WO2021189517A1 - Mthfd1抑制剂在抑制和杀灭病毒中的应用 - Google Patents

Mthfd1抑制剂在抑制和杀灭病毒中的应用 Download PDF

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WO2021189517A1
WO2021189517A1 PCT/CN2020/082809 CN2020082809W WO2021189517A1 WO 2021189517 A1 WO2021189517 A1 WO 2021189517A1 CN 2020082809 W CN2020082809 W CN 2020082809W WO 2021189517 A1 WO2021189517 A1 WO 2021189517A1
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virus
influenza
mthfd1
application according
alkyl
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PCT/CN2020/082809
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English (en)
French (fr)
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谭旭
崔进
谭文杰
黄宝英
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清华大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/22—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom rings with more than six members
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61K31/365—Lactones
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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

Definitions

  • the present invention relates to the field of medical technology, in particular to the application of MTHFD1 inhibitors (especially carolacton and its derivatives) in inhibiting and killing viruses.
  • MTHFD1 inhibitors especially carolacton and its derivatives
  • Coronavirus is a large virus family. Coronaviruses are known to infect the upper respiratory and gastrointestinal tracts of mammals and birds. They can cause colds and the Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome. , SARS) and other more serious diseases.
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • MTHFD1 Methylenetetrahydrofolate dehydrogenase (NADP+dependent)1 (MTHFD1) only encodes a single protein with 3 enzymes (5,10methylenetetrahydrofolate dehydrogenase, 5, The activities of 10-methine tetrahydrofolate cyclase and 10-formate tetrahydrofolate synthase) play a vital role in folate metabolism. There is no research report on the antiviral aspects of MTHFD1.
  • the present invention provides an application of an MTHFD1 inhibitor in the preparation of products for inhibiting and/or killing viruses.
  • the aforementioned MTHFD1 inhibitor may be an antibody or an antigen-binding fragment thereof, interfering RNA or a small molecule compound.
  • the aforementioned antigen-binding fragments can be selected from: Fab, Fab', F(ab)2, Fv, dsFv, scFv, Fd and Fd' fragments and the like.
  • the aforementioned interfering RNA may be selected from: siRNA, dsRNA, shRNA, aiRNA, miRNA, and combinations thereof.
  • the above-mentioned small molecule compound may be selected from: methotrexate, pemetrexed, trimexate, edatrexate, lometrisol, 5-fluorouracil, pratroxa, One or more of aminopterin and its salts, solvates, stereoisomers, ethers, esters, prodrugs, etc.
  • the above-mentioned small molecule compound may have the following structure:
  • R 1 , R 3 and R 4 are independently selected from: H, C1-C12 alkyl, C7-C12 aromatic hydrocarbon group;
  • R 2 is selected from: H, C1-C12 alkyl, C7-C12 aromatic hydrocarbon group and OR 8 ; wherein, R 8 is selected from: H, C1-C12 alkyl, C7-C12 aromatic hydrocarbon group;
  • R 5 , R 6 and R 7 are independently selected from: H, C1-C12 alkyl.
  • R 1 is H.
  • R 1 is selected from C1-C6 alkyl, especially C1-C3 alkyl; in one embodiment of the present invention, R 1 is methyl.
  • R 3 is H.
  • R 3 is selected from C1-C6 alkyl, especially C1-C3 alkyl; in one embodiment of the present invention, R 3 is methyl.
  • R 4 is H.
  • R 4 is selected from C1-C6 alkyl, especially C1-C3 alkyl; in one embodiment of the present invention, R 4 is methyl.
  • R 2 is OR 8 .
  • R 2 is OH
  • R 5 is selected from C1-C6 alkyl, especially C1-C3 alkyl; in one embodiment of the present invention, R 5 is methyl.
  • R 6 is selected from C1-C6 alkyl, especially C1-C3 alkyl; in one embodiment of the present invention, R 6 is methyl.
  • R 7 is selected from C1-C6 alkyl, especially C1-C3 alkyl; in one embodiment of the present invention, R 7 is methyl.
  • the bond connecting C-15 and C-16 is a single bond, and C-15 and C-16 are saturated with hydrogen atoms.
  • the bond connecting C-15 and C-16 is a double bond.
  • the bond connecting C-7 and C-8 is a single bond, and C-7 and C-8 are saturated with hydrogen atoms.
  • the bond connecting C-7 and C-8 is a double bond.
  • the above-mentioned MTHFD1 inhibitor has the following structure:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 have the above definitions of the present invention.
  • the above-mentioned MTHFD1 inhibitor has the following structure:
  • R 1 , R 2 , R 5 , R 6 and R 7 have the above definitions of the present invention.
  • MTHFD1 inhibitor has the following structure:
  • R 2 , R 5 , R 6 and R 7 have the above-mentioned definitions of the present invention.
  • the above-mentioned MTHFD1 inhibitor has the following structure:
  • the aforementioned MTHFD1 inhibitor is carolacton, which has the following structure:
  • the above-mentioned MTHFD1 inhibitor may also have the following structure:
  • the Carolacton derivative of the present invention can be obtained by the method described in, for example, WO2018220176A1.
  • viruses may be Adenoviridae, Herpesviridae (such as EBV), Papilloma vacuoleviridae, Picornaviridae, Poxviridae (such as variola virus), Hepatoviridae (such as type B) Hepatitis virus), coronavirus family (such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc.), Bonaviridae, filoviridae (E.g. Ebola virus), Orthomyxoviridae (e.g. influenza virus), Paramyxoviridae, Retroviridae (e.g. HIV), Reoviridae, Rhabdoviridae (e.g. rabies virus), yellow Virology etc.
  • Herpesviridae such as EBV
  • Papilloma vacuoleviridae Pi
  • the above-mentioned virus is a coronavirus, specifically HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc.
  • the above-mentioned virus is an orthomyxovirus, such as an influenza virus (such as influenza A virus, influenza B virus, influenza C virus, etc.).
  • influenza virus such as influenza A virus, influenza B virus, influenza C virus, etc.
  • the above-mentioned virus is a paramyxovirus, such as type 1 human parainfluenza virus (HPV), type 2 HPV, type 3 HPV, type 4 HPV, Sendai virus, mumps virus, measles virus, Respiratory syncytial virus, Newcastle disease virus, etc.
  • HPV type 1 human parainfluenza virus
  • HPV type 2 HPV
  • type 3 HPV type 4 HPV
  • Sendai virus mumps virus
  • measles virus measles virus
  • Respiratory syncytial virus Newcastle disease virus
  • the above-mentioned virus is a flavivirus, such as dengue fever virus, Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus Wait.
  • flavivirus such as dengue fever virus, Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus Wait.
  • the above-mentioned products can be used for diagnostic or therapeutic purposes, as well as for non-diagnostic or therapeutic purposes.
  • the inhibition and/or killing of the above-mentioned viruses can be carried out in vivo or in vitro.
  • the above-mentioned product is a pharmaceutical composition.
  • the MTHFD1 inhibitor can be used as the sole active ingredient, or can be used in combination with one or more other active ingredients for the same indication or different indications, wherein the MTHFD1 inhibitor and the Other active ingredients can be formulated for simultaneous, separate or sequential administration (simultaneous, separate or sequential administration).
  • the above-mentioned pharmaceutical composition further comprises pharmaceutically acceptable excipients.
  • the above-mentioned pharmaceutically acceptable excipients may include sweeteners (specifically, sucrose, xylitol, oligofructose, cyclamate, stevia, aspartame, etc.), aromatics (such as spices, flavors, etc.) ), mucilage agents (specifically, sodium alginate, gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, etc.), clarifying agents (specifically, chitosan, gelatin, etc.), preservatives (specifically For example, benzoic acid and its salts, sorbic acid and its salts, paraben series, etc.), disintegrants (specifically, low-substituted hydroxypropyl cellulose, crospovidone, sodium starch glycolate, croscarmellose Sodium methylcellulose, starch, etc.), binders (specifically, hydroxypropyl cellulose, hypromellose, povidone, copovidone, pregelatinized starch
  • the above-mentioned pharmaceutical composition can adopt any dosage form or administration form, especially oral dosage form.
  • Those skilled in the art can choose according to the situation, including, but not limited to, tablets (including sugar-coated tablets, film-coated tablets). , Sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), lozenges, syrups, solutions, emulsions, suspensions , Controlled release formulations (e.g., instant release formulations, sustained-release formulations, sustained-release microcapsules), aerosols, membranes (e.g., oral disintegrating membranes, oral mucosa-adhesive membranes), injections (e.g., subcutaneous Injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous drip, transdermal preparation, ointment, lotion, adhesive preparation, suppository (for example, rectal suppository, vaginal s
  • the above-mentioned product is a functional food composition.
  • the MTHFD1 inhibitor in the above functional food composition, can be used as the sole active ingredient, or can be used in combination with one or more other active ingredients.
  • the above-mentioned functional food composition may also include food supplements.
  • the form of the above-mentioned functional food composition can take any form, such as tablets, pills, capsules (such as soft capsules, microcapsules), candy (such as pressed tablet candies, gelatin candies, gum-based candies, etc.) ), solid beverages (such as powders, granules, etc.), liquid beverages, etc.
  • the various forms of the above-mentioned functional food composition can be prepared according to conventional production methods in the field of functional food.
  • the above-mentioned product is a disinfection product.
  • the MTHFD1 inhibitor can be used as the only active ingredient, or can be combined with one or more other active ingredients (such as chlorine-containing disinfectants, alcohol disinfectants, quaternary ammonium salt disinfectants, peroxides Disinfectants, etc.) in combination.
  • active ingredients such as chlorine-containing disinfectants, alcohol disinfectants, quaternary ammonium salt disinfectants, peroxides Disinfectants, etc.
  • the above-mentioned disinfection product may be a medical disinfection product, or may be a daily household disinfection product.
  • the above-mentioned disinfection product may also contain auxiliary materials of the disinfection product, such as one of moisturizer, fat excipient, flavoring agent, thickener, pH adjuster, skin feel adjuster, stabilizer, buffer, etc. Many kinds.
