Classifications

• • 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
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • 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
  • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
  • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/116—Heterocyclic compounds
  • A23K20/137—Heterocyclic compounds containing two hetero atoms, of which at least one is nitrogen
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • 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
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/60—Sugars; Derivatives thereof
  • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/10—General cosmetic use

Definitions

• the present invention relates to compositions that can be used as anti-influenza virus drugs, etc., and to medicines, supplements, agricultural chemicals, feeds and cosmetics that use the same.
• Influenza viruses periodically occur as epidemics among humans almost every year. Influenza A viruses in particular are known as zoonotic pathogens that widely infect mammals, including humans, and birds, and often cause severe damage to society through pandemics around the world. There have been quite a few reported cases in which viruses prevalent in poultry have been transmitted directly to humans, resulting in deaths.
• anti-influenza virus drugs for the treatment of influenza, etc. All of these act directly on specific viral proteins.
• a representative anti-influenza virus drug that has been developed is a pharmaceutical composition containing the active ingredient oseltamivir phosphate, which has inhibitory activity against neuraminidase, which is found in influenza viruses.
• Patent Document 1 discloses a pharmaceutical composition containing one or more excipients selected from sugars and sugar alcohols having an equilibrium moisture content of 1% by weight or less at 25°C and a relative humidity of 70%, and oseltamivir phosphate, in which the contents of glucose and mannose contained as impurities in the sugar and sugar alcohol are each 0.01% by weight or less relative to the sugar or sugar alcohol.
• This technology aims to provide a pharmaceutical composition containing oseltamivir phosphate that has improved storage stability, particularly storage stability against humidity, temperature, etc. in the storage environment, and in particular inhibition of discoloration during storage.
• the present invention was made in light of this background, and its purpose is to provide a new anti-influenza virus drug that inhibits the proliferation and infection mechanisms of influenza viruses, has a target that is not a specific viral protein, and prevents the emergence of drug-resistant viruses, as well as medicines, foods and beverages, supplements, agricultural chemicals, feed, and cosmetics that use the same.
• the present invention includes the following aspects.
• An anti-influenza virus composition comprising tubercidin, a tubercidin derivative, or a salt thereof as an active ingredient.
• the tubercidin derivative is a compound represented by the following general formula (1): ...
• R 1 , R 2 and R 3 are the same or different and each represents a hydrogen atom, a halogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted alkoxy group, an optionally substituted amino group, an optionally substituted amido group, a cyano group, a nitro group, a hydroxy group, a sulfone group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, an optionally substituted aliphatic heterocyclic group, or an optionally substituted aromatic heterocyclic group.
• R 1 and R 2 are the same or different and each represent a hydrogen atom, a halogen atom, an optionally substituted amino group, an optionally substituted amido group, or a cyano group;
• composition according to [2], wherein the tubercidin derivative is a compound represented by the following formula (2): ... (2)
• a medicine, a food or drink, a supplement, an agricultural chemical, a feed, or a cosmetic comprising the composition according to any one of [1] to [9].
• the present invention provides a new anti-influenza virus drug that inhibits the proliferation and infection mechanisms of influenza viruses, has a target that is not a specific viral protein, and does not result in the emergence of drug-resistant viruses, as well as medicines, food and beverages, supplements, agricultural chemicals, feed, and cosmetics that use the same.
• FIG. 1 is a schematic diagram showing an outline of the search for MTr1 inhibitory compounds.
• FIG. 1 is a photograph of a Western blot showing the generation of MTr1 knockout (KO) A549 cells by CRISPR/Cas9.
• FIG. 1 is a graph showing accumulation of IAV and IBV viruses in wild-type and MTr1 KO A549 cells.
• FIG. 13 is a graph showing strand-specific qRT-PCR analysis of IAV vRNA, mRNA, and cRNA in WT and MTr1 KO A549 cells infected with IAV PR8 for 24 hours.
• FIG. 1 is a graph showing strand-specific qRT-PCR analysis of IAV vRNA, mRNA, and cRNA in WT and MTr1 KO A549 cells infected with IAV PR8 at different moi and times.
• FIG. 13 is a graph showing qRT-PCR analysis of NT/U2-IAV hybrid RNA in WT and MTr1 KO A549 cells infected with IAV PR8 for 24 hours at each moi.
• FIG. 8 Graphical representation of qRT-PCR analysis of U2 RNA for the same samples as in FIG. 8.
• Photographs showing the results of Western blot of WT and MTr1 KO cells infected with IAV (a) shows the results of WT and MTr1 KO A549 cells infected with IAV WSN, and (b) shows the results of WT and MTr1 KO A549 cells infected with SC35M-GFP.
• FIG. 1 is a schematic diagram outlining the dependency of MTr1 on Cap-snatching virus replication.
• FIG. 1 is a schematic diagram showing a general flow chart of the primary screening.
• FIG. 1 Schematic showing the docking scores and virtual screening rankings of representative compounds. This is a graph showing the results of qRT-PCR analysis of IAV RNA in A549 cells infected with IAV PR8 strain for 24 hours.
• WST-8 cytotoxicity test
• FIG. 1 is a graph showing the results of a thermal shift assay of recombinant MTr1 using SYPRO Orange.
• (a) Another result of a thermal shift assay of recombinant MTr1 using SYPRO Orange (b) a result of an MTase assay using human MTr1 WT or MTr1 K239A with SAM, SAH, or sinefungin, and (c) a result of an MTase assay using human MTr1 with tubercidin at the indicated concentrations.
• FIG. 1 is a graphical representation showing viral replication of seasonal IAV in human lung tissue.
• FIG. 1 is a photographic representation showing viral replication and immunohistochemistry of seasonal IAV proteins in human lung tissue.
• FIG. 1 is a graph showing the results of GFP levels and cytotoxicity assay in LA-4 cells infected with IAV-GFP.
• (a) Graph showing weight change upon intranasal inoculation of mice.
• FIG. 1 is a graph showing the results of qRT-PCR analysis of each RNA in A549 cells infected with other RNA viruses.
• (a) Graph showing the results of qRT-PCR analysis of each viral RNA in MTr1 KO A549 cells infected with each virus,
• (b) Photograph showing the results of Western blot analysis of each protein in MTr1 KO A549 cells infected with each virus,
• FIG. 1 is a graph showing qRT-PCR analysis of IAV RNA in A549 cells infected with IAV PR8, (a) CAPAM KO and (b) Mtr2 KO cells.
• Graph showing the results of qRT-PCR analysis of ribosomal RNA (18S) and mRNA (GAPDH) in input and immunoprecipitated RNA from HEK-293T cells (a) ribosomal RNA (18S), (b) mRNA (GAPDH), (c) photograph showing the results of Western blot analysis of PABP immunoprecipitated PABP protein.
• FIG. 1 shows a graph of luciferase reporter assay of IAV PR8 minireplicons in MTr1 KO HEK-293T overexpressing each PA subunit.
• (a) shows the results for WT (wild type) and
• (b) shows the results for MTr1 KO cells.
• (c) shows the virus yield in the supernatant of MTr1 KO A549 cells infected with IAV HK483 PA I38T, a BXM-resistant virus.
• FIG. 1 is a graph showing luciferase reporter assay of IAV PR8 minireplicon in MTr1 KO HEK-293T cells overexpressing the PA subunits shown on the horizontal axis of each figure (a) to (d).
• FIG. 1 shows the CRISPR target sequence and mutated region in the knockout cells used.
• the anti-influenza virus composition according to the present invention and the medicines, foods and beverages, supplements, agricultural chemicals, feed, and cosmetics that use the composition, with reference to the embodiments.
• the present invention is not limited to the following embodiments.
• the anti-influenza virus composition of the present embodiment contains tubercidin, a tubercidin derivative, or a salt thereof as an active ingredient.
• Tubercidin, its derivatives and salts are known as natural organic compounds.
• the derivatives and salts can be appropriately selected as long as they are pharmacologic.
• tubercidin a compound in which R 1 , R 2 and R 3 in the formula (1) described below are all hydrogen atoms is known as tubercidin (CAS Registry Number: 69-33-0).
• a tubercidin derivative is a compound in which any site of tubercidin, preferably any one of R 1 , R 2 and R 3 in the formula (1) described below, is any one of the substituents described below.
• a salt of tubercidin or a tubercidin derivative refers to a salt of any ionic substituent of tubercidin or a tubercidin derivative, and refers broadly to pharma- ceutically acceptable salts of the compound.
• examples of pharma- ceutically acceptable salts include acid addition salts or base addition salts of the compound represented by formula (1).
• acid addition salts include acid addition salts with inorganic or organic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, trifluoroacetic acid, citric acid, or maleic acid).
