Anti-influenza virus composition, medicine, food, beverage, supplement, agricultural chemical, feed and cosmetic

Abstract

Provided is a novel anti-influenza virus agent which inhibits the growth and infection mechanisms of influenza virus and thus has a target other than specific viral proteins and which does not cause the emergence of drug-resistant viruses. Also provided are a medicine, food, beverage, supplement, agricultural chemical, feed and cosmetic using the same. An anti-influenza virus composition containing, as an active ingredient, tubercidin, a tubercidin derivative or a salt thereof, and a medicine, food, beverage, supplement, agricultural chemical, feed and cosmetic using the same.

Description

Anti-influenza virus compositions, medicines, foods and beverages, supplements, agricultural chemicals, feeds, and cosmetics

[0001] The present invention relates to compositions that can be used as anti-influenza virus drugs, etc., and to pharmaceuticals, supplements, agricultural chemicals, feed, and cosmetics using the compositions. This application claims priority to U.S. Patent Application No. 63 / 412,884, provisionally filed in the U.S. on October 4, 2022, the contents of which are incorporated herein by reference.

[0002] Influenza viruses periodically recur 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 devastating damage to society through global pandemics. There have been numerous reported cases in which viruses circulating in poultry have been directly transmitted to humans, resulting in fatalities.

[0003] Several drugs have been approved as anti-influenza virus drugs for the treatment of influenza, etc. All of these drugs act directly on specific viral proteins. For example, a representative anti-influenza virus drug that has been developed is a pharmaceutical composition containing oseltamivir phosphate as an active ingredient, which has inhibitory activity against neuraminidase possessed by influenza viruses.

[0004] For example, Patent Document 1 discloses a pharmaceutical composition containing oseltamivir phosphate and 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%, wherein 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, and the like in the storage environment, and in particular, suppression of discoloration during storage.

[0005] International Publication WO2007 / 097325

[0006] However, drugs with active ingredients that act directly on specific viral proteins may result in the emergence of drug-resistant viruses due to mutations in the viral genes. Some influenza virus strains have been found to be resistant to antiviral drugs that target neuraminidase. Therefore, there is a strong need for the development of new antiviral drugs that target other than specific viral proteins, or that have mechanisms other than directly acting on specific viral proteins, that can inhibit viral growth, and that do not result in the emergence of drug-resistant viruses.

[0007] The present invention has been made in light of the above background, and its object 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.

[0008] The present invention includes the following aspects: [1] An anti-influenza virus composition containing tubercidin, a tubercidin derivative, or a salt thereof as an active ingredient. [2] The composition according to [1], wherein the tubercidin derivative is a compound represented by the following general formula (1): ... (1) (In the above general formula (1), R １ , R ２ and R ３ are the same or different and represent 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.) [3] R １ and R ２are the same or different and each represents 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, R ３ represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted amino group, or an optionally substituted aryl group. [4] The composition according to [2]. ３ [5] The composition according to [2] or [3], wherein R represents a hydrogen atom. １ and R ２ [6] The composition according to any one of [2] to [4], wherein R are the same or different and represent a halogen atom. １ and R ２ [7] The composition according to any one of [2] to [4], wherein R are the same or different and each represent an amino group or a cyano group which may have a substituent. １ [8] The composition according to any one of [2] to [4], wherein R represents an alkyl group substituted with a halogen. １ [9] The composition according to any one of [2] to [4], wherein the tubercidin derivative is a compound represented by the following formula (2): ... (2)

[10] 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].

[0009] The present invention provides a new anti-influenza virus drug that targets a specific non-viral protein and prevents the emergence of drug-resistant viruses by inhibiting the proliferation and infection mechanisms of influenza viruses, as well as medicines, foods and beverages, supplements, agricultural chemicals, feeds, and cosmetics that use the same.

[0010] 1 is a schematic diagram outlining the search for MTr1 inhibitory compounds. This is a photograph of a Western blot showing the generation of MTr1 knockout (KO) A549 cells by CRISPR / Cas9. This is a graph showing the accumulation of IAV and IBV viruses in wild-type and MTr1 KO A549 cells. (a) This is a graph showing qRT-PCR analysis of IAV RNA in WT and MTr1 KO A549 cells infected with IAV PR8. (b) This is a graph showing qRT-PCR analysis of individual IAV RNA segments in WT and MTr1 KO A549 cells infected with IAV PR8 for 24 hours. (c) This is a photograph showing immunostaining of viral proteins in WT and MTr1 KO A549 cells infected with IAV PR8 for 19 hours. (d) qRT-PCR analysis of IBV RNA in WT and MTr1 KO A549 cells infected with IBV for 24 hours. (e) Photographs showing Western blot analysis of the indicated proteins in WT or MTr1 KO A549 cells infected with IBV for 24 hours. (f) Photographs showing Western blot analysis of each protein in WT and MTr1 KO A549 cells infected with IAV PR8 strain for 24 hours. (a) Photographs showing each immunostaining in WT and MTr1 KO A549 cells infected with IAV PR8 strain for 24 hours. (b) Graph showing the results of flow cytometry in WT and MTr1 KO A549 cells infected with IAV PR8 strain for 24 hours. Figure 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 for 24 hours. Figure 2 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 each m.o.i. and each time. Figure 3 is a graph showing qRT-PCR analysis of NT / U2-IAV hybrid RNA in WT and MTr1 KO A549 cells infected with IAV PR8 at each m.o.i. for 24 hours. (a) Schematic showing primer sequences for cap-snatching-specific qPCR.(b) qRT-PCR analysis of IAV RNA, U2, and U2-IAV RNA hybrid RNA in A549 cells infected with IAV PR8 for 24 hours. (c) qRT-PCR analysis of U2 RNA from the same samples as in Figure 8. qRT-PCR analysis and viral yield of WT and MTr1 KO cells infected with IAV. Photographs showing Western blot results of WT and MTr1 KO cells infected with IAV. (a) Results for WT and MTr1 KO A549 cells infected with IAV WSN, and (b) Results for WT and MTr1 KO A549 cells infected with SC35M-GFP. qRT-PCR analysis and viral yield of WT and MTr1 KO cells infected with IDV and THOV. (a) Virus yields in the supernatants of WT and MTr1 KO A549 cells infected with IDV. (b) qRT-PCR analysis of THOV RNA in WT and MTr1 KO A549 cells infected with THOV. Graphs showing virus yields and RNA amounts in WT and MTr1 KO A549 cells infected with various viruses. (a) Virus yields in the supernatants from WT and MTr1 KO A549 cells infected with RVFV. (b) qRT-PCR analysis of the indicated viral RNAs in WT and MTr1 KO A549 cells infected with HAZV, ISKV, STBV, or DUGV. (a) Schematic diagram showing the phylogenetic tree of the S segment of bunyaviruses. (b) Photographs showing the results of Western blot analysis of the indicated proteins in HAZV-infected WT or MTr1 KO A549 cells. (c) Graphical representation showing the results of qRT-PCR analysis of the indicated viral RNAs in A549 cells. Schematic outlining the dependency of MTr1 on cap-snatching virus replication. Schematic showing a general flow chart of the primary screen. (a) Schematic showing the 2D interaction of SAM with human MTr1 (PDB ID: 4N49). (b) Schematic representation of the screening strategy to obtain candidate MTr1 inhibitors.(c) Schematic diagram showing the docking scores and virtual screening rankings of representative compounds. (a) Graph showing the results of quantification of GFP levels in A549 cells infected with IAV-GFP SC35M strain for 19 hours. (b) Graph showing the results of another quantification of GFP levels in A549 cells infected with IAV-GFP SC35M strain for 19 hours. (c) Graph showing the results of quantification of GFP levels and cytotoxicity test (WST-8) in A549 cells infected with IAV-GFP for 19 hours. (d) Graph showing the results of qRT-PCR analysis of IAV RNA in A549 cells infected with IAV PR8 strain for 24 hours. (e) Graph showing the results of thermal shift assay of recombinant MTr1 using SYPRO Orange. (a) Another graph showing the results of a thermal shift assay of recombinant MTr1 using SYPRO Orange. (b) A graph showing the results of an MTase assay using human MTr1 WT or MTr1 K239A with SAM, SAH, or sinefungin. (c) A graph showing the results of an MTase assay using human MTr1 with the indicated concentrations of tubercidin. (a) A schematic flow chart of the three-step secondary screening process. (b) A schematic diagram showing the docking state of TFMT in the SAM-binding pocket of human MTr1. (c) A graph showing the results of a thermal shift assay of MTr1 with 1 mM TFMT or ribavirin using SYPRO Orange. (a) A graph showing the results of an MTase assay of MTr1 with TFMT or ribavirin. (b) Graph showing the results of qRT-PCR analysis of each viral RNA, (c) Graph showing the results of quantification of GFP levels in A549 cells infected with IAV-GFP, and (d) Graph showing the results of another quantification of GFP levels in A549 cells infected with IAV-GFP.(a) Graph showing the results of qRT-PCR analysis of IAV RNA (segment 7) in normal human bronchial epithelial (NHBE) cells infected with the IAV PR8 strain at an m.o.i. of 1 for 24 hours. (b) Photograph showing the results of Western blot analysis of the indicated proteins in NHBE cells infected with the IAV PR8 strain at an m.o.i. of 1 for 24 hours. (c) Photograph showing the results of immunostaining of NHBE cells infected with the IAV PR8 strain. (d) Graph showing the results of qRT-PCR analysis of HAZV RNA in HAZV-infected NHBE cells. (e) Schematic diagram of IAV infection in human lung explants. (f) Graph showing the results of viral growth and viral titration of seasonal IAV in human lung tissue from a single donor. (g) Graph showing the results of viral growth in human lung tissue and viral titration of seasonal IAV from six independent donors. Graph showing viral growth of seasonal IAV in human lung tissue. Photographs showing viral growth in human lung tissue and immunohistochemistry of seasonal IAV proteins. Graph showing the GFP level and cytotoxicity test results in LA-4 cells infected with IAV-GFP. (a) Graph showing weight change upon intranasal inoculation of mice. (b) Schematic diagram of an IAV infection model in a mouse in vivo system. (c) Graph showing the results of qRT-PCR analysis of IAV RNA from segment 5 in lung tissue. (d) Graph showing the results of qRT-PCR analysis of IAV RNA from segment 1 in lung tissue. Graph showing the results of qRT-PCR analysis of IAV RNA in A549 cells infected with the IAV PR8 strain. (a) is a photograph showing immunostaining of viral proteins in A549 cells infected with SeV or IAV. (b) is a photograph showing the results of Western blot analysis of viral proteins in A549 cells infected with SeV or IAV. (c) is a graph showing the results of qRT-PCR analysis of IFN-β RNA in A549 cells. (d) is a graph showing the results of qRT-PCR analysis of each other RNA in A549 cells. (e) 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. (c) Photograph showing the results of immunostaining of viral proteins in MTr1 KO A549 cells infected with SeV. (a) Graph showing the results of qRT-PCR analysis of viral and host RNA in MTr1 KO A549 cells infected with SeV. (b) Photograph showing optical microscope images of MTr1 KO HEK-293T cells infected with each virus. (a) Graph showing qRT-PCR analysis of NT / U2-IAV hybrid RNA in A549 cells infected with IAV PR8. (b) Schematic diagram of the N1-2'-O-Me interaction site. (c) Structures of N1 and N2 of cap1 RNA, also showing N1-2'-O-Me. (a) Schematic diagram of the structure of IAV polymerase complexed with RNA. (b) Schematic diagram showing a close-up of the RNA-binding region of PB2. (c) Schematic diagram showing a phylogenetic tree of PB2 in influenza virus and THOV and partial sequence alignment of amino acid residues (RNA cap-binding region and conserved region). (a) Schematic diagram showing a structural model of N1-m6A in RNA bound to IAV PB2. (b) Schematic diagram showing a structural model of N2-2'-O-Me in RNA bound to IAV PB2. Graphs showing qRT-PCR analysis of IAV RNA from A549 cells infected with IAV PR8. (a) Analysis of CAPAM KO cells, (b) Analysis of Mtr2 KO cells. Graphs 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) Photographs showing the results of Western blot analysis of PABP-immunoprecipitated PABP protein.(a) Photographs showing the results of Western blot analysis of input and immunoprecipitated proteins in PB2-expressing WT cells and MTr1 KO HEK-293T cells. (b) Graph showing the synergistic effect of BXM and TFMT. (a) Graph showing WST-8 assay for FIG. 41. (b) Graph showing the synergistic effect of oseltamivir and TFMT. (c) Results of calculation of combination index as a measure of synergy evaluation in (b). (d) Graph showing the synergistic effect of amantadine and TFMT. (e) Results of calculation of combination index as a measure of synergy evaluation in (d). Graphs showing luciferase reporter assay of IAV PR8 minireplicon in MTr1 KO HEK-293T overexpressing each PA subunit. (a) Results for WT (wild strain) and (b) results for MTr1 KO cells. (c) A graph showing the virus yield in the supernatant of MTr1 KO A549 cells infected with IAV HK483 PA I38T, a BXM-resistant virus. (d) A graph showing the luciferase reporter assay of IAV PR8 minireplicon in MTr1 KO HEK-293T overexpressing the PA subunit shown on the horizontal axis of each figure. (e) A graph showing the CRISPR target sequence and mutated region in the knockout cells used.

