WO2006025536A1 - Agent anti-virus du sras - Google Patents

Agent anti-virus du sras Download PDF

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Publication number
WO2006025536A1
WO2006025536A1 PCT/JP2005/016151 JP2005016151W WO2006025536A1 WO 2006025536 A1 WO2006025536 A1 WO 2006025536A1 JP 2005016151 W JP2005016151 W JP 2005016151W WO 2006025536 A1 WO2006025536 A1 WO 2006025536A1
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Prior art keywords
asp
group
leu
lys
glu
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PCT/JP2005/016151
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English (en)
Japanese (ja)
Inventor
Nobutaka Fujii
Akira Otaka
Naoki Yamamoto
Norio Yamamoto
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Kyoto University
Tokyo Medical And Dental University
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Publication of WO2006025536A1 publication Critical patent/WO2006025536A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to an anti-SARS virus agent, a polypeptide that can be used as a SARS onset preventive agent or a SARS therapeutic agent, and a method for preventing or treating SARS using the polypeptide.
  • SARS virus coronavirus that causes SARS (SARS-related coronavirus; hereinafter referred to as “SARS virus”) is a new type of virus, and an effective therapeutic agent for this virus has not yet been found.
  • SARS virus infects target cells by fusing its cell membrane with the cell membrane of the target cell. During this membrane fusion, the SARS virus is presumed to use a Spike-protein (hereinafter referred to as S-protein: SEQ ID NO: 1) which is classified as a type I membrane fusion promoting protein.
  • S-protein Spike-protein
  • S-protein has two regions with high ⁇ -helical properties (as shown in Fig. 1, they are often called HR1 and HR2 from the terminal side).
  • HR1 and HR2 from the terminal side.
  • the terminal side of the S-protein is anchored to the target cell membrane, and then the three HR1 interact to form a 3-helix bundle.
  • This three-strand a-helical coiled-coil is combined in antiparallel so that three HR2s surround the outside, forming a 6-helix bundle.
  • the SARS virus and the target cell membrane come close to each other and membrane fusion is established.
  • Non-Patent Documents 1 and 2 are examples of SARS virus S-protein HR2-derived peptides. Their anti-SARS virus activities are all at the ⁇ level and are not sufficient as antiviral agents.
  • Non-patent literature l Liu, S .; Xiao, G .; Chen, Y .; He, Y .; Niu, J .; Escalante, CR; Xiong, H .; Farmar, J .; Debnath, AK; Tien, P .; and Jiang, S. Lancet 2004, 363, 938—947.
  • Patent Literature 2 Bosch, BJ; Martina, BEE; van der Zee, R .; Lepault, J .; Hajiema, BJ Versluis, C; Heck, AJR; de Groot, R .; Osterhaus, ADME; and Rotti er, PJM Proc. Natl. Acad. Sci. USA 2004, 101, 8455-8460
  • the present invention relates to a polypeptide having an anti-SARS virus activity superior to that of a natural type, and an anti-SARS virus agent comprising the polypeptide or a pharmaceutically acceptable salt thereof as an active ingredient, or SARS.
  • the main object is to provide an agent for preventing onset or a therapeutic agent for SARS, and a method for preventing or treating SARS using the polypeptide.
  • the present invention relates to the following polypeptide; an anti-SARS virus agent comprising the polypeptide or a pharmaceutically acceptable salt thereof as an active ingredient; an agent for preventing the onset of SARS or an SARS therapeutic agent; using the polypeptide Provide a method for preventing or treating SARS.
  • A represents the same or different acidic amino acids
  • R 1 is H or acetyl group, propiol group, butyryl group, benzoyl group, benzyloxycarbonyl group, phthalyl group, formyl group, trifluoroacetyl group and benzyl group. Represents any one group selected from the group consisting of;
  • R 2a is a single bond
  • R 3a represents Ile-X 1 -X 1 -Ile-Asn-X 1 -X or a single bond;
  • R 4a represents Leu-X-X-Leu-Gin-X-X or a single bond
  • R 5a is a single bond
  • R 6 is OH, NH, methylamino group, dimethylamino group, ethylamino group, jetylamino group
  • R 3a is a single bond
  • R 2a represents a single bond
  • R 1 ⁇ 2 is a single bond
  • R 5a represents a single bond.
  • X represents the same or different acidic amino acids, and X represents the same or different basic amino acids.
  • R 1 is any one selected from the group consisting of H or acetyl group, propiol group, butyryl group, benzoyl group, benzyloxycarbonyl group, phthalyl group, formyl group, trifluoroacetyl group and benzyl group 1 Represents a group of
  • R 2b is a single bond
  • R 3b represents Ile-Ser-X-X-Asn-X-X or a single bond
  • R 4b represents Leu-lie-X-X-Gin-X-X or a single bond
  • R 5b is a single bond
  • R 6 is OH, NH, methylamino group, dimethylamino group, ethylamino group, jetylamino group
  • R 3b is a single bond
  • R 2b represents a single bond
  • R 4b is a single bond
  • R 5b represents a single bond
  • X represents the same or different acidic amino acids, and X represents the same or different basic amino acids.