• base addition salts include metal salts, such as light metal salts, and specifically include alkali metal salts such as sodium salt or potassium salt, alkaline earth metal salts such as calcium salt, and ammonium salt.
• tubercidin, a tubercidin derivative or a salt thereof can be an active ingredient of an anti-influenza virus composition. That is, it has been reported that MTr1, an RNA methyltransferase in host cells, is involved in cap snatching of influenza viruses, but the details thereof were unknown.
• the present inventors first created MTr1 knockout cells and investigated the replication ability of various cap-snatching viruses, including influenza viruses. As a result, it was found that MTr1 is essential for the replication of influenza viruses type A and type B. It was also found that the mechanism is that influenza viruses can only snatch (steal) cap structures modified by MTr1.
• MTr1 was not involved in the replication of cap-snatching viruses other than influenza viruses type A and type B. In other words, it was thought that the replication of influenza viruses could be selectively suppressed by inhibiting the function of host MTr1.
• the present inventors further screened an existing drug library (5,597 compounds) using the three-dimensional structure information of MTr1 and a computer to search for MTr1 inhibitors.
• an existing drug library (5,597 compounds) using the three-dimensional structure information of MTr1 and a computer to search for MTr1 inhibitors.
• tubercidin as a compound that inhibits the enzymatic activity of MTr1 and exhibits antiviral activity against various influenza viruses.
• An outline of the search for MTr1 inhibitor compounds is shown in FIG.
• the tubercidin derivative is preferably a compound represented by the following general formula (1): ...(1)
• R 1 , R 2 and R 3 are the same or different and each represents a hydrogen atom, a halogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, an alkynyl group which may have a substituent, an alkoxy group which may have a substituent, an amino group which may have a substituent, an amido group which may have a substituent, a cyano group, a nitro group, a hydroxy group, a sulfone group, a cycloalkyl group which may have a substituent, an aryl group which may have a substituent, an aliphatic heterocyclic group which may have a substituent, or an aromatic heterocyclic group which may have a substituent.
• R1 and R2 may be the same or different and represent a hydrogen atom, a halogen atom, an amino group which may have a substituent, an amido group which may have a substituent, or a cyano group
• R3 may represent a hydrogen atom, an alkyl group which may have a substituent, an amino group which may have a substituent, or an aryl group which may have a substituent.
• R3 may represent a hydrogen atom.
• R 1 and R 2 may be the same or different and each may represent a halogen atom.
• R 1 and R 2 may be the same or different and each may represent an amino group or a cyano group which may have a substituent.
• R 1 may represent an alkyl group substituted with a halogen.
• R 1 may represent a trifluoromethyl group.
• R 1 , R 2 and R 3 are substituents containing carbon
• the number of carbon atoms can be selected arbitrarily within a range acceptable for administration to an organism, and may be, for example, 1 to 20, 1 to 10, or 1 to 6.
• Substituents that may contain a straight or branched chain of carbon atoms of an alkyl group, an alkenyl group, an alkynyl group, or an alkoxy group may be either a straight or branched chain.
• the number of carbon atoms may be 1 to 10, 1 to 6, or 1 to 3 within the range that can be assumed by the structure of each of the above-mentioned substituents.
• the cycloalkyl group, aryl group, aliphatic heterocyclic group and aromatic heterocyclic group may have 1 to 3 rings, or 1 or 2 rings.
• alkyl group examples include, but are not limited to, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a sec-pentyl group, a tert-pentyl group, a neopentyl group, a 1-methylpentyl group, an n-hexyl group, an isohexyl group, a sec-hexyl group, a tert-hexyl group, or a neohexyl group.
• alkenyl group examples include, but are not limited to, a vinyl group, an allyl group, a propenyl group, an isopropenyl group, a 2-methyl-1-propenyl group, a 2-methylallyl group, or a 2-butenyl group.
• alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl groups.
• alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy groups.
• amino group examples include, but are not limited to, an amino group, an ethylamino group, a dimethylamino group, a butylamino group, a cyclopentylamino group, a 2-ethylhexylamino group, a dodecylamino group, an anilino group, a naphthylamino group, or a 2-pyridylamino group.
• Examples of the amide group include, but are not limited to, a methylcarbonylamino group, an ethylcarbonylamino group, a dimethylcarbonylamino group, a propylcarbonylamino group, a pentylcarbonylamino group, a cyclohexylcarbonylamino group, a 2-ethylhexylcarbonylamino group, an octylcarbonylamino group, a dodecylcarbonylamino group, a phenylcarbonylamino group, or a naphthylcarbonylamino group.
• cycloalkyl group examples include 4- to 7-membered cycloalkyl groups, specifically, but not limited to, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and the like.
• aryl groups aromatic hydrocarbon groups
• aryl groups aromatic hydrocarbon groups
• aryl groups include, but are not limited to, a phenyl group, a naphthyl group, anthryl group, an azulenyl group, an acenaphthenyl group, a fluorenyl group, a phenanthryl group, an indenyl group, a pyrenyl group, or a biphenylyl group.
• aliphatic heterocyclic group examples include, but are not limited to, those derived from an aliphatic heterocycle such as an epoxy ring, an aziridine ring, a thiirane ring, an oxetane ring, an azetidine ring, a thietane ring, a tetrahydrofuran ring, a dioxolane ring, a pyrrolidine ring, a pyrazolidine ring, an imidazolidine ring, an oxazolidine ring, a tetrahydrothiophene ring, a sulfolane ring, a thiazolidine ring, an ⁇ -caprolactone ring, an ⁇ -caprolactam ring, a piperidine ring, a hexahydropyridazine ring, a hexahydropyrimidine ring, a piperazine ring, a morph
• aromatic heterocyclic groups include, but are not limited to, pyridyl, pyrimidinyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, pyrazolyl, pyrazinyl, triazolyl, oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isothiazolyl, furazanyl, thienyl, quinolyl, benzofuryl, dibenzofuryl, benzothienyl, dibenzothienyl, indolyl, carbazolyl, carbolinyl, diazacarbazolyl, quinoxalinyl, pyridazinyl, triazinyl, quinazolinyl, and phthalazinyl groups.
• the halogen element includes a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and preferably a flu
• R 1 , R 2 and R 3 further have a substituent
• the substituent can be arbitrarily selected from the above-mentioned organic substituents, halogen elements, metal elements and the like.
• R 1 , R 2 and R 3 have a substitution site with a halogen element or a substitution site with other substituents, the substitution site can also be arbitrarily selected within the range that does not inhibit physiological functions.
• substituents include the above-mentioned or other alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, aliphatic heterocyclic groups, aromatic heterocyclic groups, heterocyclic groups, cycloalkoxy groups (e.g., cyclopentyloxy groups, cyclohexyloxy groups, etc.), aryloxy groups (e.g., phenoxy groups, naphthyloxy groups, etc.), alkylthio groups (e.g., methylthio groups, ethylthio groups, propylthio groups, pentylthio groups, hexylthio groups, octylthio groups, dodecylthio groups, etc.), cycloalkylthio groups (e.g., cyclopentylthio groups, cyclohexylthio groups, etc.), arylthio groups (e.g., phenyl
• tubercidin derivative may be a compound represented by the following formula (2): ... (2)
• the compound of the formula (2) trifluoromethyltubercidin (TFMT), is a compound that is less toxic than tubercidin among tubercidin derivatives, and is the most effective MTr1 inhibitor exhibiting anti-influenza virus activity.
• TFMT trifluoromethyltubercidin
• the present inventors have examined 115 types of tubercidin derivatives and found that the TFMT is the most effective component.
• TFMT binds to the substrate-binding pocket of MTr1 and inhibits its enzymatic activity. Furthermore, structural analysis predicts that the cap structure unmodified by MTr1 may impair interaction with influenza virus RNA polymerase (PB2 protein). In other words, TFMT suppresses the modification function of the cap structure by binding to host MTr1. The cap structure of host mRNA that is not modified by MTr1 weakens its interaction with viral RNA polymerase (PB2 protein), and as a result, TFMT is thought to inhibit cap snatching mediated by RNA polymerase and the subsequent synthesis of viral RNA.
• PB2 protein influenza virus RNA polymerase
• the anti-influenza virus composition of the present embodiment is preferably used for the treatment of influenza. That is, the anti-influenza virus composition can also be called a medicine or pharmaceutical composition used for the treatment of influenza.
• the anti-influenza virus composition may also be a medicament containing other ingredients.
• the medicament for treating influenza may also contain various other ingredients contained in conventionally known medicaments or pharmaceutical compositions, as appropriate.
• the form of the pharmaceutical in this embodiment is not particularly limited, and can be, for example, a solution, a dispersion such as a sol or gel, or a powder.
• the pharmaceutical can be administered orally, for example, in the form of a tablet, capsule, elixir, or parenterally, in the form of an enema, or the like.