  • the above-mentioned disinfection products can be sprays, powders, solutions, gels, ointments, gels, etc. involved in medical pharmaceutical preparations, and can also be made into lotions (such as hand sanitizers, laundry liquids), toothpastes, etc. , Contact lens care solution, etc., can also be attached to the solid surface of sanitary or cleaning items by coating or dipping.
  • the sanitary or cleaning items can be sanitary or cleaning gloves, paper towels or paper, cotton swabs, dust cover or medical Face masks and so on.
  • the above-mentioned disinfection products can be used for skin, hair, clothing, household appliances, and the like.
  • the present invention also provides an application of an MTHFD1 inhibitor in the preparation of a medicament for preventing and/or treating diseases or disorders caused by or related to viral infections.
  • the MTHFD1 inhibitor has the structure of the above-mentioned general formula I of the present invention; more specifically, the MTHFD1 inhibitor has the structure of the above-mentioned formula V, especially formula VI of the present invention.
  • the aforementioned diseases or conditions may include acute bronchitis, chronic bronchitis, rhinitis, sinusitis, croup, acute bronchiolitis, pharyngitis, tonsillitis, mumps, laryngitis, bronchitis, asthma, pneumonia, epidemic One or more of colds, Zika virus disease, etc.
  • the above-mentioned disease is COVID-19.
  • the above-mentioned disease is influenza.
  • the above-mentioned disease is mumps.
  • the above-mentioned disease is Zika virus disease.
  • the present invention also provides a method for preventing and/or treating diseases or disorders caused by or related to viral infections, which includes the step of administering to a subject an effective amount of the above-mentioned MTHFD1 inhibitor of the present invention.
  • the MTHFD1 inhibitor has the structure of the above-mentioned general formula I of the present invention; more specifically, the MTHFD1 inhibitor has the structure of the above-mentioned formula V, especially formula VI of the present invention.
  • the aforementioned diseases or conditions may include acute bronchitis, chronic bronchitis, rhinitis, sinusitis, croup, acute bronchiolitis, pharyngitis, tonsillitis, mumps, laryngitis, bronchitis, asthma, pneumonia, epidemic One or more of colds, Zika virus disease, etc.
  • the above-mentioned disease is COVID-19.
  • the above-mentioned disease is influenza.
  • the above-mentioned disease is mumps.
  • the above-mentioned disease is Zika virus disease.
  • the above-mentioned subject is an animal; in one embodiment of the present invention, the above-mentioned subject is a mammal, such as humans, monkeys, apes, cows, horses, pigs, seals, etc.; in another embodiment of the present invention In the method, the above-mentioned subject is a bird, such as a chicken.
  • the invention also provides the application of the destruction of MTHFD1 in inhibiting and/or killing viruses.
  • the destruction of MTHFD1 can be achieved through the inhibition of antibodies or antigen-binding fragments, interfering RNA or small molecule compounds, or through gene editing methods (such as knocking out or knocking down genes to disrupt the expression of MTHFD1).
  • MTHFD1 is a potential broad-spectrum antiviral target, and its inhibitors (especially carolacton) can effectively and broadly inhibit viruses (specifically, influenza virus, coronavirus, mumps virus, Zika virus, etc.) ) Proliferation, which may have very good application prospects in the field of anti-virus.
  • Figure 1 shows the results of knocking out MTHFD1 to inhibit influenza virus replication.
  • Use CRISPR Cas9 technology to knock out the MTHFD1 gene in 293T cells, then infect the MTHFD1 knocked-out cell line with influenza virus, and then detect the amount of viral protein by western blot.
  • FIG. 2 shows a schematic diagram of the role of the MTHFD1 gene in the one-carbon metabolic pathway.
  • the MTHFD1 gene is an enzyme with three different catalytic activities, and its catalyzed synthesis product can be used for the synthesis of purine, dTMP, and methionine.
  • Figure 3 shows the result that the level of virus replication in the MTHFD1 knockout cell line can be rescued by exogenously added inosine.
  • the MTHFD1 gene knockout 293T cell line was supplemented with inosine, ⁇ -thymidine and 5-methyltetrahydrofolate, and then simultaneously infected with influenza virus.
  • the protein level of influenza virus is detected by western blot method.
  • Figure 4 shows the results of MTHFD1 gene knockout inhibiting the genomic RNA replication of influenza virus.
  • a After influenza virus infects control cells (containing normal MTHFD1 gene) and MTHFD1 knock-out cell lines, samples are taken at different infection time points, total RNA is extracted, and cDNA is obtained by reverse transcription, and then real-time PCR is used to detect influenza virus Genomic RNA level;
  • b the results of influenza virus replicon experiments. Simultaneously transfect the three subunits of influenza virus polymerase, PB1, PB2 and PA, and influenza NP protein in control cells or MTHFD1 knock-out cell lines.
  • Simultaneous co-transfection can express influenza virus negative-strand genomic RNA ( hemagglutinin fragment) plasmid. Samples were taken at different time points after transfection, and western blot was used to detect the protein level of hemagglutinin (HA). The protein level of HA represents the level of influenza virus genome replication.
  • Figure 5 shows the results of inhibiting the replication of mumps virus and Zika virus (ZIKV) after knocking down the MTHFD1 gene by siRNA.
  • a the result of siRNA knocking down the MTHFD1 gene on the inhibition of mumps virus replication
  • b the result of siRNA knocking down the MTHFD1 gene on the inhibition of Zika virus replication
  • c the result of siRNA knocking down.
  • Figure 6 shows the results of inhibiting the replication of mumps virus and Zika virus after shRNA knocks down the MTHFD1 gene.
  • a the inhibition result of mumps virus replication after shRNA knockdown of MTHFD1 gene
  • b the inhibition result of Zika virus replication after shRNA knockdown of MTHFD1 gene
  • c the result of shRNA knockdown.
  • Figure 7 shows the results of inhibition of SARS-CoV2 by carolacton, an inhibitor of MTHFD1 gene.
  • Different concentrations of carolacton, an inhibitor of MTHFD1 gene were added to African green monkey kidney cells (Vero), and then infected with SARS-CoV2. After 48 hours of virus infection, the supernatant was collected to extract viral RNA, and the virus level was detected by RT-qPCR.
  • the Y-axis on the left represents the level of inhibition of the virus by the inhibitor. After the cells are treated with the same inhibitor, the toxicity of the drug to the cells is detected by the MTT test. The Y-axis on the right represents the cytotoxicity of the inhibitor.
  • Figure 8 shows the results of inhibition of Zika virus and mumps virus by carolacton, an inhibitor of MTHFD1 gene.
  • Different concentrations of carolacton, an inhibitor of MTHFD1 gene were added to bat kidney cells (PaKi), and then infected with Zika virus and mumps virus respectively. After 48 hours of virus infection, the virus infection rate was detected by immunofluorescence.
  • the Y-axis on the left represents the level of inhibition of the virus by the inhibitor. After the cells are treated with the same inhibitor, the toxicity of the drug to the cells is detected by the MTT test.
  • the Y-axis on the right represents the cytotoxicity of the inhibitor.
  • Figure 9 shows the results of the inosine rescue experiment, which shows that the inhibition of mumps virus by carolacton, an inhibitor of the MTHFD1 gene, can be offset by inosine added by foreign aid.
  • Figure 10 shows the results of the inosine rescue experiment, which shows that the inhibition of Zika virus by carolacton, an inhibitor of the MTHFD1 gene, can be offset by inosine added by foreign aid.
  • MTHFD1 inhibitor refers to a molecule that has an inhibitory effect on MTHFD1.
  • the inhibitory effect includes but is not limited to: inhibiting the activity of MTHFD1 and inhibiting the transcription or expression of MTHFD1 gene.
  • the MTHFD1 inhibitor includes, but is not limited to, antibodies or antigen-binding fragments thereof, interfering RNA, small molecule compounds, and the like.
  • Inhibiting the activity of MTHFD1 refers to reducing the activity of MTHFD1; specifically, compared to before the inhibition, the activity of MTHFD1 is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 95%, 99%, or even 100%.
  • Inhibiting the transcription or expression of the MTHFD1 gene refers to not transcribing the MTHFD1 gene, or reducing the transcription activity of the MTHFD1 gene, or not expressing the MTHFD1 gene, or reducing the expression activity of the MTHFD1 gene.
  • MTHFD1 genes that regulate the gene transcription or expression of MTHFD1, such as gene knockout, homologous recombination, interfering RNA and the like.
  • MTHFD1 The inhibition of gene transcription or expression of MTHFD1 can be verified by experimental methods such as PCR and Western Blot.
  • the transcription or expression of the MTHFD1 gene is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99 %, especially the MTHFD1 gene is not expressed at all.
  • alkyl refers to a linear or branched hydrocarbon chain radical that does not contain unsaturated bonds, and the hydrocarbon chain radical is connected to other parts of the molecule by a single bond.
  • Typical alkyl groups contain 1 to 12, 1 to 8, 1 to 6, or 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl Base, tert-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, etc.
  • Alkyl groups may be substituted, for example, if the alkyl group is substituted by a cycloalkyl group, it corresponds to a "cycloalkylalkyl group", such as cyclopropylmethyl; if the alkyl group is substituted by a halogen, it corresponds to a "haloalkyl group” ; If the alkyl group is substituted by an aryl group, then its corresponding "aralkyl", such as benzyl, benzhydryl or phenethyl; if the alkyl group is substituted by a heterocyclic group, then its corresponding "heterocyclyl alkyl Base”; etc.
  • Aromatic hydrocarbon group refers to a hydrocarbon group containing a benzene ring structure; "hydrocarbon group” refers to a group containing only carbon and hydrogen atoms, and usually refers to the free radical remaining after the corresponding hydrocarbon loses one hydrogen atom.
  • alkenyl refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms and at least one unsaturated bond, and the hydrocarbon chain radical is connected to other parts of the molecule by a single bond.
