Pharmaceutical composition for treating or preventing coronavirus infectious diseases

Abstract

The purpose of the present invention is to provide an antiviral agent effective against COVID-19.　The purpose can be achieved by (1) a polypeptide that contains an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, and the amino acid sequence represented by SEQ ID NO: 8, or (2) a polypeptide that has antiviral activity against coronavirus, contains an amino acid sequence in which one amino acid is substituted in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, or the amino acid sequence represented by SEQ ID NO: 8, said polypeptide having an O-glycoside-linked sugar chain.

Description

Pharmaceutical composition for treating or preventing coronavirus infection

[0001] The present invention relates to a pharmaceutical composition for treating or preventing coronavirus infection. According to the present invention, coronavirus infection can be effectively treated or prevented.

[0002] Coronaviruses are classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. Known human coronaviruses include HCoV-229E and HCoV-NL63 (alphacoronavirus), as well as HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2 (betacoronavirus). Of these coronaviruses, four, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, are known as pathogens of the common cold. Recently, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2), which cause severe pneumonia, have been discovered.

[0003] SARS-CoV-2 is the virus that causes novel coronavirus disease (COVID-19) (Non-Patent Document 1). SARS-CoV-2 is primarily transmitted via droplets, causing typical cold-like symptoms such as fever, dry cough, fatigue, and phlegm. Severe cases can lead to respiratory failure due to acute respiratory distress syndrome, acute lung injury, or interstitial pneumonia. Remdesivir, dexamethasone, and baricitinib have been approved as therapeutic agents for COVID-19. Additionally, tocilizumab, lonapreve (casirivimab / imdevimab), sotrovimab, and molnupiravir have also been approved.

[0004] J Clin Med 2020;9:1225

[0005] However, the development of more effective and safer drugs is desired. Therefore, an object of the present invention is to provide an antiviral agent effective against novel coronavirus infection.

[0006] The present inventors have conducted extensive research into antiviral agents effective against COVID-19 infection and have surprisingly found that a peptide having a specific amino acid sequence can effectively suppress coronavirus infection. The present invention is based on this finding. Therefore, the present invention provides: [1] (1) a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, and the amino acid sequence represented by SEQ ID NO: 8; or (2) a polypeptide comprising an amino acid sequence in which one amino acid is substituted in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, or the amino acid sequence represented by SEQ ID NO: 8, and having antiviral activity against coronaviruses, wherein the polypeptide has an O-glycoside-linked sugar chain; [2] the polypeptide having an O-glycoside-linked sugar chain according to [1], wherein the O-glycoside-linked sugar chain is N-acetylgalactosamine; [3] the polypeptide having an O-glycoside-linked sugar chain according to [1] or [2], wherein the polypeptide has a methyl group at the N-terminus; [4] A pharmaceutical composition for treating or preventing coronavirus infection, comprising as an active ingredient the polypeptide having an O-glycoside-linked glycan according to [1] or [2]; [5] A method for treating coronavirus infection, comprising a step of administering an effective amount of the polypeptide according to [1] or [2] to a subject in need of treatment; [6] The polypeptide according to [1] or [2], which is for treating or preventing coronavirus infection; and [7] Use of the polypeptide according to [1] or [2] in the production of a pharmaceutical composition for treating or preventing coronavirus infection.

[0007] The pharmaceutical composition of the present invention can effectively treat and prevent novel coronavirus infection (COVID-19).

[0008]

[0023] Figure 1 shows the steps of administering polypeptides in the "prophylactic administration pattern" and "full-term administration pattern" against coronavirus infection in Example 3.

[0024] Figure 2 is a graph showing the reduction in viral load by plaque assay after administration of polypeptides against coronavirus infection.

[0025] Figure 3 is a graph showing the reduction in infected cell count by fluorescent antibody assay after administration of polypeptides against coronavirus infection.