• pharma- ceutically acceptable carriers those normally used in pharmaceutical preparations can be used without any particular restrictions. More specific examples include binders, excipients, swelling agents, and solvents (solvents).
• the pharmaceutical of this embodiment may contain additives.
• additives include lubricants, sweeteners, flavoring agents, stabilizers, pH buffers, dissolution aids, antioxidants, and preservatives.
• the pharmaceutical of this embodiment can be formulated by appropriately combining the above-mentioned components and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.
• the dosage of a medicament varies depending on the type, method of use, and dosage of the drug, as well as the symptoms, body weight, age, sex, and the like of the patient, and cannot be determined in general terms; however, typically, in the case of an administration form such as injection or oral administration, the active ingredient in the anti-influenza virus composition contained in the medicament may be 0.1 ng/kg to 250 mg/kg (here, kg is per body weight of the subject to be administered), or may be 0.1 ng/kg to 100 mg/kg, or may be 1 ng/kg to 100 mg/kg, or may be 1 ng/kg to 50 mg/kg per day.
• the amount of the active ingredient in the pharmaceutical preparation may be 1 to 10% by weight, 1 to 7% by weight, or 3 to 5% by weight, based on the total weight of the pharmaceutical preparation.
• the food, drink, and supplement of this embodiment contain the anti-influenza virus agent of this embodiment.
• the target food, drink, and supplement are not particularly limited.
• Examples of the food include processed foods, etc.
• the beverage of this embodiment contains the anti-influenza virus agent of this embodiment.
• an extract can be obtained by immersing the composition of this embodiment in water or other solvent, for example, alcohol such as ethanol, or other organic solvent, and heating it at a desired temperature, or by immersing the mixed rolled product in a solvent such as water at a desired temperature. That is, the beverage of this embodiment can be obtained in the same manner as the conventionally known method of drinking tea.
• a method can be used in which the liquid or solid composition of this embodiment is added to any beverage (for example, soft drinks, alcoholic beverages).
• the agricultural chemicals, feeds, and cosmetics of this embodiment contain the anti-influenza virus agent of this embodiment.
• the above-mentioned medicines, foods, and the like or may be appropriately selected according to the embodiment of conventionally known agricultural chemicals, feeds, and cosmetics.
• a new anti-influenza virus drug by inhibiting the proliferation and infection mechanisms of influenza viruses, a new anti-influenza virus drug can be obtained that has a target that is not a specific viral protein and does not result in the emergence of drug-resistant viruses, as well as medicines, foods and beverages, supplements, agricultural chemicals, feed, and cosmetics using the same.
• MTr1 has been known as a catalyst for nucleic acid methylation, in which mammalian mRNAs and small nuclear RNAs (snRNAs) are capped at their 5' ends with 7-methylguanosine (m7G) and 2,2,7-trimethylguanosine, which are linked to RNA by a triphosphate bridge and are referred to as cap0.
• 2'-O-ribose methyltransferase MTr1 (CMTR1/FTSJD2) is an RNA methyltransferase (MTase) that is mainly localized in the nucleus and catalyzes the 2'-O-methylation of the first nucleotide (N1-2'-O-Me).
• cap0 mRNA and snRNA to generate mature caps the cap structure of host RNA is important to promote stability and translation.
• Unmethylated RNA activates the innate immune response by RIG-I-like receptors and is vulnerable to cellular restriction factors such as IFIT1.
• Fully methylated cap1 RNA evades the innate immune mechanism. Thus, viruses usually hijack or mimic the cap structure of cellular RNA.
• Orthomyxoviruses including bunyaviruses, influenza A virus (IAV), and influenza B virus (IBV), rely on a strategy called cap snatching, in which the 5' end of a fully capped cellular RNA is cleaved and attached to viral mRNA.
• IAV polymerase is composed of three subunits, PA, PB1, and PB2, and the trimeric polymerase binds to the cap structure of host RNA via PB2, initiates RNA cleavage by PA endonuclease 10-13 nucleotides downstream of the cap structure, and finally synthesizes viral mRNA using PB1 as a primer.
• non-cap-snatching viruses such as coronaviruses, poxviruses, and flaviviruses, including yellow fever virus
• non-cap-snatching viruses encode their own cap-binding 2'-O-MTases that mediate 2'-O-methylation of RNA to mimic the cellular cap and avoid recognition by antiviral sensors.
• Cellular MTr1 depletion leads to the accumulation of host cap0 RNA in the cytoplasm and has been shown to activate RIG-I and IFIT1, indicating that the methylation status of endogenous capped mRNAs at the first nucleotide is one of the key features that prevents immune activation.
• MTr1 deficiency enhances the antiviral IFN response against influenza A virus and also affects its cap-snatching effect, resulting in reduced influenza A virus replication levels. Therefore, MTr1 is an interesting host-directed target for anti-influenza A virus drugs and possibly also for drugs against other cap-snatching viruses. However, no MTr1 inhibitors have been reported, and its potential as an antiviral agonist remained unexplored.
• TFMT trifluoromethyl-tubercidin
• TFMT demonstrated efficacy both in human lung explants and in vivo in mice, and exhibited synergistic antiviral activity with approved anti-influenza drugs such as baloxavir marboxil, oseltamivir, and amantadine.
• approved anti-influenza drugs such as baloxavir marboxil, oseltamivir, and amantadine.
• Our results reveal an unexpected activity of MTr1 in viral replication and a conceptually new approach to inhibit influenza virus infection.
• the present invention has the following aspects as other embodiments.
• Another aspect of this embodiment is a compound comprising tubercidin, a tubercidin derivative or a salt thereof for use in the treatment or prevention of a disease involving an influenza virus.
• Yet another aspect of this embodiment is the use of a compound comprising tubercidin, a tubercidin derivative or a salt thereof for the manufacture of a therapeutic agent for a disease involving influenza virus.
• Yet another aspect of this embodiment is a method for producing a therapeutic agent for a disease associated with influenza virus, using a compound containing tubercidin, a tubercidin derivative, or a salt thereof.
• HEK-293T Human embryonic kidney (HEK)-293T, human alveolar adenocarcinoma A549, mouse lung adenoma LA-4, Platinum-A (Plat-A), Madin-Darby canine kidney (MDCK), and Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM ) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin at 37°C and 5% CO .
• DMEM Dulbecco's modified Eagle's medium
• FBS fetal bovine serum
• penicillin 100 U/ml bovine serum
• streptomycin 100 fetal bovine serum
• streptomycin 100 fetal bovine serum
• Expi293F suspension cells were cultured in Expi293 expression medium (Gibco, A1435101) with shaking at 37 °C and 8% CO2 .
• NHBE bronchial epithelial
• BEGM bronchial epithelial cell growth medium
• Plat-A cells were cultured in a medium supplemented with 10 ⁇ g/ml blasticidin and 1 ⁇ g/ml puromycin.
• antisera from HAZV-infected B6 mice (#WT No. 2, 170807)
• C57BL/6 mice were inoculated intraperitoneally with 105 plaque-forming units (PFU) of Hazara virus. Inoculation was repeated twice at an interval of 3 weeks. Three days after the last inoculation, blood was collected in BD Microtainers to obtain serum.
• PFU plaque-forming units
• H1N1; IAV PR8 Influenza strain A/Puerto Rico/August 1934 (H1N1; IAV PR8) was propagated in chicken eggs, and viral titers were quantified by plaque assay on MDCK cells.
• influenza A/WSN/1933 (H1N1) strain (IAV WSN) was recovered from the pDZ ambisense plasmid 23 (kindly provided by Adolfo Garcia Sastre). It was propagated in MDCK cells and titers were measured by flow cytometry using the nanobody VHH NP2 Alexa Fluor 647.
• Recombinant influenza strain A/SC35M H7N7 containing a modified NS segment encoding NS1, a P2A site, an EGFP, a T2A site, and NEP (IAV SC35M-GFP) was recovered from a pHW ambisense plasmid (from Martin Schwemmle), propagated in MDCK cells, and titers were determined by flow cytometry.
• VSV-GFP Recombinant vesicular stomatitis virus Indiana strain encoding EGFP at position 5 of the genome
• Influenza A virus strains A/Hong Kong/483/1997 (H5N1) (IAV HK483) and IAV HK483 I38T were grown and harvested in embryonated chicken eggs.
• Influenza B virus strain/Hokkaido/30-4/2014 (IBV) and Influenza D virus strain D/bovine/NE/9-5/2012 (IDV) were grown and harvested in embryonated chicken eggs.
• Viral titers in MDCK cells and allantoic fluid or cell culture supernatants were quantified under 50% tissue culture infectious dose (TCID50) using MDCK cells.