  • R 1 is any one selected from the group consisting of H or acetyl group, propiol group, butyryl group, benzoyl group, benzyloxycarbonyl group, phthalyl group, formyl group, trifluoroacetyl group and benzyl group 1 Represents a group of
  • R 2e is a single bond
  • R 3c represents lie-X-X-lie-X-X-Ser or a single bond
  • R 4c represents Leu-X -X -Leu-X -X -Leu or a single bond
  • R 5e is a single bond or the following,
  • R 6 represents any one group selected from the group consisting of OH group, amide group, methylamino group, dimethylamino group, ethylamino group, jetylamino group, and benzylamino group; provided that when R 3e is a single bond, R 2e represents a single bond, and when R 4e is a single bond, R 5e represents a single bond))
  • a polypeptide comprising the amino acid sequence represented by
  • Item 4. selected from the group consisting of glutamic acid, aspartic acid and cysteic acid
  • Item 4 The polypeptide according to Item 1 to 3, which is any one selected from the group consisting of: Item 5. Below:
  • Item 8.An anti-SARS virus comprising the polypeptide according to any one of Items 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient together with a pharmaceutically acceptable carrier, excipient or diluent. Agent.
  • Item 9 A SARS syndrome comprising the polypeptide according to any one of Items 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient together with a pharmaceutically acceptable carrier, excipient or diluent. Prophylactic or SARS treatment.
  • Item 10 A method for preventing or treating SARS, comprising administering an effective amount of the polypeptide according to any one of Items 1 to 3 or a pharmaceutically acceptable salt thereof as an anti-SARS virus agent.
  • the polypeptides described in the above items 1 to 10 refer to SR9 variants.
  • the peptide in the HR2 region that forms an a-helix has a surface that interacts with the HR1 region (hereinafter sometimes referred to as the interaction surface) and a surface that contacts the solvent (hereinafter referred to as the solvent contact surface).
  • the residue required for interaction with HR1 via a-helix formation (denoted as X) is presented on the interaction surface.
  • the present inventors believe that stabilization of ⁇ -helix structure will ultimately increase antiviral activity, and if ⁇ -helix formation promoting function is added to the solvent contact surface of HR2 peptide, stronger antiviral activity is achieved. We anticipated that we could create a peptide that we had.
  • an ⁇ -helix structure is composed of a repeating structure of 7 amino acid residues (heptad repeat sequence). Therefore, the present inventors have added the HR2 peptide to X-X-X-X-X-X-X (X is a residue that interacts with the HR1 region, and the native sequence is left as it is.
  • X and X are hydrophilic amino acids
  • A is an acidic amino acid and X
  • ABB is a basic amino acid
  • X residues gather on one side to form an interaction surface, and the other side It was thought that hydrophilic amino acid residues gathered together to form a solvent contact surface (see Fig. 2).
  • X is the same as the natural sequence, and is an amino acid residue centered on hydrophobicity. That is, X is considered to be continuously arranged in the ⁇ -helix three-dimensional structure to form a hydrophobic surface (corresponding to an interaction surface) as a whole.
  • hydrophilic amino acids X
  • a derivative containing a 7-amino acid repeat sequence was synthesized so that three Xs in this frame constitute an interaction surface, and the activity of the derivative was synthesized.
  • the residues necessary for the activity expression that is, the amino acid residues constituting the interaction surface can be identified.
  • This method is different from conventional methods used in peptide structure-activity relationship studies such as the Ala scan and D-amino acid scan to capture the active site as a point. Methodology. Below, using this X-X-X-X-X-X-X-X-X frame, it has an ⁇ -helix structure.
  • LearnCoi VMF published by PS Kim et al. (J. Mol. Biol. 1999, 290, 1031-1041) based on the amino acid sequence of SARS coronavirus S-protein.
  • SR9 35 amino acid residues 1151-1185 were predicted (see Fig. 1).
  • this 35 amino acid residue is referred to as SR9.
  • LearnCoil -VMF is a software that predicts the secondary structure of the HR1 and HR2 regions based on the tendency to take a heptad repeat sequence when the HR1 and HR2 regions form an ⁇ -helix. .
  • the present inventors confirmed anti-SARS virus activity for the SR9 peptide they found that this peptide had anti-SARS virus activity.
  • the secondary structure of the HR1 region and the HR2 region can be predicted to some extent, but sufficient information on residues necessary for the interaction cannot be obtained. Therefore, the present inventors modified SR9 peptide based on X-X-X-X-X-X-X scan.
  • the body (SR9EK1-3) was created, and it was found that these variants had higher antiviral activity than the natural type.
  • D, A and E are aligned with the same amino acid as the natural type, forming a hydrophobic surface (interaction surface) in the helical structure.
  • the amino acids located at B and C are acidic amino acids
  • the amino acids located at F and G are basic amino acids.