[0009] [1] Polypeptide The polypeptide of the present invention is (1) a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, and the amino acid sequence represented by SEQ ID NO: 8, or (2) a polypeptide comprising an amino acid sequence in which one amino acid is substituted in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, or the amino acid sequence represented by SEQ ID NO: 8, and having antiviral activity against coronaviruses, wherein the polypeptides (1) and (2) have an O-glycoside-linked sugar chain. The amino acid sequences represented by SEQ ID NOs: 1 to 11 are as follows: Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (SEQ ID NO: 1) Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (SEQ ID NO: 2) Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (SEQ ID NO: 3) Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (SEQ ID NO: 4) Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (SEQ ID NO: 5) Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (SEQ ID NO: 6) Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (SEQ ID NO: 7) Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (SEQ ID NO: 8) Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala-Thr-Ala (SEQ ID NO: 9) Pro-Ile-Val-Ser-Gln-Thr-Thr-Gly-Ile-Ala (SEQ ID NO: 10) Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Ile-Gly (SEQ ID NO: 11)

[0010] <<O-glycoside-linked sugar chain>> The polypeptide of the present invention has an O-glycoside-linked sugar chain. The O-glycoside-linked sugar chain is bound to a hydroxyl group in the side chain of threonine (Thr) or serine (Ser). Examples of the sugar chain include N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal), fucose (Fuc), mannose (Man), xylose (Xyl), and sialic acid. The O-glycoside-linked sugar chain may be a monosaccharide such as the above-mentioned GalNAc. Furthermore, the O-glycoside-linked sugar chain may be Core 1 (Gal-β1,3-GalNAc-), Core 2 (Gal-β1,3-(GlcNAc)GalNAc-), Core 3 (GlcNAc-β1,3-GalNAc-), Core 4 (GlcNAc-β1,3-(GlcNAc)GalNAc-), Core 5 (GalNAc-α1,3-GalNAc-), Core 6 ((GlcNAc)GalNAc-), Core 7 (GlcNAc-(GlcNAc)GalNAc-), or Core 8 (Gal-α1,3-GalNAc-). From the viewpoint of improving the solubility of the polypeptide of the present invention, sugar chains containing N-acetylgalactosamine (GalNAc) are preferred, and N-acetylgalactosamine (GalNAc) monosaccharides are preferred.

[0011] The polypeptides of the present invention, consisting of the 10 amino acid sequences such as those represented by SEQ ID NOS: 1 to 8, can exhibit antiviral activity against coronaviruses, and the 10 amino acid sequences have a specific structure that exhibits antiviral activity. As shown in the Examples, the binding of a sugar chain to threonine (Thr) or serine (Ser) in the 10 amino acid sequence does not inhibit the antiviral activity against coronaviruses. Therefore, the polypeptides of the present invention in which an O-glycoside-linked sugar chain is bound to the polypeptides of the present invention can exhibit excellent antiviral activity against coronaviruses.

[0012] The polypeptide of the present invention has an O-glycoside-linked sugar chain at one or more of Ser at position 4, Thr at position 5, and Thr at position 7 in the amino acid sequence represented by SEQ ID NO: 1, 2, 5, or 6. It has an O-glycoside-linked sugar chain at one or more of Ser at position 4, Thr at position 5, and Thr at position 6 in the amino acid sequence represented by SEQ ID NO: 3, 4, 7, or 8.

[0013] <<Methyl Group>> The polypeptide of the present invention may have a methyl group at the N-terminus. ３ The amino acid sequences of SEQ ID NOs: 1 to 9 having methyl groups are as follows: CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (SEQ ID NO: 1) CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (SEQ ID NO: 2) CH3-Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (SEQ ID NO: 3) CH3-Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (SEQ ID NO: 4) CH3-Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (SEQ ID NO: 5) CH3-Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (SEQ ID NO: 6) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (SEQ ID NO: 7) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (SEQ ID NO: 8) CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala-Thr-Ala (SEQ ID NO: 9) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Gly-Ile-Ala (SEQ ID NO: 10) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Ile-Gly (SEQ ID NO: 11) The N-terminal methyl group does not inhibit the antiviral activity of the polypeptide of the present invention against coronaviruses. That is, polypeptides in which the N-terminal proline is methylated can exhibit excellent antiviral activity against coronaviruses.