• TCID50 tissue culture infectious dose
• Rift Valley fever virus strain MP-12 was recovered from plasmids provided by Dr. Shinji Makino in UTMB and BSR/T7-5 cells and propagated in VeroE6 cells provided by Dr. Karl-Klaus at the Max von Pettenkofer-Institute of Virology, Conzelmann.
• Hazara virus (HAZV) strain JC280, Issyk-Kul virus (ISKV) strain LEIV315K (accession number LC495734-6), Soft tick bunya virus (STBV), strain Av-18 (accession number LC495731-3), and Dugbe virus (DUGV) strain 15AC-T25 (accession number LC579816-8) were kindly provided by Drs. Roger Hewson (Public Health England), Barbara W. Johnson (Centers for Disease Control and Prevention), Shuji Ando (National Institute of Infectious Diseases (NIID)), and Chang-Kweng Lim (NIID).
• viruses were propagated in Vero cells in DMEM medium containing 2% FBS, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin. Virus-containing supernatants were collected 3-7 days after inoculation. Infectious doses were titrated on SW-13 cells by plaque formation assay in 6-well plates.
• BXM was purchased from BLD Pharmatech (BD00808126). Ribavirin, oseltamivir phosphate and amantadine were purchased from Sigma-Aldrich (R9644, SML1606, 138576). Chemicals used in the primary screen, sinefungin and TAK-599 isavuconazonium (sulfate), were purchased from Cayman Chemical Company (13829, 23696, 23950).
• SAH S-adenosyl-L-homocysteine
• SAM S-adenosyl-L-methionine
• tubercidin GDP-D-mannose disodium salt
• 5-iodotubercidin Itu
• sertindole was purchased from Sigma-Aldrich (A9384, A4377, T0642, 07508, I100, S8072).
• m7GpppA was purchased from New England Biolabs (S1405). Tecadenoson was purchased from BLD Pharmatech (BD00781750).
• Tubercidin analogs 3-deazaadenosine (3-DZA), cladribine, clofarabine, coenzyme A, and nebularine were purchased from Cayman Chemical Company (9000785, 12085, 14125, 16147, 31329).
• 2-Fluoroadenosine and cordycepin were purchased from Sigma-Aldrich (656402, C3394).
• Vidarabine and 2'-deoxy-2-fluoroadenosine were purchased from BLD Pharmatech (BD42581, BD74284).
• 2-Aminoadenosine was purchased from Santa Cruz Biotechnology (sc-220693A).
• 1-Deazaadenosine, 8-azaadenosine, and fludarabine were purchased from Tocris (4488, 6868, 3495).
• Z295883282 was purchased from Enamine (Z295883282).
• MolPort-044-721-870 was purchased from MolPort (MolPort-044-721-870).
• Ganciclovir and acyclovir were purchased from Pharmaks (PHAR088857, PHAR100587).
• 3'-Deoxy-tubercidin and 3'-deoxy-7-bromotubercidin were purchased from Shinsei Chemical Industry Co., Ltd. (custom made).
• 96 compounds were obtained from PharmaCenter Bonn & Pharmaceutical Institute.
• CRISPR/Cas9 knockout cells CRISPR/Cas9 knockout cells
• sgRNA and Cas9 were transduced into target cells by transfection or lentiviral infection, and cells were selected by sorting using GFP or mCherry fluorescence (BD FACS Aria III) or puromycin resistance, isolated as single clones, and confirmed by Western blot and sequence analysis.
• CRISPR target sequences and mutated regions in the knockout cells used are shown in Figure 45.
• the plasmids used are shown in Tables 1 and 2.
• NP40 lysis buffer 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40
• RIPA buffer 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate
• DTT dithiothreitol
• PMSF phenylmethylsulfonyl fluoride
• vanadate 1 mM vanadate
• the supernatant was collected, mixed with 2x sample buffer (125 mM Tris-HCl pH 6.8, 10% 2-mercaptoethanol, 4% SDS, 20% glycerol, 0.01% bromophenol blue (BPB)), and boiled at 95°C for 3 min.
• 2x sample buffer 125 mM Tris-HCl pH 6.8, 10% 2-mercaptoethanol, 4% SDS, 20% glycerol, 0.01% bromophenol blue (BPB)
• the sections were separated by SDS-PAGE (buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS), blotted onto Immobileon-P PVDF membranes (Merck, IPVH00010) (buffer: 18.6 mM Tris, 144 mM glycine, 20% methanol), blocked with 5% skim milk (AppliChem, A0830), stained with primary antibodies for 1 hour at room temperature (approximately 18-23°C) or overnight at 4°C, washed three times with TBS-T (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.04% Tween 20), stained with secondary antibodies (HRP-conjugated) for 1 hour at room temperature, and washed three times with TBS-T. Bands were detected by chemiluminescence using ECL Prime reagents (Amersham, RPN2232) and an Amersham 680 Imager.
• RNA Quantification Cells were harvested with TRIzol (Invitrogen, 15596) and RNA was extracted according to the manufacturer's protocol. cDNA was purified using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 43688). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using Fast SYBR Green Master Mix (Applied Biosystems, 4385614) or TaqMan Fast Advanced Master Mix (Applied Biosystems, 4444558). The PCR primers used (SEQ ID NOs: 1 to 55 in the Sequence Listing) and the TaqMan probes used are shown in Tables 3 to 5. The product names of the antibodies used are shown in Table 6.
• GFP fluorescence in IAV SC35-GFP infected cells was quantified using FIJI (ImageJ). 8-bit images acquired with a Cell-IQ fluorescence microscope were thresholded and fluorescence quantified with "Analyze Particles”. Relative "IntDen” values were calculated as relative fluorescence units/region of interest (RFU/ROI).
• IAV PR8 titers (PFU/ml) were determined by plaque assay using MDCK cells. MDCK cells were seeded in multiwell plates one day prior to infection. Serial 1:10 dilutions of IAV-containing supernatants were added to the wells and incubated at 37°C for 1 h.
• DMEM tissue culture median infectious dose
• IAV mini-replicon reporter assay The assay was performed using the method described in Cell Host Microbe 17, 309-319.
• PB2 or PA expression plasmid was replaced with an empty expression plasmid.
• luciferase activity was measured using a Dual-Luciferase Reporter System (Promega, E1980) equipped with a TriStar2 LB942 luminometer (Berthold Technologies). Firefly luciferase activity was normalized to Renilla luciferase activity.
• Fluorescence microscopy images were acquired using an InCellis (CENiBRA) with a 10 ⁇ objective, a Cell-IQ fluorescence microscope (CM Technologies) with a 4 ⁇ objective, or a SP8 confocal microscope (Leica) with a 10 ⁇ /0.30HC PL FLUOTAR DRY objective and a HyD detector.
• CENiBRA InCellis
• CM Technologies Cell-IQ fluorescence microscope
• Leica SP8 confocal microscope
• PB2 sequences were generated by MEGA X software using the neighbor-joining method. The bootstrap method (1000 replicates) was used to evaluate the phylogeny.
• the references for PB2 sequences are as follows: IAV PR8 (GenBank: NC_002023.1), IAV WSN (GenBank: LC333182.1), IAV SC35M (GenBank: DQ266097.1), IAV HK483 (GenBank: AF258839.1), IBV (GenBank: AF101982.1), ICV (GenBank: AF170576.2), IDV (GenBank: LC318665.1), THOV (GenBank: NC_006508.1).
• Bunyavirus S segment sequence references are as follows: RVFV (GenBank: KU925457.1), Crimean-Congo hemorrhagic fever virus (CCHFV) (GenBank: M86625.1), HAZV (NCBI: NC_038711.1), ISKV (GenBank: LC495736.1), STBV (GenBank: LC495733.1), and DUGV (GenBank: KU925457.1).
• RVFV GenBank: KU925457.1
• CHFV Crimean-Congo hemorrhagic fever virus
• HAZV NCBI: NC_038711.1
• ISKV GeneBank: LC495736.1
• STBV GenBank: LC495733.1
• DUGV GeneBank: KU925457.1
• Structure-based screening of Mtr1 inhibitors Structure-based virtual screening of MTr1 inhibitors was performed using molecular docking against 5,597 bioactive compounds with molecular weights between 200 Da and 800 Da in the DrugBank database. Docking simulations were performed using the Glide SP docking program (Schrodinger, LLC) using a grid box defined by the SAM binding pocket of the crystal structure of human MTr1 (PDB ID: 4N49). After selecting only FDA-approved drugs from the results, the top 30 compounds were selected for in vitro studies.
• TFMT-Mtr1 docking Docking simulations were performed using the Glide SP docking program (Schrodinger, LLC) using a grid box defined by the SAM binding pocket of the crystal structure of human MTr1 (PDB ID: 4N49). The docking pose with the highest docking score was selected.
• Recombinant human MTr1 WT and K239A proteins were expressed in Expi293F cells using the ExpiFectamine Transfection Kit (Gibco, A14524) and purified using an affinity tag (TwinStrep-tag II).
