WO2022161436A1 - 没食子儿茶素没食子酸酯及其衍生物的医药用途 - Google Patents

没食子儿茶素没食子酸酯及其衍生物的医药用途 Download PDF

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WO2022161436A1
WO2022161436A1 PCT/CN2022/074314 CN2022074314W WO2022161436A1 WO 2022161436 A1 WO2022161436 A1 WO 2022161436A1 CN 2022074314 W CN2022074314 W CN 2022074314W WO 2022161436 A1 WO2022161436 A1 WO 2022161436A1
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coronavirus
protein
sars
cov
gcg
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PCT/CN2022/074314
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French (fr)
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张学敏
李涛
周涛
李爱玲
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南湖实验室
中国人民解放军军事科学院军事医学研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to the medical use of GCG and its derivatives.
  • the new coronavirus is the third highly pathogenic zoonotic coronavirus after the severe acute respiratory syndrome coronavirus SARS-CoV in 2002 and the Middle East respiratory syndrome coronavirus MERS-CoV in 2012. Pneumonia is also known as coronavirus disease 2019 (COVID-19).
  • the new coronavirus SARS-CoV-2 is a single-stranded positive-stranded RNA virus that encodes 29 proteins.
  • Nucleocapsid protein (N protein) as a structural protein, plays an important role in the assembly and replication of SARS-CoV-2.
  • the N protein of SARS-CoV-2 is extremely similar to that of SARS-CoV.
  • Phase separation is the process by which proteins or nucleic acids aggregate to form membraneless organelles and is a key step in the assembly of viruses. Therefore, targeting the phase separation of related viral N proteins has important antiviral significance.
  • (-)-Gallocatechol gallate is one of the natural extracts of tea.
  • GCG uses of GCG that have been disclosed so far include: anti-cancer, fat reduction, drug delivery, beauty, promoting blood circulation, inhibiting gingival bacteria, preventing and improving oxidative stress and neuronal damage, etc.
  • the purpose of the present invention is to provide new medicinal uses of GCG and its derivatives.
  • GCG The medicinal uses of GCG and its derivatives provided by the present invention are the following (a) and/or (b) and/or (c):
  • the present invention also provides medicinal uses of GCG and its derivatives, which are the following (d) and/or (e) and/or (f):
  • GCG or a derivative thereof inhibits RNA-induced phase separation of the coronavirus N protein
  • GCG or its derivative inhibits the binding of coronavirus N protein to RNA
  • GCG or its derivatives inhibit the phase separation of SARS-CoV-2 N protein.
  • GCG ((-)-Gallocatechol gallate) is one of the natural extracts of tea.
  • Chinese name gallocatechin gallate; English name: (-)-Gallocatechol gallate; CAS number: 4233-96-9; molecular formula: C 22 H 18 O 11 ; molecular weight: 458.37; its structural formula is as follows:
  • GCG epigallocatechin gallate
  • CG ((-)-Catechin gallate, catechin gallate) and the like.
  • the product may be a drug or a pharmaceutical formulation.
  • the coronavirus inhibitor can inhibit the replication of coronavirus.
  • the coronavirus can be alpha coronavirus and/or beta coronavirus, specifically selected from at least one of SARS-CoV-2 and SARS-CoV.
  • GCG or its derivatives can be used as one of the active ingredients or the only active ingredient in the preparation of medicines or pharmaceutical preparations.
  • a carrier material can also be added when preparing a drug or a drug formulation.
  • Carrier materials include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).
  • water-soluble carrier materials such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.
  • poorly soluble carrier materials such as ethyl cellulose, cholesterol stearate, etc.
  • enteric carrier materials Such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.
  • dosage forms can be prepared using these materials, including but not limited to tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Buccal tablets, suppositories, freeze-dried powder injection
  • the above dosage forms can be used for administration by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal; respiratory tract administration, such as nasal cavity; mucosal administration.
  • the present invention also provides a medicine or a pharmaceutical composition whose active ingredient is GCG or its derivatives.
  • drugs or pharmaceutical compositions can be prepared into dosage forms such as solutions, tablets, capsules or injections according to conventional methods known to those skilled in the art.
  • an effective amount of GCG or its derivatives is administered to the subject organism.
  • the dosage and method of administration of the compounds of the present invention will depend on a number of factors, including the patient's age, weight, sex, natural health, nutritional status, strength of activity of the compound, time of administration, rate of metabolism, severity of the condition, and the subjective opinion of the treating physician. judge.
  • the term "effective amount” refers to a dose that can achieve treatment, prevention, alleviation and/or amelioration of the diseases or conditions of the present invention in a subject.
  • the term "subject” may refer to patients or other animals, especially mammals, such as humans, dogs, Monkeys, cows, horses, etc.
  • the disease caused by the coronavirus may be respiratory system infection and/or multi-organ infection.
