WO2023182185A1 - Peptide having affinity for and/or inhibitory function against gingipains, and use thereof - Google Patents

Peptide having affinity for and/or inhibitory function against gingipains, and use thereof Download PDF

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Publication number
WO2023182185A1
WO2023182185A1 PCT/JP2023/010461 JP2023010461W WO2023182185A1 WO 2023182185 A1 WO2023182185 A1 WO 2023182185A1 JP 2023010461 W JP2023010461 W JP 2023010461W WO 2023182185 A1 WO2023182185 A1 WO 2023182185A1
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amino acid
peptide
acid sequence
gingipain
derivative
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PCT/JP2023/010461
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French (fr)
Japanese (ja)
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幸一郎 北村
昼也 吉田
功 大塚
充訓 原田
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メスキュージェナシス株式会社
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Publication of WO2023182185A1 publication Critical patent/WO2023182185A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • 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
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the present invention relates to peptides having affinity and/or inhibitory ability for gingipains and uses thereof. More specifically, the present invention provides a peptide having affinity and/or inhibitory ability for gingipain, a gingipain inhibitor, a gingivalis growth inhibitor, an oral composition, and a method for detecting gingipain using the peptide. Regarding.
  • Porphyromonas gingivalis is a periodontal pathogen that is considered important in the onset and progression of periodontal disease, and gingipain, a type of protease produced by Porphyromonas gingivalis, is recognized as an important factor in periodontal disease. has been done.
  • gingipains with different peptide cleavage site specificities, specifically, arginine-gingipain (Rgp), which cleaves the C-terminal side of arginine residues, and lysine-gingipains (Rgp), which cleaves the C-terminal side of lysine residues.
  • arginine-gingipain Rgp
  • lysine-gingipains Rgp
  • Kgp lysine-gingipains
  • gingipain inhibitors are expected to be an effective means for treating or preventing not only periodontal disease but also various diseases involving gingipain, and their development is progressing (Patent Documents 1 to 5). Furthermore, compounds that bind to gingipain with high affinity are expected to be applied to periodontal disease diagnostic techniques using gingipain as a marker.
  • the object of the present invention is to provide a new substance that has specific affinity and/or inhibitory ability for gingipain.
  • a peptide comprising the following amino acid sequence (I) or (II) and having a total number of amino acid residues of 4 to 15 [amino acid sequence (I): RX 1 X 2 RX 3 (amino acid sequence (I) [Amino acid sequence (II): RX 4 R ( In the amino acid sequence (II), X 4 is representing any natural amino acid residue); a peptide consisting of the amino acid sequence CRSVKWHHRVFGSC (SEQ ID NO: 11); and a peptide consisting of the amino acid sequence ITHTSRHC (SEQ ID NO: 12); and the following (a) to ( satisfies one or more of c); (a) the dissociation constant (K D ) for arginine-gingipain is 1.0 ⁇ 10 ⁇ 7 M or less; (b) the enzymatic activity of arginine-gingipain is 50% or more at 100 ⁇ M (c) provides a peptide, a derivative
  • X 1 is selected from the group consisting of A, R, V, and E
  • X 2 is selected from the group consisting of A, F, and K
  • X 3 is selected from the group consisting of R and F.
  • X 1 is selected from the group consisting of R and E
  • X 2 is selected from the group consisting of A and K
  • X 3 is R.
  • X 1 is R, X 2 is K, and X 3 is R, or X 1 is R, and X 2 is A, X 3 is R, or X 1 is E, X 2 is K, and X 3 is R.
  • X 4 is selected from the group consisting of A, P, and K. In one embodiment, X 4 in the above amino acid sequence (II) is P. In one embodiment, the peptide or derivative thereof or salt thereof does not inhibit the enzymatic activity of papain by more than 5% at 100 ⁇ M. In one embodiment, the peptide comprising the above amino acid sequence (I) or (II) is a peptide represented by SEQ ID NOs: 1 to 10. According to another aspect of the present invention, there is provided a gingipain inhibitor comprising the above-mentioned peptide, a derivative thereof, or a salt thereof. According to another aspect of the present invention, there is provided a growth inhibitor of P.