• Cell pellets were lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1 mM DTT, 2 ⁇ g/ml leupeptin, 1 mM PMSF, and 1 mM vanadate, and disrupted by three freeze-thaw cycles (liquid nitrogen to 37°C water bath).
• RNA transcription Cap0 RNA was synthesized using HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, E2050S) and the cap analog, 3'-O-Me-m7G(5')ppp(5')G (NEB, S1411) according to the manufacturer's instructions. Annealed T7 promoter-GGG-N25 DNA was used as a template (shown in the primer table in Table 5). The synthesized RNA, 3'-O-Me-m7GpppGpG (pN27), was used for the methyltransferase activity assay of human MTr1.
• Methyltransferase Activity Assay Reaction buffer 50 mM Tris-HCl pH 8.0, 5 mM KCl, 1 mM MgCl 2 , 1 mM DTT), methyltransferase (0.8 ⁇ M human MTr1, 0.8 ⁇ M human MTr1 K239A), inhibitor of interest, 10 ⁇ M 3′-O-Me-m7GpppGpG(pN27)cap0 RNA, and 1.2 ⁇ M (0.02 ⁇ Ci/ ⁇ l) adenosyl-L-methionine, S-[methyl- 3 H] (SAM[ 3 H]) (PerkinElmer, NET155V250UC) were incubated overnight at 37° C.
• Reaction samples were purified using Miniquick Oligo columns (Roche, 11814397001) to remove free SAM[3H].
• the purified samples were diluted with ULTIMA GOLD (PerkinElmer, 6013329) and methyltransferase activity was measured in disintegrations per minute (DPM) using a scintillation counter LS6500 (Beckman Coulter).
• Thermal shift assay Protein thermal stability was measured using SYPRO Orange dye (Sigma, S5692). The purchased stock solution (5000x) was diluted 50x with thermal shift assay buffer (25mM HEPES pH 7.5, 150mM NaCl) before use. Reaction mixtures were prepared by mixing 5 ⁇ l of diluted SYPRO Orange dye with 1 ⁇ g of protein and respective concentrations of compounds in a total volume of 50 ⁇ l of thermal shift assay buffer. Thermal stability was analyzed on a Step One Plus real-time PCR system with TAMRA dye detection settings. Measurements were normalized using GraphPad Prism software.
• FIG. 2 is a photograph of a Western blot showing the generation of MTr1 knockout (KO) A549 cells by CRISPR/Cas9.
• wild-type (WT) and MTr1 KO A549 cells are shown to have been infected with IAV (influenza A virus) PR8 (moi, 1) (moi: multiplicity of infection).
• FIG. 3 is a graph showing IAV and IBV virus accumulation in wild-type and MTr1 KO A549 cells.
• (a) shows the viral growth of IAV (H1N1, PR8) in WT and MTr1 KO A549 cells.
• Supernatants were harvested at the indicated time points post-infection (moi, 1) and virus titers were measured by plaque assay. The detection limit is indicated by the dotted line at the bottom.
• FIG. 4( a ) is a graphical representation showing qRT-PCR analysis of IAV RNA (segment 7) in WT and MTr1 KO A549 cells infected with IAV PR8 (moi, 1).
• Figure 4(b) is a graphical representation showing qRT-PCR analysis of individual IAV RNA segments in WT and MTr1 KO A549 cells infected with IAV PR8 at m.o.i.1 for 24 h.
• FIG. 4( c ) is a photograph showing immunostaining of viral proteins in WT and MTr1 KO A549 cells infected with IAV PR8 (moi, 10) for 19 hours.
• Figure 4(d) is a graph showing qRT-PCR analysis of IBV RNA in WT and MTr1 KO A549 cells infected with IBV at m.o.i. 1 for 24 h.
• Figure 4(e) is a photograph showing Western blot analysis of the indicated proteins in WT or MTr1 KO A549 cells infected with IBV at m.o.i.1 for 24 h. These results, in line with those described above, clearly demonstrated a loss of viral RNA levels and viral protein expression in MTr1 KO cells. Twenty-four hours prior to IAV infection, cells were infected with retroviruses expressing MTr1, MTr1 K239A, or neither. Fig.
• 4(f) is a photograph showing Western blot analysis of each protein in WT and MTr1 KO A549 cells infected with IAV PR8 strain at m.o.i. 1 for 24 hours.
• 5(a) is a photograph showing immunostaining in WT and MTr1 KO A549 cells infected with IAV PR8 strain at m.o.i. 1 for 24 hours.
• Fig. 5(b) is a graph showing the results of flow cytometry in WT and MTr1 KO A549 cells infected with IAV PR8 strain at m.o.i 1 for 24 hours.
• FIG. 6 is a graph showing strand-specific qRT-PCR analysis of IAV vRNA, mRNA, and cRNA (segment 5) in WT and MTr1 KO A549 cells infected with IAV PR8 for 24 hours at the indicated moi.
• 7 is a graph showing strand-specific qRT-PCR analysis of IAV vRNA, mRNA, and cRNA (segment 5) in WT and MTr1 KO A549 cells infected with IAV PR8 at the indicated moi and times.
• Strand-specific quantitative reverse transcription polymerase chain reaction qRT-PCR
• FIG. 9(a) is a schematic diagram showing primer sequences for cap-snatching-specific qPCR.
• Figure 9(b) is a graph showing qRT-PCR analysis of IAV RNA (segment 1), U2, and U2-IAV RNA (segment 1) hybrid RNA (cap-snatch RNA) in A549 cells infected with IAV PR8 at m.o.i 1 for 24 h. 0.1 ⁇ M BXM or 100 ⁇ M ribavirin (Rib) was added 3 h prior to infection. Non-targeting (NT)-segment 1 hybrid primers were used as controls.
• FIG. 9(c) is a graph showing qRT-PCR analysis of U2 RNA for the same samples as in FIG.
• FIG. 10 is a graph showing qRT-PCR analysis and viral yields of WT and MTr1 KO cells infected with IAV.
• 11 is a photograph showing the results of Western blot of WT and MTr1 KO cells infected with IAV, (a) showing the results of WT and MTr1 KO A549 cells infected with IAV WSN at 1 particle/cell for 24 hours, and (b) showing the results of WT and MTr1 KO A549 cells infected with SC35M-GFP at 0.1 infectious particle/cell for the indicated times.
• FIG. 12 is a graph showing qRT-PCR analysis and viral yields of WT and MTr1 KO cells infected with IDV and THOV.
• (a) shows viral yields in the supernatants of WT and MTr1 KO A549 cells infected with IDV at m.o.i. 0.1 for 48 hours.
• (b) shows the results of qRT-PCR analysis of THOV RNA in WT and MTr1 KO A549 cells infected with THOV at m.o.i. 1 for 24 hours.
• MTr1 deficiency had minimal or no effect on viral particle production and RNA replication of two other orthomyxoviruses, influenza D virus (IDV) and Thogoto virus (THOV), suggesting that the requirement for MTr1 differs among orthomyxoviruses.
• IDV influenza D virus
• THOV Thogoto virus
• RVFV Rift Valley fever virus
• HZV Hazara virus
• ISKV Issyk-Kul virus
• STBV Soft tick
• DUGV Dugbe virus
• FIG. 13 is a graph showing the viral yield and RNA amount in WT and MTr1 KO A549 cells infected with various viruses.
• (a) shows the viral yield in the supernatant from WT and MTr1 KO A549 cells infected with RVFV at m.o.i. 0.1 for 2 days.
• (b) shows the results of qRT-PCR analysis of the indicated viral RNA in WT and MTr1 KO A549 cells infected with HAZV (m.o.i., 0.1), ISKV (m.o.i., 0.01), STBV (m.o.i., 0.1), or DUGV (m.o.i., 0.1) for 24 hours.
• HAZV m.o.i., 0.1
• ISKV m.o.i.
• STBV m.o.i., 0.1
• DUGV m.o.i., 0.1
• FIG. 14( a ) is a schematic diagram showing a phylogenetic tree of the S segment of bunyaviruses.
• Figure 14(b) is a photograph showing the results of Western blot analysis of the indicated proteins in WT or MTr1 KO A549 cells infected with HAZV at m.o.i. of 0.1 for the times indicated in the figure.
• Figure 14(c) is a graph showing the results of qRT-PCR analysis of the indicated viral RNAs in A549 cells at the times and respective moi's shown in the figure, where the moi's are 1 (THOV), 0.1 (HAZV), 0.01 (ISKV), 0.1 (STBV), or 0.1 (DUGV).
• Statistics were calculated using two-tailed Student's t-test ( * p ⁇ 0.05, *** p ⁇ 0.001).