  • the respiratory infection is a respiratory infection and/or a pulmonary infection;
  • the respiratory infection can be nasopharyngitis, rhinitis, pharyngitis, bronchitis and/or bronchitis;
  • the pulmonary infection can be pneumonia;
  • the multi-organ The infection can lead to multiple organ failure.
  • the diseases caused by the coronavirus usually include viral pneumonia, severe acute respiratory syndrome, inflammatory factor storm, multiple organ failure and the like.
  • the coronavirus infection usually causes viral pneumonia, severe acute respiratory syndrome, inflammatory factor storm, multiple organ failure and other diseases.
  • the present invention also protects a method for inhibiting coronavirus infection of animals, comprising the steps of: administering GCG or a derivative thereof to recipient animals to inhibit coronavirus infection of animals.
  • the present invention also protects a method for treating a disease caused by a coronavirus, comprising the steps of: administering GCG or a derivative thereof to a recipient animal to treat a disease caused by the coronavirus.
  • the present invention also protects a method for preventing a disease caused by a coronavirus, comprising the steps of: administering GCG or a derivative thereof to a recipient animal to prevent a disease caused by the coronavirus.
  • coronaviruses can be alpha coronaviruses and/or beta coronaviruses.
  • coronaviruses can specifically be at least one of SARS-CoV-2 and SARS-CoV.
  • SARS-CoV-2 whose sequence features include but are not limited to representative viruses (Cell Host Microbe, 2020; PMID: 32183941. Nature, 2020; PMID: 32015508)
  • the inventors of the present invention have found that the N protein undergoes phase separation during SARS-CoV-2 infection.
  • GCG specifically binds to the N protein, inhibits the binding of the N protein to the viral RNA, destroys its phase separation process, and finally inhibits the replication of the virus.
  • the inventors found that the derivatives of GCG, EGCG and CG, also inhibited the intracellular phase separation of the N protein of SARS-CoV-2.
  • N protein of SARS-CoV-2 was highly similar to the N protein of SARS-CoV. Therefore, GCG and its derivatives inhibit the replication and assembly of SARS-CoV-2 and SARS-CoV-like viruses by disrupting the phase separation process of the viral N protein.
  • FIG 1 shows that GCG inhibits the replication of SARS-CoV-2.
  • GCG inhibits RNA-induced phase separation of SARS-CoV-2 N protein.
  • FIG. 3 shows the specific binding of GCG to SARS-CoV-2 N protein.
  • Figure 4 shows that GCG inhibits the binding of SARS-CoV-2 N protein to viral RNA.
  • FIG. 5 shows the phase separation of N protein when GCG inhibits SARS-CoV-2 infection.
  • Figure 7 shows that EGCG and CG inhibit the binding of SARS-CoV-2 N protein to viral RNA.
  • FIG. 8 shows that the SARS-CoV-2 N protein is highly similar to the SARS-CoV N protein sequence.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.
  • the quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.
  • H1299 Human lung adenocarcinoma cells
  • A549 Human non-small cell lung cancer cells
  • mEGFP monomeric green fluorescent protein
  • poly(I:C) polyinosinic acid cytidine
  • Hoechst nuclear dye
  • ACE2 angiotensin-converting enzyme 2
  • TRIZOL RNA extraction reagent
  • RT-qPCR real-time fluorescence quantitative PCR
  • DMSO dimethyl sulfoxide
  • PBS phosphate buffered saline
  • TBE tris(hydroxymethyl)aminomethane-boronic acid-ethylenediaminetetraacetic acid buffer
  • GCG Chinese name: gallocatechin gallate; purchased from TargetMol Company, the product catalog number is T3807.
  • EGCG Chinese name: Epigallocatechin gallate; purchased from Selleck Company, the catalog number is S2250.
  • CG Chinese name: catechin gallate; purchased from MCE company, product catalog number is HY-N0356.
  • the SARS-CoV-2 used in the examples is the Shanghai strain of novel coronavirus (nCoV-SH01). (Zhang Rong, Yi Zhigang, Wang Yuyan, et al. Isolation and identification of new coronavirus Shanghai strain (nCoV-SH01) [J]. Microorganisms and Infections, 2020(1):16-21.)
  • GCG inhibits the replication of the new coronavirus
  • the C-terminal of the human ACE2 protein coding sequence was coupled with the Flag tag protein sequence, and constructed into the pLV-EF1a-IRES-Blast lentiviral vector, and the ACE2-Flag protein was stably expressed in the lentiviral infection. in the A549 cell line.
  • the recombinant vector is a fusion protein ACE2-Flag encoding gene inserted between the BamHI and EcoRI restriction sites of the pLV-EF1a-IRES-Blast lentiviral vector; the vector expresses the fusion protein.
  • the above fusion protein ACE2-Flag is obtained by coupling the Flag tag to the C-terminal of the ACE2 protein.
  • amino acid sequence of the fusion protein ACE2-Flag is shown in SEQ ID No: 1.
