WO2020024719A1 - 二盐酸小檗胺在制备埃博拉病毒抑制剂中的应用 - Google Patents

二盐酸小檗胺在制备埃博拉病毒抑制剂中的应用 Download PDF

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WO2020024719A1
WO2020024719A1 PCT/CN2019/091838 CN2019091838W WO2020024719A1 WO 2020024719 A1 WO2020024719 A1 WO 2020024719A1 CN 2019091838 W CN2019091838 W CN 2019091838W WO 2020024719 A1 WO2020024719 A1 WO 2020024719A1
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virus
dihydrochloride
pharmaceutically acceptable
acceptable salt
berberamine
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PCT/CN2019/091838
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English (en)
French (fr)
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岑山
李泉洁
衣岽戎
施一
王寒
周金明
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中国医学科学院医药生物技术研究所
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Priority to US16/652,372 priority Critical patent/US11654141B2/en
Priority to CA3083540A priority patent/CA3083540C/en
Priority to EP19843169.4A priority patent/EP3669875A4/en
Priority to JP2020542492A priority patent/JP7085006B2/ja
Publication of WO2020024719A1 publication Critical patent/WO2020024719A1/zh
Priority to US18/132,530 priority patent/US20230321079A1/en

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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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • 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 relates to the use of berberamine dihydrochloride, in particular to the application of berberamine dihydrochloride in the preparation of an Ebola virus inhibitor.
  • Viral hemorrhagic fever is a group of natural epidemic diseases caused by viruses.
  • the main clinical features are fever, hemorrhage, and shock.
  • Such diseases are widely distributed in the world, with more severe clinical manifestations and high mortality rates.
  • Common viral hemorrhagic fevers include Ebola hemorrhagic fever, Marburg hemorrhagic fever, Lassa fever, Crimea-Congo hemorrhagic fever, Rift Valley fever, Dengue hemorrhagic fever, yellow fever and smallpox.
  • Ebola hemorrhagic fever is an acute hemorrhagic infectious disease caused by Ebola virus (EBOV) in the family Filaviridae, with a mortality rate of 90%, which is the most deadly in humans.
  • EBOV Ebola virus
  • EBOV can be divided into 5 types: Zaire (ZEBOV), Sudanebolavirus (SUDV), Tai Forest (TBFV), Bundibugyoebolavirus (BDBV), and Bundibugyoebolavirus (BDBV) and Reston ebolavirus (RESTV).
  • Zaire-type Ebola virus has the strongest pathogenicity.
  • Marburg hemorrhagic fever is an acute febrile disease caused by Marberg virus (MARV), which has severe bleeding manifestations. It belongs to the same family as Ebola hemorrhagic fever and is a highly lethal infectious disease. Marburg virus and Ebola virus belong to the family Filovirus of the family Filoviridae.
  • Lassa fever is an acute infectious disease caused by the Lassa virus (LASV), which is mainly transmitted by rodents.
  • Lassa virus belongs to the mammalian genus Mammarenavirus of the family Arenaviridae.
  • Envelope glycoprotein refers to the glycoprotein that is encoded by the virus itself and is coated on the outer layer of the virus.
  • GP is a multifunctional protein that plays a vital role in the process of virus adsorption and penetration into host cells, pathogenicity, down-regulation of host cell surface protein expression, and increased virus assembly and budding.
  • the technical problem to be solved by the present invention is how to inhibit viruses such as Ebola virus, Marburg virus and / or Lassa virus that cause viral hemorrhagic fever.
  • the present invention provides the use of berbamine dihydrochloride.
  • berbamine dihydrochloride provided by the present invention is any one of U1 to U5;
  • the virus may be an envelope glycoprotein that can pass through an activated state and berberamine dihydrochloride or a pharmaceutically acceptable Salt-bound virus;
  • the virus may be an envelope glycoprotein capable of passing through an activated state and berberamine dihydrochloride or a pharmaceutically acceptable salt thereof Combined virus
  • berberamine dihydrochloride or a pharmaceutically acceptable salt thereof in the preparation of a product for treating and / or preventing viral hemorrhagic fever (such as a drug, vaccine or pharmaceutical preparation); the viral hemorrhagic fever can be caused by Virus-induced diseases: viruses that can bind to berberamine dihydrochloride or a pharmaceutically acceptable salt thereof through an activated envelope glycoprotein;
  • the viral hemorrhagic fever may be a disease caused by the following viruses: A virus in which a membrane glycoprotein is combined with berberamine dihydrochloride or a pharmaceutically acceptable salt thereof;
  • the virus may be a virus belonging to the family Filaviridae and / or gliovirus, such as a virus that causes viral hemorrhagic fever.
  • the virus causing viral hemorrhagic fever may be Ebola virus, Marburg virus and / or Lassa virus.
  • the viral hemorrhagic fever may be Ebola hemorrhagic fever, Marburg hemorrhagic fever and / or Lassa fever.
  • the virus inhibitor, the product for treating and / or preventing viral hemorrhagic fever, and the product combined with a virus-activated envelope glycoprotein, except for containing berberamine dihydrochloride or a pharmaceutically acceptable may be included.
  • suitable carriers or excipients may be included.
  • the carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carriers Materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among these, a water-soluble carrier material is preferred.
  • Using these materials can be made into a variety of dosage forms, including but not limited to tablets, capsules, drip pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Oral tablets, suppositories, lyophilized powder injections, etc. It can be common preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. In order to form a unit dosage form into tablets, various carriers known in the art can be widely used.
  • Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, silicic acid Aluminum, etc .; humectants and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch syrup, dextrin, syrup, honey, glucose solution, gum arabic, gelatin syrup, sodium carboxymethyl cellulose , Shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc .; disintegrating agents, such as dry starch, alginate, agar powder, alginate, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, Sorbitol fatty acid ester, sodium dodecylsulfonate, methyl cellulose, ethyl cellulose, etc .
  • the tablets can also be further formed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets.
  • coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets.
  • various carriers known in the art can be widely used.
  • Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc .; binders such as gum arabic, tragacanth, gelatin , Ethanol, honey, liquid sugar, rice cereal or batter, etc .; disintegrants, such as agar powder, dried starch, alginate, sodium dodecyl sulfonate, methyl cellulose, ethyl cellulose, and the like. In order to make a unit dosage form into a suppository, various carriers known in the art can be widely used.
  • diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc .
  • binders such as gum arabic, tragacanth
  • the carrier are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like.
  • all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxidized isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc.
  • an appropriate amount of sodium chloride, glucose, or glycerol may be added to the preparation for injection, and conventional co-solvents, buffers, pH adjusters, and the like may be added.
  • a coloring agent, a preservative, a flavor, a flavoring agent, a sweetener, or other materials may be added to the pharmaceutical preparation.
  • the above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavity injection, etc .; intracavity administration, such as rectum and vagina; respiratory tract administration, such as nasal cavity; mucosal administration.
  • the invention also provides pharmaceutical compounds.
  • the pharmaceutical compound provided by the present invention is berbamine dihydrochloride or a pharmaceutically acceptable salt thereof.
  • the virus may be a virus capable of binding to berberamine dihydrochloride or a pharmaceutically acceptable salt thereof through an activated envelope glycoprotein.
  • the virus may be a virus of the family Filaviridae and / or gliovirus, such as a virus that causes viral hemorrhagic fever.
  • the virus causing viral hemorrhagic fever may be Ebola virus, Marburg virus and / or Lassa virus.
