WO2022246873A1 - 一种抗SARS-CoV-2的药物及应用 - Google Patents

一种抗SARS-CoV-2的药物及应用 Download PDF

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WO2022246873A1
WO2022246873A1 PCT/CN2021/097391 CN2021097391W WO2022246873A1 WO 2022246873 A1 WO2022246873 A1 WO 2022246873A1 CN 2021097391 W CN2021097391 W CN 2021097391W WO 2022246873 A1 WO2022246873 A1 WO 2022246873A1
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sars
cov
cells
derivatives
application
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PCT/CN2021/097391
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French (fr)
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李长志
钱朝南
陈金东
周红娟
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广州市朝利良生物科技有限公司
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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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the application belongs to the technical field of biomedicine, and relates to a new drug application of 9-methoxyferramidone and/or its derivatives, in particular to an anti-SARS-CoV-2 drug and its application.
  • Coronaviruses belong to the order Nesting Viridae, the family Coronaviridae, and the genus Coronaviridae. They are a class of RNA viruses with an envelope and a linear single-stranded positive strand genome. They are a class of viruses that exist widely in nature. Certain coronaviruses can infect humans and cause disease, such as Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and pneumonia caused by the novel coronavirus (SARS-CoV-2), whose symptoms can range from the common cold to Severe lung infection.
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • SARS-CoV-2 novel coronavirus
  • SARS-CoV-2 began to spread at the end of 2019.
  • the pneumonia caused by SARS-CoV-2 mainly manifested in fever, fatigue and dry cough.
  • a small number of patients were accompanied by symptoms such as nasal congestion, runny nose, and diarrhea.
  • the time is short, and the cycle of de novo development of therapeutic drugs is long, which is difficult to meet the treatment needs.
  • Screening drugs for the treatment of SARS-CoV-2 from existing drugs is a fast and effective way.
  • Bitterwood is a plant of the genus Bitterwood. It has the effects of clearing away heat and dampness, detoxifying and killing insects, antibacterial and anti-inflammatory, and can treat gastroenteritis, biliary tract infection, acute suppurative infection and other diseases. It has been clinically proven that alkaloids in Bitterwood are the main Active ingredients, alkaloids can be divided into three categories, which are ketone alkaloids, carbaline alkaloids and dimer alkaloids, of which 9-methoxy The content of 9-Methoxycanthin-6-one and 4-methoxy-5-hydroxyferrocetone was the highest.
  • 9-Methoxyferramidone can be used to treat systemic inflammatory response syndrome, septic shock, multiple organ dysfunction syndrome, rheumatoid arthritis, osteoarthritis, spondyloarthritis, inflammatory bowel disease, cardiac Dehydration, diabetes, systemic lupus erythematosus, scleroderma, sarcoidosis, dermatomyositis, psoriasis, acute myeloid leukemia, Parkinson's disease, Alzheimer's disease, depression, septicemia, Behcet's disease, Chronic obstructive pulmonary disease, asthma, acute pancreatitis and central nervous system injury, etc.
  • This application provides an anti-SARS-CoV-2 drug and its application, that is, the new use of 9-methoxyfermidone and/or its derivatives for anti-SARS-CoV-2.
  • the present application provides an anti-SARS-CoV-2 drug, the drug includes 9-methoxycanthin-6-one and/or its derivatives, the derivative Canthin-6-one, 9-methoxycanthin-6-one N-oxide, 5-hydroxymethyl-9- 5-Hydroxymethyl-9-methoxycanthin-6-one or 1-Hydroxy-9-methoxycanthin-6-one.
  • the drug blocks entry of SARS-CoV-2 into cells.
  • the drug blocks the combination of the Spike protein (S protein) of SARS-CoV-2 and the angiotensin converting enzyme 2 (Angiotensin Converting Enzyme 2, ACE2) of cells.
  • S protein Spike protein
  • angiotensin converting enzyme 2 Angiotensin Converting Enzyme 2, ACE2
  • the drug binds to the spike protein of SARS-CoV-2, thereby blocking the spike protein of SARS-CoV-2.
  • the drug binds to the angiotensin-converting enzyme 2 of the cells, thereby blocking the angiotensin-converting enzyme 2 of the cells.