  • a solvent contact surface is formed by the amino acids located at B, F, C and G. That is, the first variant is represented in FIG. 4A.
  • ⁇ , ⁇ and B have the same amino acids as the natural type, forming a hydrophobic surface (interaction surface) in the helical structure.
  • the amino acids located at F and G are acidic amino acids
  • the amino acids located at C and D are salts. It is a basic amino acid.
  • a solvent contact surface is formed by amino acids located at F, C, G, and D. That is, the second variant is represented in FIG. 4B.
  • G, D and A have the same amino acids as the natural type, forming a hydrophobic surface (interaction surface) in the helical structure.
  • the amino acids located at E and F are acidic amino acids
  • the amino acids located at B and C are basic amino acids.
  • a solvent contact surface is formed by the amino acids located at E, B, F and C. That is, the third variant is represented in FIG. 4C.
  • the acidic amino acid (X) is preferably glutamic acid or aspartic acid.
  • cystic acid or the like more preferably glutamic acid or aspartic acid, and particularly preferably glutamic acid.
  • the basic amino acid (X) is preferably lysine.
  • Arginine, orthine histidine, etc., more preferably lysine, arginine or orthine, still more preferably lysine or arginine, particularly preferably lysine.
  • the combination of dartamic acid and lysine is more preferable.
  • 1151 isoleucine (or 1158 Norin) is located at position A.
  • variants When variants are produced, these variants are represented by the amino acid sequences shown in Tables 1 to 8 below.
  • X and X described in the amino acid sequence are hydrophilic amino acids, and X is the same or different.
  • X represents the same or different basic amino acids.
  • A, B, C, D, E, F, and G correspond to A, B, C, D, E, F, and G in FIG. 4A.
  • R 1 is a hydrogen atom (H) derived from the N-terminal amino acid of SARS virus S-protein, or a acetyl group, propiol group, butyryl group, benzoyl group, benzyloxycarbon group, A phthalyl group, a formyl group, a trifluoroacetyl group, a benzyl group, and the like, preferably a acetyl group, a propionyl group, a petityl group, a benzoyl group, a phthalyl group, a formyl group, and a trifluoroacetyl group, more preferably It is a acetyl group.
  • H hydrogen atom
  • R 2a is any one of 7 to 7 amino acid residues (Pro-Asp-Va Asp-Leu-Gly-Asp) located at 1144 to 1150 of SARS virus S-protein. It may be.
  • the amino acid residue of R 2 a may be modified according to the same rules as in Table I above. That is, for R 2a :
  • R 2a represents a single bond
  • R 1 may be directly bonded to the N terminus of R 3 a .
  • R 3a is amino acids 1151-1157 of the SARS virus S-protein or lie-X-X
  • Single amino acid or single bond preferably Ile-X -X -Ile-Asn-X -X or single bond
  • R 1 ⁇ 2 is 1179 to 1185 amino acids of SARS virus S-protein or Leu-X ⁇
  • R 5a may be any of 1 to 15 of 15 amino acid residues located at 1186 to 1200 of the SARS virus S-protein. Of these 15 amino acid residues, amino acids located at 1186 to 1192 may be modified according to the same rules as in Table I above. However, the amino acid sequence from 1186 to 1200 of S-protein contains a lot of aromatic amino acids such as Trp and Tyr. In general, aromatic amino acids have the property of easily binding to cell membranes. Therefore, it is preferable to add the natural sequence as it is. Addition of such an aromatic cluster to the SR9 peptide variant is thought to be advantageous in terms of the interaction with the SARS virus cell membrane. That is, as R 5a : Gly- (Lys or X)-(Tyr or X)-Glu- Gin- (Tyr or X)-(lie or X)-Lys- Trp- Pro- Tr
  • any peptide or amino acid of 1 to 15 starting from Gly at the N-terminus may be mentioned.
  • R 5a represents a single bond, and R 6 may be directly bonded to the C-terminal amino acid of R 1 ⁇ 2 .
  • R 6 is OH derived from the C-terminal amino acid, NH, methylamino group, dimethyla
  • R 3a when R 3a is a single bond, R 2a is preferably a single bond.
  • R 1 ⁇ 2 is a single bond, R 5a is preferably a single bond.
  • A, B, C, D, E, F, and G correspond to A, B, C, D, E, F, and G in FIG. 4B.
  • R 1 represents a hydrogen atom (H) derived from an N-terminal amino acid, or a acetyl group, propiol group, butyryl group, benzoyl group, benzyloxycarbol group, phthalyl group, A formyl group, a trifluoroacetyl group, a benzyl group, and the like, preferably a acetyl group, a propionyl group, a butyryl group, a benzoyl group, a phthalyl group, a formyl group, and a trifluoroacetyl group, more preferably a acetyl group.
  • R 2b is one of 7 to 7 amino acid residues (Pr 0- Asp- Va As Asp- Leu- Gly- Asp) located at 1144 to 1150 of SARS virus S-protein. It may be.