[0011] Hereinafter, the anti-influenza virus composition according to the present invention and the medicines, foods and beverages, supplements, agricultural chemicals, feeds, and cosmetics using the same will be described with reference to embodiments, although the present invention is not limited to the following embodiments.

[0012] (Anti-influenza virus composition) The anti-influenza virus composition of this 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 pharmaceutically usable.

[0013] Specifically, R in formula (1) described below １ , R ２ , R ３are hydrogen atoms is known as tubercidin (CAS registration number: 69-33-0). A tubercidin derivative is a compound in which any site of tubercidin, preferably R １ , R ２ , R ３ or a substituent 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 broadly refers to a pharmaceutically acceptable salt of the compound. As used herein, pharmaceutically acceptable salts include, for example, acid addition salts or base addition salts of the compound represented by formula (1). Examples of acid addition salts include acid addition salts with inorganic or organic acids (hydrochloric acid, hydrobromic acid, sulfuric acid, trifluoroacetic acid, citric acid, maleic acid, etc.). Examples of base addition salts include metal salts, for example, 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.

[0014] Through the following intensive research, the present inventors have discovered tubercidin, a tubercidin derivative, or a salt thereof as an active ingredient for an anti-influenza virus composition. It has been reported that MTr1, an RNA methyltransferase in host cells, is involved in influenza virus cap snatching, but the details of this process remain unknown. The present inventors first generated MTr1 knockout cells and examined the replication ability of various cap-snatching viruses, including influenza viruses. The results revealed that MTr1 is essential for influenza A and B virus replication. Furthermore, the mechanism behind this was determined to be that influenza viruses can only snatch (steal) cap structures modified by MTr1. On the other hand, MTr1 was not involved in the replication of cap-snatching viruses other than influenza A and B viruses. It was therefore believed that influenza virus replication could be selectively inhibited by inhibiting the function of host MTr1.

[0015] Therefore, 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 inhibitory compounds. As a result, they discovered the natural compound tubercidin as a compound that inhibits the enzymatic activity of MTr1 and exhibits antiviral activity against various influenza viruses. An overview of this search for MTr1 inhibitory compounds is shown in Figure 1.

[0016] The tubercidin derivative is preferably a compound represented by the following general formula (1): ... (1) (In the above general formula (1), R １ , R ２ and R ３ are the same or different and each represent 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.

[0017] R １ and R ２ are 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; R ３ may represent a hydrogen atom, an optionally substituted alkyl group, an optionally substituted amino group, or an optionally substituted aryl group.

[0018] Also, R ３ may represent a hydrogen atom. １ and R ２ may be the same or different and may represent a halogen atom. １ and R ２may be the same or different and may represent an amino group or a cyano group which may have a substituent. １ may represent an alkyl group substituted with a halogen. １ may represent a trifluoromethyl group.

[0019] The R １ , R ２ and R ３ When is a substituent containing carbon, the number of carbon atoms can be selected arbitrarily within the 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 linear or branched chain of carbon atoms in an alkyl group, alkenyl group, alkynyl group, or alkoxy group may be either linear or branched. Furthermore, 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 to 2 rings.

[0020] Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, 1-methylpentyl, n-hexyl, isohexyl, sec-hexyl, tert-hexyl, and neohexyl. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, propenyl, isopropenyl, 2-methyl-1-propenyl, 2-methylallyl, and 2-butenyl. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy groups.

[0021] Examples of amino groups include, but are not limited to, amino, ethylamino, dimethylamino, butylamino, cyclopentylamino, 2-ethylhexylamino, dodecylamino, anilino, naphthylamino, and 2-pyridylamino groups. Examples of amido groups include, but are not limited to, methylcarbonylamino, ethylcarbonylamino, dimethylcarbonylamino, propylcarbonylamino, pentylcarbonylamino, cyclohexylcarbonylamino, 2-ethylhexylcarbonylamino, octylcarbonylamino, dodecylcarbonylamino, phenylcarbonylamino, and naphthylcarbonylamino groups.

[0022] Examples of cycloalkyl groups include 4- to 7-membered cycloalkyl groups. Specific examples include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl groups. Examples of aryl groups (aromatic hydrocarbon groups) include, but are not limited to, phenyl, naphthyl, anthryl, azulenyl, acenaphthenyl, fluorenyl, phenanthryl, indenyl, pyrenyl, and biphenylyl groups. Examples of the aliphatic heterocyclic group 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 ε-caprolactone ring, an ε-caprolactam ring, a piperidine ring, a hexahydropyridazine ring, a hexahydropyrimidine ring, a piperazine ring, a morpholine ring, a tetrahydropyran ring, a 1,3-dioxane ring, a 1,4-dioxane ring, a trioxane ring, a tetrahydrothiopyran ring, a thiomorpholine ring, a thiomorpholine-1,1-dioxide ring, a pyranose ring, or a diazabicyclo[2,2,2]-octane ring. Examples of 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. Examples of halogen atoms include fluorine, chlorine, bromine, and iodine atoms, and preferably fluorine and chlorine atoms.

[0023] The R １ , R ２ and R ３When R further has a substituent, the substituent can be arbitrarily selected from the above-mentioned organic substituents, halogen elements, metal elements, etc. １ , R ２ and R ３ When has a substitution site with a halogen element or a substitution site with other substituents, the substitution site can also be selected arbitrarily within the range that does not impair physiological functions.