[0014] <<Functionally Equivalent Variants>> Functionally equivalent variants are not particularly limited, as long as they are polypeptides comprising an amino acid sequence in which one amino acid has been substituted at one position in the amino acid sequences represented by SEQ ID NOs: 1 to 11 and have antiviral activity against coronaviruses. For example, relative to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5 is a functionally equivalent variant in which one amino acid has been substituted, a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6 is a functionally equivalent variant in which two amino acids have been substituted, a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7 is a functionally equivalent variant in which three amino acids have been substituted, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 is a functionally equivalent variant in which four amino acids have been substituted.

[0015] The "substitution of one amino acid" in a functionally equivalent variant is a conservative substitution that maintains the function of the polypeptide of the present invention. "Conservative substitution" can be achieved, for example, by replacing an amino acid residue with another chemically similar amino acid residue, but is not limited to this. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue with the same charge. Functionally similar amino acids that can be obtained by such substitutions are known in the art for each amino acid. Nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Without being limited thereto, such conservative substitutions can result in functionally equivalent variants that have antiviral activity against coronaviruses.

[0016] The functionally equivalent variants of the present invention are polypeptides having an amino acid sequence in which one amino acid is substituted in the amino acid sequences represented by SEQ ID NOs: 1 to 11. Therefore, "an amino acid sequence in which one amino acid is substituted in the amino acid sequences represented by SEQ ID NOs: 1 to 11" is one in which one amino acid is substituted in a sequence of 10 or 12 amino acids. Therefore, "an amino acid sequence in which one amino acid is substituted in the amino acid sequences represented by SEQ ID NOs: 1 to 11" is a polypeptide having 90% or more amino acid sequence identity to the amino acid sequences represented by SEQ ID NOs: 1 to 11.

[0017] Coronaviruses are classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. Endemic coronaviruses are known in domestic animals such as dogs, cats, cattle, pigs, chickens, horses, alpacas, and camels, as well as wild animals and birds such as beluga whales, giraffes, ferrets, suncus, bats, and sparrows. In many cases, these viruses only cause mild respiratory symptoms and diarrhea in host animals. However, fatal symptoms are known to occur in domestic animals, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and infectious bronchitis virus (IBV), in laboratory animals, mouse hepatitis virus (MHV), and feline infectious peritonitis virus (FIPV) in pets.

[0018] On the other hand, coronaviruses that infect humans include HCoV-229E and HCoV-NL63 (alphacoronavirus genus), as well as HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2 (betacoronavirus genus). Of these human coronaviruses, four, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, are known to cause mild colds. Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), and novel coronavirus (SARS-CoV-2) can cause fatal pneumonia. In particular, SARS-CoV-2 is the virus that causes the novel coronavirus disease (COVID-19). SARS-CoV-2 is primarily transmitted through droplets, causing typical cold-like symptoms such as fever, dry cough, fatigue, and phlegm. In severe cases, respiratory failure due to acute respiratory distress syndrome, acute lung injury, or interstitial pneumonia develops.

[0019] <<Antiviral Activity Against Coronaviruses>> The polypeptides of the present invention exhibit antiviral activity against coronaviruses. Coronaviruses against which the polypeptides of the present invention exhibit antiviral activity are not particularly limited, and include coronaviruses of animals including humans, and avian species. The polypeptides of the present invention exhibit excellent antiviral activity against, but are not limited to, SARS-CoV or SARS-CoV-2.