  • Typical alkenyl groups contain 2 to 12, 2 to 8, 2 to 6, or 2 to 3 carbon atoms, such as vinyl, 1-methyl-vinyl, 1-propenyl, 2-propenyl Or butenyl.
  • Alkynyl refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms and at least one carbon-carbon triple bond, and the hydrocarbon chain radical is connected to other parts of the molecule by a single bond.
  • Typical alkynyl groups contain 2 to 12, 2 to 8, 2 to about 6, or 2 to 3 carbon atoms, such as ethynyl, propynyl (e.g. 1-propynyl, 2-propynyl ) Or butynyl (e.g. 1-butynyl, 2-butynyl, 3-butynyl).
  • Cycloalkyl refers to an alicyclic hydrocarbon. Typical cycloalkyl groups contain 1 to 4 monocyclic and/or condensed rings, 3 to 18 carbon atoms, such as 3 to 10 carbon atoms or 3 to about 6 carbon atoms, such as cyclopropyl and cyclohexyl.
  • Aryl refers to monocyclic or polycyclic radicals, including polycyclic radicals containing monoaryl groups and/or condensed aryl groups. Typical aryl groups contain 1 to 3 monocyclic or condensed rings and 6 to 18 carbon ring atoms, preferably 6 to 14 carbon ring atoms, such as phenyl, naphthyl, biphenyl, indenyl, phenanthryl Or anthracene radicals.
  • Heterocyclic group includes heteroaromatic groups and heteroalicyclic groups containing 1 to 3 monocyclic and/or fused rings and 3 to 18 ring atoms. Specifically, heteroaromatic groups and heteroalicyclic groups contain 5 to about 10 ring atoms. Suitable heteroaryl groups may contain 1, 2, or 3 types of heteroatoms, which are selected from N, O or S atoms.
  • salt must be understood as any form of the compound according to the present invention, wherein said compound is in ionic form or is charged and coupled with an oppositely charged ion (cation or anion) or in solution.
  • This definition also includes quaternary ammonium salts and complexes of the molecule with other molecules and ions, especially complexes formed by ionic interactions.
  • solvate should be understood to refer to any form of the compound of the present invention, wherein the compound is connected to another molecule (usually a polar solvent) by a non-covalent bond, especially including hydrates and alcoholates, such as Methanolate.
  • solvates are hydrates.
  • prodrug uses its broad meaning and encompasses derivatives that can be transformed into the compounds of the invention in vivo.
  • examples of prodrugs include, but are not limited to, derivatives and metabolites of the compound, including biohydrolyzable moieties, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, and biohydrolyzable Carbonate esters, biohydrolyzable ureides, and biohydrolyzable phosphate analogs.
  • the prodrug with a carboxyl functional group is a lower alkyl ester of carboxylic acid. The carboxylic acid ester is easily obtained by esterification of any carboxylic acid moiety present in the molecule.
  • Prodrugs are usually prepared by known methods, such as in Burger’s “Medicinal Chemistry and Drug Discovery Sixth Edition (Donald J. Abraham ed., 2001, Wiley) and “Design and Applications of Prodrugs” (H. Bundgaard ed., 1985). , The method described in Harwood Academic Publishers).
  • any compounds referred to herein are intended to represent such specific compounds and certain variations or certain forms thereof.
  • the compounds referred to herein may have asymmetric centers and therefore exist in different enantiomeric or diastereomeric forms.
  • any given compound referred to herein represents any one of racemates, one or more enantiomeric forms, one or more diastereomeric forms, and mixtures thereof.
  • each double bond will have its own stereoisomerism, which can be the same or different from the stereoisomerism of other double bonds of the molecule.
  • the compounds referred to herein may exist atroisomers. All stereoisomers of the compounds referred to herein, including enantiomers, diastereomers, geometric isomers and atroisomers, and mixtures thereof, are within the scope of the present invention.
  • any of the compounds referred to herein may exist in the form of tautomers.
  • tautomer refers to one of two or more structural isomers of a compound. These isomers are in equilibrium and can be converted into each other. Common tautomer pairs are enamine-imine, amide-imine, keto-enol, lactam-lactam, etc.
  • the compounds of the present invention may also include isotopically labeled forms, that is, compounds that differ only in the presence of one or more isotopically-rich atoms.
  • isotopically labeled forms that is, compounds that differ only in the presence of one or more isotopically-rich atoms.
  • the compounds are all included in the scope of the present invention.
  • the compounds described in the present invention or their salts, solvates, stereoisomers, ethers, and esters are preferably in a pharmaceutically acceptable form.
  • pharmaceutically acceptable means that when the molecular body and the composition containing it are properly administered to a subject, they will not produce adverse, allergic or other adverse reactions.
  • sgMTHFD1-1 forward primer 5′-caccgAAGGGGAGTGGATCAAACCT-3′;
  • sgMTHFD1-1 reverse primer 5'-aaac AGGTTTGATCCACTCCCCTT c-3';
  • sgMTHFD1-2 forward primer 5′-caccg GAGGTTATTAGCTGCAGTGA-3′;
  • sgMTHFD1-2 reverse primer 5'-aaac TCACTGCAGCTAATAACCTC c-3'.
  • the synthesized sgRNA primer sequence was treated with T4 polynucleotide kinase at 37°C for 30 minutes to phosphorylate, denatured at 95°C for 5 minutes, and annealed at a temperature of 1.5°C/min to 25°C to obtain BsmBI (or BbsI)
  • BsmBI or BbsI
  • the double-stranded DNA fragments with sticky ends are as follows:
  • Phosphorylation, denaturation and annealing systems are:
  • the double-stranded DNA fragment was ligated with the LentiGuidepuro vector digested with BsmbI, and the ligation product was transformed into stbl3 competent cells, and spread on an ampicillin-resistant LB plate to select positive colonies and extract positive colony plasmids After analysis and sequencing, it was confirmed that the sgRNA expression vector was successfully constructed and named sgMTHFD1-1 and sgMTHFD1-2.
  • anti-MTHFD1 Proteintech, 10794-1-AP
  • anti- ⁇ actin Easybio, BE0022
  • anti-PR8M1 Genetex, GTX125928-S
  • anti-HA H1N1
  • Influenza virus RNA is reverse transcribed using specific primers.
  • Bio-Rad CFX96 fluorescence quantitative PCR instrument to detect.
  • the primer sequence is as follows:
  • Human GAPDH forward primer 5′-ACAACTTTGGTATCGTGGAAGG-3′;
  • Human GAPDH reverse primer 5′-GCCATCACGCCACAGTTTC-3′;
  • Influenza virus forward primer 5'-TTCTAACCGAGGTCGAAACGTACG-3';
  • Influenza virus reverse primer 5'-ACAAAGCGTCTACGCTGCAG-3';
  • Influenza virus specific reverse transcription primer 5'-AGCRAAAGCAGG-3';
  • P.alecto ACTIN forward primer 5’-gccagtctacaccgtctgcag-3’;
  • P.alecto ACTIN reverse primer 5’-cgtaggaatccttctggcccatg-3’;
  • P.alecto MTHFD1 forward primer 5’-gggagcgactgaagaaccaag-3’;
  • P.alecto MTHFD1 reverse primer 5’-tcttcagcagccttcagcttcac-3’;
  • ZIKV NS5 forward primer 5’-GGTCAGCGTCCTCTCTAATAAACG-3’;
  • ZIKV NS5 reverse primer 5’-GCACCCTAGTGTCCACTTTTTCC-3’.
  • the control 293T cells (MTHFD1 normal expression) and the MTHFD1 knock-out 293T cell line were plated into a 12-well plate with 2 ⁇ 10 5 cells per well. After culturing in medium containing 100 ⁇ M hypoxanthine and 16 ⁇ M ⁇ -thymidine overnight, the transfection can express the 3 subunits of influenza virus polymerase, PB1, PB2 and PA, and influenza NP protein, and the co-transfection can express at the same time Influenza virus negative-strand genomic RNA (hemagglutinin fragment) plasmid.
  • Influenza virus negative-strand genomic RNA hemagglutinin fragment
  • the medium was changed to a medium without hypoxanthine and ⁇ -thymidine, and samples were taken at different time points after transfection, and the protein level of hemagglutinin (HA) was detected by western blot.
  • the protein level of HA represents the level of influenza virus genome replication.
  • the cytotoxicity test of inhibitor is determined by MTT. Prepare the cells and add inhibitors according to the corresponding steps in Method 5.1. After the cells were cultured for 48 hours, the supernatant was discarded, and 90 ⁇ l of fresh medium and 10 ⁇ l of MTT (Solarbio) were added. After the cells were cultured for 4 hours, the supernatant was discarded, and 110 ⁇ l of DMSO was used to dissolve the cells, and the absorbance at OD490nm was measured.
  • the virus-infected cells were fixed with 4% paraformaldehyde solution for 10 minutes at room temperature, then permeabilized with 0.2% Triton X-100 at room temperature for 10 minutes, washed 3 times with PBS, and incubated with Zika virus E protein antibody (Millipore, MAB10216), 4°C overnight, wash 3 times with PBS, and then incubate the fluorescent secondary antibody AF488.
  • the nucleus was stained with DAPI.
  • the virus itself contains GFP, no primary antibody or secondary antibody treatment is required, and DAPI stains DNA directly after fixation.
  • the infection rate is detected and analyzed by the high-content imaging system (Cellomic ArrayScan VTI HCS, Thermo Scientific).
  • Mumps virus titer is determined by the TCID 50 method.
  • the level of Zika virus replication was detected by QPCR.
  • step 1.1 Refer to the sgRNA construction method in step 1.1 to connect the MTHFD1 shRNA to the PLKO.1 vector that has been digested with EcoRI and AgeI.
  • the PMD2.G and psPAX2 double plasmid lentiviral packaging system was used to co-transfect the shRNA plasmid in 293T.
  • the supernatant was harvested at 48h and inoculated into the paved Paki cells. After 48h, puromycin was used to screen and obtain positive cells with stable expression of shRNA.
  • the knockdown effect of shRNA was tested by western blot.