[0024] Specific examples of the substituent 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 group, cyclohexyloxy group, etc.), aryloxy groups (e.g., phenoxy group, naphthyloxy group, etc.), alkylthio groups (e.g., methylthio group, ethylthio group, propylthio group, pentylthio group, hexylthio group, octylthio group, dodecylthio group, etc.), cycloalkylthio groups (e.g., cyclopentylthio group, thio group, cyclohexylthio group, etc.), arylthio group (for example, phenylthio group, naphthylthio group, etc.), alkoxycarbonyl group (for example, methyloxycarbonyl group, ethyloxycarbonyl group, butyloxycarbonyl group, octyloxycarbonyl group, dodecyloxycarbonyl group, etc.), aryloxycarbonyl group (for example, phenyloxycarbonyl group, naphthyloxycarbonyl group, etc.), sulfamoyl group (for example, aminosulfonyl group, methylaminosulfonyl group, dimethylaminosulfonyl group, butylaminosulfonyl group, hexylaminosulfonyl group, sulfonyl group, cyclohexylaminosulfonyl group, octylaminosulfonyl group, dodecylaminosulfonyl group, phenylaminosulfonyl group, naphthylaminosulfonyl group, 2-pyridylaminosulfonyl group, etc.), acyl group (for example, acetyl group, ethylcarbonyl group, propylcarbonyl group, pentylcarbonyl group, cyclohexylcarbonyl group, octylcarbonyl group, 2-ethylhexylcarbonyl group, dodecylcarbonyl group, phenylcarbonyl group, naphthylcarbonyl group, pyridylcarbonyl group, etc.), acyloxy group (for example, acetyl oxy group, ethylcarbonyloxy group, butylcarbonyloxy group, octylcarbonyloxy group, dodecylcarbonyloxy group, phenylcarbonyloxy group, etc.), amido group, carbamoyl group (for example, aminocarbonyl group, methylaminocarbonyl group, dimethylaminocarbonyl group, propylaminocarbonyl group, pentylaminocarbonyl group, cyclohexylaminocarbonyl group, octylaminocarbonyl group, 2-ethylhexylaminocarbonyl group, dodecylaminocarbonyl group, phenylaminocarbonyl group, naphthylaminocarbonyl group,2-pyridylaminocarbonyl group, etc.), ureido groups (for example, methylureido group, ethylureido group, pentylureido group, cyclohexylureido group, octylureido group, dodecylureido group, phenylureido group, naphthylureido group, 2-pyridylaminoureido group, etc.), sulfinyl groups (for example, methylsulfinyl group, ethylsulfinyl group, butylsulfinyl group, cyclohexylsulfinyl group, 2-ethylhexylsulfinyl group, dodecylsulfinyl group, phenylsulfinyl group, naphthylsulfinyl group, 2-pyridylsulfinyl group, etc.), alkylsulfonyl groups (for example, methylsulfonyl group, ethylsulfonyl group, butylsulfonyl group, Examples of the substituent include, but are not limited to, an arylsulfonyl group, a cyclohexylsulfonyl group, a 2-ethylhexylsulfonyl group, a dodecylsulfonyl group, etc.), an arylsulfonyl group or a heteroarylsulfonyl group (e.g., a phenylsulfonyl group, a naphthylsulfonyl group, a 2-pyridylsulfonyl group, etc.), an amino group, a halogen atom, a fluorinated hydrocarbon group (e.g., a fluoromethyl group, a trifluoromethyl group, a pentafluoroethyl group, a pentafluorophenyl group, etc.), a cyano group, a nitro group, a hydroxy group, a mercapto group, a silyl group (e.g., a trimethylsilyl group, a triisopropylsilyl group, a triphenylsilyl group, a phenyldiethylsilyl group, etc.), or a phosphono group. Furthermore, any element of these substituents may be substituted with another element or any of the above-mentioned substituents. Any of the above can be arbitrarily selected from a range that does not inhibit physiological functions and is pharmaceutically acceptable for administration to an organism.

[0025] That is, more specifically, the tubercidin derivative may be a compound represented by the following formula (2): ... (2)

[0026] The compound of 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. The present inventors investigated 115 types of tubercidin derivatives and found TFMT as the most effective component.

[0027] Regarding the mechanism of TFMT, the inventors have predicted from three-dimensional structural analysis that TFMT binds to the substrate-binding pocket of MTr1 and inhibits its enzymatic activity. Furthermore, structural analysis predicts that the unmodified cap structure of MTr1 may impair its interaction with influenza virus RNA polymerase (PB2 protein). That is, TFMT suppresses the cap structure modification function 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 RNA polymerase-mediated cap snatching and subsequent viral RNA synthesis.

[0028] [Medicines, foods and beverages, supplements, agricultural chemicals, feeds, and cosmetics] (Medicines) The anti-influenza virus composition of this embodiment is suitable for use in treating influenza. That is, the anti-influenza virus composition can also be referred to as a medicine or pharmaceutical composition used in treating influenza. The anti-influenza virus composition can also be a medicine containing other ingredients. The medicine for treating influenza may also contain, as appropriate, various ingredients contained in conventionally known medicines or pharmaceutical compositions.

[0029] The form of the pharmaceutical of this embodiment is not particularly limited, and may be, for example, a solution, a dispersion such as a sol or gel, or a powder. The pharmaceutical may be administered orally in the form of, for example, a tablet, capsule, or elixir, or parenterally in the form of an enema.

[0030] As the pharmaceutically acceptable carrier, those usually used in pharmaceutical formulations can be used without any particular limitation, more specifically, for example, binders, excipients, swelling agents, solvents, etc.

[0031] The pharmaceutical of this embodiment may contain additives, such as lubricants, sweeteners, flavoring agents, stabilizers, pH buffers, solubilizers, antioxidants, and preservatives.

[0032] The medicament of this embodiment can be formulated by appropriately combining the above-mentioned ingredients and mixing them in a unit dose form required for generally accepted pharmaceutical practice.

[0033] The dosage of a pharmaceutical varies depending on the type, method of use, and dosage of the pharmaceutical, as well as the symptoms, body weight, age, sex, etc. of the patient, and cannot be determined in general terms, but typically, when administered by injection or oral administration, the active ingredient in the anti-influenza virus composition contained in the pharmaceutical may be 0.1 ng / kg to 250 mg / kg (where kg is the body weight of the subject), 0.1 ng / kg to 100 mg / kg, 1 ng / kg to 100 mg / kg, or 1 ng / kg to 50 mg / kg per day. Furthermore, the active ingredient in a pharmaceutical formulation may be 1 to 10 wt %, 1 to 7 wt %, or 3 to 5 wt % relative to the total weight of the pharmaceutical.

[0034] (Foods, drinks, supplements) The foods, drinks, and supplements of this embodiment contain the anti-influenza virus agent of this embodiment. The target foods, drinks, and supplements are not particularly limited. For example, a gel-like food product obtained by adding a thickening polysaccharide, gelatin, or the like to the beverage of this embodiment and cooling the product can be used. Examples of foods include processed foods.

[0035] The beverage of this embodiment contains the anti-influenza virus agent of this embodiment. For example, an extract can be obtained by immersing the composition of this embodiment in water or another solvent, for example, an alcohol such as ethanol, or another organic solvent, and then heating the resulting mixture at a desired temperature, or by immersing the rolled mixture 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 conventionally known methods for drinking tea. Alternatively, a method can be used in which the liquid or solid composition of this embodiment is added to any beverage (e.g., soft drinks, alcoholic beverages).

[0036] (Pesticides, Feeds, and Cosmetics) The pesticides, feeds, and cosmetics of this embodiment contain the anti-influenza virus agent of this embodiment. As with the above-mentioned medicines, foods, etc., they can be appropriately selected according to the embodiment of conventionally known pesticides, feeds, and cosmetics.

[0037] [Effects of this embodiment] According to this embodiment, it is possible to obtain a new anti-influenza virus drug that has a target other than a specific viral protein and does not result in the emergence of drug-resistant viruses by inhibiting the proliferation and infection mechanisms of influenza viruses, as well as medicines, foods and beverages, supplements, agricultural chemicals, feeds, and cosmetics that use the same.

[0038] MTr1 has traditionally been known as a catalyst for nucleic acid methylation. The 5'-ends of mammalian mRNAs and small nuclear RNAs (snRNAs) are capped with 7-methylguanosine (m7G) and 2,2,7-trimethylguanosine, respectively, which are linked to the RNA by a triphosphate bridge, termed cap0. 2'-O-ribose methyltransferase MTr1 (CMTR1 / FTSJD2) is an RNA methyltransferase (MTase) primarily localized in the nucleus and catalyzes the 2'-O-methylation of the first nucleotide (N1-2'-O-Me). The cap structure of cap0 mRNAs and snRNAs, as well as host RNAs, is important for generating mature caps and promoting their stability and translation. Unmethylated RNAs activate RIG-I-like receptor-mediated innate immune responses and are vulnerable to cellular restriction factors such as IFIT1. Fully methylated cap1 RNA evades innate immune mechanisms, and viruses therefore commonly hijack or mimic the cap structure of cellular RNA.

[0039] 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 fully capped cellular RNA is cleaved and attached to viral mRNA. For example, IAV polymerase is composed of three subunits: PA, PB1, and PB2. The trimeric polymerase binds to the cap structure of host RNA via PB2, initiates RNA cleavage by the PA endonuclease 10–13 nucleotides downstream of the cap structure, and finally synthesizes viral mRNA using PB1 as a primer. In contrast, non-cap-snatching viruses, such as coronaviruses (including yellow fever virus), poxviruses, and flaviviruses, encode their own cap-binding 2'-O-MTases, which mediate 2'-O-methylation of RNA to mimic the cellular cap and avoid recognition by antiviral sensors. It has been shown that cellular MTr1 depletion leads to the accumulation of host cap0 RNA in the cytoplasm, activating RIG-I and IFIT1, indicating that the methylation status of endogenous capped mRNA at the first nucleotide is one of the key features that prevents immune activation.

[0040] MTr1 deficiency has been reported to enhance the antiviral IFN response against influenza A virus, affect its cap-snatching effect, and reduce influenza A virus replication levels. Therefore, MTr1 is an interesting host-directed target for anti-influenza A virus drugs and possibly for drugs against other cap-snatching viruses. However, no MTr1 inhibitors have been reported, and its potential as an antiviral agonist remains unexplored.