  • the nucleotide sequence of the gene encoding the fusion protein ACE2-Flag is shown in SEQ ID No: 2.
  • the recombinant vector was transformed into A549 cells to obtain a post-transfection cell line, namely the A549-ACE2-Flag cell line.
  • A549-ACE2-Flag cells were plated in a 12-well plate (100,000 cells/well) 12 hours in advance to allow them to grow naturally.
  • step (3) The cells treated in step (2) were pretreated with 100 ⁇ M GCG (the solvent was DMSO) for 1 hour.
  • the wells to which DMSO was added were called control wells, and the wells containing GCG were collectively called test wells.
  • step (3) Infect the cells after step (3) with SARS-CoV-2 virus solution (1 ⁇ l/well, the virus content in SARS-CoV-2 virus solution is 100,000 PFU/ml, MOI is 1), and after 4 hours harvest cells.
  • RNA of the cells Discard the supernatant medium, lyse and extract the RNA of the cells with TRIZOL, perform reverse transcription, and use the obtained cDNA as a template, and RT-qPCR detects the replication amount of the new coronavirus RNA.
  • the primer probe sets used in RT-qPCR are as follows (the target sequence is located in the N protein gene of SARS-CoV-2):
  • Upstream primer 5'-GACCCCAAAATCAGCGAAT-3'
  • Fluorescent probe required for detection 5'-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3'
  • the presence of GCG can significantly inhibit the RNA replication of the new coronavirus.
  • GCG inhibits RNA-induced phase separation of 2019-nCoV N protein
  • the C-terminus of the N protein coding sequence of the new coronavirus was coupled with the mEGFP green fluorescent label, and constructed into the pCDX-Tet-On lentiviral vector. Through lentivirus infection, the Flag-N-mEGFP protein was stably expressed in in the H1299 cell line.
  • the recombinant vector is to insert the coding gene of fusion protein Flag-N-mEGFP between the NheI and NotI restriction sites of pCDX-Tet-On lentiviral vector; the vector expresses the fusion protein.
  • the above fusion protein Flag-N-mEGFP is obtained by coupling the N-terminus of the N protein with the Flag tag and the C-terminus with the mEGFP fluorescent protein.
  • the amino acid sequence of the fusion protein Flag-N-mEGFP is shown in SEQ ID No: 4.
  • the nucleotide sequence of the fusion protein Flag-N-mEGFP encoding gene is shown in SEQ ID No: 5.
  • the recombinant vector was transferred into H1299 cells to obtain post-transfection cells, namely H1299-Flag-N-mEGFP cells.
  • step (2) The cells in step (2) were pretreated with 10 ⁇ M GCG prepared by dissolving in DMSO for 1 hour.
  • step (4) The cells treated in step (4) were washed three times with PBS to discard the medium, and fixed with 4% paraformaldehyde for 10 minutes at room temperature.
  • GCG specifically binds to the N protein of 2019-nCoV
  • the small molecule compound GCG was dissolved in 2.5 ml of binding buffer (coupling buffer: 0.1 M NaHCO 3 pH 8.3, containing 0.5 M NaCl).
  • the N protein was able to specifically bind GCG compared to CNBr agarose without GCG.
  • GCG inhibits the binding of new coronavirus N protein to viral RNA
  • N-mEGFP protein 2 ⁇ g was premixed with 0-100 ⁇ M of GCG for 5 minutes at room temperature, and then Cy3-labeled RNA was added to incubate for 5 minutes at room temperature.
  • GCG inhibits the binding of SARS-CoV-2 N protein to RNA.
  • GCG inhibits the phase separation of N protein under SARS-CoV-2 infection
  • A549-ACE2-Flag cells were plated in a 12-well plate (100,000 cells/well) 12 hours in advance to allow them to grow naturally.
  • step (3) The A549 cells treated in step (2) were pretreated with 100 ⁇ M GCG prepared by dissolving in DMSO for 1 hour.
  • the wells to which DMSO was added were called control wells, and the wells containing GCG were collectively called test wells.
  • step (3) Infect the cells of step (3) with SARS-CoV-2 virus solution (1 ⁇ L/well, the virus content in the SARS-CoV-2 virus solution is 100,000 PFU/ml, and the MOI is 1) for 24 hours.
  • Triton X100 0.3% Triton X100 was punched at room temperature for 10 minutes.
  • the C-terminus of the N protein coding sequence of the new coronavirus was coupled with the mEGFP green fluorescent label, and constructed into the pCDX lentiviral vector, and the N-mEGFP protein was stably expressed in H1299 cells through lentivirus infection.
  • the cells stably expressing N-mEGFP were pretreated with 20 ⁇ M EGCG prepared by dissolving in DMSO, 20 ⁇ M CG or 20 ⁇ M GCG for 1 hour.