  • 9-methoxy ferricone is a ferricone alkaloid, and its molecular structure is shown in FIG. 1 .
  • the present application creatively finds that 9-methoxyfermetone and/or its derivatives can be used as anti-SARS-CoV-2 drugs, and SARS-CoV-2 infects humans mainly through the S1 subunit RBD of its Spike protein
  • the region (S-RBD) binds to ACE2 of human cells
  • 9-methoxyfermidone can bind to the Spike protein of SARS-CoV-2 and/or ACE2 of human cells to block the Spike protein of SARS-CoV-2 And/or ACE2 of human cells, thereby blocking the combination of Spike protein of SARS-CoV-2 and ACE2 of human cells, blocking SARS-CoV-2 from entering cells, and realizing the treatment of SARS-CoV-2.
  • the present application provides the application of 9-methoxyferramidone and/or its derivatives in the preparation of anti-SARS-CoV-2 drugs.
  • the present application provides the application of 9-methoxyferramidone and/or its derivatives in the preparation of a drug for blocking SARS-CoV-2 from entering cells.
  • the present application provides the application of 9-methoxyfermidone and/or its derivatives in the preparation of drugs for blocking the spike protein of SARS-CoV-2.
  • the present application provides the application of 9-methoxyferramidone and/or its derivatives in the preparation of angiotensin-converting enzyme 2 drugs that close cells.
  • the derivatives described in the second, third, fourth, and fifth aspects include iron ketone-6-one, 9-methoxy iron ketone N oxide, 5-hydroxymethyl-9-methanone Oxyfermetone or 1-hydroxy-9-methoxyfermetone.
  • the dosage forms of the medicines described in the second, third, fourth and fifth aspects include suspensions, granules, capsules, powders, tablets, emulsions, solutions, drop pills, injections, suppositories, enemas, gas Either spray, patch or drops.
  • the medicament also includes pharmaceutically acceptable auxiliary materials.
  • the auxiliary materials include carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilizers, osmotic pressure regulators, surfactants, packaging Any one or a combination of at least two of coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers, such as combinations of diluents and excipients, viscose
  • the combination of the mixture and the wetting agent, the combination of the emulsifier and the co-solvent, etc., any other combination can be selected, and will not be repeated here.
  • 9-methoxyfermetone and/or its derivatives can be used as anti-SARS-CoV-2 drugs, which can interact with the Spike protein of SARS-CoV-2 and/or human cells ACE2 binding, blocking the Spike protein of SARS-CoV-2 and/or ACE2 of human cells, thereby blocking the combination of Spike protein of SARS-CoV-2 and ACE2 of human cells, blocking SARS-CoV-2 from entering cells, and achieving Treating SARS-CoV-2.
  • This application provides a theoretical basis for the study of the use of 9-methoxyfermidone and its derivatives for the treatment of SARS-CoV-2, and provides an insertion point for the preparation of new anti-SARS-CoV-2 drugs.
  • Fig. 1 is the molecular structure figure of 9-methoxy ferricone
  • Fig. 2 is the dose-effect curve of blocking the combination of S protein and ACE2 in vitro by the drug 9-methoxyferramidone molecule of the present application;
  • Figure 3A is a fluorescence map of 293T ACE2 overexpression cells
  • Figure 3B is a graph of the ratio of green fluorescence and red fluorescence in 293T ACE2 overexpressed cells
  • Fig. 4A is the fluorescence diagram of Capan2 ACE2 overexpression cells
  • Figure 4B is a graph showing the ratio of green fluorescence to red fluorescence in Capan2 ACE2 overexpressed cells.
  • the compound that can block the binding of S protein and ACE2 is screened and verified in vitro by using the homogeneous time fluorescence resolution method.
  • the principle of the method is to bind the antibody labeled with a fluorescent group to ACE2 and S protein respectively, when 340nm excitation light is irradiated , will produce the first emission light at 620nm. If ACE2 binds to S protein, the space distance between the two fluorescent groups is relatively close, and it will also generate emission light at 665nm.