[0041] Orthomyxoviruses and bunyaviruses initiate transcription by robbing the 5' cap portion of host RNA. Here, we have found that cap modification by the host 2'-O-ribose methyltransferase MTr1 is essential for influenza A and B virus replication, but not for other cap-snatching viruses. In silico chemical screening and functional analysis revealed that trifluoromethyl-tubercidin (TFMT) inhibits MTr1 through its interaction with the S-adenosyl-L-methionine-binding pocket, blunting influenza virus replication. Mechanistically, TFMT impairs the binding of host capped RNA to the viral polymerase PB2 subunit, which is the first critical step in the cap-snatching process. Notably, TFMT demonstrated efficacy in both human lung explants and in vivo in mice, exhibiting synergistic antiviral activity with approved anti-influenza drugs, including baloxavir marboxil, oseltamivir, and amantadine. Our results reveal an unexpected activity of MTr1 in viral replication and a conceptually new approach to inhibiting influenza virus infection.

[0042] [Other Embodiments] 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 associated with influenza virus. Yet another aspect of this embodiment is use of a compound comprising tubercidin, a tubercidin derivative, or a salt thereof for the manufacture of a therapeutic agent for a disease associated with 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 comprising tubercidin, a tubercidin derivative, or a salt thereof. Yet another aspect of this embodiment is a method for producing a pharmaceutical composition for treating or preventing a disease associated with influenza virus, using a compound comprising tubercidin, a tubercidin derivative, or a salt thereof. Yet another aspect of this embodiment is a method for producing a composition for treating or preventing a disease associated with influenza virus, using a compound comprising tubercidin, a tubercidin derivative, or a salt thereof. Yet another aspect of this embodiment is a method for treating a disease associated with influenza virus, comprising administering to a subject in need thereof an effective amount of a compound comprising tubercidin, a tubercidin derivative, or a salt thereof. Yet another aspect of this embodiment is a compound comprising tubercidin, a tubercidin derivative, or a salt thereof for use in treating a disease associated with influenza virus.

[0043] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0044] [Experimental Conditions and Methods Used] (Cell Culture) 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 μg / ml streptomycin at 37°C and 5% CO. ２Expi293F suspension cells were cultured in Expi293 expression medium (Gibco, A1435101) at 37°C with shaking in 8% CO ２ Normal human bronchial epithelial (NHBE) cells (Lonza, CC-2540) were cultured in bronchial epithelial cell growth medium (BEGM; Lonza, CC-3170). Plat-A cells were cultured in a medium supplemented with 10 μg / ml blasticidin and 1 μg / ml puromycin. To obtain antisera from HAZV-infected B6 mice (#WT No. 2, 170807), C57BL / 6 mice were cultured in 10 μg / ml blasticidin and 1 μg / ml puromycin. ５ Plaque-forming units (PFU) of Hazara virus were inoculated intraperitoneally. The inoculation was repeated twice at an interval of 3 weeks. Three days after the last inoculation, blood was collected in BD Microtainers and serum was obtained.

[0045] (Viruses) Influenza strain A / Puerto Rico / August 1934 (H1N1; IAV PR8) was propagated in chicken eggs. The virus titer was quantified by plaque assay on MDCK cells. Encephalomyocarditis virus (EMCV) was propagated in L929 cells, and the virus titer was quantified using L929 cells. Sendai virus (SeV) was propagated in chicken eggs, and the virus titer was quantified by HA assay using chicken erythrocytes.

[0046] The A / WSN / 1933 (H1N1) recombinant influenza strain (IAV WSN) was recovered from the pDZ ambisense plasmid 23 (a gift from Adolfo Garcia Sastre). It was propagated in MDCK cells and titers were measured by flow cytometry using the nanobody VHH NP2 Alexa Fluor 647.

[0047] Recombinant influenza strain A / SC35M (H7N7) containing a modified NS segment encoding NS1, P2A, EGFP, T2A, and NEP (IAV SC35M-GFP) was recovered from the pHW ambisense plasmid (from Martin Schwemmle), propagated in MDCK cells, and titers were measured by flow cytometry. Recombinant vesicular stomatitis virus Indiana strain (VSV-GFP), encoding EGFP at position 5 of the genome, was infected with VACV WR vTF7.3, rescued from vTF7.3-transfected BSR T7 / 5 cells, and propagated in pVSV1(+)P5_EGFP, pL, pP, pN, and BSR T7 / 5 cells. Viral titers were measured by plaque assay using Vero cells.

[0048] Influenza A virus strains A / Hong Kong / 483 / 1997 (H5N1) (IAV HK483) and IAV HK483 I38T were propagated in embryonated chicken eggs and harvested. Influenza B virus strain Hokkaido / 30-4 / 2014 (IBV) and influenza D virus strain D / bovine / NE / 9-5 / 2012 (IDV) were propagated in embryonated chicken eggs and harvested. Virus titers in MDCK cells and allantoic fluid or cell culture supernatants were quantified at 50% tissue culture infectious dose (TCID50) using MDCK cells. Rift Valley fever virus strain MP-12 was recovered from UTMB and BSR / T7-5 cells using a plasmid provided by Dr. Shinji Makino and propagated in VeroE6 cells provided by Dr. Karl-Klaus of the Max von Pettenkofer Institute of Virology, Conzelmann.

[0049] 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 Dr. Roger Hewson (Public Health England), Dr. Barbara W. Johnson (Centers for Disease Control and Prevention), Dr. Shuji Ando (National Institute of Infectious Diseases (NIID)), and Dr. Chang-Kweng Lim (NIID). These viruses were propagated in Vero cells in DMEM medium containing 2% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin. Virus-containing supernatants were collected 3 to 7 days after inoculation. The infectious dose was titrated on SW-13 cells by plaque formation assay in 6-well plates.

[0050] Chemicals: 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 screening, sinefungin, and TAK-599 isavuconazonium sulfate, were purchased from Cayman Chemical Company (13829, 23696, 23950). S-Adenosyl-L-homocysteine ​​(SAH), S-Adenosyl-L-methionine (SAM), tubercidin, GDP-D-mannose disodium salt, 5-iodotubercidin (Itu), and sertindole were 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).

[0051] 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 Pharmakeks (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.

[0052] 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.

[0053] Figure 45 shows the CRISPR target sequence and mutation region in the knockout cells used. CRISPR targets the sgRNA sequence and genotype of all knockout cells used in this study. The plasmids used are listed in Tables 1 and 2.

[0054]

[0055]

[0056] Western blot: Cells were lysed in NP40 lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40) or RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate) containing 1 mM dithiothreitol (DTT), 2 μg / ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM vanadate. The cells were incubated on ice for 10 minutes and centrifuged at maximum speed (20,627 × g) for 15 minutes. 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 minutes. The samples 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.

[0057] (RNA quantification) Cells were harvested with TRIzol (Invitrogen, 15596), and RNA was extracted according to the manufacturer's protocol. cDNA was purified using a 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 (SEQ ID NOS: 1 to 55 in the Sequence Listing) and TaqMan probes used are shown in Tables 3 to 5. The product names of the antibodies used are shown in Table 6.

[0058]

[0059]

[0060]

[0061]

[0062] Fluorescence quantification: GFP fluorescence in IAV SC35-GFP infected cells was quantified using ImageJ (FIJI). 8-bit images acquired with a Cell-IQ fluorescence microscope were thresholded and fluorescence quantified using "Analyze Particles." Relative "IntDen" values ​​were calculated as relative fluorescence units / region of interest (RFU / ROI).

[0063] (Retrovirus Production) Semi-confluent Plat-A cells in 6-well plates were transfected with 2 μg of transfection mix or retroviral vector (pLZR backbone), 1 μg of pCMV-VSV-G-RSV-ReV, and 500 μl of PEI MAX 10 μg Opti-MEM (Thermo Fisher Scientific, 31985). After 6 hours, the medium was replaced with fresh medium. Two days after transfection, the supernatant was collected and passed through a 0.45 μm pore filter (Sigma-Aldrich, WHA10462100), and 10 μg / ml polybrene (Merck, TR-1003-G) was used to infect target cells.

[0064] (Virus Titer) IAV PR8 titer (PFU / ml) was determined by plaque assay using MDCK cells. MDCK cells were seeded into multiwell plates one day before infection. Serial 1:10 dilutions of IAV-containing supernatant were added to the wells and incubated at 37°C for 1 hour. One hour after infection, cells were washed twice with DMEM medium, followed by the addition of overlay medium (DMEM, 1.5% Avicel PH-101 (Sigma-Aldrich, 11365), 0.2% bovine serum albumin (BSA; Biomol, 01400.100), 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.5 μg / ml TPCK-treated trypsin (Thermo Fischer Scientific, 20233)). Three days after infection, cells were washed with phosphate-buffered saline (PBS) and fixed with 6% PBS-buffered formaldehyde. Fixed cells were stained with 0.5% crystal violet solution. After washing the cells with water, plaques were counted and virus titers were calculated. The titers (tissue culture median infectious dose (TCID50) / ml) of IBV, IAV HK483, and RVFV were measured using MDCK and Vero cells, respectively.

[0065] (IAV mini-replicon reporter assay) The assay was performed using the method described in Cell Host Microbe 17, 309-319. HEK-293T WT or MTr1 KO cells were transfected with IAV PB2, PB1, PA, NP, a firefly luciferase minigenome construct (negative sense), and a Renilla luciferase expression plasmid (ratio = 1:1:0.1:1:1:1:0.1). As a negative control, the PB2 or PA expression plasmid was replaced with an empty expression plasmid. One day after transfection, 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.