  • gingivalis which comprises the above-mentioned peptide, a derivative thereof, or a salt thereof.
  • an oral composition comprising the above-mentioned peptide, a derivative thereof, or a salt thereof.
  • a method for detecting gingipain which comprises detecting gingipain by an immunoassay method using the above-mentioned peptide, a derivative thereof, or a salt thereof.
  • the immunoassay is a sandwich immunoassay using two types of peptides selected from the peptides with different amino acid sequences, derivatives thereof, or salts thereof.
  • the sandwich immunoassay method is an ELISA method or an immunochromatography assay method.
  • a novel peptide having affinity and/or inhibitory ability for gingipain is provided.
  • FIG. 2 is a diagram showing the selection process of gingipain-binding peptides.
  • FIG. 2 is a diagram showing an alanine-substituted peptide of peptide “R3-1”. It is a schematic diagram explaining a competition experiment using QCM. It is a graph showing affinity changes of alanine-substituted peptides.
  • FIG. 3 is a diagram showing the results of specificity evaluation of peptide “R3-1”.
  • FIG. 3 is a diagram showing the results of specificity evaluation of peptides “K7294” and “R5-7”.
  • FIG. 3 is a diagram showing the evaluation effect of the ability to inhibit the growth of P. gingivalis bacteria.
  • ⁇ representing a numerical range includes the numerical value of the upper limit and the lower limit.
  • natural amino acids include alanine (A), leucine (L), arginine (R), lysine (K), asparagine (N), methionine (M), aspartic acid (D), and phenylalanine (F).
  • cysteine (C) proline (P), glutamine (Q), serine (S), glutamic acid (E), threonine (T), glycine (G), tryptophan (W), histidine (H), tyrosine (Y) , isoleucine (I), and valine (V).
  • A. Peptide - A peptide containing the following amino acid sequence (I) or (II) and having a total number of amino acid residues of 4 to 15 [Amino acid sequence (I): RX 1 X 2 RX 3 (In the amino acid sequence (I), X 1 , X 2 , and X 3 each independently represent any natural amino acid residue)], [Amino acid sequence (II): RX 4 R (In the amino acid sequence (II), X 4 represents any natural amino acid residue)]; - A peptide (peptide "K7226") consisting of the amino acid sequence of CRSVKWHHRVFGSC (SEQ ID NO: 11); and - A peptide (peptide "R5-7”) consisting of the amino acid sequence of ITHTSRHC (SEQ ID NO: 12); selected from A peptide, a derivative thereof, or a salt thereof that satisfies one or more of the following (a) to (c), preferably two or more, is provided
  • the dissociation constant (K D ) for Rgp is 1.0 ⁇ 10 ⁇ 7 M or less, preferably 1.0 ⁇ 10 ⁇ 8 M or less, more preferably 1.0 ⁇ 10 ⁇ 9 M or less It is.
  • Kgp enzyme activity is inhibited by 50% or more at 500 ⁇ M. In other words, the IC 50 for Kgp is 500 ⁇ M or less, preferably 400 ⁇ M or less, more preferably 300 ⁇ M or less.
  • the peptide does not inhibit the enzymatic activity of papain, which is classified as a cysteine protease like gingipain, by more than 5% at 100 ⁇ M.
  • the peptide (peptide "K7226") consisting of the amino acid sequence of SEQ ID NO: 11 may have a cyclic form at both terminal Cs due to disulfide bonds.
  • the number of R's in the amino acid sequence of the peptide is typically 1 or more, for example 1, 2, 3, or 4, and may be 5 or more.
  • X1 is selected from the group consisting of, for example, an alanine residue (A), an arginine residue (R), a valine residue (V), and a glutamic acid residue (E), preferably R and E.
  • X2 is selected from the group consisting of, for example, alanine residue (A), phenylalanine residue (F), and lysine residue (K), preferably selected from the group consisting of A and K. be done.