  • the specific operation process is as follows: 2 ⁇ L of prepared compounds of different concentrations are added to the 384-well plate, the compound to be tested is 9-methoxyferricone (hereinafter named TS-984), and the positive control group is The compound added was tannic acid (Tannic), and the compound added in the negative control group was buffer solution of equal volume, prepared PBS buffer solution containing BSA (0.1%), diluted S-RBD-His protein (purchased from novoprotein), ACE2 -Fc protein (purchased from novoprotein) to the corresponding concentration, after adding the response volume solution and incubating at 37°C for 1 h; adding the corresponding volume of tag antibody Anti 6His-Tbcryptate Gold and Anti HumanIgG-d2 (purchased from CISBIO), the preparation system is as shown in Table 1 As indicated, the detection was carried out after 30 min of reaction in the dark.
  • TS-984 was verified according to the above methodology, and the drug gradient concentrations were set as: 100 ⁇ M, 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, 6.25 ⁇ M, 3.125 ⁇ M, 1.563 ⁇ M, 0.7815 ⁇ M, 0.3908 ⁇ M, 0.195 ⁇ M, 0 ⁇ M.
  • the present embodiment verifies the effect of 9-methoxyferramidone blocking SARS-COV-2 pseudovirus from entering cells in 293T ACE2 overexpression cells.
  • SARS-CoV-2_S(D614G) protein pseudolentivirus >10 8 TU/mL, 10x100 ⁇ L, HBSS buffer, carrier VB900088-2229upx, with green fluorescent EGFP
  • Polybrene 5mg/mL, 200 ⁇ L
  • step 1) Add the virus to the 96-well plate of the cultured cells in step 1) for transfection, and set up the control group (Control), virus group (Pseudovirus), virus+TS-984 (working concentration 50 ⁇ M), virus+TS- 984 (working concentration 100 ⁇ M) and other 4 groups, each group had 2 duplicate wells, the duplicate wells were adjacent to each other, and there was a horizontal row between each group.
  • Control control group
  • virus group Pseudovirus
  • virus+TS-984 working concentration 50 ⁇ M
  • virus+TS- 984 working concentration 100 ⁇ M
  • 70 ⁇ L of fresh medium was added to the control group, virus group, virus + TS-984 ( Add 70 ⁇ L, 66.5 ⁇ L and 63 ⁇ L of the medium containing virus particles to the virus + TS-984 (working concentration 100 ⁇ M) group and the virus + TS-984 (working concentration 100 ⁇ M) group and virus + TS-984 (working concentration 100 ⁇ M) group was added corresponding amount of drug mother solution, and finally the culture plate was gently shaken so that the virus solution could cover every cell, and then placed in a carbon dioxide incubator at 37°C and 5% CO 2 for overnight cultivation;
  • Figure 3A is a fluorescent picture of different cells. From left to right, they show only red light, only green light, and mixed light showing both red and green light.
  • 293T ACE2 overexpressed cells can produce red fluorescence by themselves, and pseudoviruses can produce Green fluorescence, the cells transfected with pseudovirus can produce green fluorescence, it can be seen that each experimental group has normal red light, indicating that the cells grow normally, while the virus group has obvious green light, indicating that the pseudovirus can enter the cells normally, and adding 50 ⁇ M
  • the green fluorescence of the TS-984 experimental group was significantly weakened, and almost no green fluorescence was observed in the experimental group added with 100 ⁇ M TS-984.
  • This example verifies the effect of 9-methoxyferricone on blocking the entry of SARS-COV-2 pseudovirus into cells in Capan2ACE2 overexpressing cells.
  • the present embodiment verifies the effect of 9-methoxyferramidone blocking SARS-COV-2 pseudovirus from entering cells in 293T ACE2 overexpression cells.
  • step 1) Add the virus to the 96-well plate of the cultured cells in step 1) for transfection, and set up the control group (Control), virus group (Pseudovirus), virus+TS-984 (working concentration 50 ⁇ M), virus+TS- 984 (working concentration 100 ⁇ M) and other 4 groups, each group had 2 duplicate wells, the duplicate wells were adjacent to each other, and there was a horizontal row between each group.