  • the selected cells are used for mumps virus and Zika virus infection experiments.
  • the virus infection rate is determined by immunofluorescence and high-content imaging system analysis.
  • PaKi shMTHFD1-1 forward primer 5’-ccgg GCACATGGGAATTCCTCTACCctcgagGGTAGAGGAATTCCCATGTGCtttttg-3’;
  • PaKi sh MTHFD1-1 reverse primer 5’-AATTCAAAAAGCACATGGGAATTCCTCTACC CTCGAGGGTAGAGGAATTCCCATGTGC-3’;
  • PaKi sh MTHFD1-2 reverse primer 5’-AATTCAAAAAGCCTGCTGTCACTTAGGAAAT CTCGAG ATTTCCTAAGTGACAGCAGGC-3’.
  • virus samples to be tested were diluted 10 times in a gradient and added to the cells respectively, with 100 ⁇ l per well. After 96h, observe the CPE and calculate the virus titer according to the TCID 50 method.
  • MTHFD1 knockout cell line The replication of influenza virus in MTHFD1 knockout cell line can be rescued by exogenously added inosine.
  • MTHFD1 is an important enzyme in the one-carbon metabolic pathway, which can catalyze a three-step reaction, and its catalyzed product is involved in the synthesis of purine, ⁇ -thymidine and methionine (as shown in Figure 2).
  • the MTHFD1 knockout cell line was supplemented with purine, ⁇ -thymidine and 5-methyltetrahydrofolate, and then infected with influenza virus. The results are shown in Figure 3.
  • the virus in the experimental group with hypoxanthine added was rescued , And close to the virus level in the control group. It shows that the inhibition of the virus after MTHFD1 knockout is due to the hindered purine synthesis.
  • Knockdown of MTHFD1 by siRNA or shRNA can significantly inhibit the replication of mumps virus and Zika virus.
  • Figure 5a after knocking down MTHFD1 with siRNA, it was infected with mumps virus. After 48 hours, the supernatant was taken to test the titer. It was found that the reduction of MTHFD1 significantly inhibited the replication of mumps virus. Similarly, the QPCR method found that the knockdown of MTHFD1 also significantly inhibited Zika virus (as shown in Figure 5b).
  • Figure 5c shows that siRNA has a good knockdown effect.
  • MTHFD1 inhibitor effectively inhibits SARS-CoV2.
  • different concentrations of MTHFD1 inhibitor (carolacton) were added for 1 hour, and then SARS-CoV2 virus was added. It was found that carolacton can significantly inhibit the replication of SARS-CoV2 (as shown in Figure 7).
  • the half inhibitory concentration is 0.14 ⁇ M, which is far less than the concentration that causes half toxicity to cells (greater than 10 ⁇ M).
  • MTHFD1 inhibitor effectively inhibits mumps virus and Zika virus.
  • different concentrations of MTHFD1 inhibitor (carolacton) were added, and then infected with mumps virus or Zika virus. After 48 hours of infection, it was found that carolacton can also significantly inhibit the replication of mumps virus or Zika virus (as shown in the figure). 8).
  • the half inhibitory concentrations were 0.24 ⁇ M for mumps virus and 0.12 ⁇ M for Zika virus. Consistent with the results of the previous influenza virus rescue experiment, the inhibitory effect of carolacton on mumps virus or Zika virus can also be offset by inosine added by foreign aid (as shown in Figure 9 and Figure 10).
  • MTHFD1 is a potential broad-spectrum antiviral target, and its inhibitor carolacton has good application prospects.

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Abstract

本发明涉及医药技术领域，具体公开了MTHFD1抑制剂在抑制和杀灭病毒中的应用。本发明通过实验发现MTHFD1是一个潜在的广谱抗病毒靶标，其抑制剂(特别是carolacton及其衍生物)可有效且广谱抑制病毒(具体如流感病毒、冠状病毒、腮腺炎病毒、寨卡病毒等)增殖，在抗病毒领域可具有非常好的应用前景。

Description

MTHFD1抑制剂在抑制和杀灭病毒中的应用 技术领域
本发明涉及医药技术领域，具体涉及MTHFD1抑制剂(特别是carolacton及其衍生物)在抑制和杀灭病毒中的应用。
背景技术
人类患有的许多重要传染病都是由病毒造成的。这些疾病包括狂犬病、天花、脊髓灰质炎、肝炎、肺炎、黄热病、免疫缺陷和各种脑炎病，它们中的许多是高度传染性的且会产生急性不适，且常常是致命的，其它的例如风疹和巨细胞病毒会造成先天畸形。冠状病毒是一个大型病毒家族，已知冠状病毒会感染哺乳动物和鸟类的上呼吸道和胃肠道，其可引起感冒以及中东呼吸综合征(MERS)和严重急性呼吸综合征(Severe Acute Respiratory Syndrome，SARS)等较严重疾病。
[根据细则26改正25.05.2020]　
[根据细则26改正25.05.2020]　
亚甲基四氢叶酸脱氢酶1(methylenetetrahydrofolate dehydrogenase(NADP+dependent)1，MTHFD1)仅编码一种单一的蛋白，具有3种酶(5,10亚甲基四氢叶酸脱氢酶、5,10次甲基四氢叶酸环化水解酶和10甲酸四氢叶酸合成酶)的活性，其在叶酸代谢中起着至关重要的作用。目前尚无关于MTHFD1在抗病毒方面的研究报道。
发明内容
本发明提供一种MTHFD1抑制剂在制备抑制和/或杀灭病毒的产品中的应用。
具体地，上述MTHFD1抑制剂可以为抗体或其抗原结合片段、干扰RNA或小分子化合物。
具体地，上述抗原结合片段可选自：Fab、Fab'、F(ab)2、Fv、dsFv、scFv、Fd和Fd'片段等。
具体地，上述干扰RNA可选自：siRNA、dsRNA、shRNA、aiRNA、 miRNA及其组合。
在本发明的一个实施方式中，上述小分子化合物可以选自：甲氨蝶呤、培美曲塞、曲美沙特、依达曲沙、洛美曲索、5-氟尿嘧啶、普拉曲沙、氨基蝶呤及其盐、溶剂化物、立体异构体、醚、酯、前药等中的一种或多种。
在本发明的另一个实施方式中，上述小分子化合物可具有如下结构：
Figure PCTCN2020082809-appb-000001
其中，