  • the invention also provides a method for inhibiting a virus from infecting an animal.
  • the method for inhibiting virus-infected animals comprises administering berberine dihydrochloride or a pharmaceutically acceptable salt thereof to a recipient animal to inhibit virus-infected animals;
  • the virus may be an envelope sugar capable of passing an activated state Viruses whose protein binds to berbamine dihydrochloride or a pharmaceutically acceptable salt thereof.
  • the virus may be a virus of the family Filaviridae and / or gliovirus, such as a virus that causes viral hemorrhagic fever.
  • the virus causing viral hemorrhagic fever may be Ebola virus, Marburg virus and / or Lassa virus.
  • the invention also provides methods for treating and / or preventing viral hemorrhagic fever.
  • the method for treating and / or preventing viral hemorrhagic fever comprises administering berberamine dihydrochloride or a pharmaceutically acceptable salt thereof to a recipient animal to treat and / or prevent viral hemorrhagic fever; said virus Hemorrhagic fever can be a disease caused by a virus capable of binding to berberamine dihydrochloride or a pharmaceutically acceptable salt thereof through an activated envelope glycoprotein.
  • the animal may be a mammal, such as a human; the animal may also be other animals, such as birds, infected by the virus other than mammals.
  • the term "pharmaceutically acceptable salt” means, within the scope of reliable medical judgment, suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., and reasonably The effect / risk ratio is proportional to the salt.
  • Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in S.M.Berge, etal., J. Pharmaceuticals, Science, 1977, 66: 1.
  • the Ebola virus may be a Zaire-type, Sudan-type, Tay forest-type, Bendibugio-type and / or Leston-type Ebola virus.
  • the inhibitory virus may also be referred to as an anti-virus.
  • the inhibiting virus may be inhibiting a virus from invading a cell.
  • the inhibition of virus invasion into the cell may be a virus-activated envelope glycoprotein (GPcl) -mediated virus entry into the cell.
  • GPcl virus-activated envelope glycoprotein
  • an Ebola-activated envelope glycoprotein (EBOV-GPcl) is used as a target, and an antiviral active compound having the ability to bind to EBOV-GPcl is obtained through structure-based virtual screening.
  • the compound is a small dihydrochloride Lamine.
  • Berberine dihydrochloride can specifically inhibit the entry of Ebola recombinant virus by binding to the target protein EBOV-GPcl, and achieve the effect of resisting Ebola virus infection.
  • the half-maximum effect concentration (EC50) of berberamine dihydrochloride against EBOV was 0.49 ⁇ M, indicating that berberamine dihydrochloride has a strong inhibitory effect on EBOV.
  • Figure 1 Structural formula of berbamine dihydrochloride.
  • FIG. 2 Berberamine dihydrochloride specifically inhibits the entry of EBOV-Zaire GP / HIV-luc recombinant virus in Example 1.
  • VSVG indicates VSV-G / HIV-luc
  • Ebola-GP indicates EBOV.
  • DMSO represents blank control treatment
  • TET represents tetrandrine treatment
  • 1-22 represents 22 compound treatments respectively, of which 10 is berberamine dihydrochloride treatment.
  • FIG. 3 Cell growth experiments in Example 2 verify the effect of berberamine dihydrochloride on the growth of 293T cells; in Figure 3, DMSO represents blank control treatment, TET represents tetrandrine treatment, and EEI-10 represents berberamine dihydrochloride treatment .
  • Figure 4 The inhibitory effect of berberamine dihydrochloride on EBOV-Zaire GP / HIV-luc recombinant virus in Example 3 has a good dose-dependent effect.
  • FIG. 5 The experiment of the drug action time point in Example 4 shows that berberamine dihydrochloride acts on the entry stage of the virus; in Figure 5, TET stands for tetrandrine treatment, EEI-10 means berberamine dihydrochloride treatment, and RT means according to law Wei Lun handles.
  • Figure 6 Berberamine dihydrochloride inhibits both MARV-GP / HIV-luc recombinant virus and LASV-GP / HIV-luc recombinant virus in Example 5.
  • Figure 7 Kinetic binding curves of different concentrations of berberamine dihydrochloride and target protein GPcl in vitro by biofilm layer optical interference technique.
  • Berberamine dihydrochloride is a known compound and can be purchased from commercially available products. The specific means of acquisition are the prior art, which is not particularly limited in the present invention. Berberamine dihydrochloride in the following examples is a product of TargetMol.
  • the eukaryotic expression vector pcDNA3.1 (+) in the following examples is a product of Invitrogen.
  • the HIV-luc plasmid pNL4-3Luc (RE-) (Ma, L., etal. (23 May 2018)) carrying the luciferase reporter gene in the following examples.
  • RE- The HIV-luc plasmid pNL4-3Luc
  • the biological material is only used for the experiment of repeating the present invention, and cannot be used for other purposes.
  • Ebola virus envelope glycoprotein enters the lysosome and will be digested.
  • the activated envelope glycoprotein after digestion can interact with endosomes.
  • the receptor-human-derived cholesterol transporter Niemann-Pick C1, NPC1 directly interacts, thereby triggering the membrane fusion process between the virus and the host cell.
  • EBOV-GPcl the activated glycoprotein of Ebola virus
  • NPC1-C the receptor-human-derived cholesterol transporter
  • the inventors constructed a pharmacophore model based on the hydrogen bonding, electrostatic interaction, and hydrophobic interaction between the active polypeptide and EBOV-GPcl, and established a virtual screening method for Ebola virus entry inhibitors targeting EBOV-GPcl In order to find a small molecule compound that specifically binds EBOV-GPcl, thereby inhibiting the binding of EBOV-GPcl and NPC1-C, and thus inhibiting the replication of Ebola virus.
  • This model was used to screen the database. After scoring multiple software, the target compound was finally obtained, and its biological activity was tested. Finally, an antiviral active compound with EBOV-GPcl as the target was obtained.
  • the compound was berbamine dihydrochloride. Has the ability to combine with EBOV-GPcl.
  • the present invention uses pseudovirus technology as a safe and effective research method to evaluate the biological activity of small molecule compounds in vitro.
  • the most virulent Zaire-type EBOV GP protein was used to coat the HIV core to prepare the replication-deficient pseudovirus EBOV-GP / HIV-luc, and the antiviral activity of the samples was determined by fluorescent reporter gene detection technology.
  • the specificity of small molecule compounds was analyzed using the VSVG / HIV-luc recombinant virus model. After cytotoxicity was ruled out, the action mechanism of small molecule compounds was further verified using drug action time point experiments.
  • the biofilm-layer-based optical interference technology based on optical fiber biosensors was used to determine the binding ability of small molecule compounds to the target protein GPcl in vitro to verify the targeting of small molecule compounds.
  • the specific experimental methods and results are as follows.
  • Example 1 EBOV enters an inhibitor screening model to verify that berberine dihydrochloride can specifically inhibit EBOV activity.
  • Zaire-EBOV GP was co-expressed with HIV core plasmid (pNL4-3.Luc) using cell-level recombinant virus technology to prepare a recombinant virus, and a high-throughput screening model of EBOV entry inhibitor targeting GP protein was used to evaluate the compounds Antiviral activity.