[0066] Flow cytometry analysis: Detached cells were fixed with 4% paraformaldehyde in PBS for 10 minutes, washed twice with PBS, permeabilized with 0.05% Triton X-100, washed twice with 2% FBS in PBS, and blocked with 0.05% Tween 20 in PBS (PBS-T), 5 mg / ml BSA for 30 minutes. Cells were stained with anti-NP nanobody for 1 hour at room temperature, washed three times with 2% FBS in PBS, and NP-expressing cells were measured using Canto II (BD) and analyzed using FlowJo 10.6.2 software (Tree Star).

[0067] (Cytotoxicity Assay) To assess cell viability, WST-8 (CCK8) tetrazolium salt (Abcam, ab228554) was used according to the manufacturer's instructions.

[0068] Immunostaining: Cells were fixed with 4% paraformaldehyde in PBS for 10 minutes, washed twice with PBS, permeabilized with 0.05% Triton X-100, washed twice with PBST, blocked with 5 mg / ml BSA in PBST for 30 minutes, and stained with primary antibody in 5 mg / ml BSA at room temperature for 1 hour, washed three times with PBS-T for 10 minutes each, stained with secondary antibody in 5 mg / ml BSA for 30 minutes, washed once in PBS containing 1 μg / ml Hoechst 33342 (Thermo Fisher Scientific, 62249) for 10 minutes, and washed twice with PBS-T for 10 minutes each.

[0069] Fluorescence Microscopy Fluorescence microscopy images were acquired using an InCellis (CENiBRA) with a 10x objective, a Cell-IQ fluorescence microscope (CM Technologies) with a 4x objective, or an SP8 confocal microscope (Leica) with a 10x / 0.30HC PL FLUOTAR DRY objective and a HyD detector.

[0070] (Phylogenetic tree construction) Phylogenetic trees were generated using MEGA X software using the neighbor-joining method. The bootstrap method (1000 replicates) was used to assess phylogeny. 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), and 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).

[0071] Structure-based virtual screening of Mtr1 inhibitors was performed using molecular docking against 5,597 bioactive compounds with molecular weights ranging from 200 Da to 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 human Mtr1 crystal structure (PDB ID: 4N49). After selecting only FDA-approved drugs from the results, the top 30 compounds were selected for in vitro studies.

[0072] (TFMT-Mtr1 docking) Docking simulations were performed using the Glide SP docking program (Schrodinger, LLC) with 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.

[0073] (Production and Purification of Recombinant Proteins) 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 μg / ml leupeptin, 1 mM PMSF, and 1 mM vanadate, and disrupted by three freeze-thaw cycles (liquid nitrogen to 37°C water bath). After centrifugation to remove cell debris, the supernatant was mixed with Strep-Tactin Superflow Agarose (Merck, 71592-4) in an open column (Pierce, 29920), washed, and eluted with biotin-containing buffer (Merck, 71613-3, [100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 2.5 mM desthiobiotin, pH 8.0]). Protein concentration was determined by generating a standard curve using BSA standard protein (Pierce, 23210) by SDS-PAGE with Coomassie blue staining.

[0074] In vitro RNA transcription. Cap0 RNA was synthesized using the 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 (see primer list in Table 5). The synthesized RNA, 3'-O-Me-m7GpppGpG (pN27), was used in a methyltransferase activity assay of human MTr1.

[0075] (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 μM human MTr1, 0.8 μM human MTr1 K239A), the inhibitor of interest, 10 μM 3′-O-Me-m7GpppGpG(pN27)cap0 RNA, and 1.2 μM (0.02 μCi / μl) adenosyl-L-methionine, S-[methyl- ３ H] (SAM [ ３ The reaction mixture was incubated overnight at 37°C with a Miniquick Oligo column (Roche, 11814397001) to remove free SAM [H]. The purified sample was diluted with ULTIMA GOLD (PerkinElmer, 6013329), and methyltransferase activity was measured in disintegrations per minute (DPM) using a scintillation counter LS6500 (Beckman Coulter).

[0076] 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 (25 mM HEPES pH 7.5, 150 mM NaCl) before use. Reaction mixtures were prepared by mixing 5 μl of diluted SYPRO Orange dye with 1 μg of protein and the respective compound concentrations in a total volume of 50 μl of thermal shift assay buffer. Thermal stability was analyzed using a Step One Plus real-time PCR system with TAMRA dye detection settings. Measurements were normalized using GraphPad Prism software.

[0077] Human lung explants: IAV infection in human lung explants was performed as described in Emerg Microbes Infect 8, 1763-1776. Tumor-free human lung tissue blocks obtained from patients undergoing lung surgery at the University Hospital Münster were used at approximately 100 mg / well (12-well plate). The explants were cultured in Roswell Park Memorial Institute 1640 medium (RPMI) containing 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.1% BSA at 33°C and 5% CO. ２ The lungs were incubated overnight at 25°C. 10 μM TFMT was added 1 hour before IAV infection. Medium containing IAV was instilled into the lung blocks, and 1 hour post-infection, the tissues were washed and placed in fresh medium containing 10 μM TFMT. Supernatants were collected for viral titration by plaque assay, and lung tissues were immunostained using anti-IAV NP antibodies. All patients provided written consent to donate their lung tissue for scientific purposes. Ethical approval was provided by the Ethics Committee of the Arztekammer Westphalen-Lippe (AZ:2016-265-f-S).

[0078] (Synergistic Effect) Evaluation of synergistic effect was carried out as described in Pharmacol. Rev. 58, 621-681.

[0079] Statistics: Statistics were calculated using two-tailed Student's t-test for cell line assays, Mann-Whitney test for mouse in vivo assays, and Wilcoxon matched-pairs signed-rank test for human lung explant assays ( ＊ p<0.05, ＊＊ p<0.01, ＊＊＊ p<0.001).

[0080] Test Example 1: MTr1-dependent cap structure modification To establish a functional antiviral screening system for MTr1 inhibitors, we first confirmed that MTr1 is required for the replication of various cap-snatching viruses.

[0081] 2 is a photograph of a Western blot showing the generation of MTr1 knockout (KO) A549 cells by CRISPR / Cas9, in which wild-type (WT) and MTr1 KO A549 cells were infected with IAV (influenza A virus) PR8 (m.o.i., 1) (m.o.i.: multiplicity of infection).

[0082] These cells were infected with two orthomyxoviruses, IAV (PR8, H1N1) and IBV (influenza B virus) (Victoria), and the replication levels were monitored. Figure 3 is a graph showing the accumulation of IAV and IBV viruses 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 collected at the indicated time points post-infection (m.o.i., 1) and virus titers were measured by plaque assay. The detection limit is indicated by the dotted line at the bottom. (b) shows the viral yield in the supernatants from WT and MTr1 KO A549 cells infected with IBV at an m.o.i. of 0.1 for 48 hours. It is noteworthy from these results that IAV and IBV accumulated in MTr1 KO A549 cells. IBV accumulation was significantly impaired.

[0083] Figure 4(a) is a graph 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 graph showing qRT-PCR analysis of individual IAV RNA segments in WT and MTr1 KO A549 cells infected with IAV PR8 at an moi of 1 for 24 hours. Figure 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 an moi of 1 for 24 hours. 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 hours. These results, along with those described above, clearly indicated a loss of viral RNA levels and viral protein expression in MTr1 KO cells. Twenty-four hours before IAV infection, cells were infected with retroviruses expressing MTr1, MTr1 K239A, or neither. Figure 4(f) is a photograph showing Western blot analysis of each protein in WT and MTr1 KO A549 cells infected with the IAV PR8 strain at m.o.i. 1 for 24 hours. Figure 5(a) is a photograph showing immunostaining in WT and MTr1 KO A549 cells infected with the IAV PR8 strain at m.o.i. 1 for 24 hours. Figure 5(b) is a graph showing the results of flow cytometry in WT and MTr1 KO A549 cells infected for 24 hours with IAV PR8 strain at an m.o.i. of 1. These results show that IAV replication in MTr1 KO cells was restored by re-expression of MTr1, but not by re-expression of the catalytically inactive mutant MTr1 K239A.

[0084] Figure 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. Figure 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 for the indicated time. Strand-specific quantitative reverse transcription polymerase chain reaction (qRT-PCR) detected only IAV vRNA, but not mRNA or cRNA, in MTr1 KO cells.

[0085] In line with previous findings, we investigated the defect in the cap-snatching step of IAV replication in MTr1 KO cells by quantifying the levels of IAV mRNA (segment 1) specifically snatched from U2 spliceosomal snRNA by qRT-PCR using U2 segment 1 (PB2) hybrid primers. Figure 8 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 the indicated m.o.i. Figure 9(a) is a schematic diagram showing the primer sequences for cap-snatching-specific qPCR. Figure 9(b) shows the results of cap-snatching-specific qPCR analysis of IAV PR8 at the indicated m.o.i. Figure 9(c) 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 i1 for 24 hours. 0.1 μM BXM or 100 μM ribavirin (Rib) was added 3 hours before infection. Non-targeting (NT)-segment 1 hybrid primers were used as controls. Figure 9(c) is a graph showing qRT-PCR analysis of U2 RNA from the same samples as Figure 8.