  • step (4) The cells treated in step (4) were washed three times with PBS to discard the medium, and fixed with 4% paraformaldehyde for 10 minutes at room temperature.
  • N protein 1 ⁇ g was premixed with 100 ⁇ M of GCG, EGCG or CG for 5 minutes at room temperature, and then Cy3-labeled RNA was added and incubated for 5 minutes at room temperature.
  • EGCG and CG also inhibited the binding of SARS-CoV-2 N protein to RNA.
  • the N protein of SARS-CoV-2 is highly similar to the N protein of SARS.
  • N protein sequence of SARS-CoV-2 is extremely similar to that of SARS.
  • GCG and its derivatives can target the N protein of the virus, inhibit its phase separation, and further inhibit the replication of related viruses.
  • GCG and its derivatives inhibit the replication and assembly of SARS-CoV-2 and SARS-CoV-like viruses by disrupting the phase separation process of the viral N protein.
  • GCG or its derivatives can be used to prepare products for preventing and/or treating coronavirus-induced diseases or coronavirus infections.

Abstract

本发明公开了一种GCG及其衍生物新的医药用途。本发明所提供的医药用途为：(a)GCG或其衍生物在制备治疗冠状病毒所致疾病或冠状病毒感染的产品中的应用；(b)GCG或其衍生物在制备预防冠状病毒所致疾病或冠状病毒感染的产品中的应用；(c)GCG或其衍生物在制备冠状病毒抑制剂中的应用。本发明的发明人研究发现，病毒感染时，SARS-CoV-2的N蛋白质发生相分离。GCG通过与N蛋白质特异性结合，抑制N蛋白质与病毒RNA的结合，破坏其相分离，最终抑制病毒的复制。

Description

没食子儿茶素没食子酸酯及其衍生物的医药用途 技术领域
本发明属于生物医药技术领域，具体涉及一种GCG及其衍生物的医药用途。
背景技术
新冠病毒是继2002年的严重急性呼吸系统综合症冠状病毒SARS-CoV、2012年中东呼吸系统综合症冠状病毒MERS-CoV之后的第三种高致病性的人畜共患冠状病毒，其造成的肺炎也称为2019年冠状病毒病(COVID-19)。
新冠病毒SARS-CoV-2是一种单股正链RNA病毒，编码29个蛋白质。核衣壳蛋白质(N蛋白质)作为一种结构蛋白质在新冠病毒的组装和复制中发挥重要作用。另外，SARS-CoV-2与SARS-CoV的N蛋白质存在极高相似性。
相分离是蛋白质或核酸聚集形成无膜细胞器的过程，是病毒的装配的关键步骤。因此，靶向相关病毒N蛋白质的相分离，具有重要的抗病毒意义。
(-)-Gallocatechol gallate(GCG)是茶的天然提取物之一。中文名称：没食子儿茶素没食子酸酯；英文名称：(-)-Gallocatechol gallate；CAS号：4233-96-9；分子式：C 22H 18O 11；分子量：458.37；其结构式如下所示：
Figure PCTCN2022074314-appb-000001
目前已经公开的GCG的用途包括：抗癌、减脂、药物递送、美容、促进血液循环、抑制牙龈细菌、预防及改善氧化应激和神经元损伤等。
发明公开
本发明的目的是提供GCG及其衍生物新的医药用途。
本发明所提供的GCG及其衍生物的医药用途，为以下(a)和/或(b)和/或(c)：
(a)GCG或其衍生物在制备治疗冠状病毒所致疾病或冠状病毒感染的产品中的应用；
(b)GCG或其衍生物在制备预防冠状病毒所致疾病或冠状病毒感染的产品中的应用；
(c)GCG或其衍生物在制备冠状病毒抑制剂中的应用。
本发明还提供了GCG及其衍生物的医药用途，为以下(d)和/或(e)和/或(f)：
(d)GCG或其衍生物抑制RNA诱导的冠状病毒N蛋白质的相分离；
(e)GCG或其衍生物抑制冠状病毒N蛋白质与RNA的结合；
(f)GCG或其衍生物抑制SARS-CoV-2 N蛋白质的相分离。
GCG((-)-Gallocatechol gallate)是茶的天然提取物之一。中文名称：没食子儿茶素没食子酸酯；英文名称：(-)-Gallocatechol gallate；CAS号：4233-96-9；分子式：C 22H 18O 11；分子量：458.37；其结构式如下所示：
Figure PCTCN2022074314-appb-000002
本发明所述GCG的衍生物包括EGCG(Epigallocatechin gallate，表没食子儿茶素没食子酸酯)、CG((-)-Catechin gallate，儿茶素没食子酸盐)等。