  • the specific steps are:
  • 293T cells were cultured. After the cells were filled with the culture flask, the old medium was discarded and digested with a digestive solution containing 0.25% trypsin and 0.02% EDTA. After the cells become round, discard the digestion solution, add 10% FBS (purchased from GIBCO) high-sugar DMEM medium (GIBICO) immediately, and gently blow the bottom of the bottle with a pipette to completely remove the cells from the bottom of the bottle and disperse them into a single unit. Cell suspension. After counting, the cell concentration was adjusted to 2.2 ⁇ 10 5 cells / ml with a medium, and the cells were seeded in a 6-well plate at 2 mL / well.
  • FBS purchased from GIBCO
  • GIBICO high-sugar DMEM medium
  • transfection was performed.
  • the amount of plasmid 2 ⁇ g pZEBOV-GP and 3 ⁇ g HIV-luc plasmid pNL4-3Luc (RE-) carrying a luciferase reporter gene.
  • the transfection reagent was Lipofectamine 2000 (Invitrogen). According to the instruction manual, transfection was performed to generate an Ebola pseudotype virus, and the Ebola pseudotype virus was named EBOV-Zaire GP / HIV-luc.
  • Supernatants containing pseudotyped virus were collected 48 hours after transfection, pooled, clarified from floating cells and cell debris by low speed centrifugation, and filtered through a 0.45 ⁇ m pore size filter. Pseudoviral particles were quantified by measuring virus-associated HIV p24 levels using an ELISA assay.
  • pZEBOV-GP is the 5900- A recombinant expression plasmid of Zaire-EBOV-expressing glycoprotein (GP) obtained by inserting the 8305-position into the vector pcDNA3.1 (+).
  • EBOV-Zaire GP / HIV-luc pseudovirus particles were incubated with 293T cells into 96-well plates. After 48 hours, cells were collected and lysed to measure firefly luciferase activity. The value of luciferase activity represents a viral infection.
  • the compound was dissolved in DMSO and mixed with EBOV-Zaire and GP / HIV-luc pseudoviruses, respectively, and added to 293T cells so that the content of the compound was 10 ⁇ M. After 48 hours, 293T cells were lysed, and the inhibitory rate of the compound on the virus was evaluated by measuring the luciferase activity. Solvent DMSO was used as a blank control, and EBOV entry inhibitor tetrandrine (TET) was also used as a control. Powder tetrandrine was dissolved in DMSO and mixed with EBOV-Zaire GP / HIV-luc pseudovirus to add to 293T cells. The content of powder tetrandrine was 1 ⁇ M.
  • Virus inhibition rate of the compound 1-relative luciferase activity.
  • Relative luciferase activity refers to luciferase activity relative to a blank control. The luciferase activity actually represents virus infectivity.
  • EBOV inhibitors are broad-spectrum antiviral drugs.
  • VSVG Vesicular stomatitis virus glycoprotein
  • EBOV-GPcl play similar roles in vesicular stomatitis virus coat glycoprotein and play an important role in the recognition of viruses and receptors. Therefore, a pseudovirus VSV-G / HIV- luc performs specific analysis of compounds. After excluding the cytotoxic factors, the luciferase principle was also used to detect the inhibitory activity of the compound on the VSV-G / HIV-luc pseudovirus. The method was the same as above. If the compound only has a significant inhibitory effect on EBOV-GPcl-mediated virus entry, but does not inhibit VSV or the inhibition rate is very low, it indicates that the compound is specific for EBOV.
  • the preparation method of the pseudovirus VSV-G / HIV-luc expressing VSV-GP differs from the preparation method of EBOV-Zaire GP / HIV-luc only by the pZEBOV in the preparation method of EBOV-Zaire GP / HIV-luc -GP is replaced by pVSV-GP, the other operations are exactly the same.
  • pVSV-GP is an expression vesicular sex mouth obtained by inserting positions 14-1567 of the vesicular stomatitis virus coat glycoprotein GP gene (GenBankAccession No. V01214.1 (Update DateFeb 4, 2011) into the vector pcDNA3.1 (+) Recombinant Expression Plasmid of Meningitis Virus Coat Glycoprotein.
  • Example 2 The antiviral activity of berbamine dihydrochloride has nothing to do with its cytotoxicity.
  • cell counting kit-8 (CCK-8) was used to evaluate the effect of berbamine dihydrochloride on the growth of 293T cells.
  • CCK-8 kit is a kit for detecting cell proliferation, cell survival and cytotoxicity. It is a WST-8 (water-soluble tetrazolium salt, chemical name: 2- (2-methoxy-4-nitrophenyl) ) -3- (4-nitrophenyl) -5- (2,4-disulfobenzene) -2H-tetrazole monosodium salt) is a fast and sensitive detection kit widely used as an alternative to the MTT method.
  • the kit uses water-soluble tetrazolium salt-WST-8. In the presence of an electron coupling reagent, it can be reduced by some dehydrogenases in the mitochondria to form an orange-yellow formazan.
  • the light absorption value measured by the enzyme-linked immunosorbent detector at a wavelength of 450nm can indirectly reflect the number of living cells. The specific steps are:
  • 293T cells were cultured in a 96-well plate and incubated with berberamine dihydrochloride (dissolved with DMSO).
  • the content of berberamine dihydrochloride in the culture medium was 10 ⁇ M, 2.5 ⁇ M, and 0.625 ⁇ M, respectively.
  • the cell supernatant was replaced with a cell culture solution containing 10% CCK-8 reagent, and the cells were further cultured for 1 h in a 37 ° C, 5% CO 2 incubator.
  • the optical density (OD) value of each well at 450 nm was recorded on a microplate reader (Thermo, Varioskan Flash).
  • tetrandrine Using tetrandrine (TET) as a control, 293T cells were cultured in 96-well plates and incubated with tetrandrine (dissolved with DMSO). The contents of tetrandrine in the culture medium were 10 ⁇ M, 2.5 ⁇ M, and 0.625 ⁇ M, respectively. After 48 hours, the cell supernatant was replaced with a cell culture solution containing 10% CCK-8 reagent, and the cells were further cultured for 1 h in a 37 ° C, 5% CO 2 incubator. The optical density (OD) value of each well at 450 nm was recorded on a microplate reader (Thermo, Varioskan Flash).
  • OD optical density
  • Solvent DMSO was used as a blank control (DMSO). The OD450nm of the blank control was recorded as 100% cell viability.
  • Example 3 The inhibitory effect of berberamine dihydrochloride on EBOV has a good dose-dependent effect.
  • berberine dihydrochloride was dissolved in DMSO and mixed with EBOV-Zaire GP / HIV-luc in Example 1 and added to 293T cells to make the content of berberamine dihydrochloride separately. It is 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 ⁇ M.
  • 293T cells were lysed and the anti-EBOV activity of berbamine dihydrochloride was evaluated by measuring luciferase activity.
  • the solvent DMSO was used as a blank control (DMSO), and the luciferase activity of the blank control was taken as 100% of cell viability. The experiment was repeated three times, and the results are shown in Figure 4.
  • Berberamine dihydrochloride significantly inhibited the EBOV-Zaire GP / HIV-luc pseudovirus activity in a dose-dependent manner.
  • the half-maximal effect concentration (EC50) of berberamine dihydrochloride against EBOV was 0.49 ⁇ M.
  • Example 4 It is determined through the experiment of drug action time point that berberamine dihydrochloride acts on the entry stage of the virus.
  • the content (final concentration) is 1 ⁇ 10 -5 mol ⁇ L -1 ), and EBOV enters the inhibitor tetrandrine (TET) (dissolved in DMSO, and the content in the medium is 1 ⁇ 10 -7 mol ⁇ L -1 ), non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) (dissolved in DMSO, the content in the medium is 1 ⁇ 10 -9 mol ⁇ L -1 ) as controls, and DMSO is Solvent control; 48h after infection, detection of reporter gene luciferase activity reflects the level of recombinant virus replication.
