor of coronavirus infection including 2019 novel coronavirus, and an application of thymoquinone in preparation of a medicament for preventing or treating coronavirus infection.

The application, wherein the coronavirus comprises 2019 novel coronavirus (COVID-19), Severe Acute Respiratory Syndrome (SARS) virus and common cold human coronavirus NL63(HCoV-NL 63).

The application, wherein the coronavirus is 2019 novel coronavirus (COVID-19).

The use wherein the coronavirus infection is coronavirus pneumonia or common cold.

The application is that the coronavirus infection comprises 2019 novel coronavirus COVID-19 pneumonia, severe acute respiratory syndrome SARS, human coronavirus NL63 pneumonia and common cold.

The use of (b), wherein the coronavirus infection is 2019 novel coronavirus pneumonia.

The inventor finds a novel application of thymoquinone through a large number of researches, wherein the thymoquinone can be combined with a coronavirus receptor ACE2 so as to block viruses from entering cells, and the thymoquinone is EC (endothelial cell death) at a cell level₅₀＝4.999uM,CC₅₀＝35.10uM。

The compounds of the present invention may be formulated into various suitable pharmaceutical preparation forms. Can be used alone or mixed with medicinal adjuvants (such as excipient, diluent, etc.) to make into oral tablet, capsule, granule or syrup, or powder for injection or solution.

Compared with the prior art, the invention has the following technical effects:

at the cellular level, thymoquinone can bind to a coronavirus receptor ACE2 to prevent viruses from entering cells, so that the aims of treating coronavirus pneumonia infection and common cold and inhibiting the EC50 ═ 4.999uM of SARS-CoV-2 pseudovirus are achieved.

Drawings

FIG. 1 ACE2& thymoquinone binding sites

Fig. 1 shows AI docking results: binding site for thymoquinone and ACE2

Thymoquinone conjugated ACE2(pdb:6vw1)

Thymoquinone docks to ACE2 protein, with the dotted line indicating hydrogen bonding

FIG. 2 ACE2& thymoquinone SPR sensorgram

FIG. 2 shows the affinities of thymoquinone and 2019 for the novel coronavirus receptor ACE2

FIG. 3 EC of thymoquinone in inhibiting SARS-CoV-2 pseudovirus₅₀And CC₅₀

FIG. 3 shows the determination of replication-deficient pseudovirus thymoquinone EC50 of VSV-dG-fLuc for the outer membrane of the novel coronavirus (SARS-CoV-2).

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The technical scheme of the present invention will be further described below with reference to specific experimental operations and data.

The inhibitory effect of thymoquinone on individual subtypes of coronavirus is demonstrated by the following assay. Sources of thymoquinone: thymoquinone powder was purchased from alatin, cat # (T115128).

Example 1AI predicted binding site

The ACE2 protein (PDB:6vw1) was downloaded from PDB (http:// www.rcsb.org /). All hetero atoms and ACE2 ligands were removed. The Mpro docking grid was maximized for thymoquinone docking. The PDB file is converted to macromolecules in the PDBQT format prior to virtual screening. The position of the grid (ligand docking search space) is as described above. Then, Autodock Vina 1.1.2 was used for subsequent molecular docking. Protein-ligand interactions can be observed using Pymol version 1.7.4.5. ACE2 protein proximity hit ligand

Are highlighted as potential interaction residues involved in protein-ligand interactions. The results are shown in FIGS. 1-3.

The experimental results are as follows:

1. the binding sites for thymoquinone to the ACE2 protein are ILE291 and PHE 438.

The results are shown in FIG. 1

Example 2 experiment of SPR protein, the coronavirus receptor ACE2

Experimental materials and instruments

biacore T200(GE healthcare, Uppsala, Sweden), CM5 chip (GE healthcare, Uppsala, Sweden), thymoquinone (alatin), ACE2 his tag protein (novoprotein).

Model was biacore T200 from GE corporation, ACE2 protein (novoprotein) was coupled to CM5 chips via amino groups in an amount of 15861.6RU, respectively, and affinity of thymoquinone and ACE2 protein at 25 ℃ was measured. Thymoquinone was injected at a flow rate of 30. mu.L/min at concentrations of 100uM,50uM,25uM,12.5uM,6.25uM,3.125uM, 1.5625uM,0uM for a sample injection time of 180s and a dissociation time of 300 s. The data analysis mode is steady state analysis. The results are shown in FIG. 2. The SPR results are summarized in table 1 below.

TABLE 1 affinity of thymoquinone for binding to the coronavirus receptor ACE2 protein at 25 deg.C

Protein	KD(M)	Rmax(RU)	Chi²(RU²)	chi
					ACE2	3.214*10^-5	11.58	0.328	0.573

Example 3

Replication-deficient pseudovirus infection assay of VSV-dG-fLuc of the outer Membrane of the novel coronavirus (SARS-CoV-2) — assay of the Effect of thymoquinone to inhibit the entry of Coronavirus into cells

Experimental Material

The glycoprotein (G) of VSV was replaced by GFP fluorescent protein in VSV-dG-GFP. A pseudoviral packaging system for VSV-dG-fLuc (Firefo luciferase) Vesicular Stomatitis Virus (VSV) was obtained from reverse genetics plasmid rescue (Rescue), and the relevant plasmids were purchased from Karafast. Wherein pVSV- Δ G-Luciferase is used for transcription replication-deficient recombinant VSV genomes, wherein the glycoprotein (G) of VSV is replaced by Firefly Luciferase (fLuc), and pBS-N, pBS-P, pBS-L, pBS-G is used as an auxiliary vector to aid in first round of virus rescue.