所述产品可为药物或药物制剂。
所述冠状病毒抑制剂能够抑制冠状病毒的复制。
所述冠状病毒可为α属冠状病毒和/或β属冠状病毒，具体的选自SARS-CoV-2、SARS-CoV中的至少一种。
上述应用中，制备药物或药物制剂时，GCG或其衍生物可作为有效成分之一，也可作为唯一有效成分。
上述应用中，制备药物或药物制剂时，还可加入载体材料。
载体材料包括但不限于水溶性载体材料(如聚乙二醇、聚乙烯吡咯烷酮、有机酸等)、难溶性载体材料(如乙基纤维素、胆固醇硬脂酸酯等)、肠溶性载体材料(如醋酸纤维素酞酸酯和羧甲乙纤维素等)。使用这些材料可以制成多种剂型，包括但不限于片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、口含片、栓剂、冻干粉针剂等。可以是普通制剂、缓释制剂、控释制剂及各种微粒给药系统。使用上述剂型可以经注射给药，包括皮下注射、静脉注射、肌肉注 射和腔内注射等；腔道给药，如经直肠和阴道；呼吸道给药，如经鼻腔；粘膜给药。
本发明还提供了一种药物或药物组合物，其活性成分为GCG或其衍生物。
所述药物或药物组合物具有下述至少一种功效：
1)治疗冠状病毒所致疾病或冠状病毒感染；
2)预防冠状病毒所致疾病或冠状病毒感染；
3)抑制冠状病毒。
上述药物或药物组合物可以按照本领域技术人员已知的常规方法制成溶液剂、片剂、胶囊或注射剂等剂型。
利用本发明提供的GCG或其衍生物预防和/或治疗冠状病毒引起的感染时，给予受试者生物体有效量的GCG或其衍生物。
本发明化合物的使用剂量和使用方法取决于诸多因素，包括患者的年龄、体重、性别、自然健康状况、营养状况、化合物的活性强度、服用时间、代谢速率、病症的严重程度以及诊治医生的主观判断。
本发明中，术语“有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。
本发明中，术语“受试者”可以指患者或者其它接受本发明组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。
本发明中，所述冠状病毒所致疾病可为呼吸系统感染和/或多器官感染。
所述呼吸系统感染为呼吸道感染和/或肺部感染；所述呼吸道感染可为鼻咽炎、鼻炎、咽喉炎、气管炎和/或支气管炎；所述肺部感染可为肺炎；所述多器官感染可为多器官功能衰竭。
本发明中，所述冠状病毒所致疾病通常包括病毒性肺炎、严重急性呼吸综合征、炎症因子风暴、多器官功能衰竭等。
本发明中，所述冠状病毒感染通常引起病毒性肺炎、严重急性呼吸综合征、炎症因子风暴、多器官功能衰竭等疾病。
本发明还保护一种抑制冠状病毒感染动物的方法，包括如下步骤：给受体动物施用GCG或其衍生物以抑制冠状病毒感染动物。
本发明还保护一种治疗冠状病毒所致疾病的方法，包括如下步骤：给受体动物施用GCG或其衍生物进行治疗冠状病毒所致疾病。
本发明还保护一种预防冠状病毒所致疾病的方法，包括如下步骤：给受体动物 施用GCG或其衍生物进行预防冠状病毒所致疾病。
以上任一所述冠状病毒可为α属冠状病毒和/或β属冠状病毒。
以上任一所述冠状病毒具体可为SARS-CoV-2、SARS-CoV中的至少一种。
SARS-CoV-2，其序列特征包括但不局限于代表病毒(Cell Host Microbe,2020；PMID:32183941.Nature,2020；PMID:32015508)
本发明的发明人研究发现，SARS-CoV-2感染时N蛋白质发生相分离。GCG通过与N蛋白质特异性结合，抑制N蛋白质与病毒RNA的结合，破坏其相分离过程，最终抑制病毒的复制。
同时发明人研究发现，GCG的衍生物EGCG，CG同样抑制SARS-CoV-2的N蛋白质在细胞内的相分离。
序列比对结果显示，SARS-CoV-2的N蛋白质与SARS-CoV的N蛋白质具有高度相似性。因此，GCG及其衍生物通过破坏病毒N蛋白质的相分离过程，从而抑制SARS-CoV-2及SARS-CoV类似病毒的复制与组装。
附图说明
图1为GCG抑制SARS-CoV-2的复制。
图2为GCG抑制RNA诱导的SARS-CoV-2 N蛋白质的相分离。
图3为GCG与SARS-CoV-2 N蛋白质特异性结合。
图4为GCG抑制SARS-CoV-2 N蛋白质与病毒RNA的结合。
图5为GCG抑制SARS-CoV-2感染时N蛋白质的相分离。
图6为EGCG和CG抑制RNA诱导的SARS-CoV-2 N蛋白质的相分离。
图7为EGCG和CG抑制SARS-CoV-2 N蛋白质与病毒RNA的结合。
图8为SARS-CoV-2 N蛋白质与SARS-CoV N蛋白质序列存在高度相似性。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中的名词注释如下：
H1299：人肺腺癌细胞；A549：人非小细胞肺癌细胞；
mEGFP：单体绿色荧光蛋白质；poly(I:C)：聚肌苷酸胞苷酸；
Hoechst：细胞核染料；ACE2：血管紧张素转化酶2；
TRIZOL：RNA提取试剂；RT-qPCR：实时荧光定量PCR；
DMSO：二甲基亚砜；PBS：磷酸盐缓冲液；
TBE：三羟甲基氨基甲烷-硼酸-乙二胺四乙酸缓冲液；
GCG：中文名称：没食子儿茶素没食子酸酯；购自TargetMol公司，产品目录号为T3807。
EGCG：中文名称：表没食子儿茶素没食子酸酯；购自Selleck公司，产品目录号为S2250。
CG：中文名称：儿茶素没食子酸酯；购自MCE公司，产品目录号为HY-N0356。
实施例中所用的SARS-CoV-2为新型冠状病毒上海株(nCoV-SH01)。(张荣,易志刚,王玉燕,等.新型冠状病毒上海株(nCoV-SH01)的分离和鉴定[J].微生物与感染,2020(1):16-21.)