[0020] Antiviral activity against coronaviruses can be measured according to the Examples and the like of the present specification. That is, whether or not the polypeptide of the present invention has antiviral activity against coronaviruses can be determined by coronavirus plaque assay, quantification by PCR or the like of mRNA, or measurement of the number of infected cells by fluorescent antibody assay. For example, when a sample containing the polypeptide of the present invention shows a reduction in the number of plaques of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to the number of plaques in a negative control not containing the polypeptide of the present invention, the polypeptide of the present invention can be determined to have "antiviral activity against coronaviruses." Furthermore, the polypeptide of the present invention can be determined to have "antiviral activity against coronaviruses" when the mRNA level in infected cells or the culture supernatant of infected cells containing the polypeptide of the present invention is reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to the mRNA level in infected cells or the culture supernatant of infected cells of a negative control not containing the polypeptide of the present invention. Furthermore, the polypeptide of the present invention can be determined to have "antiviral activity against coronaviruses" when the number of infected cells in infected cells by fluorescent antibody assay containing the polypeptide of the present invention is reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to the number of infected cells in a negative control not containing the polypeptide of the present invention. The type of coronavirus used in the plaque assay, mRNA quantification, or infected cell count measurement is not particularly limited, and any of the above-mentioned animal or avian coronaviruses, including human coronaviruses, can be used as appropriate.

[0021] <<Pharmaceutical Composition>> [1] Pharmaceutical Composition for Treating or Preventing Coronavirus Infection The pharmaceutical composition for treating or preventing coronavirus infection of the present invention is (1) a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO:1, the amino acid sequence represented by SEQ ID NO:2, the amino acid sequence represented by SEQ ID NO:3, the amino acid sequence represented by SEQ ID NO:4, the amino acid sequence represented by SEQ ID NO:5, the amino acid sequence represented by SEQ ID NO:6, the amino acid sequence represented by SEQ ID NO:7, and the amino acid sequence represented by SEQ ID NO:8, or (2) a polypeptide comprising an amino acid sequence in which one amino acid is substituted in the amino acid sequence represented by SEQ ID NO:1, the amino acid sequence represented by SEQ ID NO:2, the amino acid sequence represented by SEQ ID NO:3, the amino acid sequence represented by SEQ ID NO:4, the amino acid sequence represented by SEQ ID NO:5, the amino acid sequence represented by SEQ ID NO:6, the amino acid sequence represented by SEQ ID NO:7, or the amino acid sequence represented by SEQ ID NO:8, and having antiviral activity against coronaviruses, wherein the polypeptides (1) and (2) comprise a polypeptide having an O-glycoside-linked sugar chain as an active ingredient.

[0022] Coronaviruses that can be treated with the pharmaceutical composition of the present invention include human coronaviruses HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2. Furthermore, animal or avian coronaviruses include porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus (FIPV), canine coronavirus (CCV), bovine coronavirus (BCoV), equine coronavirus (EcoV), infectious bronchitis virus (IBV), avian deltacoronavirus, porcine deltacoronavirus, porcine respiratory coronavirus (PRCoV), feline coronavirus (FECoV), mouse hepatitis virus (MHV), porcine hemagglutinating encephalomyelitis virus (HEV), rat coronavirus (RCV), turkey coronavirus (TCoV), and rabbit coronavirus.

[0023] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills, and parenteral preparations such as injections, topical liquids, ointments, suppositories, topical creams, or eye drops. Oral preparations can be prepared according to conventional methods using excipients such as gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, silicic acid anhydride, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, flow enhancers, diluents, preservatives, colorants, flavorings, flavorings, stabilizers, moisturizers, antiseptics, or antioxidants. Parenteral preparations include, for example, injections. In preparing an injection, in addition to the active ingredient, any of the following may be used: a water-soluble solvent such as physiological saline or Ringer's solution; a water-insoluble solvent such as vegetable oil or fatty acid ester; an isotonic agent such as glucose or sodium chloride; a solubilizing agent; a stabilizer; a preservative; a suspending agent; or an emulsifier.