• RNA replication levels and virus yields in the supernatants of MTr1 KO cells infected with these bunyaviruses were very small or not different from those of wild-type (WT) cells.
• FIG. 15 is a schematic outlining the dependency of MTr1 on cap-snatching virus replication. The above data indicate that host-specific inhibitors of MTr1 specifically inhibit IAV and IBV among Capsnatch viruses, regardless of subtype or strain.
• MTr1 is an attractive target for anti-IAV and anti-IBV drugs, ideally effective against highly pathogenic avian IAV strains such as H5N1.
• MTr1 is known to transfer methyl groups from the methyl donor S-adenosyl-L-methionine (SAM) to the cap0 RNA acceptor, generating cap1 RNA and S-adenosyl-L-homocysteine (SAH) as by-products. Therefore, compounds that specifically bind to the SAM-binding pocket of MTr1 may act as inhibitors.
• SAM S-adenosyl-L-methionine
• SAH S-adenosyl-L-homocysteine
• FIG. 16 is a schematic diagram showing a general flow chart of the primary screening.
• Figure 17(a) is a schematic diagram showing the 2D interaction of SAM with human MTr1 (PDB ID: 4N49). The image was generated using MOE software.
• Figure 17(b) is a schematic diagram of the screening strategy to obtain potential MTr1 inhibitors. Generated at BioRender.com using the MTr1 structure from PDB (ID: 4N49). 5,597 compounds from DrugBank were docked into the SAM binding pocket of the crystal structure of human MTr1.
• FIG. 17(c) is a schematic diagram showing the docking scores and virtual screening rankings of representative compounds.
• the compounds are listed in order of calculated affinity score, and the in silico screening method confirmed that SAM, SAH, and the SAM analog sinefungin were ranked in the top 10 of high affinity binders.
• 18 is a graph showing the results of qRT-PCR analysis of IAV RNA (segment 7) in A549 cells infected with IAV PR8 strain at m.o.i. 1 for 24 h, with tubercidin added at the indicated concentrations 3 h prior to infection.
• 19(a) is a graph showing the results of quantification of GFP levels in A549 cells infected with IAV-GFP SC35M strain (moi, 0.1) for 19 hours. Each compound was added at 100 ⁇ M 3 hours before infection.
• 19(b) is a graph showing the results of another quantification of GFP levels in A549 cells infected with IAV-GFP SC35M strain (moi, 0.1) for 19 hours, where each compound was added at 0.1, 1, 10, or 100 ⁇ M 3 hours prior to infection.
• 19(c) is a graph showing the quantification of GFP levels and the results of a cytotoxicity assay (WST-8) in A549 cells infected with IAV-GFP (moi, 0.1) for 19 hours, in which tubercidin was added at the indicated concentrations 3 hours prior to infection.
• WST-8 cytotoxicity assay
• 19(d) is a graph showing the results of qRT-PCR analysis of IAV RNA (segment 7) in A549 cells infected with IAV PR8 strain at m.o.i 1 for 24 hours. Three hours prior to infection, tubercidin was added at the concentrations indicated. As shown in the figure, tubercidin was also effective against IBV infection.
• FIG. 20 is a graph showing the results of a thermal shift assay of recombinant MTr1 using SYPRO Orange. Assays were performed at the concentrations shown and with tubercidin or ribavirin as a control. 21(a) is a graph showing another result of a thermal shift assay of recombinant MTr1 using SYPRO Orange. The assay was performed at the concentrations shown and with SAM or SAH. These results confirmed that tubercidin directly binds to recombinant MTr1 by thermal shift assay.
• FIG. 20 is a graph showing the results of a thermal shift assay of recombinant MTr1 using SYPRO Orange. Assays were performed at the concentrations shown and with tubercidin or ribavirin as a control. 21(a) is a graph showing another result of a thermal shift assay of recombinant MTr1 using SYPRO Orange. The assay was performed at the concentrations shown and with SAM or SAH.
• FIG. 21(b) is a graph showing the results of an MTase assay using human MTr1 WT or MTr1 K239A with 0.1 mM SAM, SAH, or sinefungin.
• FIG. 21(c) is a graph showing the results of an MTase assay using human MTr1 containing the indicated concentrations of tubercidin. Statistics were calculated using two-tailed Student's t-test ( *** p ⁇ 0.001).
• tubercidin inhibits the MTase activity of recombinant MTr1 protein in a dose-dependent manner and is an MTr1 inhibitor that suppresses IAV replication.
• Tubercidin has been known to have multiple activities, including antiviral and anticancer activities, but is also known to exhibit a certain degree of toxicity in the body. Therefore, to identify better tubercidin-related compounds, we evaluated 115 tubercidin-related compounds by antiviral drug assays in a three-stage screening procedure: we sought compounds with specific anti-IAV and anti-IBV activity and minimal or no toxicity in vitro and in vivo.
• FIG. 22(a) is a schematic flow chart of the three-step secondary screening process.
• IAV-GFP IAV-GFP
• each compound was added at concentrations of 100 ⁇ M (step 1), 10 ⁇ M (step 2), and 1 ⁇ M (step 3).
• qRT-PCR analysis of HAZV RNA in HAZV-infected A549 cells (moi, 0.1) was also performed in step 2.
• step 1 cells were infected with IAV in the presence of individual tubercidin-related compounds and 13 compounds with anti-IAV effects were identified.
• step 2 anti-IAV activity was investigated at lower concentrations to rule out off-target inhibition of viral infection or toxicity and counterscreened for activity against HAZV.
• step 3 we discovered four compounds in step 2 that inhibited IAV replication without reducing HAZV replication.
• step 3 it was evaluated which of the identified compounds could inhibit at the lowest concentration, and finally trifluoromethyltubercidin (TFMT) was identified as the most effective compound.
• TFMT trifluoromethyltubercidin
• FIG. 22(b) is a schematic diagram showing the docking state of TFMT in the SAM-binding pocket of human MTr1.
• FIG. 22( c ) is a graph showing the results of a thermal shift assay of MTr1 with 1 mM TFMT or ribavirin using SYPRO Orange.
• FIG. 23(a) is a graph showing the results of an MTase assay of MTr1 with TFMT or Ribavirin at the respective concentrations shown. The results in the figure confirmed that TFMT inhibited the MTase activity of the recombinant MTr1 protein.
• FIG. 23(b) is a graph showing the results of qRT-PCR analysis of each viral RNA, in which qRT-PCR analysis of the indicated viral RNAs is shown in A549 cells infected for 24 hours with IAV (moi, 1), IBV (moi, 1), HAZV (moi, 0.1), and STBV (moi, 0.1).
• IAV qRT-PCR analysis of the indicated viral RNAs
• TFMT showed antiviral activity against IAV and IBV, but not against HAZV or STBV, which is in exact agreement with the phenotype of MTr1 deficiency described above.
• 23(c) is a graph showing the results of GFP level quantification for A549 cells infected with IAV-GFP, showing IAV replication levels and GFP level quantification in a cytotoxicity assay (WST-8) in A549 cells infected with IAV-GFP (moi, 0.1) for 19 hours.
• WST-8 cell viability assay As measured by WST-8 cell viability assay, the IC50 of TFMT against IAV infection was 0.30 ⁇ M, and no significant in vitro toxicity was observed within the effective concentration range.
• Figure 23(d) is a graph showing the results of another GFP level quantification for A549 cells infected with IAV-GFP. Quantification of GFP levels in A549 cells infected with IAV-GFP (moi, 0.1) for 19 hours is shown, and TFMT was added at 10 ⁇ M before or at the indicated time points after infection. Each compound shown was added 3 hours before infection in B, D, I, and J. Statistics were calculated using two-tailed Student's t-test ( *** p ⁇ 0.001). Notably, TFMT treatment also significantly inhibited IAV replication when administered 3-4 hours post-infection, although the effect was reduced or absent when the drug was administered later.
• FIG. 24(a) is a graph showing the results of qRT-PCR analysis of IAV RNA (segment 7) in normal human bronchial epithelial (NHBE) cells infected for 24 hours with IAV PR8 strain at m.o.i. 1.
• TFMT was added 3 hours prior to infection.
• Figure 24(b) is a photographic representation showing the results of Western blot analysis of the indicated proteins in NHBE cells infected with IAV PR8 strain at m.o.i.1 for 24 hours. As shown in the figure, IAV (H1N1, PR8) RNA and protein levels were significantly reduced by TFMT treatment in a dose-dependent manner.
• Fig. 24(c) is a photograph showing the results of immunostaining of NHBE cells infected with IAV PR8 strain. Immunostaining of IAV NP was performed in the presence of 10 ⁇ M TFMT 24 hours after infection (moi 2). The bar in the figure is 100 ⁇ m. Histological analysis also revealed that IAV NP levels in TFMT-treated NHBE cells were significantly reduced without cytotoxicity.