实施例中涉及的实验在包括生物安全三级实验室(BSL-3)等实验室进行。
实施例1、
1、GCG抑制新冠病毒的复制
实验方法：
(1)将人源的ACE2蛋白质编码序列的C端，偶联Flag标签蛋白质序列，并构建到pLV-EF1a-IRES-Blast慢病毒载体上，通过慢病毒感染，将ACE2-Flag蛋白质稳定表达在A549细胞系中。
具体方法如下：
重组载体为将融合蛋白ACE2-Flag的编码基因插入pLV-EF1a-IRES-Blast慢病毒载体的BamHI和EcoRI酶切位点间；该载体表达融合蛋白质。
上述融合蛋白ACE2-Flag为ACE2蛋白质C端偶联Flag标签得到的。
该融合蛋白质ACE2-Flag的氨基酸序列如SEQ ID No：1所示。
该融合蛋白质ACE2-Flag编码基因的核苷酸序列如SEQ ID No：2所示。
pLV-EF1a-IRES-Blast慢病毒载体全序列图谱如SEQ ID No：3所示。
将重组载体转入A549细胞中，得到转染后细胞系，即A549-ACE2-Flag细胞系。
(2)提前12个小时将A549-ACE2-Flag细胞平铺在12孔板中(100,000个细胞/孔)，使其自然贴壁生长。
(3)将步骤(2)处理后的细胞用100μM GCG(溶剂为DMSO)预处理1个小时。加入DMSO的孔称为对照孔，加入含GCG的孔统称为供试孔。
(4)用SARS-CoV-2病毒液(1μl/孔，SARS-CoV-2病毒液中的病毒含量为100,000PFU/ml，MOI为1)感染完成步骤(3)的细胞，4个小时后收细胞。
弃上清培养基，用TRIZOL裂解并提取细胞的RNA，进行反转录，将得到cDNA作为模板，RT-qPCR检测新冠病毒RNA的复制量。
RT-qPCR采用的引物探针组如下(靶序列位于SARS-CoV-2的N蛋白质基因)：
上游引物：5’-GACCCCAAAATCAGCGAAAT-3’
下游引物：5’-TCTGGTTACTGCCAGTTGAATCTG-3’
检测所需荧光探针：5’-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3’
结果：
如图1所示，GCG存在的情况下，能够显著抑制新冠病毒的RNA复制。
2、GCG抑制RNA诱发的新冠病毒N蛋白质的相分离
实验方法：
(1)将新冠病毒的N蛋白质编码序列的C端，偶联mEGFP绿色荧光标签，并构建到pCDX-Tet-On慢病毒载体上，通过慢病毒感染，将Flag-N-mEGFP蛋白质稳定表达在H1299细胞系中。
具体方法如下：
重组载体为将融合蛋白Flag-N-mEGFP的编码基因插入pCDX-Tet-On慢病毒载体的NheI和NotI酶切位点间；该载体表达融合蛋白质。
上述融合蛋白Flag-N-mEGFP为N蛋白质N端偶联Flag标签，C端偶联mEGFP荧光蛋白质得到的。
该融合蛋白Flag-N-mEGFP的氨基酸序列如SEQ ID No：4所示。
该融合蛋白Flag-N-mEGFP编码基因的核苷酸序列如SEQ ID No：5所示。
将重组载体转入H1299细胞中，得到转染后细胞，即H1299-Flag-N-mEGFP细胞。
(2)提前12个小时在细胞培养皿中平铺细胞，使其贴壁均匀生长。
(3)将步骤(2)中的细胞，用DMSO溶解制成的10μM GCG预处理1个小时。
(4)用poly(I:C)处理细胞3个小时，模拟病毒感染时RNA的释放。
(5)将步骤(4)处理完毕的细胞，用PBS清洗三次，以弃去培养基，并用4％的多聚甲醛室温固定10分钟。
(6)用Hoechst对细胞核进行染色，室温15分钟。
(7)用PSB清洗三次，每次5分钟，并用封片剂将细胞贴片。
(8)用ZEISS LSM 880显微镜进行成像观察。
结果：
如图2所示，未处理GCG的细胞，在poly(I:C)处理下，N蛋白质发生了明显的点状聚集。GCG处理组，在poly(I:C)处理下，未见到N蛋白质的点状聚集，说明GCG抑制了RNA诱导的新冠病毒N蛋白质的相分离。
3、GCG与新冠病毒N蛋白质特异性结合
实验方法：