  • Control control group
  • virus group Pseudovirus
  • virus+TS-984 working concentration 50 ⁇ M
  • virus+TS- 984 working concentration 100 ⁇ M
  • 70 ⁇ L of fresh medium was added to the control group, virus group, virus + TS-984 ( Add 70 ⁇ L, 66.5 ⁇ L and 63 ⁇ L of the medium containing virus particles to the virus + TS-984 (working concentration 100 ⁇ M) group and the virus + TS-984 (working concentration 100 ⁇ M) group and virus + TS-984 (working concentration 100 ⁇ M) group was added corresponding amount of drug mother solution, and finally the culture plate was gently shaken so that the virus solution could cover every cell, and then placed in a carbon dioxide incubator at 37°C and 5% CO 2 for overnight cultivation;
  • Figure 4A is a fluorescent picture of different cells. From left to right, only red light, only green light, and a mixture of red and green light are displayed respectively.
  • Capan2ACE2 overexpressed cells can produce red fluorescence by themselves, and pseudoviruses can produce green Fluorescence, the cells transfected with pseudovirus can produce green fluorescence, it can be seen that each experimental group has normal red light, indicating that the cells grow normally, while the virus group has obvious green light, indicating that the pseudovirus can enter the cell normally, and adding 50 ⁇ M TS The green fluorescence of the -984 experimental group was significantly weakened, and almost no green fluorescence was observed in the experimental group added with 100 ⁇ M TS-984.
  • 9-methoxyfermetone and/or its derivatives can be used as anti-SARS-CoV-2 drugs, which can be combined with SARS-CoV-2 Spike protein and /or ACE2 binding of human cells, blocking the Spike protein of SARS-CoV-2 and/or ACE2 of human cells, thereby blocking the combination of Spike protein of SARS-CoV-2 and ACE2 of human cells, blocking SARS-CoV-2 Enter the cell, and realize the treatment of SARS-CoV-2.