  • the amino acid residues 1 to 7 of R 2b may be modified according to the same rules as in Table II above. Snow B, as follows:
  • the peptide or amino acid represented by these is mentioned, Preferably, it is Leu-X-X. Or
  • R 2b represents a single bond, and R 1 may be directly bonded to the i ⁇ N end.
  • R 3b is the amino acid 1151-1157 of SARS virus S-protein or the terminal Ser of the 7 amino acid sequences represented by lie-Ser-X-X-Asn-X-X Count from X 1 ⁇
  • R 4b is 1179 to 1185 amino acids of SARS virus S-protein or 7 amino acid sequences represented by Leu-Ile-X-X-Gin-X-X, counted from Leu. 1-7 amino acids
  • it is a single bond, preferably Leu-Ile-X-X-Gln-X-X or a single bond.
  • R 5b may be any of 1 to 15 of 15 amino acid residues located at 1186 to 1200 of the SARS virus S-protein. These 15 amino acid residues have the property of easily binding to the cell membrane, so it is preferable to add them as they are in the natural sequence. However, for the amino acids located at 1186 to 1189, follow the same rules as in Table II above. It is preferable to modify Yes. That is, for R bb :
  • any peptide or amino acid sequence of 1 to 15 starting from N-terminal Gly is mentioned, preferably Gly-Lys-X-X.
  • R 5b represents a single bond
  • R 6 is
  • R 6 is OH derived from the C-terminal amino acid, NH, methylamino group, dimethyla
  • Mino group ethylamino group, jetylamino group, benzylamino group, etc.
  • NH is more preferable.
  • R 3b when R 3b is a single bond, R 2b is preferably a single bond.
  • R 4b when R 4b is a single bond, R 5b is preferably a single bond.
  • A, B, C, D, E, F, and G correspond to A, B, C, D, E, F, and G in FIG. 4C.
  • R 1 represents a hydrogen atom (H) derived from 1151 isoleucine of SARS virus S-protein, or a acetyl group, propiol group, butyryl group, benzoyl group, benzoxycarbon group, A phthalyl group, a formyl group, a trifluoroacetyl group, a benzyl group, and the like, preferably a acetyl group, a propionyl group, a petityl group, a benzoyl group, a phthalyl group, a formyl group, and a trifluoroacetyl group, more preferably In the acetyl group is there.
  • H hydrogen atom
  • R is a deviation from 1 to 7 of 7 amino acid residues (Pr 0- Asp- Va ⁇ Asp- Leu- Gly- Asp) located at 1144 to 1150 of SARS virus S-protein.
  • This amino acid residue of R 2c may be modified according to the same rules as in Table III above. That is, for R:
  • R 2e represents a single bond
  • R 1 may be directly bonded to the R 3 c N terminal.
  • R 3c is the amino acid 1151-1157 of the SARS virus S-protein or Ile-X ⁇
  • R 4e represents amino acids 1179 to 1185 of the SARS virus S-protein or Leu-X
  • R 5c may be any of 1 to 15 of 15 amino acid residues located at 1186 to 1200 of the SARS virus S-protein. Since these 15 amino acid residues have the property of easily binding to the cell membrane, it is preferable to add them in their native sequence, but the amino acids located at 1186 to 1192 are modified according to the same rules as above. It is preferable. That is, for R 5e :
  • any one of 1 to 15 peptides or amino acids starting from N-terminal Gly is mentioned, preferably Gly-X-X or Gly-X-X-Glu-X-X-lie.
  • R 5 represents a single bond, and R 6 may be directly bonded to the C-terminal amino acid of R 1 ⁇ 2 .
  • R 6 is OH derived from the C-terminal amino acid, NH, methylamino group, dimethyla
  • R 3e when R 3e is a single bond, R 2e is preferably a single bond.
  • R 4e when R 4e is a single bond, R 5e is preferably a single bond.
  • the proline located at 1144 of the SARS virus S-protein should not be used if there is a possibility that the a-helix structure may be destroyed by use. If there is no possibility of destruction, it can be used. Further, this proline may be substituted with a hydrophobic amino acid such as isoleucine or leucine.
  • the modified SR9 peptide of the present invention preferably has 21 to 42 amino acids, more preferably 35 to 42 residues.
  • SR9 peptide variant strength When composed of 21 amino acid residues, it is preferably an amino acid strength located in SARS virus S-proteins 1158 to 1178, modified according to the laws of the first to third variants.
  • SR9 peptide variant When composed of 28 amino acid residues, from the amino acids located in SARS virus S-protein 1151-1178 or 1158-1185 modified according to the rules of the first to third variants It is preferable to become.
  • the ⁇ -helix part preferably contains an amino acid sequence obtained by modifying 35 amino acid residues located in SARS virus S-protein 1151-1185. That's right.
  • the amino acid used in the modified form of the present invention is preferably L form (L amino acid), but D form may also be used.
  • D form it is preferable to convert all optically active amino acids to the D form.