  • X 3 is selected, for example, from the group consisting of arginine residue (R) and phenylalanine residue (F), and is preferably R.
  • X 1 is R, X 2 is K, and X 3 is R. In one embodiment, in amino acid sequence (I), X 1 is R, X 2 is A, and X 3 is R. In one embodiment, in amino acid sequence (I), X 1 is E, X 2 is K, and X 3 is R.
  • the total number of amino acid residues of the peptide containing the amino acid sequence (I) is typically 5 to 15, preferably 5 to 14, and may be, for example, 5 to 12.
  • the number of amino acid residues added to the N-terminus of the amino acid sequence (I) is equal to the number of amino acid residues added to the C-terminus. can be greater than or equal to the number of The number of amino acid residues added to the N-terminal side of the amino acid sequence (I) is typically 1 to 10, and may be 1 to 8, for example. The number of amino acid residues added to the C-terminal side of the amino acid sequence (I) is typically 1 to 6, and may be 1 to 5, for example.
  • X 4 is selected from the group consisting of, for example, an alanine residue (A), a proline residue (P), and a lysine residue (K), and is preferably P.
  • the total number of amino acid residues of the peptide containing the amino acid sequence (II) is typically 4 to 15, preferably 5 to 14, for example 5 to 10.
  • the number of amino acid residues added to the N-terminal side of the amino acid sequence (II) is equal to the number of amino acid residues added to the C-terminal side.
  • the number of amino acid residues added to the N-terminal side of the amino acid sequence (II) is typically 1 to 12, and may be 1 to 8, for example.
  • the number of amino acid residues added to the C-terminal side of the amino acid sequence (II) is typically 1 to 4, and may be 1 to 3, for example.
  • the peptide comprising the amino acid sequence (II) comprises the amino acid sequence represented by LRX 4 R, and may be a peptide comprising the amino acid sequence represented by KLRX 4 R or ILRX 4 R, for example.
  • the above peptide can be synthesized by chemical synthesis methods such as solid phase synthesis, stepwise extension, and liquid phase synthesis. Among these, solid phase synthesis is preferred. Examples of the solid phase synthesis method include Fmoc synthesis method and Boc synthesis method.
  • a therapeutic, prophylactic, or diagnostic agent for diseases associated with gingipain can be provided, which contains the above-mentioned peptide as an active ingredient.
  • the amount of the peptide in the therapeutic, prophylactic, or diagnostic agent may be an effective amount capable of exerting its function, and may be appropriately set depending on the intended use, dosage form, and the like. Examples of diseases related to gingipains include periodontal disease, type 2 diabetes, cardiovascular disease, pneumonia, rheumatoid arthritis, Alzheimer's disease, premature birth, and low birth weight.
  • a peptide may be in the form of a derivative or salt as long as the effects of the present invention can be obtained. Therefore, in this specification, unless otherwise specified, a peptide may be a derivative of the peptide or a salt of the peptide or its derivative.
  • peptide derivatives include those in which functional groups such as the N-terminal amino group, C-terminal carboxyl group, side chain carboxyl group, amino group, guanidino group, hydroxyl group, and thiol group are substituted with various substituents. It will be done.
  • Substituents are not particularly limited, and include, for example, alkyl groups, acyl groups, hydroxyl groups, amino groups, alkylamino groups, nitro groups, amide groups, sulfonyl groups, halogens, and various protective groups. These substituents may be further substituted with halogen such as fluorine. Further, the substitution may be the introduction of a label such as a fluorescent label or a biotin label.
  • the peptide salt is preferably a pharmacologically acceptable salt.
  • Pharmaceutically acceptable salts include acid addition salts and base addition salts.
  • acid addition salts include inorganic acid salts and organic acid salts.
  • inorganic acid salts include hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, and the like.
  • organic acid salts include citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, Examples include paratoluenesulfonate.
  • Examples of base addition salts include inorganic base salts and organic base salts.