(1)将小分子化合物GCG溶解至2.5ml结合缓冲液(coupling buffer:0.1M NaHCO 3pH 8.3，包含0.5M NaCl)中。
(2)使用溴化氰(CNBr)-活化琼脂糖凝胶珠(C500099，生工)与GCG溶液在4℃状态下偶联过夜。
(3)使用纯化重组的N蛋白质(带有Flag标签)与CNBr-GCG琼脂糖凝胶珠进行4℃偶联6小时。
(4)通过免疫印迹法对N蛋白质进行检测。
结果：
如图3所示，与不加GCG的CNBr琼脂糖凝胶相比，N蛋白质能够特异性结合GCG。
4、GCG抑制新冠病毒N蛋白质与病毒RNA的结合
实验方法：
(1)2μg的N-mEGFP蛋白质与0-100μM的GCG室温预混合5分钟，随后加入Cy3标记的RNA室温共孵育5分钟。
(2)使用8％的非变性聚丙烯酰胺凝胶，在0.5×TBE缓冲液中进行电压为200V时间为1小时的电泳。
(3)利用Bio-Rad公司的ChemiDocMP成像仪成像观察。
结果：
如图4所示，GCG抑制新冠病毒N蛋白质与RNA的结合。
5、GCG抑制新冠病毒感染下N蛋白质的相分离
实验方法：
(1)将人源的ACE2蛋白质编码序列的C端，偶联Flag标签蛋白质序列，并构建到pLV-EF1a-IRES-Blast慢病毒载体上，通过慢病毒感染，将ACE2-Flag蛋白质稳 定表达在A549细胞系中。(具体构建方法同“GCG抑制新冠病毒的复制”)
(2)提前12个小时将A549-ACE2-Flag细胞平铺在12孔板中(100,000个细胞/孔)，使其自然贴壁生长。
(3)将步骤(2)处理后的A549细胞用DMSO溶解制成的100μM GCG预处理1个小时。加入DMSO的孔称为对照孔，加入含GCG的孔统称为供试孔。
(4)用SARS-CoV-2病毒液(1μL/孔，SARS-CoV-2病毒液中的病毒含量为100,000PFU/ml，MOI为1)感染步骤(3)的细胞24小时。
(5)用PBS清洗三次，以弃去培养基，并用4％的多聚甲醛室温固定10分钟。
(6)0.3％Triton X100室温打孔10分钟。
(7)用5％的羊血清室温封闭1小时。
(8)用特异性新冠病毒N蛋白质抗体进行免疫荧光染色。
(9)用ZEISS LSM 880显微镜进行成像观察。
结果：
如图5所示，SARS-CoV-2感染A549-ACE2-Flag细胞，新冠病毒的N蛋白质发生明显相分离聚集。在GCG处理组中这一现象被明显抑制，说明GCG抑制了SARS-CoV-2 N蛋白质的相分离。
6、EGCG和CG抑制新冠病毒N蛋白质的相分离
实验方法：
(1)将新冠病毒的N蛋白质编码序列的C端，偶联mEGFP绿色荧光标签，并构建到pCDX慢病毒载体上，通过慢病毒感染，将N-mEGFP蛋白质稳定表达在H1299细胞中。
(2)提前12个小时在细胞培养皿中平铺细胞，使其贴壁均匀生长。
(3)将稳定表达了N-mEGFP的细胞，用DMSO溶解制成的20μM EGCG，20μM CG或20μM GCG预处理1个小时。
(4)用poly(I:C)处理细胞3个小时，模拟病毒感染时RNA的释放。
(5)将步骤(4)处理完毕的细胞，用PBS清洗三次，以弃去培养基，并用4％的多聚甲醛室温固定10分钟。
(6)用Hoechst对细胞核进行染色，室温15分钟。
(7)用PSB清洗三次，每次5分钟，并用封片剂将细胞贴片。
(8)用ZEISS LSM 880显微镜进行成像观察。
结果：
如6所示，在poly(I:C)处理下，N蛋白质发生了明显的点状聚集。EGCG，CG和GCG处理组，在poly(I:C)处理下，未见到N蛋白质的点状聚集，说明EGCG和CG同样抑制了RNA诱导的新冠病毒N蛋白质的相分离。
7.EGCG和CG抑制新冠病毒N蛋白质与病毒RNA的结合
实验方法：
(1)1μg的N蛋白质与100μM的GCG，EGCG或CG室温预混合5分钟，随后加入Cy3标记的RNA室温共孵育5分钟。
(2)使用8％的非变性聚丙烯酰胺凝胶，在0.5×TBE缓冲液中进行电压为200V时间为1小时的电泳。
(3)利用Bio-Rad公司的ChemiDocMP成像仪成像观察。
结果：
如图7所示，EGCG和CG同样抑制新冠病毒N蛋白质与RNA的结合。
8.SARS-CoV-2的N蛋白质与SARS的N蛋白质具有高度相似性。