  • modified SR9 (SR9EK1-3) according to the present invention include the following amino acid sequences (Ac represents a acetyl group).
  • the N-terminal is acetylated and the C-terminal is amidated, because a stable ⁇ -helix structure can be formed.
  • the heel end and the C terminus are preferably not bonded to each other and are acyclic.
  • one or two variants of the present invention form a trimer with two or one HR2 peptide and inhibit membrane fusion of SARS virus and target cells. This may prevent infection with SARS virus.
  • the variant of the present invention can be produced by a known polypeptide synthesis method, particularly a liquid phase synthesis method or a solid phase synthesis method.
  • the DNA encoding the variant of the present invention is inherited. Child recombination technology
  • It can also be synthesized by a method of introducing it into a host cell by surgery and expressing the target protein.
  • the carboxyl group of an N-protected amino acid in which the amino group of the amino acid corresponding to the C-terminus is protected with a urethane-type protecting group such as a 9-fluorenylmethyloxycarboxyl (Fmoc) group Is bound to an insoluble rosin having an amino group.
  • a urethane-type protecting group such as a 9-fluorenylmethyloxycarboxyl (Fmoc) group
  • Fmoc 9-fluorenylmethyloxycarboxyl
  • the insoluble resin having an amino group is not particularly limited, but Fmoc-NH-SA L resin (4- (2 ', 4'-dimethoxyphenol-Fmoc-aminoethyl) Preference is given to phenoxy linker oil.
  • the desired product can be obtained directly by cleavage.
  • An amino acid having a protecting group can be used for the synthesis of the modified product of the present invention.
  • the protected amino acid can be obtained by protecting the functional group with a known protecting group by a known method, and a commercially available protected amino acid can also be used.
  • the protecting group known ones can be used.
  • an Fmoc group or the like can be used as described above.
  • a known method such as DIPCDI (diisopropylcarposimide) -HOBt (1-hydroxybenzotriazole) method can be used.
  • the condensation reaction can be carried out in a known solvent, and examples thereof include organic solvents such as dimethylformamide.
  • the reagent for removing the protecting group of the amino group is not particularly limited, and examples thereof include known reagents such as pipericin Z dimethylformamide, and such a reagent can cleave protecting groups such as the Fmoc group. .
  • the degree of progress of the condensation reaction at each stage of the synthesis can be confirmed by a known method such as a -hydrin reaction method.
  • the modified product of the present invention thus obtained can be obtained by publicly known methods such as extraction, recrystallization, various chromatographies (gel filtration, ion exchange, distribution, adsorption, etc.), electrophoresis, countercurrent distribution, and the like. Isolation and purification can be carried out by means, and isolation and purification by reverse phase high performance liquid chromatography is particularly preferred.
  • the present invention comprises an anti-SARS virus agent containing a pharmaceutically acceptable carrier, excipient, diluent, etc., containing as an active ingredient a variant obtained by the above method or a pharmaceutically acceptable salt thereof, To provide a preventive agent for SARS or a therapeutic agent for SARS.
  • Variants of the present invention also include pharmaceutically acceptable salts thereof.
  • such salts include nontoxic alkali metal salts such as sodium, potassium, lithium, calcium and magnesium, alkaline earth metal salts and the like prepared by methods well known to those skilled in the art.
  • the above salts include non-toxic acid addition salts obtained by reacting the modified product of the present invention with an appropriate organic acid or inorganic acid.
  • acid addition salts include hydrochloride, hydrobromide, sulfate, acetate, valerate, laurate, lactate, phosphate, p-toluenesulfonate (tosylate), and kenate. Maleate, fumarate, succinate, tartrate, glycolate, benzenesulfonate and methanesulfonate.
  • the anti-SARS virus agent, the SARS onset prevention agent, or the SARS therapeutic agent of the present invention contains a pharmacologically effective amount of the above-mentioned variant as an active ingredient, and contains this together with a suitable pharmaceutical carrier or diluent.
  • a pharmaceutical composition or a pharmaceutical preparation is included.
  • two or more of the variants obtained by the above methods may be used in combination as an active ingredient!
  • Examples of the pharmaceutical carrier that can be used in the pharmaceutical composition include a filler, a bulking agent, a binder, a disintegrant, a surfactant, and a lubricant that are usually used depending on the use form of the preparation.
  • examples of such diluents or excipients may be appropriately selected and used depending on the dosage unit form of the resulting preparation.
  • Various preparation forms can be selected according to the purpose of treatment, and typical ones are tablets, pills, powders, solutions, suspensions, capsules, suppositories, injections (solutions, suspensions). , Sprays, aerosols, inhalants, sustained-release microcapsules Illustrative examples of such agents.
  • Particularly preferred pharmaceutical preparations of the present invention include various components that can be used in normal protein preparations, such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH adjusters, interfaces. It is prepared by appropriately using an activator, phospholipid and the like.
  • Examples of the stabilizer include human serum albumin, ordinary amino acids, saccharides, cellulose derivatives and the like, and these can be used alone or in combination with a surfactant or the like. In particular, according to this combination, the stability of the active ingredient may be further improved.