Figure PCTCN2020082809-appb-000002
代表单键或双键；
R 1、R 3和R 4独立地选自：H、C1-C12烷基、C7-C12芳烃基；
R 2选自：H、C1-C12烷基、C7-C12芳烃基和OR 8；其中，R 8选自：H、C1-C12烷基、C7-C12芳烃基；
R 5、R 6和R 7独立地选自：H、C1-C12烷基。
在本发明的一个实施例中，R 1为H。
在本发明的另一个实施例中，R 1选自C1-C6烷基，特别是C1-C3烷基；在本发明的一个实施例中，R 1为甲基。
在本发明的一个实施例中，R 3为H。
在本发明的另一个实施例中，R 3选自C1-C6烷基，特别是C1-C3烷基；在本发明的一个实施例中，R 3为甲基。
在本发明的一个实施例中，R 4为H。
在本发明的另一个实施例中，R 4选自C1-C6烷基，特别是C1-C3烷基；在本发明的一个实施例中，R 4为甲基。
在本发明的一个实施方式中，R 2为OR 8。
在本发明的一个实施例中，R 2为OH。
具体地，R 5选自C1-C6烷基，特别是C1-C3烷基；在本发明的一个实施例中，R 5为甲基。
具体地，R 6选自C1-C6烷基，特别是C1-C3烷基；在本发明的一个实施例中，R 6为甲基。
具体地，R 7选自C1-C6烷基，特别是C1-C3烷基；在本发明的一个实施例中，R 7为甲基。
在本发明的一个实施方式中，连接C-15和C-16的键为单键，且C-15和C-16被氢原子饱和。
在本发明的另一个实施方式中，连接C-15和C-16的键为双键。
在本发明的一个实施方式中，连接C-7和C-8的键为单键，且C-7和C-8被氢原子饱和。
在本发明的另一个实施方式中，连接C-7和C-8的键为双键。
具体地，上述MTHFD1抑制剂具有如下结构：
Figure PCTCN2020082809-appb-000003
其中，R 1、R 2、R 3、R 4、R 5、R 6和R 7具有本发明上述定义。
具体地，上述MTHFD1抑制剂具有如下结构：
Figure PCTCN2020082809-appb-000004
其中，R 1、R 2、R 5、R 6和R 7具有本发明上述定义。
更具体地，上述MTHFD1抑制剂具有如下结构：
Figure PCTCN2020082809-appb-000005
其中，R 2、R 5、R 6和R 7具有本发明上述定义。
在本发明的一个实施例中，上述MTHFD1抑制剂具有如下结构：
Figure PCTCN2020082809-appb-000006
具体地，上述MTHFD1抑制剂为carolacton，其具有如下结构：
Figure PCTCN2020082809-appb-000007
具体地，上述MTHFD1抑制剂还可如下结构：
Figure PCTCN2020082809-appb-000008
Figure PCTCN2020082809-appb-000009
本发明所述的Carolacton衍生物可以采用例如WO2018220176A1中所记载的方法获得。
具体地，上述病毒可以为腺病毒科、疱疹病毒科(如EBV)、乳头多瘤空泡病毒科、小RNA病毒科、痘病毒科(如天花病毒)、嗜肝DNA病毒科(如乙型肝炎病毒)、冠状病毒科(如HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV、SARS-CoV-2等)、玻那病毒科、丝状病毒科(如埃博拉病毒)、正粘病毒科(如流感病毒)、副粘病毒科、反转录病毒科(如HIV)、呼肠孤病毒科、弹状病毒科(如狂犬病毒)、黄病毒科等。
在本发明的一个实施方式中，上述病毒为冠状病毒，具体如HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV、SARS-CoV-2等。
在本发明的另一个实施方式中，上述病毒为正粘病毒，如流感病毒(如甲型流感病毒、乙型流感病毒、丙型流感病毒等)。
在本发明的另一个实施方式中，上述病毒为副粘病毒，如1型人副流感病毒(HPV)、2型HPV、3型HPV、4型HPV、仙台病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒、新城疫病毒等。
在本发明的另一个实施方式中，上述病毒为黄病毒，如登革热病毒、寨卡病毒、乙型脑炎病毒、基孔肯亚病毒、黄热病病毒、丙型肝炎病毒、西尼罗病毒等。
具体地，上述产品可用于诊断或治疗目的，也可用于非诊断或治疗目的。
具体地，上述病毒的抑制和/或杀灭可在体内进行，也可在体外进行。
在本发明的一个实施方式中，上述产品为药物组合物。
具体地，上述药物组合物中，MTHFD1抑制剂可以作为唯一的活性成分，也可与一种或多种其它用于相同适应症或不同适应症的活性成分联用，其中，MTHFD1抑制剂与该其他活性成分可配制用于同时、单独或顺序给药(simultaneous,separate or sequential administration)。
具体地，上述药物组合物还包含药学上可接受的辅料。
具体地，上述药学上可接受的辅料可以包括甜味剂(具体如，蔗糖、木糖醇、低聚果糖、甜蜜素、甜菊糖、阿斯巴甜等)、芳香剂(如香料、香精等)、胶浆剂(具体如，海藻酸钠、阿拉伯胶、明胶、甲基纤维素、羧甲基纤维素钠等)、澄清剂(具体如，壳聚糖、明胶等)、防腐剂(具体如，苯甲酸及其盐、山梨酸及其盐、尼泊金类系列等)、崩解剂(具体如，低取代羟丙基纤维素、交聚维酮、羧基乙酸淀粉钠、交联羧甲基纤维素钠、淀粉等)、粘结剂(具体如，羟丙基纤维素、羟丙甲纤维素、聚维酮、共聚维酮、预胶凝淀粉等)、润滑剂(具体如，硬脂酸、硬脂酸镁、富马酰硬脂酸钠等)、润湿剂(具体如，聚氧乙烯山梨糖醇酐脂肪酸酯、泊洛沙姆、聚氧乙烯蓖麻油衍生物等)、悬浮剂(具体如，羟丙甲纤维素、羟丙基纤维素、聚维酮、 共聚维酮、羧甲基纤维素钠、甲基纤维素等)、稳定剂(具体如，柠檬酸、富马酸、琥珀酸等)、填充剂(具体如，淀粉、蔗糖、乳糖、微晶纤维素等)、粘合剂(具体如，纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮等)等中的一种或多种。
具体地，上述药物组合物可以采用任意的剂型或给药形式，特别是口服剂型，本领域技术人员可以根据情况选用，包括，但不限于，片剂(包括糖衣片剂、膜包衣片剂、舌下片剂、口腔崩解片、口腔片剂等等)、丸剂、粉剂、颗粒剂、胶囊剂(包括软胶囊、微胶囊)、锭剂、糖浆剂、溶液剂、乳剂、混悬剂、控制释放制剂(例如，瞬时释放制剂、缓释制剂、缓释微囊)、气雾剂、膜剂(例如，口服崩解膜剂、口腔粘膜-粘附膜剂)、注射剂(例如，皮下注射、静脉注射、肌内注射、腹膜内注射)、静脉滴注剂、透皮吸收制剂、软膏剂、洗剂、粘附制剂、栓剂(例如，直肠栓剂、阴道栓剂)、小药丸、鼻制剂、肺制剂(吸入剂)、眼睛滴剂等等、口服或胃肠外制剂(例如，静脉内、肌内、皮下、器官内、鼻内、皮内、滴注、脑内、直肠内等给药形式，给药至病灶附近和直接给药至病变处)。
在本发明的另一个实施方式中，上述产品为功能性食品组合物。
具体地，上述功能性食品组合物中，MTHFD1抑制剂可以作为唯一的活性成分，也可与一种或多种其它活性成分联用。
具体地，上述功能性食品组合物还可包含食品辅料。
具体地，上述功能性食品组合物的形式可以采用任意的形式，如，片剂、丸剂、胶囊剂(如软胶囊、微胶囊)、糖果(如压片糖果、凝胶糖果、胶基糖果等)、固体饮料(如粉剂、颗粒剂等)、液体饮料等。
上述功能性食品组合物的各种形式可以按照功能性食品领域的常规生产方法制备。
在本发明的另一个实施方式中，上述产品为消毒产品。
具体地，上述消毒产品中，MTHFD1抑制剂可以作为唯一的活性成分， 也可与一种或多种其它活性成分(例如含氯消毒剂、醇类消毒剂、季铵盐类消毒剂、过氧化物类消毒剂等)联用。
具体地，上述消毒产品可以是医用消毒产品，也可以为日常家用消毒产品。
具体地，上述消毒产品还包含可包含消毒产品辅料，如保湿剂、赋脂剂、赋香剂、增稠剂、pH调节剂、肤感调节剂、稳定剂、缓冲剂等中的一种或多种。
具体地，上述消毒产品可以为医学药物制剂涉及的喷雾剂、粉末剂、溶液剂、凝胶剂、膏剂、啫喱等任何形式，也可以被制成洗液(如洗手液、洗衣液)、牙膏、隐形眼镜护理液等形式，也可以通过涂布或浸渍等方式附着于卫生或清洁物品的固体表面，该卫生或清洁物品可以为卫生或清洁手套、纸巾或纸、棉签、防尘罩或医用面罩等。
具体地，上述消毒产品可用于皮肤、毛发、衣物、家居等。
本发明还提供一种MTHFD1抑制剂在制备预防和/或治疗由病毒感染引起或病毒感染相关的疾病或病症的药物中的应用。
特别是，在上述应用中，MTHFD1抑制剂具有本发明上述通式Ⅰ所示结构；更具体地，MTHFD1抑制剂具有本发明上述式Ⅴ特别是式Ⅵ的结构。
具体地，上述疾病或病症可包括急性支气管炎、慢性支气管炎、鼻炎、鼻窦炎、哮吼、急性细支气管炎、咽炎、扁桃体炎、腮腺炎、喉炎、气管炎、哮喘、肺炎、流行性感冒、寨卡病毒病等中的一种或多种。
在本发明的一个实施例中，上述疾病为COVID-19。
在本发明的一个实施例中，上述疾病为流行性感冒。
在本发明的一个实施例中，上述疾病为腮腺炎。
在本发明的一个实施例中，上述疾病为寨卡病毒病。
本发明还提供一种预防和/或治疗由病毒感染引起或病毒感染相关的疾病或病症的方法，其包括对受试者给予有效量的本发明上述MTHFD1抑制 剂的步骤。
特别是，在上述应用中，MTHFD1抑制剂具有本发明上述通式Ⅰ所示结构；更具体地，MTHFD1抑制剂具有本发明上述式Ⅴ特别是式Ⅵ的结构。
具体地，上述疾病或病症可包括急性支气管炎、慢性支气管炎、鼻炎、鼻窦炎、哮吼、急性细支气管炎、咽炎、扁桃体炎、腮腺炎、喉炎、气管炎、哮喘、肺炎、流行性感冒、寨卡病毒病等中的一种或多种。
在本发明的一个实施例中，上述疾病为COVID-19。
在本发明的一个实施例中，上述疾病为流行性感冒。
在本发明的一个实施例中，上述疾病为腮腺炎。
在本发明的一个实施例中，上述疾病为寨卡病毒病。
具体地，上述受试者为动物；在本发明的一个实施方式中，上述受试者为哺乳动物，如人类、猴、猿、牛、马、猪、海豹等；在本发明的另一个实施方式中，上述受试者为禽类，如鸡。
本发明还提供了MTHFD1的破坏在抑制和/或杀灭病毒中的应用。特别是，MTHFD1的破坏可以为通过抗体或其抗原结合片段、干扰RNA或小分子化合物抑制作用实现，也可以通过基因编辑的方法(例如敲除或敲低基因破坏MTHFD1表达)实现。