实验方法：
(1)从UniProt数据库(https://www.uniprot.org/)下载SARS-CoV-2(Entry：P0DTC9)与SARS-CoV(Entry：P59595)的N蛋白(Nucleoprotein)的氨基酸序列数据文件(.fasta格式)
(2)将上述序列文件输入MAFFT软件(https://mafft.cbrc.jp/alignment/software/)进行序列比对，得到比对后的序列数据文件(.fasta格式)
(3)将比对后的序列数据文件输入MEGA软件(https://www.megasoftware.net/)，使用MEGA软件的”Analysis-Distance-Compute Pairwise Distances..”计算SARS-CoV-2与SARS的氨基酸序列差异。其中，”Model/Method”参数选择”p-distance”，其输出结果为nd/n，即序列间存在差异的氨基酸数量与序列全长的氨基酸数量之比。
(4)将通过公式1计算SARS-CoV-2与SARS的氨基酸序列的相似百分比(Percent similarity)：
Figure PCTCN2022074314-appb-000003
结果：
如图8所示，SARS-CoV-2的N蛋白质序列与SARS的N蛋白质序列具有极高相似性。
以上数据说明，GCG及其衍生物可以靶向病毒的N蛋白质，抑制其相分离，进一步抑制相关病毒的复制。
工业应用
GCG及其衍生物通过破坏病毒N蛋白质的相分离过程，从而抑制SARS-CoV-2及SARS-CoV类似病毒的复制与组装。GCG或其衍生物可用于制备预防和/或治疗冠状病毒所致疾病或冠状病毒感染的产品。

Claims (10)

  1. 没食子儿茶素没食子酸酯或其衍生物的应用，为以下(a)和/或(b)和/或(c)：
    (a)没食子儿茶素没食子酸酯或其衍生物在制备治疗冠状病毒所致疾病或冠状病毒感染的产品中的应用；
    (b)没食子儿茶素没食子酸酯或其衍生物在制备预防冠状病毒所致疾病或冠状病毒感染的产品中的应用；
    (c)没食子儿茶素没食子酸酯或其衍生物在制备冠状病毒抑制剂中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述产品为药物或药物制剂。
  3. 根据权利要求1或2所述的应用，其特征在于：所述冠状病毒为α属冠状病毒和/或β属冠状病毒；所述冠状病毒抑制剂能够抑制冠状病毒的复制；
    所述没食子儿茶素没食子酸酯的衍生物包括表没食子儿茶素没食子酸酯、儿茶素没食子酸盐。
  4. 根据权利要求3所述的应用，其特征在于：所述冠状病毒选自SARS-CoV-2、SARS-CoV中的至少一种。
  5. 没食子儿茶素没食子酸酯或其衍生物的应用，为以下(d)和/或(e)和/或(f)：
    (d)没食子儿茶素没食子酸酯或其衍生物抑制RNA诱导的冠状病毒N蛋白质的相分离；
    (e)没食子儿茶素没食子酸酯或其衍生物抑制冠状病毒N蛋白质与RNA的结合；
    (f)没食子儿茶素没食子酸酯或其衍生物抑制SARS-CoV-2 N蛋白质的相分离。
  6. 抑制冠状病毒感染动物的方法，包括如下步骤：给受体动物施用没食子儿茶素没食子酸酯或其衍生物以抑制冠状病毒感染动物。
  7. 根据权利要求6所述的方法，其特征在于：所述冠状病毒为α属冠状病毒和/或β属冠状病毒；所述冠状病毒具体选自SARS-CoV-2、SARS-CoV中的至少一种。
  8. 治疗冠状病毒所致疾病的方法，包括如下步骤：给受体动物施用没食子儿茶素没食子酸酯或其衍生物进行治疗冠状病毒所致疾病。
  9. 预防冠状病毒所致疾病的方法，包括如下步骤：给受体动物施用没食子儿茶素没食子酸酯或其衍生物进行预防冠状病毒所致疾病。
  10. 根据权利要求8或9所述的方法，其特征在于：所述冠状病毒为α属冠状病毒和/或β属冠状病毒；所述冠状病毒具体选自SARS-CoV-2、SARS-CoV中的至少一种。
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