  • the amino acid is not particularly limited, and may be any of glycine, cysteine, glutamic acid and the like.
  • sugar examples include, but are not limited to, monosaccharides such as glucose, mannose, galactose, and fructose, sugar alcohols such as mannitol, inositol, and xylitol, disaccharides such as sucrose, maltose, and lactose, and dextran.
  • monosaccharides such as glucose, mannose, galactose, and fructose
  • sugar alcohols such as mannitol, inositol, and xylitol
  • disaccharides such as sucrose, maltose, and lactose
  • dextran examples include, but are not limited to, monosaccharides such as glucose, mannose, galactose, and fructose, sugar alcohols such as mannitol, inositol, and xylitol
  • disaccharides such as sucrose, maltose, and lactose
  • dextran examples include, but are not limited
  • any of anionic surfactant, cationic surfactant, or non-ionic surfactant can be used without particular limitation.
  • polyoxyethylene glycol sorbitan alkyl ester polyoxyethylene alkyl Ether type, sorbitan monoacyl ester type, fatty acid glyceride type and the like can be used.
  • Examples of the cellulose derivative that can be used include, but are not limited to, methylcellulose, ethylcellulose, hydroxyethinoresenorelose, hydroxypropinoresenorelose, hydroxypropinoresethylcellulose, sodium carboxymethylcellulose, and the like.
  • the addition amount of the saccharide is suitably about O.OOlmg or more, preferably about 0.01 to 10 mg per 1 ⁇ g of the active ingredient.
  • the amount of the surfactant added is about O.OOOOlmg or more, preferably about 0.0001 to 0.01 mg, per l / zg of the active ingredient.
  • the amount of human serum albumin added is suitably about O.OOlmg or more, preferably about 0.001 to 0.1 mg, per 1 g of active ingredient.
  • the amino acid is suitably about 0.001 to 10 mg per gram of active ingredient.
  • the amount of added calories of the cellulose derivative is about O.OOOOlmg or more, preferably about 0.001 to 0.1 mg per 1 ⁇ g of the active ingredient. A range is appropriate.
  • the amount of the peptide variant as an active ingredient contained in the pharmaceutical preparation of the present invention is a force appropriately selected from a wide range. Usually about 0.00001 to 70% by weight, preferably about 0.0001 to 20% by weight The amount is preferably about 0.0001 to 10% by weight, more preferably about 0.0001 to 5% by weight.
  • buffering agents examples include phosphoric acid, acetic acid, taenoic acid, ⁇ - aminocaproic acid, glutamic acid and sputum or a salt corresponding thereto (for example, alkali metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, and the like) Examples thereof include alkaline earth metal salts).
  • alkali metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, and the like
  • alkaline earth metal salts examples of the isotonic agent include sodium chloride, potassium chloride salt, saccharides, glycerin and the like.
  • chelating agents examples include sodium edetate and citrate.
  • the pharmaceutical preparation of the present invention can be used as a solution preparation.
  • it is dissolved in a buffer solution containing water, saline, etc. at the time of use, and adjusted to an appropriate concentration before use. It is also possible to do.
  • the pharmaceutical preparation of the present invention may be a solid dosage form such as a tablet, pill, powder, powder, granule or capsule, or a liquid dosage form such as a solution, suspension, emulsion, syrup or elixir. May be prepared. These are further classified into oral preparations, parenteral preparations, nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments, etc., depending on the administration route, and can be prepared, molded or prepared according to ordinary methods. it can.
  • lactose sucrose, sodium chloride sodium, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, caustic acid, potassium phosphate, etc. are used as the above-mentioned preparation carrier.
  • Excipient water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polybulurpyrrolidone and other binders, sodium carboxymethylcellulose, carboxymethylcellulose calcium, low Degree of substitution Hydroxypropyl cellulose, dried starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, disintegrator, polyoxyethylene sorbitan fat Absorption promotion of surfactants such as fatty acid esters, sodium lauryl sulfate, monoglycerides of stearic acid, disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, quaternary ammonia base, sodium lauryl sulfate Moisturizers such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal key acid, lubricants such as purified
  • the tablets can be made into tablets with ordinary coatings, if necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, or double tablets or multilayer tablets.
  • excipients such as glucose, lactose, denpun, cacao butter, hydrogenated vegetable oil, kaolin, talc, gum arabic powder, tragacanth powder, gelatin, ethanol, etc.
  • binders, disintegrants such as laminaran and agar.
  • Capsules are prepared according to conventional methods. Usually, the active ingredient of the present invention is mixed with the various pharmaceutical carriers exemplified above and filled into hard gelatin capsules, soft capsules and the like.
  • Liquid dosage forms for oral administration include conventional inert diluents such as pharmaceutically acceptable solutions, emulsions, suspensions, syrups, elixirs, etc., including water, and wetting agents, Auxiliaries such as emulsions and suspensions can be included, and these are prepared according to conventional methods.