• 24(d) is a graph showing the results of qRT-PCR analysis of HAZV RNA in HAZV-infected NHBE cells.
• NHBE cells infected with HAZV for 24 hours at moi of 0.1 were analyzed.
• TFMT treatment did not inhibit HAZV replication, indicating that the specific efficacy of this compound against this particular virus is retained in human primary cells.
• Figure 24(e) is a schematic diagram of IAV infection in human lung explants. Lung tissue was infected with IAV (H1N1, 2019 seasonal isolate) and viral titers in the supernatant were measured by plaque assay at the indicated time points.
• FIG. 24( f ) is a graph showing the results of viral replication and viral titration of seasonal IAV in human lung tissue from a single donor.
• FIG. 24( g ) is a graph showing the results of viral growth in human lung tissue and viral titration of seasonal IAV in six independent donors.
• Figure 26 is a photograph showing viral growth and immunohistochemistry of seasonal IAV (H1N1 from 2019) proteins in the human lung tissue. Tissues were stained using anti-NP antibody. TFMT was added at 10 ⁇ M before and after infection. Consistent with the viral titers, neither IAV NP-positive cells nor morphological changes were observed in IAV-infected lung tissues treated with TFMT and infected with IAV, indicating the high ability of TFMT to abolish the replication of seasonal IAV isolates ex vivo, suggesting its potential application in clinical treatment.
• FIG. 27 is a graph showing the results of GFP levels and cytotoxicity assays in LA-4 cells infected with IAV-GFP.
• IAV-GFP (moi, 1) was infected for 19 hours, followed by quantification of GFP levels and cytotoxicity assays (WST-8).
• TFMT was added at the indicated concentrations 3 hours prior to infection.
• Statistics were calculated using the Wilcoxon matched-pairs signed rank test for human lung explant assays ( * p ⁇ 0.05). The figure confirms that TFMT retains inhibitory activity in the IAV-infected mouse cell line LA-4, despite being less potent (IC50: 7.7 ⁇ M) than in human cells.
• 28(a) is a graph showing changes in body weight when mice were inoculated intranasally.
• C57BL/6 mice were intranasally administered 2 mg/kg of TFMT or tubercidin on days 0 and 1 (arrows in the figure) after infection.
• tubercidin is known to have a certain degree of toxicity, and treatment with tubercidin caused significant weight loss in mice, the results showed that the selected derivative TFMT did not cause weight loss or cytotoxicity.
• FIG. 28(b) is a schematic diagram of an IAV infection model in a mouse in vivo system. IAV WSN and TFMT were intranasally administered as shown in the figure. TFMT was administered on days 0 and 1 of IAV WSN infection.
• FIG. 28( c ) is a graph showing the results of qRT-PCR analysis of segment 5 of IAV RNA in lung tissue.
• FIG. 28( d ) is a graph showing the results of qRT-PCR analysis of segment 1 of IAV RNA in lung tissue.
• qRT-PCR analysis of IAV RNA 5x103 PFU was performed in lung tissues of IAV-infected C57BL/6 mice on day 2.
• TFMT was administered intranasally at 2 mg/kg on days 0 and 1 of infection.
• Statistics were calculated using two-tailed Student's t-test for NHBE cell assays, Mann-Whitney test for mouse in vivo assays, and Wilcoxon matched-pairs signed-rank test for human lung ex vivo assays. ( * p ⁇ 0.05, ** p ⁇ 0.01).
• Two days postinfection with the indicated TFMT treatments significantly reduced IAV NP and PB2 mRNA levels in mouse lungs, indicating that trifluoromethyl substitution of tubercidin eliminates in vivo toxicity but retains anti-IAV efficacy.
• TFMT treatment demonstrated the potential to inhibit IAV replication in all systems tested, including human cell lines and NHBE cells in vitro, human lung explants ex vivo, and mice in vivo.
• FIG. 29 is a graph showing the results of qRT-PCR analysis of IAV RNA in A549 cells infected with IAV PR8 strain at m.o.i.1 for 24 hours and analyzed for IAV RNA (segment 7).
• the effect of TFMT treatment on IAV replication was independent of IFIT1-dependent sequestration of RNA or RIG-I/MDA5 signaling.
• FIG. 30(a) is a photograph showing immunostaining of viral proteins in A549 cells infected with SeV or IAV. Immunostaining of viral proteins was performed for A549 cells infected with SeV (1 ⁇ 10 ⁇ 5 HAU/cell) or IAV PR8 (moi, 10) for 20 hours in the indicated genotypes.
• 30(b) is a photograph showing the results of Western blot analysis of viral proteins in A549 cells infected with SeV or IAV. A549 cells infected with IAV PR8 strain at m.o.i.1 for 26 hours were subjected to detection of the indicated proteins in the indicated genotypes.
• 31 is a graph showing the results of qRT-PCR analysis of IFN- ⁇ RNA in A549 cells.
• IFN- ⁇ RNA was analyzed in A549 cells infected with IAV PR8 strain at the indicated time points in the figure, in the presence or absence of 10 ⁇ M TFMT, with IAV: m.o.i. 1.
• 32 is a graph showing the results of qRT-PCR analysis of each of the other RNAs in A549 cells.
• Each of the RNAs shown in the figure was analyzed in A549 cells infected or not with IAV PR8 strain at the indicated time points at m.o.i.1 in the presence or absence of 10 ⁇ M TFMT.
• Treatment with TFMT did not induce IFN- ⁇ or antiviral ISGs, further emphasizing that the observed antiviral effect was not dependent on innate immune activation.
• FIG. 33 is a graph showing the results of qRT-PCR analysis of each RNA in A549 cells infected with other RNA viruses.
• SeV, VSV, or EMCV RNA was analyzed in A549 cells 24 hours after infection with SeV (1 ⁇ 10 ⁇ 6 HAU/cell), VSV-GFP (moi, 1), or EMCV (moi, 0.01). Consistent with the above results, the replication levels of IFN-sensitive non-cap-snatching RNA viruses, such as Sendai virus (SeV), VSV, and EMCV, were not altered by treatment with TFMT.
• SeV Sendai virus
• VSV Sendai virus
• EMCV Sendai virus
• 34(a) is a graph showing the results of qRT-PCR analysis of each viral RNA in MTr1 KO A549 cells infected with each virus. Each viral RNA in the figure was analyzed in WT and MTr1 KO A549 cells infected with SeV (1 ⁇ 10 ⁇ 6 HAU/cell), VSV (moi, 1), or EMCV (moi, 0.1) for 26 hours. In (a) to (c), TFMT was added at 10 ⁇ M 3 hours before infection.
• 34(b) is a photograph showing the results of Western blot analysis of each protein in MTr1 KO A549 cells infected with each virus.
• 34(c) is a photograph showing the results of immunostaining of viral proteins in MTr1 KO A549 cells infected with SeV. Viral proteins were analyzed in WT and MTr1 KO A549 cells infected with SeV (1 ⁇ 10 ⁇ 6 HAU/cell) for 12 hours.
• 35(a) is a graph showing the results of qRT-PCR analysis of viral and host RNA in MTr1 KO A549 cells infected with SeV.
• Viral and host RNA were analyzed in WT and MTr1 KO A549 cells infected with SeV (1 ⁇ 10 ⁇ 6 HAU/cell) at the time points indicated in the figure.
• 35(b) is a photograph showing optical microscope images of MTr1 KO HEK-293T cells infected with each virus.
• WT and MTr1 KO HEK-293 cells infected with IAV PR8 (moi, 1), IBV (moi, 1), VSV (moi, 10), SeV (1 ⁇ 10 ⁇ 6 HAU/cell), and EMCV (moi, 0.001) were observed 2 days after infection.
• the bar in the figure is 50 ⁇ m. Examination of infection of these viruses in MTr1 KO cells confirmed no differences in viral replication levels.
• FIG. 36(a) is a graph showing qRT-PCR analysis of NT/U2-IAV hybrid RNA in A549 cells infected with IAV PR8.
• NT/U2-IAV hybrid RNA was analyzed in A549 cells infected with IAV PR8 for 24 hours at m.o.i.1.
• the expression levels of IAV mRNA (segment 1) specifically snatched from U2 spliceosomal snRNA were reduced by TFMT treatment, similar to MTr1 deficiency.
• FIG. 36(b) is a schematic diagram of the N1-2'-O-Me interaction site.
• a structural model (PDB ID: 6RR7) of N1-2'-O-Me in RNA bound to IAV PB2 is shown.
• the circled area indicated by the arrow indicates the methyl group at the N1-2'-O position.
• FIG. 36(c) shows the structures of N1 and N2 of cap1 RNA, similarly showing N1-2′-O-Me.