  • the present application illustrates the detailed method of the present application through the above-mentioned examples, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the application must rely on the above-mentioned detailed method to be implemented.
  • Those skilled in the art should understand that any improvement to the present application, the equivalent replacement of each raw material of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

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Abstract

一种抗SARS-CoV-2的药物及应用。所述药物包括9-甲氧基铁屎米酮和/或其衍生物，所述衍生物包括铁屎米-6-酮、9-甲氧基铁屎米酮N氧化物、5-羟甲基-9-甲氧基铁屎米酮或1-羟基-9-甲氧基铁屎米酮。9-甲氧基铁屎米酮和/或其衍生物可以作为抗SARS-CoV-2的药物进行使用，其能够与SARS-CoV-2的Spike protein和/或人体细胞的ACE2结合，从而阻断SARS-CoV-2的Spike protein与人体细胞的ACE2结合，阻断SARS-CoV-2进入细胞，用于治疗SARS-CoV-2。

Description

一种抗SARS-CoV-2的药物及应用 技术领域
本申请属于生物医药技术领域，涉及一种9-甲氧基铁屎米酮和/或其衍生物的药物新用途，具体涉及一种抗SARS-CoV-2的药物及应用。
背景技术
冠状病毒属于套式病毒目、冠状病毒科、冠状病毒属，是一类具有囊膜、基因组为线性单股正链的RNA病毒，是自然界广泛存在的一类病毒。某些冠状病毒会感染人类并引起疾病，比如中东呼吸综合征(MERS)、严重急性呼吸综合征(SARS)和新型冠状病毒(SARS-CoV-2)引起的肺炎，其症状可从普通感冒到重症肺部感染。
SARS-CoV-2开始流行于2019年底，SARS-CoV-2引起的肺炎主要以发热、乏力和干咳为主要表现，少数患者伴有鼻塞、流涕、腹泻等症状，由于SARS-CoV-2出现时间较短，从头开发治疗药物的周期较长，难以满足治疗需求，从现有药物中筛选治疗SARS-CoV-2药物是一种快速有效的途径。
苦木为苦木科苦木属植物，具有清热燥湿、解毒杀虫、抗菌消炎之功效，可治疗胃肠炎、胆道感染、急性化脓性感染等疾病，临床证明苦木中生物碱为主要有效成分，生物碱可分为三类，分别为铁屎米酮类生物碱、咔巴啉类生物碱和二聚体类生物碱，其中以铁屎米酮类生物碱中的9-甲氧基铁屎米酮(9-Methoxycanthin-6-one)和4-甲氧基-5-羟基铁屎米酮含量最高。
9-甲氧基铁屎米酮可用于治疗全身炎症反应综合症、脓毒性休克、多器官功能障碍综合征、类风湿性关节炎、骨性关节炎、脊柱关节炎、炎性肠病、心力衰竭、糖尿病、系统性红斑狼疮、硬皮病、结节病、皮肌炎、银屑病、急性髓性白血病、帕金森氏症、早老性痴呆、抑郁症、败血性、白塞氏病、慢性阻塞性肺病、哮喘、急性胰腺炎和中枢神经损伤等。
目前现有技术中尚未有记载9-甲氧基铁屎米酮具有抗新型冠状病毒作用的报道。
发明内容
本申请提供一种抗SARS-CoV-2的药物及应用，即9-甲氧基铁屎米酮和/或其衍生物用于抗SARS-CoV-2的新用途。
第一方面，本申请提供一种抗SARS-CoV-2的药物，所述药物包括9-甲氧基铁屎米酮(9-Methoxycanthin-6-one)和/或其衍生物，所述衍生物包括铁屎米-6-酮(Canthin-6-one)、9-甲氧基铁屎米酮N氧化物(9-Methoxycanthin-6-one N-oxide)、5-羟甲基-9-甲氧基铁屎米酮(5-Hydroxymethyl-9-methoxycanthin-6-one)或1-羟基-9-甲氧基铁屎米酮(1-Hydroxy-9-methoxycanthin-6-one)。
本申请中，所述药物阻断SARS-CoV-2进入细胞。
本申请中，所述药物阻断SARS-CoV-2的刺突蛋白(Spike protein，S蛋白)与细胞的血管紧张素转换酶2(Angiotensin Converting Enzyme 2，ACE2)结合。
本申请中，所述药物与SARS-CoV-2的刺突蛋白结合，进而封闭SARS-CoV-2的刺突蛋白。
本申请中，所述药物与细胞的血管紧张素转换酶2结合，进而封闭细胞的血管紧张素转换酶2。