  • liquid dosage forms for parenteral administration such as sterile aqueous or non-aqueous solutions, emulsions, suspensions, etc.
  • diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol, ethoxy Vegetable oils such as hydrogenated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester and olive oil can be used, and injectable organic esters such as ethyl oleate can be added.
  • solubilizers, buffering agents, wetting agents, emulsifiers, suspending agents, preservatives, dispersing agents and the like can be added to these. Sterilization can be achieved, for example, by bacteria retention
  • bactericide irradiation treatment and heat treatment. They should also be dissolved in sterile water or an appropriate sterilizable medium immediately before use. It can also be prepared in the form of a sterilized solid composition.
  • polyethylene glycol, cocoa butter, higher alcohol, esters of higher alcohol, gelatin, semi-synthetic dalyceride and the like can be used as a pharmaceutical carrier.
  • a composition for spraying, aerosol, inhalation, nasal or sublingual administration can be prepared according to a conventional method using a well-known standard excipient.
  • the pharmaceutical preparation of the present invention may contain a colorant, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals as necessary.
  • the administration method of the above pharmaceutical preparation is determined according to various preparation forms that are not particularly limited, the age, sex and other conditions of the patient, the degree of disease, and the like.
  • tablets, pills, solutions, suspensions, emulsions, granules, and capsules are administered orally, and injections are administered intravenously alone or mixed with normal fluids such as glucose and amino acids.
  • it is administered intramuscularly, intradermally, subcutaneously or intraperitoneally, suppositories are administered rectally, vaginal agents are administered intravaginally, nasal agents are administered intranasally, and sublingual agents are administered orally.
  • the dosage of the above pharmaceutical preparation is not particularly limited and is appropriately selected from a wide range according to the desired therapeutic effect, administration method, treatment period, patient age, sex, and other conditions.
  • the dosage is about 0.01 ⁇ g to 100 mg, preferably about 0.1 mg to 10 mg per 1 kg body weight per day, and the preparation is divided into once or several times a day. Can be administered.
  • the anti-SARS virus agent, SARS onset preventive agent or SARS therapeutic agent of the present invention is a variant of a polypeptide obtained by predicting the amino acid sequence of the HR2 peptide of SARS virus. Effective for infections caused by viruses.
  • the anti-SARS virus agent of the present invention can also be applied to coronaviruses other than SARS virus. it can.
  • the SARS preventive agent or SARS therapeutic agent, or the anti-SARS virus agent of the present invention is bowed by infection with coronaviruses such as nasal cold (nasal discharge, nasal congestion, sneezing, etc.) in addition to SARS. I can be widely applied to disease caused.
  • coronaviruses such as nasal cold (nasal discharge, nasal congestion, sneezing, etc.) in addition to SARS.
  • coronaviruses such as nasal cold (nasal discharge, nasal congestion, sneezing, etc.) in addition to SARS.
  • XX -X -XXX -X scan used to obtain the variant of the present invention was used.
  • antiviral agents can be made relatively quickly against viruses that rapidly spread by mutation.
  • the viral gene is decoded by a conventionally known method, and then the amino acid region relating to membrane fusion. Confirm. Then use the X-X -X -X-X-X -X scan for the peptide region sequence.
  • antiviral agents By synthesizing tides and evaluating antiviral activity, antiviral agents can be obtained.
  • a polypeptide having an extremely high anti-SARS virus activity, an anti-SARS virus agent and a SARS onset-preventing agent or SARS therapeutic agent comprising the polypeptide as an active ingredient, and the polypeptide are used. It was possible to provide a method for preventing or treating SARS.
  • the variant of the present invention has a high antiviral activity against other coronaviruses other than SARS virus. May have.
  • the amino acid sequence information (SEQ ID NO: 1) of SARS virus S-protein was obtained from CDC (US Department of Health and Welfare, Center for Disease Control and Prevention). The sequence was predicted using LearnCon-VMF, and the possible sequence (SR9) was derived as the HR2 region.
  • SR9 35-residue peptide covering the HR2 region was synthesized.
  • XX -X -XXX -X scan SR9 EK1 (SEQ ID NO: 6), SR9EK2 (SEQ ID NO: 7) and SR9EK3 (SEQ ID NO: 8) were designed and peptide synthesis was performed.
  • Fmoc-NH-Rink-ArgoGel resin (ARGONAUT TECHNOLOGY INC.), A protected peptide coagulant was constructed by the usual Fmoc type solid phase synthesis method.
  • the N-terminal was acetylated by stirring acetic anhydride (10 eq.) And pyridine (10 eq.) In a DMF solvent for 2 hr.
  • TT assay it means the concentration of the compound necessary to suppress the cytopathic effect of virus infection by 90% of the control value, and in the case of real time RT-PCR, the amount of virus is suppressed by 90% of the control value.
  • concentration of the compound needed to and EC (MT
  • T assay it means the concentration of the compound necessary to suppress the cytopathic effect due to virus infection by 99% of the control value, and in the case of real time RT-PCR, the amount of virus is the control value of 99%.