[0086] To determine whether MTr1 is required only for the PR8 strain of IAV (H1N1) or whether other orthomyxoviruses are similarly dependent on this host factor, we also examined the replication efficiency of various IAV strains and other viruses belonging to the orthomyxovirus family. Figure 10 is a graph showing qRT-PCR analysis and virus yields of WT and MTr1 KO cells infected with IAV. (a) qRT-PCR analysis of IAV RNA (segment 7) or GFP RNA (inserted in segment 8) in WT and MTr1 KO A549 cells infected with IAV WSN (1 particle / cell) or IAV SC35M-GFP (H7N7) (1 particle / cell) for 24 hours. (c) qRT-PCR analysis of IAV RNA (segment 7) or GFP RNA (inserted in segment 8) in WT and MTr1 KO A549 cells infected with IAV WSN (1 particle / cell) or IAV SC35M-GFP (H7N7) (1 particle / cell) for 24 hours. Figure 11 shows the viral yields in the supernatants of WT and MTr1 KO A549 cells infected with IAV at 0.1 for 48 hours. Figure 11 is a photograph showing Western blot results for WT and MTr1 KO cells infected with IAV. (a) shows the results for WT and MTr1 KO A549 cells infected with IAV WSN at 1 particle / cell for 24 hours, and (b) shows the results for WT and MTr1 KO A549 cells infected with SC35M-GFP at 0.1 infectious particles / cell for the indicated times. The results in the figure demonstrate that not only PR8 (H1N1), but also WSN (H1N1), SC35M (H7N7), and the highly pathogenic avian IAV HK483 (H5N1) exhibited replication defects in MTr1 KO cells.

[0087] Figure 12 is a graph showing qRT-PCR analysis and virus yields of WT and MTr1 KO cells infected with IDV and THOV. (a) Virus yields in the supernatants of WT and MTr1 KO A549 cells infected with IDV at an m.o.i. of 0.1 for 48 hours. (b) qRT-PCR analysis of THOV RNA in WT and MTr1 KO A549 cells infected with THOV at an m.o.i. of 1 for 24 hours. As shown in the figure, 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.

[0088] Next, we investigated whether MTr1 functions as a general host factor for cap-snatching viruses. In addition to orthomyxoviruses, bunyaviruses are also well known to possess cap-snatching activity. Therefore, we investigated various bunyaviruses, including Rift Valley fever virus (RVFV), Hazara virus (HAZV), Issyk-Kul virus (ISKV), and soft tick virus (STBV), as well as Dugbe virus (DUGV).

[0089] Figure 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 an m.o.i. of 0.1 for 2 days. (b) shows the results of qRT-PCR analysis of the indicated viral RNAs 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. Figure 14(a) is a schematic diagram showing the phylogenetic tree of the S segment of bunyaviruses. Figure 14(b) shows the viral yield in the supernatant from WT and MTr1 KO A549 cells infected with RVFV at an m.o.i. of 0.1 for 2 days. Figure 14(a) 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 the indicated times and respective moi. Figure 14(c) is a graph showing the results of qRT-PCR analysis of the indicated viral RNAs in A549 cells at the indicated times and respective moi. The moi is 1 (THOV), 0.1 (HAZV), 0.01 (ISKV), 0.1 (STBV), or 0.1 (DUGV), respectively. Statistics were calculated using a two-tailed Student's t-test ( ＊ p<0.05, ＊＊＊ p<0.001).

[0090] As shown in the figure, the RNA replication levels and virus yields in the supernatants of MTr1 KO cells infected with these bunyaviruses were significantly less or not different from those of wild-type (WT) cells. These data suggest that the cap-snatching activity of these bunyaviruses and two orthomyxoviruses, IDV and THOV, is independent of MTr1-dependent cap modification.

[0091] Figure 15 is a schematic diagram outlining the dependency of MTr1 on cap snatching virus replication. The data presented above demonstrate that host-specific inhibitors of MTr1 specifically inhibit IAV and IBV, among cap snatching viruses, regardless of subtype or strain.

[0092] [Test Example 2: Verification of MTr1 Inhibition by TFMT through In Silico Compound Screening] Given the specific and potent restriction of IAV and IBV replication by MTr1 deficiency, 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 a methyl group 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 have the potential to act as inhibitors.

[0093] Figure 16 is a schematic diagram showing the general flow chart of the primary screening. Figure 17(a) is a schematic diagram showing the 2D interaction between SAM and 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 MTr1 inhibitor candidates. The MTr1 structure from PDB was used to generate the image (ID: 4N49) at BioRender.com. 5,597 compounds from DrugBank were docked into the SAM-binding pocket of the human MTr1 crystal structure. Figure 17(c) is a schematic diagram showing the docking scores and virtual screening rankings of representative compounds.

[0094] As shown, 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 rank among the top 10 high affinity binders.

[0095] Next, the anti-IAV activity of 12 commercially available compounds with the highest affinity scores among the top 30 was examined. Figure 18 is a graph showing the results of qRT-PCR analysis of IAV RNA (segment 7) in A549 cells infected with the IAV PR8 strain at an moi of 1 for 24 hours. The indicated concentrations of tubercidin were added 3 hours before infection. Figure 19(a) is a graph showing the results of quantification of GFP levels in A549 cells infected with the IAV-GFP SC35M strain (moi, 0.1) for 19 hours. 100 μM of each compound was added 3 hours before infection. Figure 19(b) is a graph showing the results of another quantification of GFP levels in A549 cells infected with the IAV-GFP SC35M strain (moi, 0.1) for 19 hours. Each compound was added at 0.1, 1, 10, or 100 μM 3 hours before infection. Figure 19(c) is a graph showing the quantification of GFP levels and the results of a cytotoxicity test (WST-8) in A549 cells infected with IAV-GFP (moi, 0.1) for 19 hours. Three hours before infection, tubercidin was added at the concentrations shown. Interestingly, tubercidin, an adenosine analog, was identified as the most effective compound, demonstrating dose-dependent inhibition at 0.1 to 100 μM with minimal toxicity.

[0096] 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 an m.o.i. of 1 for 24 hours. Three hours before infection, tubercidin was added at the concentrations shown in the figure. As shown in the figure, tubercidin also had an effect on IBV infection.

[0097] Figure 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, using tubercidin or ribavirin as a control. Figure 21(a) is a graph showing another result of a thermal shift assay of recombinant MTr1 using SYPRO Orange. Assays were performed at the concentrations shown, using SAM or SAH. These results confirmed that tubercidin directly binds to recombinant MTr1 by thermal shift assay. Figure 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. Figure 21(c) is a graph showing the results of an MTase assay using human MTr1 with the indicated concentrations of tubercidin. Statistics were calculated using a two-tailed Student's t-test ( ＊＊＊ p<0.001).

[0098] Importantly, these results indicate that tubercidin inhibits the MTase activity of recombinant MTr1 protein in a dose-dependent manner, and that tubercidin is an MTr1 inhibitor that suppresses IAV replication.

[0099] Tubercidin has been known to have multiple activities, including antiviral and anticancer activities, but it is also known to exhibit certain toxicity in vivo. Therefore, to identify superior tubercidin-related compounds, we evaluated 115 tubercidin-related compounds using 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.

[0100] Figure 22(a) shows a schematic flowchart of the three-step secondary screening process. For quantification of GFP levels in A549 cells infected with IAV-GFP (moi, 0.1) for 19 hours, each compound was added at concentrations of 100 μM (step 1), 10 μM (step 2), and 1 μM (step 3). qRT-PCR analysis of HAZV RNA in HAZV-infected A549 cells (moi, 0.1) was also performed in step 2.

[0101] In step 1, cells were infected with IAV in the presence of individual tubercidin-related compounds, and 13 compounds with anti-IAV effects were identified. In step 2, to rule out off-target inhibition of viral infection or toxicity, anti-IAV activity at lower concentrations was investigated and counterscreened for activity against HAZV. Interestingly, similar to the results in MTr1 KO cells described below, step 2 found four compounds that inhibited IAV replication without reducing HAZV replication. In step 3, we evaluated which of the identified compounds could inhibit IAV replication at the lowest concentration, and ultimately identified trifluoromethyltubercidin (TFMT) as the most effective compound.

[0102] Next, the binding of TFMT to the SAM-binding pocket of MTr1 and recombinant MTr1 was confirmed by in silico docking and in vitro thermal shift assays, respectively. Figure 22(b) is a schematic diagram showing the docking state of TFMT in the SAM-binding pocket of human MTr1. Figure 22(c) is a graph showing the results of a thermal shift assay of MTr1 with 1 mM TFMT or ribavirin using SYPRO Orange. Figure 23(a) is a graph showing the results of an MTase assay of MTr1 with the indicated concentrations of TFMT or ribavirin. The results in the figure confirmed that TFMT inhibits the MTase activity of the recombinant MTr1 protein.

[0103] The specificity of the antiviral activity was also investigated using two additional viruses, IBV and STBV. Figure 23(b) is a graph showing the results of qRT-PCR analysis of each viral RNA. qRT-PCR analysis of the indicated viral RNAs in A549 cells infected with IAV (moi, 1), IBV (moi, 1), HAZV (moi, 0.1), and STBV (moi, 0.1) for 24 hours is shown. Importantly, TFMT exhibited antiviral activity against IAV and IBV, but not against HAZV or STBV. This is in agreement with the phenotype of MTr1 deficiency described above.

[0104] 23(c) is a graph showing the results of GFP level quantification for A549 cells infected with IAV-GFP. The graph shows the IAV replication level of A549 cells infected with IAV-GFP (moi, 0.1) for 19 hours and GFP level quantification in a cytotoxicity test (WST-8). As measured by a WST-8 cell viability assay, the IC50 of TFMT against IAV infection was 0.30 μM, and no significant in vitro toxicity was observed within the effective concentration range.

[0105] Figure 23(d) is a graph showing the results of another quantification of GFP levels in 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 μM 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 a two-tailed Student's t-test ( ＊＊＊ p<0.001). Notably, TFMT treatment significantly inhibited IAV replication even when administered 3-4 hours after infection, although the effect was reduced or absent when the drug was administered later.