[0024] When using a pharmaceutical composition, the dosage can be determined appropriately depending on, for example, the type of active ingredient used, the type of disease, the patient's age, sex, weight, and level of health, or the administration method, and can be administered orally or parenterally. For example, when the pharmaceutical composition of the present invention is orally ingested, the intake amount is preferably 0.01 to 100 mg / kg of polypeptide per day for an adult. Note that the above administration method is only one example, and other administration methods may also be used. The administration method, dosage, administration period, administration interval, etc. of the pharmaceutical composition to humans are desirably determined through controlled clinical trials.

[0025] Furthermore, the administration form is not limited to pharmaceuticals, and various forms, such as functional foods, health foods (including beverages), or feed, can be used. The pharmaceutical composition containing the polypeptide can be produced using known pharmaceutical production methods, except that the composition contains a polypeptide as an active ingredient.

[0026] The pharmaceutical composition of the present invention may contain other ingredients. Examples of the other ingredients include food ingredients and food additives such as edible fats and oils, water, emulsifiers such as edible oils and fats, water, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, glycerin organic acid fatty acid esters, polyglycerin fatty acid esters, calcium stearoyl lactylate, sodium stearoyl lactylate, and polyoxyethylene sorbitan fatty acid esters; thickening and stabilizing agents such as locust bean gum, carrageenan, alginic acids, pectin, xanthan gum, crystalline cellulose, carboxymethylcellulose, methylcellulose, agar, glucomannan, gelatin, starch, and modified starch; salting agents such as salt and potassium chloride; acidulants such as acetic acid, lactic acid, and gluconic acid; sugars or sugar alcohols, sweeteners such as stevia and aspartame; coloring agents such as beta-carotene, caramel, and red koji pigment; antioxidants such as tocopherol and tea extract; flavorings; pH adjusters; food preservatives; and shelf-life enhancers. It is also possible to add functional materials such as various vitamins, coenzyme Q, plant sterols, or milk fat globule membranes. The total content of these other ingredients in the pharmaceutical composition of the present invention is preferably 8% by mass or less, more preferably 40% by mass or less, and even more preferably 20% by mass or less.

[0027] The pharmaceutical composition of the present invention can be administered to humans, but may also be administered to animals other than humans, including pets such as dogs, cats, and rabbits; livestock such as cows, horses, chickens, turkeys, and pigs; laboratory animals such as mice and rats; and animals kept in zoos, etc.

[0028] <<Method for Treating Coronavirus Infection>> The method for treating coronavirus infection of the present invention comprises the step of administering an effective amount of the polypeptide to a subject in need of treatment. That is, the polypeptide of the present invention can be used in the method for treating coronavirus infection. Cancer can be treated by administering an effective amount of the pharmaceutical composition to a human or animal.

[0029] <Polypeptide for treating coronavirus infection> The polypeptide of the present invention is for treating coronavirus infection. The polypeptide can be used in a method for treating coronavirus infection. That is, the present specification discloses a polypeptide for treating coronavirus infection.

[0030] <<Use of Polypeptide in the Production of a Pharmaceutical Composition>> The polypeptide can be used in the production of a pharmaceutical composition. That is, the present specification discloses the use of the polypeptide in the production of a pharmaceutical composition. The pharmaceutical composition is, but is not limited to, a pharmaceutical composition for treating coronavirus infection.

[0031] <<Action>> The mechanism by which the polypeptides of the present invention have antiviral activity against coronaviruses has not been fully elucidated, but is presumed as follows. However, the present invention is not limited by the presumption below. The polypeptides of the present invention are presumed to exhibit antiviral activity against coronaviruses due to the structure commonly present in the amino acid sequences of SEQ ID NOS: 1 to 11. Furthermore, it is presumed that the inclusion of a sugar chain enhances the antiviral activity against coronaviruses. In particular, it is presumed that N-acetylgalactosamine (GalNAc) itself is effective in enhancing antiviral activity against coronaviruses. Furthermore, it is presumed that the polypeptides of the present invention inhibit the process of coronavirus particle formation and / or the process of virus budding due to the structure and sugar chain commonly present in the amino acid sequences of SEQ ID NOS: 1 to 11. Furthermore, it is presumed that the excellent water solubility of the polypeptides of the present invention is due to the presence of an O-glycoside-linked sugar chain at serine or threonine in the amino acid sequences of SEQ ID NOS: 1 to 8. The amino acid sequences of SEQ ID NOS: 1 to 8 contain many hydrophobic amino acids, and therefore, it is presumed that the attachment of an O-glycoside-linked sugar chain to serine or threonine will result in excellent water solubility overall. In particular, N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc) bonded to the hydroxylase exhibits excellent water solubility, with N-acetylgalactosamine (GalNAc) exhibiting particularly excellent water solubility.