Rescue and amplification of VSV-dG-fLuc replication-defective viruses containing the VSVG outer Membrane

BHK21 cells were attached to 3.5cm dish, and the next day when the cells reached 90% density, and infected with a poxvirus recombinantly expressing T7 RNA polymerase (Vaccinium virus-T7, vv-T7) at a titer of MOI ═ 5 (DMEM, serum-free) for 45 minutes at 37 ℃. After washing the cells once with PBS, the cells that had been infected with vv-T7 were transfected with the mixed reverse genetics plasmid (pVSV-. DELTA.G-Luciferase: pBS-N: pBS-P: pBS-G: pBS-L ═ 5:3:5:8:1) transfected with Lipo3000, the solution was changed after 12 hours, the supernatant containing the primary VSV-dG-fLuc virus was collected after 48 hours, and the residual vv-T7 was removed by filtration through a 0.22 μm filter. To further amplify the virus, HEK293T cells were transfected with pMD2.G overexpressing VSV G protein (ATCC: Plasmid #12259), 24 hours later, primary supernatants were combined with DMEM + 10% serum 1: 1 for infection, collecting the supernatant after 24-48 hours, centrifuging at 12000 rpm for 2 minutes, retaining the supernatant and packaging, determining the virus titer by plaque method, and storing at-80 ℃.

Packaging of VSV-dG-fLuc replication-deficient pseudovirus containing novel coronavirus (SARS-CoV-2) outer membrane

HEK293T cells or Vero-E6 cells were transfected with plasmids (pCAGGS-SARS2-S-dc) overexpressing the S outer membrane protein of SARS-CoV-2 (18 amino acids truncated at the C-terminus, increasing the packaging efficiency). After 24 hours, the transfected cells were infected with VSV-dG-ffluc virus containing the VSVG outer membrane at MOI ═ 10 (1:30 dilution) for 1 hour at 37 ℃, followed by addition of a solution containing 1: 300 dilutions of rat antiserum against VSVG (CharningSha laboratories) in DMEM + 10% FBS medium were used to completely neutralize residual VSV-dG-fLuc pseudovirus containing the VSVG outer membrane. The virus-containing supernatants were harvested after 24-48 hours, centrifuged at 12000 rpm for 2 minutes, retained and aliquoted, assayed for viral titer by CPE using limiting dilution (Infections Unit) and stored at-80 ℃.

Determination of half effective concentration of drug by infection with SARS-CoV-2 pseudovirus based on VSV (EC50)

BHK21-ACE2-gLuc (bag) cells simultaneously overexpressing ACE2-3xFlag and Gaussia luciferase (gLuc) were mixed with different concentrations of drug and different pseudoviruses (MOI ═ 0.1), respectively, and adhered to 96-well plates in DMEM + 10% FBS medium. After 24 hours of infection, GFP-expressing cells were photographed by fluorescence microscopy after infection; or, in order to measure the intracellular luciferase intensity of firefly, the culture supernatant was removed, 20. mu.l of 1 × passive lysine buffer (Promega) was added thereto, and the mixture was left at room temperature for 10 minutes and passed through One-gloluciferase kit (Promega) and

20/20 bioluminescence assay apparatus (Promega). Cytotoxicity (CC50) or evaluation of Gaussia luciferase activity in the supernatant was carried out according to the method of detection of QUANTI-Luc by Invovagen^TMAnd (5) carrying out reagent instruction. The results of the experiment are shown in FIG. 3. Thymoquinone inhibits SARS-CoV-2 pseudovirus with EC50 ═ 4.999 uM.

Claims

1. Application of thymoquinone in preparing medicine for preventing and treating coronavirus infection is provided.

2. Use according to claim 1, characterized in that: the coronavirus includes 2019 novel coronavirus COVID-19, severe acute respiratory syndrome SARS virus, and human coronavirus NL 63.

3. Use according to claim 1, characterized in that: the coronavirus comprises 2019 novel coronavirus COVID-19 and SARS virus of severe acute respiratory syndrome.

4. Use according to claim 1, characterized in that: the coronavirus is a 2019 novel coronavirus COVID-19.

5. Use according to any one of claims 1 to 4, characterized in that: the coronavirus infection is coronavirus pneumonia or common cold.

6. Use according to any one of claims 1-2, characterized in that: the coronavirus infection comprises 2019 novel coronavirus COVID-19 pneumonia, severe acute respiratory syndrome SARS, human coronavirus NL63 pneumonia, and common cold.

7. Use according to any one of claims 1 to 3, characterized in that: the coronavirus infection comprises 2019 novel coronavirus COVID-19 pneumonia and severe acute respiratory syndrome SARS.

8. Use according to any one of claims 1 to 4, characterized in that: the coronavirus infection is 2019 novel coronavirus COVID-19 pneumonia.

9. Use according to any one of claims 1 to 4, characterized in that: thymoquinone binds to the coronavirus receptor ACE 2.

10. Use according to claim 1, characterized in that: when in use, the oral preparation is adopted, and contains thymoquinone with effective treatment amount and pharmaceutically acceptable carrier auxiliary materials for preparing the oral preparation.