  • TET inhibitor tetrandrine
  • EVF non-nucleoside reverse transcriptase inhibitor efavirenz
  • berberamine dihydrochloride By measuring the failure time of the drug during a single infection of EBOV, the action of the drug can be initially determined. As shown in FIG. 5, berberamine dihydrochloride showed a very strong inhibitory effect in the early stage of virus entry, and had no inhibitory effect on virus infection after the virus completed the adsorption process. This is consistent with the action time of EBOV entering the inhibitor tetrandrine. The non-nucleoside reverse transcriptase inhibitor efavirenz still inhibited the virus at 6h. These results indicate that berberamine dihydrochloride works after the virus binds to the host and before the virus and the host undergo membrane fusion.
  • Example 5 Evaluation of compounds using Marburg recombinant virus and Lassa recombinant virus models.
  • Ebola virus belongs to the family of filamentous viruses. Based on recombinant virus technology, two other filamentous recombinant virus models have been established, namely Marburg recombinant virus (MARV-GP / MAR-GP / HIV-luc). Recombinant virus (LASV-GP / HIV-luc). Among them, the preparation method of the MARV-GP pseudovirus MARV-GP / HIV-luc and the LASV-GP pseudovirus LASV-GP / HIV-luc are all different from those of the EBOV-Zaire GP / HIV-luc. The method is to replace pZEBOV-GP with pMARV-GP and pLASV-GP in the preparation method of EBOV-Zaire GP / HIV-luc, and other operations are exactly the same.
  • pMARV-GP is an expression vector obtained by inserting the Marburg virus coat glycoprotein GP gene (GenBank Accession No. NC_001608.3) (Update Date 12-NOV-2014) at position 5941-7986 into the vector pcDNA3.1 (+). Recombinant expression plasmid for the glycoprotein of the paulovirus coat.
  • pLASV-GP is the expressed Lassa virus obtained by inserting the 1872-3347 position of the GP gene of the Lassa virus coat glycoprotein (GenBank Accession No. J04324.1) (Update Date Jun 23, 2010) into the vector pcDNA3.1 (+) Recombinant expression plasmid of coat glycoprotein.
  • the MARV-GP / HIV-luc and LASV-GP / HIV-luc recombinant virus models were used to determine the half-maximal effect concentration of berbamine dihydrochloride against Marburg virus and Lassa virus.
  • Berberamine dihydrochloride can inhibit the entry of Marburg virus and Lassa virus into the host, with EC50 of 0.99 ⁇ M and 2.64 ⁇ M, respectively.
  • This study suggests that berberamine dihydrochloride has a broad-spectrum antiviral effect. It can be seen from the results of the protein sequence comparison that the sequence homology of the GP proteins of the two strains of Ebola virus and Marburg virus is only 23%. The use of multiple strains of virus models to evaluate compounds will facilitate the discovery of broad-spectrum antiviral drugs and will assist in the study of the mechanism of action of the drugs.
  • Example 6 The in vitro determination of the binding capacity of berberamine dihydrochloride with the target protein Gpc1 using a biofilm layer optical interference technique.
  • glycoprotein GP on the surface of the Ebola virus envelope was digested by the host protease Cathepsin in the endosome to become the activated glycoprotein GPcl, exposing the receptor binding site.
  • a fiber-layer biosensor-based biolayer interferometry (BLI) technology was used to determine berberamine dihydrochloride and the target protein in vitro. GPcl binding ability.
  • BLI technology can track the interactions between biomolecules in real time and is ideal for studying the interactions between proteins and other biomolecules. The specific steps are:
  • the target protein GPcl needs to be biotinylated first.
  • Mix Biotin EZ-LinkTM NHS-LC-LC-Biotin, Cat. # 21343, ThermoScientificTM
  • ThermoScientificTM the purified target protein GPcl at a molar ratio of 3: 1
  • react for 1 hour at room temperature through a desalting column Zeba TM Spin Desalting Columns, Cat. # 89883, Thermo
  • the combination experiment uses an Octet RED96 (ForteBio, Inc., CA, USA) instrument.
  • the experiment is mainly performed by the following steps: 1) detecting the baseline and immersing the SSA sensor in a buffer solution for 120s to reach equilibrium; 2) the biotinylated The target protein GPcl is incubated on the sensor. The sensor probe is moved to the biotinylated GPcl protein solution (50 ⁇ g / ml) and left for 600s to fix the protein on the SSA sensor. 3) Close the sensor and move the sensor to a 5 ⁇ M organism. (EZ-Biocytin, Cat.
  • Thermo was blocked for 60s in the solution; 4) The second detection baseline was moved to the buffer solution and allowed to stand for 120s to reach equilibrium; 5) Combined to move the sensor to the compound solution The Kon value is measured after standing for 60s; 5) Dissociate and move the sensor to the buffer solution for 60s to obtain the Koff value.
  • the buffer used in the experiment was PBS (for solubilizing protein) and PBS + 5% DMSO (for solubilizing berberamine dihydrochloride). In this experiment, loading and detection are performed separately.
  • the first microplate contains 3 columns, the first column is PBS as the baseline, the second column is the biotinylated target protein GPcl, and the third column is 5 ⁇ M biocytin is used for blocking.
  • the second microplate is detected.
  • the first to sixth columns are PBS + 5% DMSO, and the seventh to 12th columns are berbamine dihydrochloride from low to high. Concentration gradient (31.25 ⁇ M-500 ⁇ M). In this process, five different concentrations of berbamine dihydrochloride solution were used to obtain the final kinetic curve. ForteBio data analysis software DataAnalysis9.0 was used to analyze the experimental data.
  • the dissociation rate constant KD Koff / Kon.
  • the abscissa in FIG. 7 is the response time in seconds.
  • the ordinate is the signal intensity of the interaction between GPcl and the compound berbamine dihydrochloride, and the unit is nm. The results show that berberamine dihydrochloride can bind to GPcl protein.
  • the present invention takes EBVO as an example to specifically explain the antiviral mechanism of the technical scheme of the present invention
  • the scope of protection of the present invention on the use of berberamine dihydrochloride or a pharmaceutically acceptable salt thereof is not limited to EBOV.
  • Any virus that applies the above-mentioned antiviral mechanism is within the scope of the virus described in the present invention.
  • it can be the other four subtypes of EBOV, as well as Marburg virus (MARV) and Lassa virus (LASA). And other filamentous viruses.