[0032] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0033] Example 1 In this synthesis example, peptides were synthesized in which the N-terminal proline of each of the amino acid sequences shown in SEQ ID NOs: 1 to 8 was methylated, a peptide in which the amino acid sequence shown in SEQ ID NO: 1 (proline unmethylated), and a peptide in which the N-terminal proline of each of the amino acid sequences shown in SEQ ID NO: 9 was methylated. Peptide synthesis was outsourced to Greiner / Fasmax. The amino acid sequence shown in SEQ ID NO: 9 is an amino acid sequence in which threonine and alanine have been added to the C-terminus of the amino acid sequence shown in SEQ ID NO: 1. CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 1; SEQ ID NO: 1) CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (hereinafter referred to as peptide 2; SEQ ID NO: 2) CH3-Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 3; SEQ ID NO: 3) CH3-Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (hereinafter referred to as peptide 4; SEQ ID NO: 4) CH3-Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 5; SEQ ID NO: 5) CH3-Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (hereinafter referred to as peptide 6; SEQ ID NO: 6) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 7; SEQ ID NO: 7) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (hereinafter referred to as peptide 8; SEQ ID NO: 8) CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala-Thr-Ala (hereinafter referred to as peptide 9; SEQ ID NO: 9) Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 10; SEQ ID NO: 1) Amino acids were synthesized according to the standard 9-fluorenylmethoxycarbonyl (Fmoc) method.Specifically, Fmoc amino acids were activated with an HBTU / HOBT solution (HBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBT: 1-Hydroxybenzotriazole), and then condensed with N,N'-Diisopropylethylamine (DIEA). The synthesized amino acids were cleaved from the resin as follows: A TFA (trifluoroacetic acid) solution (4.125 mL TFA, 0.25 mL HO, 0.375 g phenol, 0.125 mL ethanedithiol, and 0.25 mL thioanisole) was prepared and added to the resin. The mixture was incubated at room temperature for 2 hours, and then precipitated with cold ether to obtain crude peptides. The resulting crude peptides were purified using RP-HPLC and lyophilized. The purity of the purified peptides was examined by HPLC and MS under the following conditions.・HPLC conditions A Buffer: 0.1% TFA / H2O, B Buffer: 0.1% TFA / Acetonitrile Column: SunFire C18 Column, 5 μm, 4.6 x 150 mm Flow rate: 1 mL / min Wavelength: 220 nm ・MALDI-TOF-MS.

[0034] The peptides 1 to 10 exhibited cell fusion activity and apoptosis-inducing activity against RFL cells or RM4 cells.