  • Examples of inorganic base salts include sodium salts, potassium salts, calcium salts, magnesium salts, and ammonium salts.
  • Examples of the organic base salt include organic base salts such as triethylammonium salt, triethanolammonium salt, pyridinium salt, and diisopropylammonium salt.
  • the peptide described in Section A can exhibit a gingipain inhibitory effect. Therefore, according to another aspect of the present invention, there is provided a gingipain inhibitor comprising the peptide described in Section A.
  • the gingipain to be inhibited may be one or both of Rgp and Kgp.
  • As the peptide a peptide having an IC 50 of, for example, 500 ⁇ M or less, preferably 300 ⁇ M or less, and more preferably 100 ⁇ M or less against the gingipain to be inhibited can be preferably used.
  • the gingipain inhibitor may be composed only of the peptide, or may contain other components.
  • Other ingredients include a pharmacologically acceptable carrier, gingipain inhibitors other than the peptide (for example, gabexate mesylate, leupeptin, antipain, KYT-01, E-64, KYT-36, KYT-41, hinokitiol). , copper chlorophyllin metal salt), etc.
  • the content of the peptide in the gingipain inhibitor may be any effective amount that provides the gingipain inhibitory effect of the peptide, and may be, for example, 0.1% to 100% by weight, preferably 1% to 100% by weight.
  • a gingipain inhibitor can be used as a therapeutic, preventive, or diagnostic agent for diseases related to gingipain. Diseases related to gingipain are as described in Section A.
  • the peptide described in section A can exhibit the effect of inhibiting the growth of Gingivalis bacteria. Therefore, according to another aspect of the present invention, there is provided a growth inhibitor of P. gingivalis, which comprises the peptide described in Section A.
  • the P. gingivalis growth inhibitor may be composed only of the peptide, or may contain other components. Other components include a pharmacologically acceptable carrier, a growth inhibitor of G. gingivalis bacteria other than the peptide (for example, an antibiotic such as minocycline), and the like.
  • the content of the peptide in the P. gingivalis growth inhibitor may be any effective amount that provides the peptide with the effect of inhibiting the growth of P. gingivalis, for example, 0.1% to 100% by weight, preferably 1% to 100% by weight. It can be.
  • an oral composition comprising the peptide described in Section A. Due to the inclusion of the peptide, the oral composition can exhibit an inhibitory effect on gingipain activity and/or an inhibitory effect on the growth of Gingivalis bacteria.
  • the amount of peptide to be blended in the oral composition may be an effective amount that achieves the effect of inhibiting gingipain activity and/or the effect of inhibiting the growth of gingivalis bacteria.
  • the amount of the peptide etc. blended in the oral composition may vary depending on the dosage form of the composition, but for example, 0.1% to 80% by weight, preferably 0.5% to 50% by weight, more preferably 1% by weight. % to 50% by weight.
  • Oral compositions include, for example, dentifrices such as toothpastes, moisturizing dentifrices, powdered dentifrices, liquid dentifrices, and liquid dentifrices, mouthwashes, mouth fresheners, oral sprays, It may be an oral ointment, a gargle, or the like.
  • the oral composition can be, for example, a tablet, gum, gummy, candy, or drink.
  • dentifrices may contain abrasives, thickeners, binders, surfactants, fragrances, sweeteners, colorants, preservatives, active ingredients, and the like.
  • abrasives examples include silica-based abrasives such as silica gel, precipitated silica, aluminosilicate, and zirconosilicate, dibasic calcium phosphate dihydrate and anhydrate, tribasic calcium phosphate, quaternary calcium phosphate, calcium pyrophosphate, calcium carbonate, and water.
  • examples include aluminum oxide, alumina, magnesium carbonate, tertiary magnesium phosphate, zeolite, hydroxyapatite, and synthetic resin abrasives.
  • the amount of abrasive is adjusted depending on the dosage form, and is, for example, 2% to 40% by weight, preferably 10% to 30% by weight in toothpaste, and may be 0% by weight in liquid dentifrice.