  • % Means the concentration of the compound necessary to suppress).
  • SR9EK1 is several tens to one hundred times higher than SR9 (natural SR9) and has antiviral activity.
  • SR9EK2 also has antiviral activity several times to ten times or more than SR9.
  • SR9EK3 Although the antiviral activity of SR9EK3 is lower than that of SR9EK1 and SR9EK2, it has about 1 to 2 times the activity of SR9EK3.
  • FIG. 6 is a schematic diagram showing the positional relationship of SR9.
  • FIG. 2 shows the interaction surface and solvent contact surface of the HR2 peptide variant.
  • FIG. 3 is a diagram showing the positional relationship of 7 amino acid residues in an a-helix.
  • [4A] A diagram showing the structure of the first variant. In the figure, black circles represent amino acid residues (X residues) that interact with HR1, white circles represent acidic amino acids, and squares represent basic amino acids.
  • [4B] A diagram showing the structure of the second variant. In the figure, black circles represent amino acid residues (X residues) that interact with HR1, white circles represent acidic amino acids, and squares represent basic amino acids.
  • [4C] A diagram showing the structure of the third variant. In the figure, black circles represent amino acid residues (X residues) that interact with HR1, white circles represent acidic amino acids, and squares represent basic amino acids.
  • SEQ ID NO: 1 is the amino acid sequence of the S-protein of SARS virus.
  • SEQ ID NO: 2 is the amino acid sequence of SR9.
  • SEQ ID NO: 3 is the amino acid sequence set forth in Table I.
  • SEQ ID NO: 4 is the amino acid sequence set forth in Table II.
  • SEQ ID NO: 5 is the amino acid sequence set forth in Table III.
  • SEQ ID NO: 6 is the amino acid sequence of SR9EK1.
  • SEQ ID NO: 7 is the amino acid sequence of SR9EK2.
  • SEQ ID NO: 8 is the amino acid sequence of SR9EK3.
  • SEQ ID NO: 9 is a SARS virus ORF-1 forward primer.
  • SEQ ID NO: 10 is a SARS virus ORF-1 reverse primer.
  • SEQ ID NO: 11 is a DNA probe used in real time RT-PCR.

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Abstract

La présente invention a pour objet un polypeptide d’activité anti-virus du SRAS supérieure à celle de composés naturels, obtenu par un balayage de type X-XA-XA-X―X-XB-XB en considérant la séquence d'acides aminés du peptide HR2 du virus du SRAS. La présente invention décrit également un agent anti-virus du SRAS contenant ce polypeptide ou un sel de qualité pharmaceutique de ce dernier en tant que composant actif, ainsi qu’une méthode de traitement prophylactique ou thérapeutique utilisant ledit polypeptide.
PCT/JP2005/016151 2004-09-02 2005-09-02 Agent anti-virus du sras WO2006025536A1 (fr)

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JP2008063275A (ja) * 2006-09-07 2008-03-21 Kyoto Univ 抗ｆｉｖ剤
WO2008050830A1 (fr) * 2006-10-25 2008-05-02 Kyoto University Agent anti-vih
CN111732637A (zh) * 2020-05-25 2020-10-02 上海交通大学 抑制新型冠状病毒SARS-CoV-2感染宿主细胞的多肽及其应用
CN113908254A (zh) * 2021-10-19 2022-01-11 山西锦波生物医药股份有限公司 一种干粉吸入剂及其制备方法和用途
CN114437184A (zh) * 2021-08-16 2022-05-06 中国科学院微生物研究所 一种抗新型冠状病毒的多肽及其应用

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CN108822190B (zh) * 2018-05-23 2021-11-09 中国人民解放军军事科学院军事医学研究院 多肽及其药物组合物和用途
KR20240039156A (ko) 2021-08-20 2024-03-26 가부시끼가이샤 레조낙 바이러스 감염 억제제

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008063275A (ja) * 2006-09-07 2008-03-21 Kyoto Univ 抗ｆｉｖ剤
WO2008050830A1 (fr) * 2006-10-25 2008-05-02 Kyoto University Agent anti-vih
CN111732637A (zh) * 2020-05-25 2020-10-02 上海交通大学 抑制新型冠状病毒SARS-CoV-2感染宿主细胞的多肽及其应用
CN114437184A (zh) * 2021-08-16 2022-05-06 中国科学院微生物研究所 一种抗新型冠状病毒的多肽及其应用
WO2023020298A1 (fr) * 2021-08-16 2023-02-23 中国科学院微生物研究所 Polypeptide pour le nouveau coronavirus résistant et son utilisation
CN113908254A (zh) * 2021-10-19 2022-01-11 山西锦波生物医药股份有限公司 一种干粉吸入剂及其制备方法和用途
CN113908254B (zh) * 2021-10-19 2024-05-28 山西锦波生物医药股份有限公司 一种干粉吸入剂及其制备方法和用途

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