本申请中9-甲氧基铁屎米酮为一种铁屎米酮类生物碱，其分子结构如图1所示。本申请创造性地发现9-甲氧基铁屎米酮和/或其衍生物可以作为抗SARS-CoV-2药物进行使用，SARS-CoV-2感染人类主要是通过其Spike protein的S1亚基RBD区域(S-RBD)与人体细胞的ACE2结合，9-甲氧基铁屎米酮能够与SARS-CoV-2的Spike protein和/或人体细胞的ACE2结合，封闭SARS-CoV-2的Spike protein和/或人体细胞的ACE2，从而阻断SARS-CoV-2的Spike protein与人体细胞的ACE2结合，阻断SARS-CoV-2进入细胞，实现了治疗SARS-CoV-2。
第二方面，本申请提供9-甲氧基铁屎米酮和/或其衍生物在制备抗SARS-CoV-2的药物中的应用。
第三方面，本申请提供9-甲氧基铁屎米酮和/或其衍生物在制备阻断SARS-CoV-2进入细胞的药物中的应用。
第四方面，本申请提供9-甲氧基铁屎米酮和/或其衍生物在制备封闭SARS-CoV-2的刺突蛋白的药物中的应用。
第五方面，本申请提供9-甲氧基铁屎米酮和/或其衍生物在制备封闭细胞的 血管紧张素转换酶2的药物中的应用。
本申请中，第二、三、四、五方面中所述的衍生物包括铁屎米-6-酮、9-甲氧基铁屎米酮N氧化物、5-羟甲基-9-甲氧基铁屎米酮或1-羟基-9-甲氧基铁屎米酮。
优选地，第二、三、四、五方面中所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、乳剂、溶液剂、滴丸剂、注射剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的任意一种。
优选地，所述药物还包括药学上可接受的辅料。
优选地，所述辅料包括载体、稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合，所述至少两种的组合例如稀释剂和赋形剂的组合、粘合剂和润湿剂的组合、乳化剂和助溶剂的组合等，其他任意的组合方式均可选择，在此便不再一一赘述。
相对于现有技术，本申请具有以下有益效果：
本申请创造性地发现9-甲氧基铁屎米酮和/或其衍生物可以作为抗SARS-CoV-2的药物进行使用，其能够与SARS-CoV-2的Spike protein和/或人体细胞的ACE2结合，封闭SARS-CoV-2的Spike protein和/或人体细胞的ACE2，从而阻断SARS-CoV-2的Spike protein与人体细胞的ACE2结合，阻断SARS-CoV-2进入细胞，实现了治疗SARS-CoV-2。本申请为研究使用9-甲氧基铁屎米酮及其衍生物治疗SARS-CoV-2提供了理论依据，为制备新的抗SARS-CoV-2的药物提供了一个嵌入点。
附图说明
图1为9-甲氧基铁屎米酮分子结构图；
图2为本申请药物9-甲氧基铁屎米酮分子在体外阻断S蛋白与ACE2结合的量效曲线图；
图3A为293T ACE2过表达细胞荧光图；
图3B为293T ACE2过表达细胞绿色荧光和红色荧光比值图；
图4A为Capan2 ACE2过表达细胞荧光图；
图4B为Capan2 ACE2过表达细胞绿色荧光和红色荧光比值图。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例体外验证9-甲氧基铁屎米酮阻断S蛋白与ACE2结合的效果。
采用均相时间荧光分辨方法在体外筛选和验证能够阻断S蛋白与ACE2结合的化合物，其方法原理为将带有荧光基团标记的抗体分别于ACE2和S蛋白结合，当340nm激发光照射时，会产生620nm的第一发射光，如果ACE2与S蛋白结合，两个荧光基团空间距离较近，还会产生665nm的发射光，当有小分子化合物能够阻断ACE2和S蛋白结合时，只有620nm一种激发光，通过荧光检测即可筛选出有效的阻断ACE2和S蛋白结合的小分子化合物。
由于SARS-CoV-2病毒感染人类主要是通过Spike protein的S1亚基RBD区域与ACE2受体结合，因此，选择了Fc标记的ACE-2和His标记的S-RBD，并分别用检测试剂anti-human IgG-XL665和anti-6his-tb gold进行标记，构建ACE2和S-RBD结合复合体模型。
具体操作流程为：在384孔板中加入2μL配置好的不同浓度的化合物，待测化合物组加入的化合物为9-甲氧基铁屎米酮(下文中命名为TS-984)，阳性对照组加入的化合物为单宁酸(Tannic)，阴性对照组加入的化合物为等体积的缓冲液，配制含BSA(0.1％)的PBS缓冲液，稀释S-RBD-His蛋白(购自novoprotein)，ACE2-Fc蛋白(购自novoprotein)到相应浓度，加入响应体积溶液后37℃孵育1h后；加入相应体积的标签抗体Anti 6His-Tbcryptate Gold与Anti HumanIgG-d2(购自CISBIO)，配制体系按表1所示，避光反应30min后进行检测。