  • thickening agent examples include sugar alcohols such as sorbitol, xylitol, maltitol, and lactitol, and polyhydric alcohols such as glycerin, propylene glycol, and polyethylene glycol.
  • sugar alcohols such as sorbitol, xylitol, maltitol, and lactitol
  • polyhydric alcohols such as glycerin, propylene glycol, and polyethylene glycol.
  • the blending amount is usually 5% to 50% by weight, preferably 20% to 45% by weight.
  • binders examples include cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxyethylcellulose, gums such as xanthan gum and gum arabic, organic binders such as carrageenan, polyvinyl alcohol, and sodium polyacrylate, gelling silica, and gel. Inorganic binders such as oxidizable aluminum silica, vegum, and laponite can be mentioned.
  • the blending amount is usually 0.1% to 5% by weight in toothpastes, and 0% to 5% by weight in liquid toothpastes and mouthwashes.
  • anionic surfactants nonionic surfactants, cationic surfactants, and amphoteric surfactants can be blended.
  • anionic surfactants include alkyl sulfates such as sodium lauryl sulfate, N-acyl sarcosinates such as sodium N-lauroyl sarcosine and sodium N-myristoyl sarcosine, N-acyl glutamates such as sodium N-palmitoyl glutamate, Examples include sodium N-methyl-N-acyl taurate, sodium N-methyl-N-acylalanine, and sodium ⁇ -olefin sulfonate.
  • nonionic surfactants include sugar fatty acid esters such as sucrose fatty acid ester and maltose fatty acid ester, sugar alcohol fatty acid esters such as maltitol fatty acid ester and lactitol fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, hexaglyceryl monolaurate, Polyglycerin fatty acid esters such as hexaglyceryl monomyristate, decaglyceryl monolaurate, decaglyceryl monomyristate, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, polyoxyethylene Polyoxyethylene fatty acid esters such as hydrogenated castor oil, polyoxyethylene higher alcohol ethers such as polyoxyethylene lauryl ether, fatty acid alkanolamides such as lauric acid diethanolamide, polyoxyethylene polyoxypropylene copoly
  • cationic surfactants include alkylammonium and alkylbenzylammonium salts
  • amphoteric surfactants include betaine-based surfactants such as alkyl betaines, fatty acid amidopropyl betaines, and alkylimidazolinium betaines.
  • the blending amount of the surfactant in the oral composition is, for example, 0.001% to 10% by weight, preferably 0.1% to 5% by weight.
  • Fragrances include peppermint oil, spearmint oil, anise oil, eucalyptus oil, wintergreen oil, cassia oil, clove oil, thyme oil, sage oil, lemon oil, orange oil, peppermint oil, cardamom oil, coriander oil, mandarin oil, Lime oil, lavender oil, rosemary oil, laurel oil, chamomile oil, caraway oil, marjoram oil, bay oil, lemongrass oil, origanum oil, pine needle oil, neroli oil, rose oil, jasmine oil, grapefruit oil, sweetie oil
  • Natural fragrances such as oil, yuzu oil, iris concrete, absolute peppermint, absolute rose, orange flower, etc., and processing of these natural fragrances (front distillation cut, rear distillation cut, fractional distillation, liquid-liquid extraction, essence formation, powdered fragrances, etc.), and menthol, carvone, anethole, cineole, methyl salicylate, cinnamic aldehyde
  • the fragrance material is preferably used in an amount of 0.000001% to 1% by weight in the oral composition. Further, it is preferable that the perfume for flavoring using the above-mentioned perfume material is used in the oral composition in an amount of 0.1% to 2% by weight.
  • sweeteners include saccharin sodium, stevioside, paramethoxycinnamic aldehyde, perilartine, and the like.
  • coloring agent include Blue No. 1, Yellow No. 4, and titanium dioxide.
  • preservative examples include benzoic acid or its salts such as paraoxybenzoic acid ester and sodium benzoate.