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Abstract

二盐酸小檗胺在制备埃博拉病毒抑制剂中的应用。以埃博拉病毒激活态的包膜糖蛋白(EBOV-GPcl)为靶点，通过基于结构的虚拟筛选得到具有与EBOV-GPcl结合能力的抗病毒的活性化合物，该化合物为二盐酸小檗胺。二盐酸小檗胺能够通过与靶蛋白EBOV-GPcl结合从而特异性抑制埃博拉重组病毒的进入，达到抗埃博拉病毒感染的效应。二盐酸小檗胺抗EBOV的半最大效应浓度(EC50)为0.49μM，说明二盐酸小檗胺对EBOV具有较强的抑制作用。

Description

二盐酸小檗胺在制备埃博拉病毒抑制剂中的应用
本申请要求于2018年08月01日提交中国专利局、申请号为201810863809.5、发明名称为“二盐酸小檗胺在制备埃博拉病毒抑制剂中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及二盐酸小檗胺的用途，特别涉及二盐酸小檗胺在制备埃博拉病毒抑制剂中的应用。
背景技术
病毒性出血热是一组由病毒所引起的自然疫源性疾病，以发热、出血和休克为主要临床特征。此类疾病在世界上分布很广，临床表现多较严重，病死率很高，目前世界上已发现十多种。常见病毒性出血热包括埃博拉出血热、马堡出血热、拉沙热、克里米亚-刚果出血热、裂谷热、登革出血热、黄热病及天花等。
埃博拉出血热(埃博拉病毒病)是由丝状病毒科的埃博拉病毒(Ebola virus，EBOV)所引起的一种急性出血性传染病，死亡率高达90％，是人类最致命的病毒性传染病之一。EBOV可以分为5个种:扎伊尔型(Zaire ebolavirus,ZEBOV)、苏丹型(Sudanebolavirus,SUDV)、塔伊森林型(Tai Forest ebolavirus,TAFV)、本迪布焦型(Bundibugyoebolavirus,BDBV)和莱斯顿型(Reston ebolavirus,RESTV)。其中，扎伊尔型埃博拉病毒致病力最强。
马尔堡出血热是由马尔堡病毒(Marberg virus,MARV)引起的一种急性发热性疾病，有严重的出血表现。它和埃博拉出血热属于同一家族，都是高致死性传染病。马尔堡病毒和埃博拉病毒属于丝状病毒科(Filoviridae)的丝状病毒属(Filovirus)。
拉沙热是由拉沙病毒(LASV)引起，主要经啮齿类动物传播的一种急性传染病。拉沙病毒属于沙粒病毒科(Arenaviridae)哺乳类沙粒病毒属(Mammarenavirus)。
包膜糖蛋白(Glycoprotein,GP)指由病毒自身编码的、包被在病毒外层的糖蛋白。GP是一种多功能蛋白质，在病毒的吸附和穿入宿主细胞、致病性、下调宿主细胞表面蛋白质表达和增加病毒装配和出芽过程中起着至 关重要的作用。
目前对病毒性出血热主要采取对症和支持治疗，尚没有经系统临床验证有效的特异性治疗药物和疫苗。
发明内容
本发明所要解决的技术问题是如何抑制埃博拉病毒、马尔堡病毒和/或拉沙病毒等引起病毒性出血热的病毒。
为了解决以上技术问题，本发明提供了二盐酸小檗胺的用途。
二盐酸小檗胺的结构式如图1所示，其CAS登录号为6078-17-7。
本发明所提供的二盐酸小檗胺的用途为U1至U5中的任一种；
U1.二盐酸小檗胺或其药学上可接受的盐在制备病毒抑制剂中的应用；所述病毒可为能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒；
U2.二盐酸小檗胺或其药学上可接受的盐在抑制病毒中的应用；所述病毒可为能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒；
U3.二盐酸小檗胺或其药学上可接受的盐在制备治疗和/或预防病毒性出血热产品(如药物、疫苗或药物制剂)中的应用；所述病毒性出血热可为由如下病毒所致疾病：能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒；
U4.二盐酸小檗胺或其药学上可接受的盐在治疗和/或预防病毒性出血热中的应用；所述病毒性出血热可为由如下病毒所致疾病：能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒；
U5.二盐酸小檗胺或其药学上可接受的盐在制备与病毒激活态的包膜糖蛋白结合的产品(如药物、疫苗或药物制剂)中的用途。
上述用途中，所述病毒可为丝状病毒科和/或沙粒病毒科的病毒，如引起病毒性出血热的病毒。所述引起病毒性出血热的病毒可为埃博拉病毒、马尔堡病毒和/或拉沙病毒。
上述用途中，所述病毒性出血热可为埃博拉出血热、马堡出血热和/或拉沙热。
上述用途中，所述病毒抑制剂、所述治疗和/或预防病毒性出血热产 品和与病毒激活态的包膜糖蛋白结合的产品，除含有二盐酸小檗胺或其药学上可接受的盐外，还可含有适宜的载体或赋形剂。这里的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)。其中优选的是水溶性载体材料。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。为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯、山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。为了将单位给药剂型制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等；粘合剂如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。为了将单位给药剂型制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。为了将单位给药剂型制成注射用制剂，如溶液剂、乳剂、冻干粉针剂和混悬剂，可以使用本领域常用的所有稀释剂，例如，水、乙醇、聚乙二醇、1,3-丙二醇、乙 氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。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。
本发明还提供了药用化合物。
本发明所提供的药用化合物为二盐酸小檗胺或其药学上可接受的盐。
上述药用化合物中，所述药用化合物可用于抑制病毒感染动物。所述病毒可为能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒。所述病毒可为丝状病毒科和/或沙粒病毒科的病毒，如引起病毒性出血热的病毒。所述引起病毒性出血热的病毒可为埃博拉病毒、马尔堡病毒和/或拉沙病毒。
本发明还提供了抑制病毒感染动物的方法。
本发明所提供的抑制病毒感染动物的方法，包括给受体动物施用二盐酸小檗胺或其药学上可接受的盐以抑制病毒感染动物；所述病毒可为能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒。进一步，所述病毒可为丝状病毒科和/或沙粒病毒科的病毒，如引起病毒性出血热的病毒。所述引起病毒性出血热的病毒可为埃博拉病毒、马尔堡病毒和/或拉沙病毒。
本发明还提供了治疗和/或预防病毒性出血热的方法。
本发明所提供的治疗和/或预防病毒性出血热的方法，包括给受体动物施用二盐酸小檗胺或其药学上可接受的盐进行治疗和/或预防病毒性出血热；所述病毒性出血热可为由如下病毒所致疾病：能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒。