本发明发明人通过实验发现MTHFD1是一个潜在的广谱抗病毒靶标，其抑制剂(特别是carolacton)可有效且广谱抑制病毒(具体如流感病毒、冠状病毒、腮腺炎病毒、寨卡病毒等)增殖，在抗病毒领域可具有非常好的应用前景。
附图说明
图1所示为敲除MTHFD1抑制流感病毒复制的结果。使用CRISPR Cas9技术在293T细胞中敲除MTHFD1基因，然后用流感病毒感染MTHFD1敲除的细胞系，再通过western blot检测病毒蛋白量。
图2所示为MTHFD1基因在一碳代谢通路中的作用示意图。MTHFD1基因是具有3个不同催化活性酶，其催化的合成产物可以用于嘌呤、dTMP，以及蛋氨酸的合成。
图3所示为MTHFD1敲除细胞系中的病毒复制水平可以被外源添加的肌苷拯救的结果。在MTHFD1基因敲除的293T细胞系中分别补充肌苷、β-胸苷和5甲基四氢叶酸，然后同时感染流感病毒。流感病毒蛋白水平由western blot方法检测。
图4所示为MTHFD1基因敲除抑制流感病毒的基因组RNA复制的结果。其中，a：流感病毒感染对照细胞(含有正常MTHFD1基因)和MTHFD1敲除的细胞系后，在不同感染时间点取样，提取总RNA，反转录得到cDNA后，通过real time PCR来检测流感病毒基因组RNA水平；b：流感病毒复制子实验结果。在对照细胞或MTHFD1敲除的细胞系中同时转染能够表达流感病毒聚合酶的3个亚基，PB1、PB2和PA，以及流感NP蛋白，同时共转染能够表达流感病毒负链基因组RNA(hemagglutinin片段)的质粒。于转染后不同时间点取样，通过western blot来检测hemagglutinin(HA)的蛋白水平。HA的蛋白水平代表了流感病毒基因组复制的水平。
图5所示为siRNA敲低MTHFD1基因后抑制腮腺炎病毒和寨卡病毒(ZIKV)复制的结果。其中，a：siRNA敲低MTHFD1基因后对腮腺炎病毒复制的抑制结果；b：siRNA敲低MTHFD1基因后对寨卡病毒复制的抑制结果；c：siRNA的敲低结果。
图6所示为shRNA敲低MTHFD1基因后抑制腮腺炎病毒和寨卡病毒复制的结果。其中，a：shRNA敲低MTHFD1基因后对腮腺炎病毒复制的抑制结果；b：shRNA敲低MTHFD1基因后对寨卡病毒复制的抑制结果；c：shRNA的敲低结果。
图7所示为MTHFD1基因的抑制剂carolacton抑制SARS-CoV2的结果。在非洲绿猴肾细胞(Vero)中加入不同浓度的MTHFD1基因的抑制剂 carolacton，随后感染SARS-CoV2。病毒感染48h后，收取上清提取病毒RNA，经过RT-qPCR检测病毒水平。左边的Y轴代表了抑制剂对病毒的抑制水平。同样的抑制剂处理细胞后，药物对细胞的毒性通过MTT实验检测，右边的Y轴代表了抑制剂的细胞毒性。
图8所示为MTHFD1基因的抑制剂carolacton抑制寨卡病毒和腮腺炎病毒的结果。在蝙蝠肾细胞(PaKi)中加入不同浓度的MTHFD1基因的抑制剂carolacton，随后分别感染寨卡病毒和腮腺炎病毒。病毒感染48h后，通过免疫荧光检测病毒感染率。左边的Y轴代表了抑制剂对病毒的抑制水平。同样的抑制剂处理细胞后，药物对细胞的毒性通过MTT实验检测，右边的Y轴代表了抑制剂的细胞毒性。
图9所示为肌苷拯救实验结果，其表明MTHFD1基因的抑制剂carolacton对腮腺炎病毒的抑制可以被外援添加的肌苷抵消。
图10所示为肌苷拯救实验结果，其表明MTHFD1基因的抑制剂carolacton对寨卡病毒的抑制可以被外援添加的肌苷抵消。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
在本发明中，术语“MTHFD1抑制剂”是指对MTHFD1具有抑制效果的分子，该抑制效果包括但不限于：抑制MTHFD1的活性，抑制MTHFD1基因转录或表达。该MTHFD1抑制剂包括但不限于，抗体或其抗原结合片段、干扰RNA、小分子化合物等。
抑制MTHFD1活性是指使MTHFD1活力下降；具体地，相比抑制前，MTHFD1活力下降至少5％，10％，20％，30％，40％，50％，60％，70％，80％，90％，95％，99％，甚至100％。
抑制MTHFD1基因转录或表达是指：使MTHFD1的基因不转录，或降 低MTHFD1的基因的转录活性，或者使MTHFD1的基因不表达，或降低MTHFD1的基因的表达活性。
本领域技术人员可以使用常规方法对MTHFD1的基因转录或表达进行调节，如基因敲除、同源重组、干扰RNA等。
MTHFD1的基因转录或表达的抑制可以通过PCR及Western Blot等实验手段检测表达量验证。
优选地，与野生型相比，MTHFD1基因转录或表达降低至少5％，10％，20％，30％，40％，50％，60％，70％，80％，90％，95％，99％，特别是MTHFD1基因完全没有表达。
在本发明中，术语“烷基”指的是直链或支链的且不含不饱和键的烃链自由基，且该烃链自由基以单键与分子其它部分连接。典型的烷基基团含有1至12个、1至8个、1至6个或1至3个碳原子，如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、正己基、异己基、正庚基、异庚基等。烷基可以被取代，例如，如果烷基被环烷基取代，其相应为“环烷基烷基”，如环丙基甲基；如果烷基被卤素取代，那么其相应为“卤代烷基”；如果烷基被芳基取代，那么其相应为“芳烷基”，如苄基、二苯甲基或苯乙基；如果烷基被杂环基取代，那么其相应为“杂环基烷基”；等。
“芳烃基”是指含有苯环结构的烃基；“烃基”是指只含碳、氢两种原子的基团，通常指相应的烃失去一个氢原子后剩余的自由基。
上述基团可以在一个或多个可用的位置被一个或多个合适的基团所取代，所述基团如：OR'、＝O、SR'、SOR'、SO 2R'、OSO 2R'、OSO 3R'、NO 2、NHR'、N(R') 2、＝N-R'、N(R')COR'、N(COR') 2、N(R')SO 2R'、N(R')C(＝NR')N(R')R'、N 3、CN、卤素、COR'、COOR'、OCOR'、OCOOR'、OCONHR'、OCON(R') 2、CONHR'、CON(R') 2、CON(R')OR'、CON(R')SO 2R'、PO(OR') 2、PO(OR')R'、PO(OR')(N(R')R')、C 1-C 12烷基、C 3-C 10环烷基、C 2-C 12烯基、C 2-C 12炔基、芳基和杂环基，其中每个R'基团各自独立地选自：氢、 OH、NO 2、NH 2、SH、CN、卤素、COH、CO烷基、COOH、C 1-C 12烷基、C 3-C 10环烷基、C 2-C 12烯基、C 2-C 12炔基、芳基和杂环基。其中，这些基团本身被取代，取代基可选自前述列表。
“烯基”指的是至少含两个碳原子、至少一个不饱和键的直链或支链的烃链自由基，且该烃链自由基以单键与分子其它部分连接。典型的烯基基团含有2至12个、2至8个、2至6个或2至3个碳原子，如乙烯基、1-甲基-乙烯基、1-丙烯基、2-丙烯基或丁烯基。
“炔基”指的是含至少两个碳原子、至少一个碳碳三键的直链或支链的烃链自由基，且该烃链自由基以单键与分子其它部分连接。典型的炔基基团含有2至12个、2至8个、2至约6个或2至3个碳原子，如乙炔基、丙炔基(例如1-丙炔基、2-丙炔基)或丁炔基(例如1-丁炔基、2-丁炔基、3-丁炔基)。
“环烷基”指的是脂环烃。典型的环烷基含1至4个单环和/或稠环、含3至18个碳原子，如3至10个碳原子或3至约6个碳原子，如环丙基、环己基。
“芳基”指的是单环或多环自由基，包括含单芳基基团和/或稠芳基基团的多环自由基。典型的芳基基团包含1至3个单环或稠环及6至18个碳环原子，优选6至14个碳环原子，如苯基、萘基、联苯基、茚基、菲基或蒽基自由基。
“杂环基”包括含1至3个单环和/或稠环及3至18个环原子的杂芳香族基团和杂脂环基团。具体地，杂芳香族基团和杂脂环基团含5至约10个环原子。合适的杂芳基可以含1、2或3种杂原子，该杂原子选自N、O或S原子。
术语“盐”须理解为根据本发明的化合物的任意形式，其中所述的化合物为离子形式或者为带电荷的且与带相反电荷的离子(阳离子或阴离子)耦合或在溶液中。该定义还包括季铵盐和该分子与其它分子和离子的复合物，特别是通过离子相互作用形成的复合物。
术语“溶剂化物”应理解为是指本发明的化合物的任意形式，其中所述化合物通过非共价键与另一个分子相连(通常为极性溶剂)，特别是包括水化物和醇化物，例如甲醇化物。特别是，溶剂化物为水化物。
术语“前药”使用其广义含义，并涵盖在体内可转化成本发明化合物的衍生物。前药的例子包括但不限于该化合物的衍生物和代谢物，包括可生物水解的部分，如可生物水解的酰胺、可生物水解的酯、可生物水解的氨基甲酸酯、可生物水解的碳酸酯、可生物水解的酰脲和可生物水解的磷酸酯类似物。优选地，具有羧基官能团的前体药物为羧酸的低级烷基酯。所述的羧酸酯易由存在于分子中的任何羧酸部分进行酯化得到。前药通常可由已知方法来制备，如在Burger“Medicinal Chemistry and Drug Discovery第六版(Donald J.Abraham ed.，2001，Wiley)和“Design and Applications of Prodrugs”(H.Bundgaard ed.，1985，Harwood Academic Publishers)中描述的方法。
在这里所涉及的任何化合物均旨在代表这样的特定化合物及其某些变形或某些形式。特别地，在这里所涉及的化合物可能具有不对称中心，并因此存在不同的对映体或非对映体形式。由此，本文涉及的任何给定的化合物代表外消旋物的任意一种、一种或多种对映体形式、一种或多种非对映体形式、及其混合物。同样地，也可能存在双键的立体异构体或几何异构体，由此在一些情况中，分子可能存在为(E)-异构体或(Z)-异构体(反式和顺式异构体)。如果分子包含多个双键，那么每个双键将具有其自身的立体异构现象，其可以与所述分子的其它双键的立体异构现象相同或不同。此外，本文中涉及的化合物可存在阿托异构体。本文涉及的化合物的所有立体异构体，包括对映体、非对映异构体、几何异构体和阿托异构体、及其混合物，都在本发明的范围内。
此外，本文所涉及的任何化合物可以互变异构体形式存在。特别地，术语互变异构体是指化合物的两个或多个结构异构体中的一个，这些异构体间存在平衡，可以相互转换。常见的互变异构体对为烯胺-亚胺、酰胺-亚胺酸、 酮-烯醇、内酰胺-内酰亚胺等。