• Figure 37(a) is a schematic diagram of the structure of IAV polymerase complexed with RNA.
• IAV polymerase (PDB ID: 6RR7)
• Figure 37(b) is a schematic diagram showing an enlarged view of the RNA-binding domain of PB2, with the target sites of MTr1, MTr2, or CAPAM indicated.
• FIG. 37(c) is a schematic diagram showing a phylogenetic tree of PB2 in influenza viruses and THOV and partial sequence alignment of amino acid residues (RNA cap binding region and conserved region). The above figure shows a computational model of the hypothetical addition of N1-2'-O-Me based on the reported structure of IAV polymerase complex with cap0 RNA (PDB ID: 6RR7).
• N1-2'-O-Me is located in close proximity (within 3 ⁇ ) to two amino acids, Q257 and I261, in the ⁇ -helical structure of PB2. Therefore, methylation is presumed to increase the affinity between the helix of PB2 and the host mRNA through the hydrophobic effect and van der Waals interactions between the hydrophobic amino acids I260 and I261.
• N1-2'-O-methyl groups on capped RNA in MTr1 KO cells appears to prevent proper interaction with the PB2 subunit of IAV polymerase.
• two other well-known methylations occur in the mature cap structure: N2-2'-O-Me and N1-m6A. These methylation reactions are catalyzed by the methyltransferases MTr2 and Cap-specific adenosine methyltransferase (CAPAM, PCIF1), respectively.
• CAPAM Cap-specific adenosine methyltransferase
• FIG. 38( a ) is a schematic diagram showing a structural model of N1-m6A in RNA bound to IAV PB2.
• FIG. 38(b) is a schematic diagram showing a structural model of N2-2′-O-Me in RNA bound to IAV PB2.
• the possible steric clash between N1-m6A and N2-2′-O-methylation was simulated, where the circled site indicated by the arrow indicates the additional methyl group.
• no significant interaction between PB2 and N2-2'-O-Me or N1-m6A could be observed.
• FIG. 39 is a graph showing qRT-PCR analysis of IAV RNA in A549 cells infected with IAV PR8.
• (a) CAPAM KO and (b) Mtr2 KO cells are shown.
• A549 cells infected with IAV PR8 at m.o.i.1 for 24 hours were analyzed for IAV RNA (segment 7) in the indicated genotypes. Consistent with the above data, no IAV replication defects could be observed in CAPAM or MTr2 KO cells.
• FIG. 40 is a graph showing the results of qRT-PCR analysis of ribosomal RNA (18S) and mRNA (GAPDH) in input and immunoprecipitated RNA from HEK-293T cells.
• RNA was 10 ng each.
• FIG. 40( c ) is a photograph showing the results of Western blot analysis of PABP-immunoprecipitated PABP protein.
• RT-PCR we were able to confirm that mRNA was enriched in the PABP immunoprecipitated fraction.
• FIG. 41( a ) is a photographic diagram showing the results of Western blot analysis of input and immunoprecipitated proteins in PB2-expressing WT cells and MTr1 KO HEK-293T cells.
• MTr1-deficient cells the amount of PB2 coprecipitated with PABP immunoprecipitation was significantly reduced, indicating that the presence of MTr1-dependent N1-2'-O-Me greatly increases the affinity of PB2 for the cap, a result consistent with the results of structural modeling of mRNA in cells.
• Baloxavir marboxil (BXM) is an approved anti-IAV drug that targets the active site of the polymerase PA subunit. 18, 19 Because TFMT affects the cap-binding activity of different subunits of the IAV polymerase PB2, we investigated its potential synergistic effects on anti-IAV activity.
• Figure 41(b) is a graph showing the synergistic effect of BXM and TFMT. Relative fluorescence units of GFP are shown in A549 cells infected with IAV SC35M-GFP at 0.1 infectious particles/cell for 19 hours. TFMT and/or BXM were added at the indicated concentrations 3 hours prior to infection.
• FIG. 41(c) shows the combination index calculated as a measure of synergy evaluation.
• FIG. 41(c) shows the combination index calculated as a measure of synergy evaluation.
• FIG. 42(a) is a graph showing the WST-8 assay for FIG. Interestingly, a strong synergistic effect was observed with combined treatment of TFMT and BXM without cytotoxicity. Furthermore, combinations of TFMT with the neuraminidase inhibitor oseltamivir and the m2 inhibitor amantadine also showed synergistic effects.
• Figure 42(b) is a graph showing the synergistic effect of oseltamivir and TFMT. qRT-PCR analysis of IAV RNA (segment 7) in A549 cells infected with IAV PR8 strain at m.o.i. of 0.01 for 55 h is shown.
• FIG. 42(c) shows the results of calculating the combination index as a measure of the synergy evaluation in the previous figure.
• Figure 42(d) is a graph showing the synergistic effect of amantadine and TFMT. Relative fluorescence units of GFP are shown in A549 cells infected with IAV SC35M-GFP at 0.1 infectious particles/cell for 19 hours. TFMT and/or amantadine were added at the indicated concentrations 3 hours prior to infection.
• FIG. 42(e) shows the results of calculating the combination index as a measure of the synergy evaluation in the previous figure. These data suggest the potential use of TFMT in combination therapy.
• FIG. 43 is a graph showing luciferase reporter assay of IAV PR8 minireplicons in MTr1 KO HEK-293T overexpressing each PA subunit. (a) shows the results for WT (wild type) and (b) shows the results for MTr1 KO cells. Results are shown for WT and MTr1 KO HEK-293T cells overexpressing IAV PB2, PB1, NP, and the indicated PA. 0.1 ⁇ M BXM was added 1 hour before transfection.
• Figure 43(c) is a graph showing virus yields in the supernatants of MTr1 KO A549 cells infected with BXM-resistant virus IAV HK483 PA I38T.
• IAV HK483 PA I38T was infected for 48 hours at an m.o.i of 0.1, and the yields in the supernatants of WT and MTr1 KO A549 cells are shown.
• Statistics were calculated using two-tailed Student's t-test ( ** p ⁇ 0.01, *** p ⁇ 0.001). IAV minireplicon assays and infection with BXM-resistant H5N1 viruses revealed that MTr1 deficiency also inhibited replication of BXM-resistant viruses.
• FIG. 44 is a graph showing luciferase reporter assays of IAV PR8 minireplicons in MTr1 KO HEK-293T cells overexpressing the PA subunits shown on the horizontal axis of each figure (a) to (d), using cells overexpressing IAV PB1, PA, NP, and PB2 as indicated in the figure. It is noteworthy to mention that all tested mutants with changes in two of the interacting amino acids I260 or I261 did not support IAV replication in MTr1 KO cells.
• the modeling approach of the present embodiment suggests that the conserved amino acids are important for IAV replication in WT cells, suggesting that the IAV PB2 subunit is unlikely to adapt and overcome the lack of the N1-2'-O-Me capped RNA structure for efficient replication.
• TFMT induces MTr1 deficiency and accumulation of cap0 RNA, which impairs PB2 binding to host capped RNA and reduces the effectiveness of IAV polymerase to initiate cap snatching and RNA synthesis.
• TFMT showed strong synergy with BXM, as both drugs target distinct polymerase subunits, PB2 and PA, respectively.
• this TFMT-dependent restriction of IAV is independent of innate immune responses and restriction by RIG-I and IFIT1, and does not affect the replication of interferon-sensitive viruses such as VSV and EMCV.
• comparison of PB2 between influenza viruses and THOV revealed that the primary structure of the cap RNA-binding region (N1-2'-O-Me interacting amino acids) of IAV PB2 is well conserved in IBV PB2 but not in ICV, IDV, or THOV PB2.
• IAV requires 10-13 nucleotides for cap snatching, whereas THOV reportedly snatches the m7G cap residue at the 5' end. This different approach of viral polymerases to cap snatching may explain the specificity of TFMT restriction for IAV and IBV.
• This compound is not only an inhibitor with a new mechanism of action, but also belongs to a new class of antiviral drugs with specific efficacy against influenza A and B.
• Currently approved drugs are available for IAV treatment against viral proteins, although mutant viruses resistant to them have been reported for each of the existing drugs.
• host-directed antiviral drugs are less likely to induce drug resistance, as demonstrated by the MEK inhibitor ATR-002, which has broad efficacy against RNA viruses such as influenza virus and SARS-CoV-2 by directly affecting viral replication and modulating inflammation.
• the efficacy of the host-directed MTr1 inhibitor TFMT may provide a new therapeutic approach against influenza A and B viruses.
• the present invention provides a new anti-influenza virus drug that inhibits the proliferation and infection mechanisms of influenza viruses, has a target that is not a specific viral protein, and does not result in the emergence of drug-resistant viruses, as well as medicines, foods and beverages, supplements, agricultural chemicals, feed, and cosmetics that use the same.

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