本发明中，所述动物可为哺乳动物，如人；所述动物还可为除哺乳动物以外的所述病毒感染的其他动物，如禽。
本发明中，术语“药学上可接受的盐”指在可靠的医学判断范围内，适合用于与人类和低等动物的组织接触而不出现过度的毒性、刺激、过敏反 应等，且与合理的效果/风险比相称的盐。药学可接受的盐是本领域公知的。例如，S.M.Berge,etal.,J.Pharmaceutical Sciences,1977,66:1中对药学可接受的盐进行了详细描述。
本发明中，所述埃博拉病毒可为扎伊尔型、苏丹型、塔伊森林型、本迪布焦型和/或莱斯顿型埃博拉病毒。
本发明中，所述抑制病毒也可称为抗病毒。所述抑制病毒可为抑制病毒侵入细胞。所述抑制病毒侵入细胞可为抑制病毒激活态的包膜糖蛋白(GPcl)介导的病毒进入细胞。
本发明以埃博拉病毒激活态的包膜糖蛋白(EBOV-GPcl)为靶点，通过基于结构的虚拟筛选得到具有与EBOV-GPcl结合能力的抗病毒的活性化合物，该化合物为二盐酸小檗胺。二盐酸小檗胺能够通过与靶蛋白EBOV-GPcl结合从而特异性抑制埃博拉重组病毒的进入，达到抗埃博拉病毒感染的效应。二盐酸小檗胺抗EBOV的半最大效应浓度(EC50)为0.49μM，说明二盐酸小檗胺对EBOV具有较强的抑制作用。
说明书附图
图1：二盐酸小檗胺的结构式。
图2：实施例1中二盐酸小檗胺对EBOV-Zaire GP/HIV-luc重组病毒的进入具特异性抑制作用；图2中，VSVG表示VSV-G/HIV-luc，Ebola-GP表示EBOV-Zaire GP/HIV-luc，病毒感染率＝1-抑制率，DMSO表示空白对照处理，TET表示粉防己碱处理，1-22分别表示22种化合物处理，其中10为二盐酸小檗胺处理。
图3：实施例2中细胞生长实验验证二盐酸小檗胺对293T细胞生长的影响；图3中，DMSO表示空白对照处理，TET表示粉防己碱处理，EEI-10表示二盐酸小檗胺处理。
图4：实施例3中二盐酸小檗胺对EBOV-Zaire GP/HIV-luc重组病毒的抑制作用具有良好的剂量依赖性。
图5：实施例4中药物作用时间点实验表明二盐酸小檗胺作用于病毒的进入阶段；图5中，TET表示粉防己碱处理，EEI-10表示二盐酸小檗胺处理，RT表示依法韦仑处理。
图6：实施例5中二盐酸小檗胺对MARV-GP/HIV-luc重组病毒和 LASV-GP/HIV-luc重组病毒均有抑制作用。
图7：生物膜层光学干涉技术体外测定不同浓度的二盐酸小檗胺与靶蛋白GPcl的动力学结合曲线。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为本领域技术人员公知的常规方法或按照厂商所建议的条件。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
二盐酸小檗胺为一种已知化合物，可购自市售产品，具体的获取手段为现有技术，本发明对此不作特别限定。下述实施例中的二盐酸小檗胺为TargetMol公司产品。
下述实施例中的真核表达载体pcDNA3.1(+)为Invitrogen公司产品。
下述实施例中的携带荧光素酶报告基因的HIV-luc质粒pNL4-3Luc(R-E-)(Ma,L.,etal.(23May 2018)."Identification of small molecule compounds targeting theinteraction of HIV-1Vif and human APOBEC3G by virtual screening andbiological evaluation."Sci Rep 8(1):8067)，公众可以从中国医学科学院医药生物技术研究所获得该生物材料。该生物材料只为重复本发明的实验所用，不可作为其它用途使用。
前人的研究表明埃博拉病毒包膜糖蛋白(EBOV-GP)进入溶酶体后会发生酶切，酶切之后的激活态的包膜糖蛋白(Primed GP,GPcl)可以和内吞体受体-人源胆固醇转运蛋白(Niemann-Pick C1，NPC1)直接相互作用，从而引发病毒与宿主细胞内的膜融合过程。在本发明中，发明人根据EBOV-GPcl(埃博拉病毒激活态的包膜糖蛋白)与NPC1-C相互作用，采用全新药物设计方法设计合成与EBOV-GPcl特异性结合并且能抑制埃博拉病毒进入细胞的活性多肽。发明人根据活性多肽与EBOV-GPcl之间的氢键、静电相互作用和疏水相互作用等构建药效基团模型，建立了一个靶向EBOV-GPcl的埃博拉病毒进入抑制剂的虚拟筛选方法，以期找到与 EBOV-GPcl特异性结合的小分子化合物，从而抑制EBOV-GPcl与NPC1-C的结合，进而抑制埃博拉病毒的复制。利用此模型对数据库进行筛选，经过多软件打分最终得到目标化合物，对其进行生物学活性检测，最终得到以EBOV-GPcl为靶点抗病毒的活性化合物，该化合物为二盐酸小檗胺，其具有与EBOV-GPcl结合的能力。
EBOV被列为危险性四级病毒，因此本发明使用假病毒技术这种安全有效的研究手段对小分子化合物进行体外水平的生物学活性评价。利用毒性最强的扎伊尔型EBOV的GP蛋白包裹HIV核心制备复制缺陷型假病毒EBOV-GP/HIV-luc，通过荧光报告基因检测技术来判断样品的抗病毒活性。与此同时，采用VSVG/HIV-luc重组病毒模型分析小分子化合物的特异性。在排除细胞毒性后，采用药物作用时间点实验进一步验证小分子化合物的作用机制。最后利用基于光纤生物传感器的生物膜层光学干涉技术体外测定小分子化合物与靶蛋白GPcl的结合能力，验证小分子化合物的靶向性。具体实验方法和结果如下。
实施例1、EBOV进入抑制剂筛选模型验证二盐酸小檗胺能够特异性抑制EBOV活性。
利用细胞水平重组病毒技术，将Zaire-EBOV的GP与HIV核心质粒(pNL4-3.Luc)共表达，制备重组病毒，应用靶向GP蛋白的EBOV进入抑制剂的高通量筛选模型评价化合物的抗病毒活性。具体步骤为：
取293T细胞进行培养，待细胞长满培养瓶后，弃旧培养基，用含0.25％胰酶和0.02％EDTA的消化液消化。待细胞变圆，弃消化液，立即加入含10％FBS(购自GIBCO)的高糖DMEM培养基(GIBICO)，用吸管轻轻吹打瓶底，使细胞完全脱离瓶底且使之分散为单细胞悬液。计数后，用培养基将细胞浓度调整为2.2×10 5个/ml，接种于6孔板，2mL/孔。24h后(细胞丰度约为70％)转染，质粒用量：2μgpZEBOV-GP与3μg携带荧光素酶报告基因的HIV-luc质粒pNL4-3Luc(R-E-)，转染试剂为Lipofectamine2000(Invitrogen公司),根据使用说明书进行转染，产生埃博拉假型病毒，将该埃博拉假型病毒命名为EBOV-Zaire GP/HIV-luc。在转染后48小时收集含有假型病毒的上清液，合并，通过低速离心从漂浮的细胞和细胞碎片中澄清，并通过0.45μm孔径的过滤器过滤。通过使用 ELISA测定法测量病毒相关的HIV p24水平来定量假病毒颗粒。
其中，pZEBOV-GP是将Zaire ebolavirus isolate H.sapiens-wt/GIN/2014/Makona-Gueckedou-C07的GP基因(GenBank Accession No.KJ660347.2(Update Date Dec18,2014 01:25PM)的第5900-8305位插入到载体pcDNA3.1(+)得到的表达Zaire-EBOV的糖蛋白(GP)的重组表达质粒。
将EBOV-Zaire GP/HIV-luc假病毒颗粒与293T细胞一起温育到96孔板中。48小时后，收集细胞并裂解以测量萤火虫萤光素酶活性。萤光素酶活性的值代表病毒感染。