表1
  阴性对照组 阳性对照组 待测化合物组
S-RBD-His(0.88μg/mL) 5μL 5μL 5μL
ACE2-Fc(1.15μg/mL) 5μL 5μL 5μL
缓冲液 2μL —— ——
化合物 —— 2μL 2μL
Anti 6His-TbcryptateGold 5μL 5μL 5μL
Anti HumanIgG-d2 5μL 5μL 5μL
将TS-984按照以上方法学进行验证，设置药物梯度浓度分别为：100μM，50μM，25μM，12.5μM，6.25μM，3.125μM，1.563μM，0.7815μM，0.3908μM，0.195μM，0μM。
结果如图2所示，随着TS-984浓度增加，650nm荧光/620nm荧光的比值逐渐降低并趋向于0，说明体系中650nm荧光不断减少并趋于消失，表明TS-984能够有效的阻断ACE2和S-RBD结合，且EC 50＝16.38μM。
实施例2
本实施例在293T ACE2过表达细胞中验证9-甲氧基铁屎米酮阻断SARS-COV-2假病毒进入细胞的效果。
具体过程为：
(1)实验对象：293T ACE2过表达细胞(红色荧光mCherry)；
(2)实验材料：SARS-CoV-2_S(D614G)蛋白假慢病毒(>10 8TU/mL，10x100μL，HBSS缓冲液，载体为VB900088-2229upx，具有绿色荧光EGFP)；Polybrene(5mg/mL，200μL)，以上两种材料均购于云舟生物；
(3)实验药物：本申请药物9-甲氧基铁屎米酮，即TS-984。
(4)实验方法：
1)SARS-COV-2假病毒转导前1天(第0天)，将293T ACE2接种到新的无菌96孔板中，每孔接种的细胞密度为3x10 5/mL(计数仪计数)，共接种12个孔，每孔100μL，分别接种到96孔板的第1、2、4、5竖排，放置于37℃、5％CO 2的二氧化碳培养箱中培养过夜；
2)SARS-COV-2假病毒转导当天(第1天)，首先在冰上融解冻结的病毒液，轻柔吹打几下，混匀融解后的病毒颗粒，再取50μL的病毒液加至450μL含有5μg/mL的Polybrene的新鲜的完全培养基(DMEM+10％FBS)中，使病毒液的体积比为10％，轻柔混匀，然后吸出原有培养基，向细胞中加入完全培 养基，得到含病毒颗粒的培养基；
3)向步骤1)的培养细胞的96孔板中加入病毒进行转染，实验共设置对照组(Control)、病毒组(Pseudovirus)、病毒+TS-984(工作浓度50μM)、病毒+TS-984(工作浓度100μM)等4个组，每组2个复孔，复孔相邻，每组之间间隔一横排，其中，对照组加入70μL新鲜的培养基，病毒组、病毒+TS-984(工作浓度50μM)组和病毒+TS-984(工作浓度100μM)组分别加入上述含病毒颗粒的培养基70μL、66.5μL和63μL，病毒+TS-984(工作浓度50μM)组和病毒+TS-984(工作浓度100μM)组加入相应量的药物母液，最后轻柔地摇动培养板以使病毒液都能覆盖每一处细胞，然后放置于37℃、5％CO 2的二氧化碳培养箱中培养过夜；
4)SARS-COV-2假病毒转导第2天，病毒转染24h后拍照，比较各组荧光表达情况，结果如图3A和图3B所示。
图3A为不同细胞荧光图片，从左到右分别为只显示红光、只显示绿光和同时显示红光和绿光的混合光，293T ACE2过表达细胞自身能产生红色荧光，假病毒能产生绿色荧光，转染假病毒的细胞能够产生绿色荧光，可见每个实验组均有正常红光，表明细胞正常生长，而病毒组具有明显的绿光，表明假病毒能够正常进入细胞，而加入50μM TS-984的实验组的绿色荧光明显减弱，加入100μM TS-984的实验组几乎观察不到绿色荧光，进一步地，由图3B绿光和红光的比值(EGFP/mCherry)结果可知，随着加入TS-984的浓度的增加，绿色荧光逐渐降低，即感染假病毒的细胞数量大大降低，综合上述表明9-甲氧基铁屎米酮可以有效阻断SARS-COV-2假病毒进入293T ACE2过表达细胞，且成剂量依赖性。
实施例3
本实施例在Capan2ACE2过表达细胞中验证9-甲氧基铁屎米酮阻断SARS-COV-2假病毒进入细胞的效果。
本实施例在293T ACE2过表达细胞中验证9-甲氧基铁屎米酮阻断SARS-COV-2假病毒进入细胞的效果。
具体过程为：
(1)实验对象：Capan2ACE2过表达细胞(红色荧光)；
(2)实验材料：SARS-CoV-2_S(D614G)蛋白假慢病毒(>10 8TU/mL，10x100μL，HBSS缓冲液，载体为VB900088-2229upx，具有绿色荧光)；Polybrene (5mg/mL，200μL)，以上两种材料均购于云舟生物；
(3)实验药物：本申请药物9-甲氧基铁屎米酮，即TS-984。
(4)实验方法：