除非另有说明，本发明的化合物还可包括同位素标记的形式，即区别仅在于存在一种或多种富含同位素的原子的化合物。例如，具有仅用氘或氚来替代至少一个氢原子、或者使用富含 13C或 14C的碳来替代至少一个碳、或者使用富含 15N的氮来替代至少一个氮的现有结构的化合物均包含在本发明范围内。
本发明中所述的化合物或其盐、溶剂化物、立体异构体、醚、酯优选为药学上可接受的形式。其中，“药学上可接受的”是指当分子本体及包含其的组合物适当地给予受试者时，它们不会产生不利的、过敏的或其它不良反应。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
材料和方法
1、MTHFD1敲除细胞系的构建
1.1、sgRNA表达载体构建
sgRNA引物序列：
sgMTHFD1-1正向引物:5′-caccg AAGGGGAGTGGATCAAACCT-3′；
sgMTHFD1-1反向引物：5′-aaac AGGTTTGATCCACTCCCCTT c-3′；
sgMTHFD1-2正向引物：5′-caccg GAGGTTATTAGCTGCAGTGA-3′；
sgMTHFD1-2反向引物：5′-aaac TCACTGCAGCTAATAACCTC c-3′。
将合成的sgRNA引物序列用T4多聚核苷酸激酶于37℃处理30分钟使其磷酸化，95℃变性5分钟、以1.5℃/分钟降温至25℃退火后，得到具有BsmBI(或BbsI)粘性末端的双链DNA片段，如下：
正向：5′-CACCGNNNNNNNNNNNNNNNNNNNN；
反向：CNNNNNNNNNNNNNNNNNNNNCAAA-5′。
磷酸化、变性及退火体系为：
1μl 100μM正向寡核苷酸链；
1μl 100μM反向寡核苷酸链；
1μl 10×T4Ligation Buffer(NEB)；
6.5μl ddH 2O；
0.5μl T4多聚核苷酸激酶。
将双链DNA片段和用BsmbI酶切过的LentiGuidepuro载体进行连接，将连接产物转化到stbl3感受态细胞中，涂布于带有氨苄青霉素抗性的LB平板上，筛选阳性菌落，提取阳性菌落质粒进行分析及测序，确定sgRNA表达载体构建成功，命名为sgMTHFD1-1和sgMTHFD1-2。
1.2、MTHFD1敲除细胞系的构建
将293T细胞铺到6孔细胞培养板中，7x10 5细胞每孔。培养过夜后，转染MTHFD1sgRNA质粒和表达Cas9的质粒。转染48h后，细胞传代，通过嘌呤霉素和blasticidin来筛选出转染并表达成功的阳性细胞。筛选大约一周后(未转染的对照细胞全部死亡)，将细胞使用胰酶消化，计数后铺到96孔板中，培养基中加入100μM的次黄嘌呤和16μM的β-胸苷。大约2周后，细胞长起来后转移至24孔板，或6孔板。通过western blot检测MTHFD1蛋白水平，挑选出成功敲除的细胞系备用。
2、Western blot
将细胞收集后，使用RIPA裂解液(Beyotime，P0013C)冰上裂解。4℃12000rpm，10min离心取上清，加入loading buffer后于95℃水浴10分钟。通过10％SDS-PAGE分离蛋白，并转印至PVDF膜，5％脱脂牛奶(PBST配置)室温封闭1h，分别孵育对应蛋白的抗体。anti-MTHFD1(Proteintech，10794-1-AP)，anti-βactin(Easybio，BE0022)，anti-PR8M1(Genetex，GTX125928-S)，anti-HA(H1N1)(Genetex，GTX117951-S)。使用HRP偶联 的二抗，通过曝光仪检测目的条带。
3、Real time PCR
使用试剂盒提取RNA后，反转录得到cDNA。流感病毒RNA使用特异性引物反转录。使用SYBR Green qPCR master mix(Vazyme，Q311-02)来做real time PCR。使用Bio-Rad CFX96荧光定量PCR仪器来检测。
引物序列如下：
Human GAPDH正向引物：5′-ACAACTTTGGTATCGTGGAAGG-3′；
Human GAPDH反向引物：5′-GCCATCACGCCACAGTTTC-3′；
流感病毒正向引物：5′-TTCTAACCGAGGTCGAAACGTACG-3′；
流感病毒反向引物：5′-ACAAAGCGTCTACGCTGCAG-3′；
流感病毒特异反转录引物：5′-AGCRAAAGCAGG-3′；
P.alecto ACTIN正向引物:5’-gccagtctacaccgtctgcag-3’；
P.alecto ACTIN反向引物:5’-cgtaggaatccttctggcccatg-3’；
P.alecto MTHFD1正向引物:5’-gggagcgactgaagaaccaag-3’；
P.alecto MTHFD1反向引物:5’-tcttcagcagccttcagcttcac-3’；
ZIKV NS5正向引物:5’-GGTCAGCGTCCTCTCTAATAAACG-3’；
ZIKV NS5反向引物:5’-GCACCCTAGTGTCCACTTTTTCC-3’。
4、流感病毒复制子实验
将对照293T细胞(MTHFD1正常表达)和MTHFD1敲除的293T细胞系铺到12孔板中，每孔2x10 5细胞。含有100μM的次黄嘌呤和16μM的β-胸苷培养基培养过夜后，转染能够表达流感病毒聚合酶的3个亚基，PB1、PB2和PA，以及流感NP蛋白，同时共转染能够表达流感病毒负链基因组RNA(hemagglutinin片段)的质粒。转染前将培养基换成不含次黄嘌呤和β-胸苷的培养基，于转染后不同时间点取样，通过western blot来检测hemagglutinin(HA)的蛋白水平。HA的蛋白水平代表了流感病毒基因组复制的水平。
5、MTHFD1抑制剂实验
5.1、对于SARS-CoV2抑制实验，将Vero细胞铺到96孔板中，10000个细胞/孔，培养过夜后形成单层细胞。换液成2％FBS的DMEM培养基，并分别加入0，0.05μM，0.1μM，0.2μM，0.4μM，和0.8μM的carolacton。每孔共100μl含抑制剂的培养液。处理1h后，加入病毒SARS-CoV2，病毒使用2％FBS DMEM培养基稀释，每孔加入100μl。病毒感染48h后收上清提取RNA，测定病毒量。
对于寨卡病毒和腮腺炎病毒抑制实验，将蝙蝠(P.alecto)肾细胞PaKi铺到96孔板中，3000个细胞每孔。培养过夜后，分别加入0，0.03μM，0.06μM，0.125μM，0.25μM，0.5μM，1μM的carolacton。同时分别加入寨卡病毒和腮腺炎病毒。对于肌苷拯救实验，于此步补充肌苷。病毒感染48h后，通过高内涵成像系统检测分析感染率。
5.2、抑制剂(药物)的细胞毒性实验通过MTT测定。按照方法5.1对应的步骤准备细胞并加入抑制剂。细胞继续培养48h后，弃掉上清液，加入90μl新鲜培养基和10μl MTT(Solarbio)，继续培养4h后，弃掉上清，使用DMSO 110μl溶解，测定OD490nm吸光度。
6.免疫荧光检测病毒感染率
将病毒感染的细胞用4％的多聚甲醛溶液室温固定10min,再用0.2％的Triton X-100室温通透10min，用PBS洗3次，孵育寨卡病毒E蛋白抗体(Millipore,MAB10216)，4℃过夜，PBS洗3次，再孵育荧光二抗AF488。细胞核用DAPI染色。对于腮腺炎病毒，病毒自身带有GFP，无需一抗二抗处理，固定完直接DAPI染DNA。感染率由高内涵成像系统(Cellomic ArrayScan VTI HCS，Thermo Scientific)检测和分析。
7.siRNA和shRNA实验
7.1 siRNA转染实验
将20000个PaKi细胞铺到12孔板中，培养过夜后，使用lipo2000将siRNA转染进入细胞，48h后，感染腮腺炎病毒或者寨卡病毒。腮腺炎病毒滴度测定 通过TCID 50方法。寨卡病毒复制水平通过QPCR检测。
si-MTHFD1序列：
正向5’-GUUCCAAGUGACAUUGAUAUAUCAC-3’；
反向5’-GUGAUAUAUCAAUGUCACUUGGAACAG-3’。
7.2 shRNA稳定敲除实验
参照步骤1.1中的sgRNA构建方法，将MTHFD1 shRNA连接到EcoRI和AgeI双酶切过后的PLKO.1载体上。得到测序正确的质粒后，通过PMD2.G和psPAX2双质粒慢病毒包装系统，在293T中共转染shRNA质粒，于48h收获上清，并接种到铺好的Paki细胞。48h后使用嘌呤霉素筛选得到shRNA稳定表达的阳性细胞。shRNA敲低效果通过western blot检测。筛选好的细胞用于腮腺炎病毒和寨卡病毒感染实验。病毒感染率由免疫荧光和高内涵成像系统分析测定。
MTHFD1 shRNA序列:
PaKi shMTHFD1-1正向引物:5’-ccgg GCACATGGGAATTCCTCTACCctcgagGGTAGAGGAATTCCCATGTGCtttttg-3’；
PaKi shMTHFD1-1反向引物:5’-AATTCAAAAAGCACATGGGAATTCCTCTACC CTCGAGGGTAGAGGAATTCCCATGTGC-3’；
PaKi shMTHFD1-2正向引物:5’-ccggGCCTGCTGTCACTTAGGAAATctcgag ATTTCCTAAGTGACAGCAGGC tttttg-3’；
PaKi shMTHFD1-2反向引物:5’-AATTCAAAAAGCCTGCTGTCACTTAGGAAAT CTCGAG ATTTCCTAAGTGACAGCAGGC-3’。
8.病毒滴度测定
将10000个PaKi细胞铺到96孔板中，培养过夜形成单层。将待测病毒样品10倍梯度稀释，分别加到细胞中，每孔100μl。96h后，观察CPE，根据TCID 50方法计算病毒滴度。
实验结果
1、MTHFD1敲除后抑制流感病毒复制的结果如图1所示，在两个独立的MTHFD1敲除的293T细胞系中，分别感染流感病毒，12h后，western blot结果检测到敲除细胞系中流感病毒M1蛋白水平远远低于对照细胞。同时看到MTHFD1蛋白被很好的敲除。说明MTHFD1基因是流感病毒正常复制所需要的。