将化合物用DMSO溶解后分别与EBOV-Zaire GP/HIV-luc假病毒混合加入到293T细胞中，使化合物含量为10μM。48小时后，裂解293T细胞，通过测量荧光素酶活性评估化合物对病毒的抑制率。以溶剂DMSO为空白对照，同时引入EBOV进入抑制剂粉防己碱(tetrandrine，TET)作为对照。粉防己碱用DMSO溶解后与EBOV-Zaire GP/HIV-luc假病毒混合加入到293T细胞中，粉防己碱的含量为1μM。48小时后，裂解293T细胞，通过测量荧光素酶活性评估化合物对病毒的抑制率。化合物对病毒的抑制率＝1-相对荧光素酶活性。相对荧光素酶活性是指相对于空白对照的荧光素酶活性。荧光素酶活性实际代表的就是病毒感染性。
目前已知的EBOV抑制剂大多是广谱性抗病毒药物，为了寻找针对EBOV的窄谱抑制剂，需要对筛到的活性化合物进行特异性分析。由于水泡性口膜炎病毒外壳糖蛋白(vesicular stomatitis virus glycoprotein,VSVG)和EBOV-GPcl作用相似，在病毒和受体的识别中发挥重要作用，因此使用表达VSVG的假病毒VSV-G/HIV-luc对化合物进行特异性分析。在排除细胞毒性因素后，同样利用荧光素酶原理检测化合物对VSV-G/HIV-luc假病毒的抑制活性，方法同上。若化合物只对EBOV-GPcl介导的病毒进入有明显抑制作用，而对VSV没有抑制或抑制率很低，则说明此化合物对EBOV具有特异性。
实验重复三次，结果如图2所示,化合物二盐酸小檗胺对EBOV-Zaire GP/HIV-luc假病毒的抑制率高于80％，相同浓度下对VSV-G/HIV-luc假病毒几乎没有抑制作用。这说明二盐酸小檗胺对EBOV-Zaire GP/HIV-luc 假病毒具有特异性抑制作用。作为阳性对照的EBOV进入抑制剂粉防己碱(TET)具有与二盐酸小檗胺相似的选择性抑制作用。
其中，表达VSV-GP的假病毒VSV-G/HIV-luc的制备方法与EBOV-Zaire GP/HIV-luc的制备方法的区别仅在于将EBOV-Zaire GP/HIV-luc的制备方法中的pZEBOV-GP替换为pVSV-GP，其它操作完全相同。pVSV-GP是将水泡性口膜炎病毒外壳糖蛋白GP基因(GenBankAccessionNo.V01214.1(Update Date Feb4,2011)的第14-1567位插入到载体pcDNA3.1(+)得到的表达水泡性口膜炎病毒外壳糖蛋白的重组表达质粒。
实施例2、二盐酸小檗胺的抗病毒活性与其细胞毒性无关。
为了排除由于化合物毒性而产生的非特异性差异，采用细胞计数试剂盒-8(cellCounting Kit-8，CCK-8)评估二盐酸小檗胺对293T细胞生长的影响。
CCK-8试剂盒是检测细胞增殖、细胞存活和细胞毒性的试剂盒，是一种基于WST-8(水溶性四唑盐，化学名：2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐)的广泛应用快速高灵敏度检测试剂盒，为MTT法的替代方法，试剂盒中采用水溶性四唑盐-WST-8，在电子耦合试剂存在的情况下，可以被线粒体内的一些脱氢酶还原生成橙黄色的formazan。细胞增殖越多越快，则颜色越深；细胞毒性越大，则颜色越浅。对于同样的细胞，颜色的深浅和细胞数目呈良好线性关系。通过酶联免疫检测仪在450nm波长处测定其光吸收值，可间接反映活细胞数量。具体步骤为：
将293T细胞在96孔板中培养并与二盐酸小檗胺(用DMSO溶解)孵育，二盐酸小檗胺在培养基中的含量分别为10μM、2.5μM、0.625μM。48小时后，细胞上清更换为含有10％CCK-8试剂的细胞培养液，细胞在37℃、5％CO 2孵箱中继续培养1h。在微孔板读数器(Thermo，Varioskan Flash)上记录450nm处每孔的光密度(OD)值。
以粉防己碱(TET)作为对照，将293T细胞在96孔板中培养并与粉防己碱(用DMSO溶解)孵育，粉防己碱在培养基中的含量分别为10μM、2.5μM、0.625μM。48小时后，细胞上清更换为含有10％CCK-8试剂的细 胞培养液，细胞在37℃、5％CO 2孵箱中继续培养1h。在微孔板读数器(Thermo，Varioskan Flash)上记录450nm处每孔的光密度(OD)值。
以溶剂DMSO为空白对照(DMSO)。将空白对照的OD450nm记作细胞活力为100％。
实验重复三次。结果如图3所示，在最高浓度10μM(远远高于测得的IC50值)，二盐酸小檗胺对细胞活性均无明显影响。由此说明，二盐酸小檗胺的抗病毒活性与其细胞毒性无关。
实施例3、二盐酸小檗胺对EBOV的抑制作用具有良好的剂量依赖性。
参照实施例1中的方法，将二盐酸小檗胺用DMSO溶解后分别与实施例1中的EBOV-Zaire GP/HIV-luc混合后加入到293T细胞中，使二盐酸小檗胺的含量分别为0.15625、0.3125、0.625、1.25、2.5、5、10、20μM。48小时后，裂解293T细胞，通过测量荧光素酶活性评估二盐酸小檗胺的抗EBOV活性。以溶剂DMSO为空白对照(DMSO)，将空白对照的荧光素酶活性作为细胞存活力为100％。实验重复三次，结果如图4所示，二盐酸小檗胺呈剂量依赖性地显著抑制EBOV-Zaire GP/HIV-luc假病毒活性。二盐酸小檗胺抗EBOV的半最大效应浓度(EC50)为0.49μM。
实施例4、通过药物作用时间点实验确定二盐酸小檗胺作用于病毒的进入阶段。
二盐酸小檗胺对EBOV-Zaire GP/HIV-luc假病毒的特异性抑制作用表明它们可能充当EBVO进入抑制剂。为了验证这点，通过药物作用时间点(Time of addition，TOA)实验研究二盐酸小檗胺在病毒感染周期中的作用阶段。具体步骤为：
感染前一天，将293T细胞按细胞数6×10 4个/孔接种到96孔板中,分别加入实施例1的EBOV-Zaire GP/HIV-luc 50μL感染细胞。在感染(-1小时)，感染期间(0小时)以及感染后于2、4、6、8、10、12、14和16h时间点加入二盐酸小檗胺(用DMSO溶解，在培养基中的含量(终浓度)为1×10 -5mol·L -1),以EBOV进入抑制剂粉防己碱(tetrandrine，TET)(用DMSO溶解，在培养基中的含量为1×10 -7mol·L -1)、非核苷类逆转录酶抑制剂依法韦仑(efavirenz，EFV)(用DMSO溶解，在培养基中的含量为 1×10 -9mol·L -1)为对照，DMSO为溶剂对照；感染48h后，检测报告基因荧光素酶活性反映重组病毒复制水平。
通过测定EBOV单次感染时药物的失效时间可初步判断药物的作用环节。如图5所示，二盐酸小檗胺在病毒进入的早期表现出非常强的抑制作用，在病毒完成吸附过程之后对病毒感染无抑制作用。这与EBOV进入抑制剂粉防己碱的作用时间一致。非核苷类逆转录酶抑制剂依法韦仑在6h时依然对病毒有抑制作用。这些结果表明二盐酸小檗胺是在病毒与宿主结合之后，病毒与宿主发生膜融合之前发挥作用。
实施例5、应用马尔堡重组病毒和拉沙重组病毒模型对化合物进行评价。
埃博拉病毒属于丝状病毒家族，基于重组病毒技术，建立了另外两株丝状重组病毒模型，分别是马尔堡重组病毒(表达MARV-GP的假病毒MARV-GP/HIV-luc)和拉沙重组病毒(表达LASV-GP的假病毒LASV-GP/HIV-luc)。其中，表达MARV-GP的假病毒MARV-GP/HIV-luc以及表达LASV-GP的假病毒LASV-GP/HIV-luc的制备方法均与EBOV-Zaire GP/HIV-luc的制备方法的区别仅在于将EBOV-Zaire GP/HIV-luc的制备方法中的pZEBOV-GP分别替换为pMARV-GP以及pLASV-GP，其它操作完全相同。