[0035] In this synthesis example, a polypeptide was synthesized in which a sugar chain was bound to the serine at position 4, the threonine at position 6, or the threonine at position 7 in the amino acid sequence represented by SEQ ID NO: 7, and a polypeptide was synthesized in which a sugar chain was bound to the threonine at position 6 in the amino acid sequences represented by SEQ ID NOs: 10 and 11. Peptide synthesis was outsourced to Peptide Institute, Inc., and the peptides were produced according to standard methods. CH3-Pro-Ile-Val-Ser(GalNAcα)-Gln-Thr-Thr-Ala-Ile-Ala (peptide 7-GalNAc4; SEQ ID NO: 7) CH3-Pro-Ile-Val-Ser-Gln-Thr(GalNAcα)-Thr-Ala-Ile-Ala (peptide 7-GalNAc6; SEQ ID NO: 7) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr(GalNAcα)-Ala-Ile-Ala (peptide 7-GalNAc7; SEQ ID NO: 7) CH3-Pro-Ile-Val-Ser-Gln-Thr(-α-O-Man)-Thr-Ala-Ile-Ala (peptide 7-Man6; SEQ ID NO: 7) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr(GlcNAcα)-Ala-Ile-Ala (peptide 7-GlcNAc7; SEQ ID NO: 7) CH3-Pro-Ile-Val-Ser-Gln-Thr(GalNAcα)-Thr-Gly-Ile-Ala (peptide 11-GalNAc6; SEQ ID NO: 10) CH3-Pro-Ile-Val-Ser-Gln-Thr(GalNAcα)-Thr-Ala-Ile-Gly (peptide 12-GalNAc6; SEQ ID NO: 11)

[0036] Similarly, for peptides 1 to 6 and peptide 8, polypeptides can be produced in which a sugar chain is bound to the fourth serine, the sixth threonine, or the seventh threonine in the amino acid sequence.

[0037] The five peptides, peptide 7, peptide 11, and peptide 12, exhibited cell fusion activity and apoptosis-inducing activity against RFL cells or RM4 cells.

[0038] Example 3 In this example, the antiviral effect of the peptide 7-GalNAc7 against coronavirus was examined. Human iPS cell lines (HILC: HiLung Co., Ltd.) were used and differentiated into lung progenitor cells using a stepwise differentiation induction method. The progenitor cells were purified using a cell separation method based on labeling with a specific surface antigen, carboxypeptidase M, and differentiated into mature airway epithelial cells using an air-liquid interface culture method in airway epithelial differentiation medium. SARS-CoV-2 (Omicron strain, BA.1) was used as the virus. Peptide 7-GalNAc7 was adjusted to 10,000 μg / mL and added to the medium to a concentration of 100 μg / mL (100-fold dilution). Distilled water was added to the medium at a 100-fold dilution as a vehicle. Specifically, as shown in Figure 1, the "prophylactic administration pattern" involved adding the polypeptide on days -2, -1, and 0. In addition, in the "whole-period administration pattern," polypeptide was added on days -2, -1, 0, 1, 2, 3, and 4. The virus was diluted to an MOI of 0.5 and inoculated into the cells. The cells were incubated at 37°C for 2 hours. The virus solution was washed away, and the polypeptide was added on day 0. The medium was replaced with fresh medium every day until 4 days later, and culture supernatants on days 0, 1, 2, 3, and 4 were collected for plaque assay and stored at -80°C. Cells for fluorescent antibody testing were collected on days 0, 2, and 4 and fixed with 4% paraformaldehyde.

[0039] The plaque assay was performed using VeroE6 / TMPRSS2. Samples were serially diluted 10-fold with 2% FCS-supplemented DMEM (GIBCO), and 100 μL of each was inoculated into a 6-well plate and incubated at 37°C for 1 hour. The samples were removed and overlaid with 0.8% sea plaque agarose (Lonza)-supplemented DMEM (5% FBS-supplemented DMEM). After 3-4 days, plaques were visually counted and the viral titer was calculated. As shown in Figure 2 , in the prophylactic administration pattern, the addition of the polypeptide significantly suppressed viral load on days 1, 2, and 3. In addition, in the full-period administration pattern, the addition of the polypeptide significantly suppressed viral load on days 1 and 2.