2、MTHFD1敲除细胞系中流感病毒的复制可以被外源添加的肌苷拯救。MTHFD1是一碳代谢通路中的重要的酶，能够催化三步反应，其催化的产物参与嘌呤，β-胸苷和蛋氨酸的合成(如图2所示)。在MTHFD1敲除的细胞系中分别补充嘌呤、β-胸苷和5-甲基四氢叶酸，随后感染流感病毒，结果如图3所示，添加了次黄嘌呤的实验组中的病毒得到拯救，并与对照组中病毒水平接近。说明MTHFD1敲除后对病毒的抑制是因为嘌呤合成受阻所致。
3、MTHFD1敲除后抑制流感病毒基因组RNA复制。由于嘌呤是病毒RNA合成的原料，发明人猜测MTHFD1敲除后，细胞嘌呤合成受阻，导致病毒RNA合成被抑制。通过检测病毒感染后基因组RNA增殖曲线(如图4a所示)，发现感染4h后，对照细胞中病毒基因组RNA开始显著并持续增高，而MTHFD1敲除的细胞系中病毒RNA水平始终处于很低的水平，符合基因组RNA复制受阻的趋势。随后经过流感病毒复制子实验，进一步明确MTHFD1敲除后导致流感病毒基因组复制受阻，从而抑制病毒增殖(如图4b所示)。
4.通过siRNA或者shRNA敲低MTHFD1可以显著抑制腮腺炎病毒和寨卡病毒复制。如图5a所示，使用siRNA敲低MTHFD1后，感染腮腺炎病毒，48h后取上清检测滴度，发现MTHFD1的减少对腮腺炎病毒的复制形成显著抑制。同样地，通过QPCR方法检测发现MTHFD1的敲低也对寨卡病毒有明显 抑制(如图5b所示)。图5c显示siRNA有很好的敲低效果。
类似地，使用shRNA先建立MTHFD1稳定敲低的细胞系，再感染腮腺炎病毒或者寨卡病毒，再通过免疫荧光和高内涵成像分析系统检测病毒感染率。结果显示，与对照(MTHFD1正常表达)细胞系相比，MTHFD1稳定敲低后，病毒感染受到显著抑制(如图6a和6b所示)。图6c的western blot结果显示shRNA有很好的敲低效果。
5.MTHFD1抑制剂有效抑制SARS-CoV2。在Vero细胞中分别加入不同浓度的MTHFD1的抑制剂(carolacton)，1h，再加入病毒SARS-CoV2，发现carolacton可以显著抑制SARS-CoV2的复制(如图7所示)。半数抑制浓度为0.14μM，远远小于对细胞造成一半毒性的浓度(大于10μM)。
6.MTHFD1抑制剂有效抑制腮腺炎病毒和寨卡病毒。在PaKi细胞中，分别加入不同浓度的MTHFD1的抑制剂(carolacton)，再感染腮腺炎病毒或者寨卡病毒，感染48h后，发现carolacton也可以显著抑制腮腺炎病毒或者寨卡病毒的复制(如图8所示)。半数抑制浓度分别为腮腺炎病毒：0.24μM，寨卡病毒0.12μM。和前面流感病毒拯救实验结果一致的是，carolacton对腮腺炎病毒或者寨卡病毒的抑制作用同样可以被外援添加的肌苷给抵消(如图9和图10所示)。
总结：MTHFD1是一个潜在的广谱抗病毒靶标，其抑制剂carolacton有很好的应用前景。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。

Claims (23)

  1. 一种MTHFD1抑制剂在制备抑制和/或杀灭病毒的产品中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述MTHFD1抑制剂选自：抗体或其抗原结合片段、干扰RNA和小分子化合物。
  3. 如权利要求2所述的应用，其特征在于，所述小分子化合物选自：甲氨蝶呤、培美曲塞、曲美沙特、依达曲沙、洛美曲索、5-氟尿嘧啶、普拉曲沙、氨基蝶呤及其盐、溶剂化物、立体异构体、醚、酯、前药中的一种或多种。
  4. 如权利要求2所述的应用，其特征在于，所述小分子化合物具有如下结构：
    Figure PCTCN2020082809-appb-100001
    其中，
    Figure PCTCN2020082809-appb-100002
    代表单键或双键；
    R 1、R 3和R 4独立地选自：H、C1-C12烷基、C7-C12芳烃基；
    R 2选自：H、C1-C12烷基、C7-C12芳烃基和OR 8；其中，R 8选自：H、C1-C12烷基、C7-C12芳烃基；
    R 5、R 6和R 7独立地选自：H、C1-C12烷基。
  5. 如权利要求4所述的应用，其特征在于，所述小分子化合物具有如下结构：
    Figure PCTCN2020082809-appb-100003
  6. 如权利要求4所述的应用，其特征在于，所述小分子化合物具有如下结构：
    Figure PCTCN2020082809-appb-100004
  7. 如权利要求4所述的应用，其特征在于，所述小分子化合物具有如下结构：
    Figure PCTCN2020082809-appb-100005
  8. 如权利要求4所述的应用，其特征在于，所述小分子化合物选自如下结构：
    Figure PCTCN2020082809-appb-100006
    Figure PCTCN2020082809-appb-100007
  9. 如权利要求1-8任一项所述的应用，其特征在于，所述病毒为腺病毒科、疱疹病毒科、乳头多瘤空泡病毒科、小RNA病毒科、痘病毒科、嗜肝DNA病毒科、冠状病毒科、玻那病毒科、丝状病毒科、正粘病毒科、副粘病毒科、反转录病毒科、呼肠孤病毒科、弹状病毒科或黄病毒科；
    优选地，所述病毒为流感病毒；
    优选地，所述流感病毒为甲型流感病毒、乙型流感病毒或丙型流感病毒；
    优选地，所述病毒选自：1型HPV、2型HPV、3型HPV、4型HPV、仙台病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒、新城疫病毒中的一种或多种；
    优选地，所述病毒选自：HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV、SARS-CoV-2中的一种或多种；
    优选地，所述病毒选自：登革热病毒、寨卡病毒、乙型脑炎病毒、基孔肯亚病毒、黄热病毒、丙型肝炎病毒、西尼罗病毒的一种或多种。
  10. 如权利要求9所述的应用，其特征在于，所述产品为药物组合物、功能性食品组合物或消毒产品。
  11. 如权利要求9所述的应用，其特征在于，所述应用为MTHFD1抑制剂在制备预防和/或治疗由病毒感染引起或病毒感染相关的疾病或病症的药物中的应用。
  12. 如权利要求11所述的应用，其特征在于，所述疾病或病症包括急性支气管炎、慢性支气管炎、鼻炎、鼻窦炎、哮吼、急性细支气管炎、咽炎、扁桃体炎、腮腺炎、喉炎、气管炎、哮喘、肺炎、流行性感冒、寨卡病毒病中的一种或多种；
    优选地，所述疾病为COVID-19、流行性感冒、腮腺炎或寨卡病毒病。
  13. 一种具有通式Ⅰ所示结构的化合物在制备抑制和/或杀灭病毒的产品中的应用
    Figure PCTCN2020082809-appb-100008
    其中，
    Figure PCTCN2020082809-appb-100009
    代表单键或双键；
    R 1、R 3和R 4独立地选自：H、C1-C12烷基、C7-C12芳烃基；
    R 2选自：H、C1-C12烷基、C7-C12芳烃基和OR 8；其中，R 8选自：H、C1-C12烷基、C7-C12芳烃基；
    R 5、R 6和R 7独立地选自：H、C1-C12烷基。
  14. 如权利要求13所述的应用，其特征在于，所述化合物具有如下结构：
    Figure PCTCN2020082809-appb-100010
  15. 如权利要求13或14所述的应用，其特征在于，所述R 1、R 3和R 4独立地选自：H和C1-C6烷基；和/或，所述R 2为OR 8。
  16. 如权利要求13或14所述的应用，其特征在于，所述R 5、R 6和R 7独立地选自：H和C1-C6烷基。
  17. 如权利要求16所述的应用，其特征在于，所述化合物具有如下结构：
    Figure PCTCN2020082809-appb-100011
  18. 如权利要求13所述的应用，其特征在于，所述化合物具有如下结构：
    Figure PCTCN2020082809-appb-100012
  19. 如权利要求13所述的应用，其特征在于，所述化合物选自如下结构：
    Figure PCTCN2020082809-appb-100013
    Figure PCTCN2020082809-appb-100014
  20. 如权利要求13所述的应用，其特征在于，所述病毒为腺病毒科、疱疹病毒科、乳头多瘤空泡病毒科、小RNA病毒科、痘病毒科、嗜肝DNA病毒科、冠状病毒科、玻那病毒科、丝状病毒科、正粘病毒科、副粘病毒科、反转录病毒科、呼肠孤病毒科、弹状病毒科或黄病毒科；
    优选地，所述病毒为流感病毒；
    优选地，所述流感病毒为甲型流感病毒、乙型流感病毒或丙型流感病毒；
    优选地，所述病毒选自：1型HPV、2型HPV、3型HPV、4型HPV、仙台病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒、新城疫病毒中的一种或多种；
    优选地，所述病毒选自：HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV、SARS-CoV-2中的一种或多种；
    优选地，所述病毒选自：登革热病毒、寨卡病毒、乙型脑炎病毒、基孔肯亚病毒、黄热病毒、丙型肝炎病毒、西尼罗病毒的一种或多种。
  21. 如权利要求13所述的应用，其特征在于，所述产品为药物组合物、功能性食品组合物或消毒产品。
  22. 如权利要求13所述的应用，其特征在于，所述应用为具有通式Ⅰ所示结构的化合物在制备预防和/或治疗由病毒感染引起或病毒感染相关的疾病或病症的药物中的应用。
  23. 如权利要求22所述的应用，其特征在于，所述疾病或病症包括急性支气管炎、慢性支气管炎、鼻炎、鼻窦炎、哮吼、急性细支气管炎、咽炎、扁桃体炎、腮腺炎、喉炎、气管炎、哮喘、肺炎、流行性感冒、寨卡病毒病中的一种或多种；
    优选地，所述疾病为COVID-19、流行性感冒、腮腺炎或寨卡病毒病。
PCT/CN2020/082809 2020-03-23 2020-04-01 Mthfd1抑制剂在抑制和杀灭病毒中的应用 WO2021189517A1 (zh)

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