[0106] Test Example 3: Verification of Efficacy in Normal Human Bronchial Epithelial Cells, Human Lung Explants, and Mice We next examined the anti-IAV activity of TFMT in normal human bronchial epithelial (NHBE) cells. Figure 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 with the IAV PR8 strain at m.o.i. of 1 for 24 hours. In Figures 24(a) to (d), TFMT was added 3 hours before infection. Figure 24(b) is a photograph showing the results of Western blot analysis of the indicated proteins in NHBE cells infected with the IAV PR8 strain at m.o.i. of 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.

[0107] Figure 24(c) is a photograph showing the results of immunostaining of NHBE cells infected with IAV PR8 strain. Immunostaining of IAV NP was performed 24 hours post-infection (m.o.i.) in the presence of 10 μM TFMT. The bar in the figure represents 100 μm. Histological analysis also revealed that IAV NP levels in TFMT-treated NHBE cells were significantly reduced without cytotoxicity.

[0108] Figure 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 at an moi of 0.1 for 24 hours were analyzed. Importantly, TFMT treatment did not inhibit HAZV replication, indicating that the specific efficacy of this compound against certain viruses is maintained in human primary cells.

[0109] Because this compound was effective in human NHBE cells, we evaluated TFMT in human lung explants ex vivo. Figure 24(e) shows a schematic diagram of IAV infection in human lung explants. Lung tissue was infected with IAV (H1N1, a seasonal isolate from 2019), and viral titers in the supernatant were measured by plaque assay at the indicated time points.

[0110] Figure 24(f) is a graph showing the results of viral growth in human lung tissue from a single donor and viral titration of seasonal IAV. Figure 24(g) is a graph showing the results of viral growth in human lung tissue from six independent donors and viral titration of seasonal IAV. Figure 25 is a graph showing viral growth of seasonal IAV (H1N1 from 2019) in human lung tissue (n=6). After infection, supernatants were collected at the indicated time points, and viral titers were measured by plaque assay. TFMT was added at 10 μM before and after infection. While the titer of untreated samples increased to over 10 PFU / ml at 48 or 72 hours after infection, titers from TFMT-treated lung explants remained below 10 PFU / ml, ranging from 100 to 1,000. Furthermore, when titers from all six independent donors were combined, there was a significant difference between the control and TFMT-treated reduced IAV titers in the culture supernatants.

[0111] 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 an anti-NP antibody. TFMT was added at 10 μM before and after infection. Consistent with the viral titer, neither IAV NP-positive cells nor morphological changes were observed in IAV-infected lung tissues treated with TFMT and infected with IAV. This demonstrates the high ability of TFMT to neutralize the replication of seasonal IAV isolates ex vivo, suggesting its potential application in clinical treatment.

[0112] To investigate the in vivo efficacy of this compound, further validation was performed using a mouse system. Figure 27 is a graph showing the results of GFP levels and cytotoxicity tests in LA-4 cells infected with IAV-GFP. IAV-GFP (m.o.i., 1) was infected for 19 hours, and quantification of GFP levels and cytotoxicity tests (WST-8) were performed. TFMT was added at the concentrations shown 3 hours before 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 than in human cells (IC50: 7.7 μM).

[0113] Next, in vivo toxicity in mice was evaluated by intranasal inoculation once daily for two days. Figure 28(a) is a graph showing weight changes following intranasal inoculation of mice. C57BL / 6 mice were intranasally administered 2 mg / kg of TFMT or tubercidin on days 0 and 1 (arrows in the figure) post-infection. Tubercidin is known to have a certain degree of toxicity, and treatment with tubercidin caused significant weight loss in mice. In contrast, the selected derivative, TFMT, did not cause weight loss or cytotoxicity.

[0114] Furthermore, the effects of TFMT treatment under various conditions were investigated. Figure 28(b) is a schematic diagram of an IAV infection model in a mouse in vivo system. IAV WSN and TFMT were introduced intranasally as shown in the figure. TFMT was administered on days 0 and 1 of IAV WSN infection. Figure 28(c) is a graph showing the results of qRT-PCR analysis of IAV RNA segment 5 in lung tissue. Figure 28(d) is a graph showing the results of qRT-PCR analysis of IAV RNA segment 1 in lung tissue. qRT-PCR analysis of IAV RNA in lung tissue of IAV-infected C57BL / 6 mice on day 2 (5x10 ３ PFU) were administered intranasally on days 0 and 1 of infection. 2 mg / kg of TFMT was administered intranasally. Statistics were calculated using the Student's two-tailed t-test for the NHBE cell assay, the Mann-Whitney test for the mouse in vivo assay, and the Wilcoxon matched-pairs signed-rank test for the human lung ex vivo assay. ＊ p<0.05, ＊＊p<0.01). TFMT treatment significantly reduced IAV NP and PB2 mRNA levels in mouse lungs 2 days post-infection with the indicated TFMT treatments, indicating that trifluoromethyl substitution of tubercidin eliminates in vivo toxicity while retaining anti-IAV efficacy.

[0115] Taken together, 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.

[0116] [Test Example 4: Inhibition of Host Capped RNA and Viral Polymerase Interactions through Synergistic Antiviral Activity of TFMT and Approved Anti-Influenza Drugs] Next, we further explored the mechanism by which TFMT treatment specifically inhibits IAV and IBV replication but not other viral replication. The following studies demonstrated that TFMT inhibits IAV replication independently of the innate immune response, the interaction between the IAV polymerase subunit PB2 and capped RNA, and its synergistic effect with BXM. Furthermore, we demonstrated that MTr1 is essential for IAV replication in a manner independent of IFIT1 or RIG-I, but is dispensable for the replication of SeV, VSV, and EMCV. We also demonstrated structural modeling and mutagenesis of influenza virus polymerase.

[0117] 29 is a graph showing the results of qRT-PCR analysis of IAV RNA in A549 cells infected with IAV PR8 strain. The IAV PR8 strain was infected at an m.o.i. of 1 for 24 hours, and IAV RNA (segment 7) was analyzed. The effect of TFMT treatment on IAV replication was independent of IFIT1-dependent sequestration of RNA or RIG-I / MDA5 signaling.

[0118] Furthermore, the IAV replication level in RIG-I / MTr1 or IFIT1 / MTr1 double KO cells was examined. Figure 30(a) is a photograph showing immunostaining of viral proteins in A549 cells infected with SeV or IAV. SeV (1 x 10 －５ A549 cells infected with SeV (100 HAU / cell) or IAV PR8 (m.o.i., 10) for 20 hours were immunostained for viral proteins of the indicated genotypes. Figure 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. of 1 for 26 hours were immunostained for the indicated proteins of the indicated genotypes. As shown in the figure, IAV replication was not detected, as in MTr1 KO cells, indicating no involvement of the immune receptor RIG-I or the antiviral protein IFIT1 in the TFMT-dependent suppression of IAV replication by MTr1 blockade.

[0119] Figure 31 is a graph showing the results of qRT-PCR analysis of IFN-β RNA in A549 cells. IFN-β 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 μM TFMT, at an m.o.i. of 1. Figure 32 is a graph showing the results of qRT-PCR analysis of other RNAs in A549 cells. The indicated RNAs were analyzed in A549 cells infected with or without IAV PR8 strain at the indicated time points in the presence or absence of 10 μM TFMT, at an m.o.i. of 1. Treatment with TFMT did not induce IFN-β or antiviral ISGs, further emphasizing that the observed antiviral effect is not dependent on innate immune activation.

[0120] Figure 33 is a graph showing the results of qRT-PCR analysis of each RNA in A549 cells infected with other RNA viruses. －６SeV, VSV, or EMCV RNA (segment 7) was analyzed in A549 cells 24 hours after infection with Sendai virus (SeV), 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.

[0121] 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. －６ WT and MTr1 KO A549 cells infected with SeV (1 × 10 HAU / cell), VSV (moi, 1), or EMCV (moi, 0.1) for 26 hours were analyzed for each viral RNA shown in the figure. Note that in (a) to (c), TFMT was added at 10 μM 3 hours before infection. Figure 34(b) is a photograph showing the results of Western blot analysis of each protein in MTr1 KO A549 cells infected with each virus. SeV (1 × 10 －６ The cells were infected with SeV (1 × 10 HAU / cell) or VSV GFP (moi, 1) at the time points indicated in the figure, and SARS-CoV-2 (moi, 0.1) was analyzed at 24 hours after infection. Figure 34(c) is a photograph showing the results of immunostaining of viral proteins in MTr1 KO A549 cells infected with SeV. SeV (1 × 10 －６ We analyzed viral proteins in WT and MTr1 KO A549 cells infected with SeV (1 x 10 HAU / cell) for 12 hours. Figure 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. SeV (1 x 10 HAU / cell) was administered at the indicated time points. －６We analyzed viral and host RNA in WT and MTr1 KO A549 cells infected with IAV PR8 (moi, 1), IBV (moi, 1), VSV (moi, 10), SeV (1 x 10), and HIV-1 (1 x 10). Figure 35(b) is a photograph showing optical microscope images of MTr1 KO HEK-293T cells infected with each virus. －６ WT and MTr1 KO HEK-293 cells infected with 1000 HAU / cell and EMCV (moi, 0.001) were observed on day 2 post-infection. The bar in the figure represents 50 μm. Examination of these virus infections in MTr1 KO cells confirmed that there was no difference in the level of viral replication.

[0122] Figure 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 an moi of 1. The expression level of IAV mRNA (segment 1), specifically deprived from U2 spliceosomal snRNA, was reduced by TFMT treatment, similar to MTr1 deficiency. These results indicate that TFMT treatment inhibits IAV replication not by immunomodulation but by directly affecting IAV cap-snatching activity. To further clarify the mechanism by which the loss of N1-2'-O-Me from host cap RNA during TFMT treatment causes IAV cap-snatching defects, we analyzed the N1-2'-O-Me interaction site in the IAV polymerase PB2 subunit. Figure 36(b) is a schematic diagram of the N1-2'-O-Me interaction site. A structural model (PDB ID: 6RR7) of the N1-2'-O-Me in the RNA bound to IAV PB2 is shown. The circled area indicated by the arrow indicates the methyl group at the N1-2'-O position. Figure 36(c) shows the structures of N1 and N2 of cap1 RNA, similarly showing the N1-2'-O-Me.