[0040] The number of infected cells was measured using the fluorescent antibody method as follows. The medium in the insert and well was removed, and 600 μL of PBS(-) was added to the well side and 150 μL to the insert side to wash the cells. 500 μL of 4% paraformaldehyde was added to the well side and 100 μL to the insert, and the cells were left to stand at room temperature for 15 minutes to fix them. The 4% paraformaldehyde in the insert and well was removed, and 600 μL of PBS(-) was added to the well side and 150 μL to the insert side to wash the cells. This process was repeated three times. 600 μL of permeabilization solution (0.2% Triton X-100 / PBS(-)) was added to the well side through the gap between the insert and well, and 150 μL to the insert side, and the cells were left to stand at room temperature for 15 minutes to permeabilize the membrane. The permeabilization solution in the insert and well was removed, and 600 μL of PBS(-) was added to the well side and 150 μL to the insert side to wash the cells. This process was repeated twice. 600 μL of blocking solution (1% BSA, 5% Donley Serum PBS(-)) was added to the well side and 150 μL to the insert side, and the wells were left to stand at room temperature for 30 minutes for blocking. Fluorescent immunostaining was performed according to standard methods using the following primary and secondary antibodies. Fluorescence images were acquired using an all-in-one microscope BZ-X710 (KEYENCE). Quantitative analysis was performed using the analysis software (BZ-H3C / Hybrid Cell Count) provided with the BZ-X710. Each cell was compartmentalized using the EpCAM-positive portion of the cell membrane, and the number of SARS-CoV-2 Nucleocapsid-positive compartments was counted to calculate the percentage. As shown in Figure 3, addition of the polypeptide reduced the infected cell rate in both the "prophylactic administration pattern" and the "full-term administration pattern."

[0041] Example 4 In this example, the solubility of peptide 7 synthesized in Example 1, and peptides 7-GalNAc6, 7-GalNAc7, 7-Man6, and 7-GlcNAc7 synthesized in Example 2 was investigated. Each peptide was added to 1x PBS at a concentration of 50 mg / mL, dissolved by ultrasound, and centrifuged for 1 hour. The supernatant was diluted 10-fold with 1x PBS and analyzed by HPLC. As shown in Table 2, the sugar-linked polypeptides showed excellent solubility, especially the GalNAc-linked polypeptide.

[0042] The polypeptide of the present invention can be effectively used for the prevention and treatment of coronavirus infections.

Claims

1. (1) A polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO:1, the amino acid sequence represented by SEQ ID NO:2, the amino acid sequence represented by SEQ ID NO:3, the amino acid sequence represented by SEQ ID NO:4, the amino acid sequence represented by SEQ ID NO:5, the amino acid sequence represented by SEQ ID NO:6, the amino acid sequence represented by SEQ ID NO:7, and the amino acid sequence represented by SEQ ID NO:8, or (2) A polypeptide comprising an amino acid sequence in which one amino acid is substituted in the amino acid sequence represented by SEQ ID NO:1, the amino acid sequence represented by SEQ ID NO:2, the amino acid sequence represented by SEQ ID NO:3, the amino acid sequence represented by SEQ ID NO:4, the amino acid sequence represented by SEQ ID NO:5, the amino acid sequence represented by SEQ ID NO:6, the amino acid sequence represented by SEQ ID NO:7, or the amino acid sequence represented by SEQ ID NO:8, and having antiviral activity against coronaviruses, wherein the polypeptide has an O-glycoside-linked sugar chain.

2. A polypeptide having an O-glycoside-linked sugar chain according to claim 1, wherein the O-glycoside-linked sugar chain is N-acetylgalactosamine.

3. A polypeptide having an O-glycoside-linked sugar chain according to claim 1 or 2, wherein the polypeptide has a methyl group at the N-terminus.

4. A pharmaceutical composition for treating or preventing coronavirus infections, comprising the polypeptide having an O-glycoside-linked glycan according to claim 1 or 2 as an active ingredient.

5. A method for treating coronavirus infection, comprising administering an effective amount of the polypeptide of claim 1 or 2 to a subject in need of treatment.

6. The polypeptide of claim 1 or 2, for use in treating or preventing coronavirus infection.

7. Use of a polypeptide according to claim 1 or 2 in the manufacture of a pharmaceutical composition for the treatment or prevention of coronavirus infections.

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