pMARV-GP是将马尔堡病毒外壳糖蛋白GP基因(GenBank Accession No.NC_001608.3)(Update Date 12-NOV-2014)的第5941-7986位插入到载体pcDNA3.1(+)得到的表达马尔堡病毒外壳糖蛋白的重组表达质粒。
pLASV-GP是将拉沙病毒外壳糖蛋白GP基因(GenBank Accession No.J04324.1)(Update Date Jun23,2010)的第1872-3347位插入到载体pcDNA3.1(+)得到的表达拉沙病毒外壳糖蛋白的重组表达质粒。
参照实施例3的方法，应用MARV-GP/HIV-luc和LASV-GP/HIV-luc重组病毒模型测定二盐酸小檗胺抗马尔堡病毒和拉沙病毒的半最大效应浓度。如图6所示，二盐酸小檗胺可抑制马尔堡病毒和拉沙病毒进入宿主，EC50分别为0.99μM和2.64μM。本研究提示，二盐酸小檗胺具有广谱抗病毒作用。通过蛋白序列比对结果可见，两株埃博拉病毒与马尔堡病毒GP蛋白的序列同源性仅为23％。应用多株病毒模型评价化合物将有利于 广谱抗病毒药物的发现，并将有助于药物的作用机制研究。
实施例6、采用生物膜层光学干涉技术体外测定二盐酸小檗胺与靶蛋白GPc l的结合能力。
埃博拉病毒囊膜表面糖蛋白GP在内吞体里经过宿主蛋白酶Cathepsin的酶切处理，变成激活态糖蛋白GPcl，暴露出受体结合位点。为了验证二盐酸小檗胺是通过与靶蛋白GPcl结合从而特异性抑制病毒的进入，利用基于光纤生物传感器的生物膜层光学干涉(BioLayer Interferometry,BLI)技术体外测定二盐酸小檗胺与靶蛋白GPcl的结合能力。BLI技术能够实时跟踪生物分子间的相互作用，是研究蛋白质和其它生物分子相互作用的理想选择。具体步骤为：
由于实验选用Super streptavidin(SSA)生物传感器，首先需要对靶蛋白GPcl进行生物素化处理。将生物素(EZ-Link TM NHS-LC-LC-Biotin，Cat.#21343,ThermoScientific TM)与纯化后的靶蛋白GPcl按摩尔比3:1进行混合，室温反应1小时后过脱盐柱(Zeba TM Spin Desalting Columns，Cat.#89883,Thermo)除去未反应的生物素，得到生物素化的靶蛋白GPcl。
结合实验采用Octet RED96(ForteBio，Inc.，CA，USA)仪器，主要通过以下步骤进行实验：1)检测基线将SSA传感器浸没在缓冲溶液中静置120s以达平衡；2)将生物素化的靶蛋白GPcl孵育到传感器上将传感器探针移动到生物素化后的GPcl蛋白溶液(50μg/ml)中静置600s，使蛋白固定在SSA传感器上；3)封闭传感器将传感器移动到含5μM生物胞素(EZ-Biocytin,Cat.#28022,Thermo)的溶液中封闭60s；4)第二次检测基线将传感器移动到缓冲溶液中静置120s以达平衡；5)结合将传感器移动到化合物溶液中静置60s测量Kon值；5)解离将传感器移动到缓冲溶液中静置60s以获得Koff值。实验所用缓冲液为PBS(用于溶解蛋白)和PBS+5％DMSO(用于溶解二盐酸小檗胺)。本次实验将上样和检测分开进行，第一块微孔板含有3列，第1列为PBS用作基线，第2列为生物素化的靶蛋白GPcl用于上样，第3列为5μM生物胞素用于封闭，传感器上样之后检测第2块微孔板，其第1至第6列为PBS+5％DMSO，第7至第12列为二盐酸小檗胺由低到高浓度梯度(31.25μM-500μM)。在此过程中使用5种不同浓度的二盐酸小檗胺溶液获得最终的动力学曲线。使用 ForteBio数据分析软件DataAnalysis9.0分析实验数据。解离速率常数KD＝K off/K on。
图7中的横坐标为反应时间，单位为秒。纵坐标为GPcl与化合物二盐酸小檗胺的相互作用的信号强度，单位为nm。结果表明二盐酸小檗胺能够与GPcl蛋白结合。
虽然本发明以EBVO为例具体说明本发明技术方案的抗病毒机理，但本发明对二盐酸小檗胺或其药学上可接受的盐的用途的保护范围并不限于EBOV。任何适用上述抗病毒机理的病毒均在本发明所述病毒的范围之内，例如可以是EBOV的其他四种亚型以及马尔堡病毒(Marburg virus,MARV)、拉沙病毒(Lassa virus,LASV)等其它丝状病毒。
以上对本发明进行了详述。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。

Claims (10)

  1. 二盐酸小檗胺或其药学上可接受的盐的用途，其特征在于：所述用途为U1或U2；
    U1.二盐酸小檗胺或其药学上可接受的盐在制备病毒抑制剂中的应用；所述病毒为能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒；
    U2.二盐酸小檗胺或其药学上可接受的盐在抑制病毒中的应用；所述病毒为能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒。
  2. 根据权利要求1所述的用途，其特征在于：所述病毒为丝状病毒科和/或沙粒病毒科的病毒，如埃博拉病毒、马尔堡病毒和/或拉沙病毒。
  3. 二盐酸小檗胺或其药学上可接受的盐的用途，其特征在于：所述用途为U3或U4；
    U3.二盐酸小檗胺或其药学上可接受的盐在制备治疗和/或预防病毒性出血热产品中的应用；所述病毒性出血热为由如下病毒所致疾病：能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒；
    U4.二盐酸小檗胺或其药学上可接受的盐在治疗和/或预防病毒性出血热中的应用；所述病毒性出血热为由如下病毒所致疾病：能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒。
  4. 根据权利要求3所述的用途，其特征在于：所述病毒性出血热为埃博拉出血热、马堡出血热和/或拉沙热。
  5. 二盐酸小檗胺或其药学上可接受的盐在制备与病毒激活态的包膜糖蛋白结合的产品中的用途。
  6. 根据权利要求5所述的用途，其特征在于：所述病毒为丝状病毒科和/或沙粒病毒科的病毒，如埃博拉病毒、马尔堡病毒和/或拉沙病毒。
  7. 药用化合物，其特征在于：所述药用化合物为二盐酸小檗胺或其药学上可接受的盐。
  8. 根据权利要求7所述的药用化合物，其特征在于：所述药用化合物用于抑制病毒感染动物；所述病毒为能通过激活态的包膜糖蛋白与二盐酸 小檗胺或其药学上可接受的盐结合的病毒。
  9. 抑制病毒感染动物的方法，包括给受体动物施用二盐酸小檗胺或其药学上可接受的盐以抑制病毒感染动物；所述病毒为能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒。
  10. 治疗和/或预防病毒性出血热的方法，包括给受体动物施用二盐酸小檗胺或其药学上可接受的盐进行治疗和/或预防病毒性出血热；所述病毒性出血热为由如下病毒所致疾病：能通过激活态的包膜糖蛋白与二盐酸小檗胺或其药学上可接受的盐结合的病毒。
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CN110314160B (zh) * 2019-08-22 2023-05-26 辽宁大学 小檗胺在制备预防和治疗糖尿病肾病药物中的应用
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