[0123] 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 region of PB2. The target sites for MTr1, MTr2, or CAPAM are indicated in the figure. Figure 37(c) is a schematic diagram showing the phylogenetic tree of PB2 in influenza virus and THOV and a 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 the IAV polymerase complex with cap0 RNA (PDB ID: 6RR7). This model revealed that N1-2'-O-Me is located very close (within 3 Å) to two amino acids, Q257 and I261, in the α-helical structure of PB2. Therefore, methylation is presumed to enhance the affinity between the PB2 helix and host mRNA through a hydrophobic effect and van der Waals interactions between the hydrophobic amino acids I260 and I261. Therefore, the absence of N1-2'-O-methyl groups on capped RNA in MTr1 KO cells may prevent proper interaction with the PB2 subunit of IAV polymerase. In addition to N1-2'-O-Me, two other well-known methylations, N2-2'-O-Me and N1-m6A, occur in the mature cap structure. These methylation reactions are catalyzed by the methyltransferase MTr2 and the Cap-specific adenosine methyltransferase (CAPAM, PCIF1), respectively.

[0124] Figure 38(a) is a schematic diagram showing a structural model of N1-m6A in RNA bound to IAV PB2. Figure 38(b) is a schematic diagram showing a structural model of N2-2'-O-Me in RNA bound to IAV PB2. The possibility of steric clash between N1-m6A and N2-2'-O-methylation was simulated. In the diagram, the circled area indicated by the arrow indicates the additional methyl group. Notably, no significant interaction between PB2 and N2-2'-O-Me or N1-m6A was observed.

[0125] Figure 39 is a graph showing qRT-PCR analysis of IAV RNA in A549 cells infected with IAV PR8. (a) Analysis of CAPAM KO and (b) analysis of Mtr2 KO cells are shown. IAV RNA (segment 7) was analyzed in the indicated genotypes of A549 cells infected with IAV PR8 at an m.o.i. of 1 for 24 hours. Consistent with the above data, no IAV replication defect was observed in CAPAM or MTr2 KO cells.

[0126] To experimentally demonstrate that depletion of MTr1 affects the interaction between PB2 and cap RNA, we performed poly(A)-binding protein (PABP)-mediated mRNA immunoprecipitation. Figure 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. (a) Ribosomal RNA (18S) and (b) mRNA (GAPDH). Each RNA was 10 ng. Figure 40(c) is a photograph showing the results of Western blot analysis of PABP-immunoprecipitated PABP protein. First, RT-PCR was used to confirm that mRNA was enriched in the PABP immunoprecipitated fraction.

[0127] Next, we performed PABP immunoprecipitation using IAV PB2-overexpressing control cells or MTr1 KO cells. Figure 41(a) is a photograph showing the results of Western blot analysis of input and immunoprecipitated proteins in PB2-expressing WT and MTr1 KO HEK-293T cells. In MTr1-deficient cells, the amount of PB2 co-precipitated with PABP immunoprecipitates was significantly reduced, indicating that the presence of MTr1-dependent N1-2'-O-Me significantly increases the affinity of PB2 for the cap. This result is consistent with the results of intracellular mRNA structural modeling.

[0128] Baloxavir marboxil (BXM) is an approved anti-IAV drug that targets the active site of polymerase PA subunits 18 and 19. Because TFMT affects the cap-binding activity of different subunits of IAV polymerase PB2, potential synergistic effects on anti-IAV activity were investigated. Figure 41(b) is a graph showing the synergistic effect of BXM and TFMT. The relative fluorescence units of GFP were 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. Figure 41(c) shows the calculated combination index as a measure of synergy evaluation. Figure 42(a) is a graph showing the WST-8 assay for Figure 41. Interestingly, potent synergistic effects were 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 demonstrated synergistic activity. 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 an m.o.i. of 0.01 for 55 hours was performed. TFMT and / or oseltamivir were added at the indicated concentrations 1 hour prior to infection. Figure 42(c) shows the results of calculating the combination index as a measure of synergy evaluation in the previous figure. Figure 42(d) is a graph showing the synergistic effect of TFMT with amantadine. The graph shows the relative fluorescence units of GFP 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 before infection. Figure 42(e) shows the results of calculating the combination index as a measure of synergy evaluation in the previous figure. These data suggest the potential use of TFMT in combination therapy.

[0129] An I38T mutation has been reported in the PA subunit of BXM-resistant IAV18. Therefore, we investigated whether this BXM-resistant virus carrying the I38T mutation in the PA subunit could replicate in MTr1 KO cells. Figure 43 is a graph showing luciferase reporter assays of IAV PR8 minireplicons in MTr1 KO HEK-293T cells overexpressing each PA subunit. (a) Results for WT (wild-type strain) and (b) results for MTr1 KO cells are shown. Results for WT and MTr1 KO HEK-293T cells overexpressing IAV PB2, PB1, NP, and the indicated PA are shown. 0.1 μM 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 IAV HK483 PA I38T, a BXM-resistant virus. IAV HK483 PA I38T was infected at an m.o.i. of 0.1 for 48 hours, and the yields in the supernatants of WT and MTr1 KO A549 cells are shown. Statistics were calculated using a 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.

[0130] Considering that viral polymerase activity is highly sensitive to MTr1 deletion, the question arises as to whether certain IAV strains can overcome the stringent requirement for N1-2'-O-Me at the PB2 cap-binding site. Figure 44 is a graph showing luciferase reporter assays of IAV PR8 minireplicons in MTr1 KO HEK-293T cells overexpressing the PA subunits indicated on the horizontal axis of each panel (a)–(d). Cells overexpressing IAV PB1, PA, NP, and the indicated PB2 were used. It is noteworthy to note that all mutants tested with changes in two of the interacting amino acids, I260 or I261, did not support IAV replication in MTr1 KO cells. Rather, 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 an N1-2′-O-Me capped RNA structure for efficient replication.

[0131] Taken together, these results highlight the potential of TFMT as an MTr1 inhibitor that specifically inhibits cap-snatching, thereby suppressing the replication of various IAV and IBV strains, including seasonal H1N1 isolates and highly pathogenic, BXM-resistant avian IAVs.

[0132] Mechanistically, TFMT induces MTr1 deficiency and the accumulation of cap0 RNA, which impairs PB2 binding to host capped RNA and reduces the effectiveness of IAV polymerase in cap snatching and initiating RNA synthesis. TFMT exhibited strong synergy with BXM, as both drugs target distinct polymerase subunits, PB2 and PA, respectively.

[0133] Notably, 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. Interestingly, comparison of PB2 between influenza virus 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. Furthermore, 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 polymerase to cap snatching may explain the specificity of TFMT restriction for IAV and IBV.

[0134] These results also report a comprehensive identification of TFMT as a novel, highly specific, and non-toxic MTr1 inhibitor that specifically restricts the replication of the cap-snatching-dependent viruses IAV and IBV. In contrast to the MTr1 inhibitor tubercidin identified in the primary screen, TFMT treatment is tolerated in vitro and in vivo without toxicity or activation of the host immune response.

[0135] This compound is not only an inhibitor with a novel mechanism of action, but also belongs to a new class of antiviral drugs with specific efficacy against influenza A and B. Currently approved drugs for the treatment of IAV against viral proteins are available, although resistant mutant viruses have been reported for each of the existing drugs. In contrast, host-directed antivirals 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 and SARS-CoV-2 by directly affecting viral replication and modulating inflammation. Considering immune activation or toxicity, the efficacy of the host-directed MTr1 inhibitor TFMT could provide a new therapeutic approach against influenza A and B viruses.

[0136] While preferred embodiments of the present invention have been described and illustrated, it should be understood that these are exemplary of the present invention and should not be considered limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the present invention. Accordingly, the present invention is not to be deemed limited by the foregoing description, but is limited only by the scope of the appended claims.

[0137] According to the present invention, by inhibiting the mechanisms of influenza virus proliferation and infection, it is possible to obtain a new anti-influenza virus drug that has a target other than 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.

Claims

1. An anti-influenza virus composition comprising tubercidin, a tubercidin derivative or a salt thereof as an active ingredient.

2. 2. The composition according to claim 1, wherein the tubercidin derivative is a compound represented by the following general formula (1): 【Chemical 1】 (In the above general formula (1), R １ , R ２ and R ３ are the same or different and each represent 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.

3. R １ and R ２ are the same or different and each 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, R ３ The composition according to claim 2 , wherein represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted amino group, or an optionally substituted aryl group.

4. R ３ The composition according to claim 2 or 3, wherein represents a hydrogen atom.

5. R １ and R ２ The composition according to claim 2 or 3, wherein each of the is the same or different and represents a halogen atom.

6. R １ and R ２ The composition according to claim 2 or 3, wherein each of the groups may be the same or different and represents an amino group or a cyano group which may have a substituent.

7. R １ The composition according to claim 2 or 3, wherein represents an alkyl group substituted with a halogen.

8. R １ The composition according to claim 2 or 3, wherein represents a trifluoromethyl group.

9. The composition according to claim 2, wherein the tubercidin derivative is a compound represented by the following formula (2): 【Chemistry 2】

10. A medicine, food or drink, supplement, agricultural chemical, feed, or cosmetic, comprising the composition according to any one of claims 1 to 3.

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