1)SARS-COV-2假病毒转导前1天(第0天)，将Capan2ACE2接种到新的无菌96孔板中，每孔接种的细胞密度为3x10 5/mL(计数仪计数)，共接种12个孔，每孔100μL，分别接种到96孔板的第1、2、4、5竖排，放置于37℃、5％CO 2的二氧化碳培养箱中培养20h；
2)SARS-COV-2假病毒转导当天(第1天)，首先在冰上融解冻结的病毒液，轻柔吹打几下，混匀融解后的病毒颗粒，再取50μL的病毒液加至450μL含有5μg/mL的Polybrene的新鲜的完全培养基(DMEM+10％FBS)中，使病毒液的体积比为10％，轻柔混匀，然后吸出原有培养基，向细胞中加入完全培养基，得到含病毒颗粒的培养基；
3)向步骤1)的培养细胞的96孔板中加入病毒进行转染，实验共设置对照组(Control)、病毒组(Pseudovirus)、病毒+TS-984(工作浓度50μM)、病毒+TS-984(工作浓度100μM)等4个组，每组2个复孔，复孔相邻，每组之间间隔一横排，其中，对照组加入70μL新鲜的培养基，病毒组、病毒+TS-984(工作浓度50μM)组和病毒+TS-984(工作浓度100μM)组分别加入上述含病毒颗粒的培养基70μL、66.5μL和63μL，病毒+TS-984(工作浓度50μM)组和病毒+TS-984(工作浓度100μM)组加入相应量的药物母液，最后轻柔地摇动培养板以使病毒液都能覆盖每一处细胞，然后放置于37℃、5％CO 2的二氧化碳培养箱中培养过夜；
4)SARS-COV-2假病毒转导第2天，病毒转染24h后拍照，比较各组荧光表达情况，结果如图4A和图4B所示。
图4A为不同细胞荧光图片，从左到右分别为只显示红光、只显示绿光和同时显示红光和绿光的混合光，Capan2ACE2过表达细胞自身能产生红色荧光，假病毒能产生绿色荧光，转染假病毒的细胞能够产生绿色荧光，可见每个实验组均有正常红光，表明细胞正常生长，而病毒组具有明显的绿光，表明假病毒能够正常进入细胞，而加入50μM TS-984的实验组的绿色荧光明显减弱，加入100μM TS-984的实验组几乎观察不到绿色荧光，进一步地，由图4B绿光和红光的比值(EGFP/mCherry)结果可知，随着加入TS-984的浓度的增加，绿色荧光逐渐降低，即感染假病毒的细胞数量大大降低，综合上述表明9-甲氧基铁屎 米酮可以有效阻断SARS-COV-2假病毒进入Capan2ACE2过表达细胞，且成剂量依赖性。
综上所述，本申请创造性地发现9-甲氧基铁屎米酮和/或其衍生物可以作为抗SARS-CoV-2的药物进行使用，其能够与SARS-CoV-2的Spike protein和/或人体细胞的ACE2结合，封闭SARS-CoV-2的Spike protein和/或人体细胞的ACE2，从而阻断SARS-CoV-2的Spike protein与人体细胞的ACE2结合，阻断SARS-CoV-2进入细胞，实现了治疗SARS-CoV-2。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (10)

  1. 一种抗SARS-CoV-2的药物，其包括9-甲氧基铁屎米酮和/或其衍生物；
    所述衍生物包括铁屎米-6-酮、9-甲氧基铁屎米酮N氧化物、5-羟甲基-9-甲氧基铁屎米酮或1-羟基-9-甲氧基铁屎米酮。
  2. 根据权利要求1所述的药物，其中，所述药物阻断SARS-CoV-2的刺突蛋白与细胞的血管紧张素转换酶2结合。
  3. 根据权利要求1所述的药物，其中，所述药物封闭SARS-CoV-2的刺突蛋白。
  4. 根据权利要求1所述的药物，其中，所述药物封闭细胞的血管紧张素转换酶2。
  5. 9-甲氧基铁屎米酮和/或其衍生物在制备抗SARS-CoV-2的药物中的应用。
  6. 9-甲氧基铁屎米酮和/或其衍生物在制备阻断SARS-CoV-2进入细胞的药物中的应用。
  7. 9-甲氧基铁屎米酮和/或其衍生物在制备封闭SARS-CoV-2的刺突蛋白的药物中的应用。
  8. 9-甲氧基铁屎米酮和/或其衍生物在制备封闭细胞的血管紧张素转换酶2的药物中的应用。
  9. 根据权利要求5-8任一项所述的应用，其中，所述衍生物包括铁屎米-6-酮、9-甲氧基铁屎米酮N氧化物、5-羟甲基-9-甲氧基铁屎米酮或1-羟基-9-甲氧基铁屎米酮。
  10. 根据权利要求5-8任一项所述的应用，其中，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、乳剂、溶液剂、滴丸剂、注射剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的任意一种。
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