  • the oral composition contains nonionic disinfectants such as isopropylmethylphenol, cationic disinfectants such as cetylpyridinium chloride, chlorhexidine hydrochloride, benzalkonium chloride, and benzethonium chloride, tranexamic acid, and epsilon aminocaprone.
  • nonionic disinfectants such as isopropylmethylphenol
  • cationic disinfectants such as cetylpyridinium chloride, chlorhexidine hydrochloride, benzalkonium chloride, and benzethonium chloride, tranexamic acid, and epsilon aminocaprone.
  • Anti-inflammatory agents such as allantoin, glycyrrhetinic acid, glycyrrhizic acid, enzymes such as dextranase, mutanase, amylase, protease, fluoride such as sodium fluoride, sodium monofluorophosphate, potassium salt of orthophosphoric acid, sodium Water-soluble phosphoric acid compounds such as salts, copper compounds such as copper gluconate and sodium copper chlorophyllin, inorganic salts such as sodium chloride, potassium nitrate, aluminum lactate, zinc chloride, zinc citrate, and strontium chloride, vitamins such as tocopherol acetate, Zeolite, azulene, dihydrocholesterol, chlorophyll, Japanese angelica soft extract, plant extracts such as thyme, scutellariae, clove, and hamamelis, anti-tartar agents, anti-plaque agents, etc. may be blended. These components can be blended in an effective amount within a range that
  • a method for detecting gingipains which comprises detecting gingipains by an immunoassay using the peptide described in Section A.
  • the gingipain to be detected may be one or both of Rgp and Kgp.
  • the dissociation constant of the peptide for gingipain to be detected is preferably 1.0 ⁇ 10 ⁇ 8 M or less, more preferably 1.0 ⁇ 10 ⁇ 9 M or less.
  • immunoassay methods include immunoblotting, enzyme-linked immunosorbent assay (EIA, ELISA), radiation immunoassay (RIA), fluorescent antibody method, and immunochromatography.
  • EIA enzyme-linked immunosorbent assay
  • ELISA enzyme-linked immunosorbent assay
  • RIA radiation immunoassay
  • fluorescent antibody method fluorescent antibody method
  • immunochromatography immunochromatography
  • the immunoassay method is preferably a sandwich immunoassay method, and can be, for example, a sandwich ELISA or a sandwich immunochromatography method.
  • Sandwich immunoassay is a method for qualitative or quantitative analysis of a test substance by immobilizing a capture antibody on a support and binding the labeled antibody and capture antibody to the test substance in a sandwich-like manner. be.
  • peptides A and B which are the peptides described in Section A and have different amino acid sequences, are used (as a result, peptides A and B each target different sites of the gingipain to be detected).
  • peptides A and B each target different sites of the gingipain to be detected).
  • unlabeled peptide A is immobilized as a capture antibody (primary antibody) on a carrier such as a 96-well microtiter plate or polystyrene beads, or biotin-labeled peptide A is immobilized on a 96-well avidin-coated well.
  • Peptide B is immobilized on a microtiter plate and labeled with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), a fluorescent substance such as FITC, or a labeling substance such as biotin as a labeled antibody (secondary antibody).
  • HRP horseradish peroxidase
  • ALP alkaline phosphatase
  • FITC fluorescent substance
  • secondary antibody a labeling substance
  • the labeled antibody may be labeled with biotin, and the labeled avidin or streptavidin may be bound to the biotin to develop color.
  • the binding of gingipain and the capture antibody or the labeled antibody is carried out under temperature conditions of, for example, 0°C to 37°C, preferably 4°C to 25°C, and for a reaction time of, for example, 30 minutes to 24 hours, preferably 60 minutes to 120 minutes. It can be carried out.
  • a stand peptide is interposed between the support and the peptide to alleviate the influence of the support and improve the measurement accuracy of gingipain. can be improved.
  • kits for detecting or measuring gingipains According to another aspect of the present invention, at least two types of peptides having different amino acid sequences among the peptides described in Section A (as a result, the two types of peptides each have A kit for detecting or measuring gingipain is provided.
  • the gingipain to be detected or measured may be one or both of Rgp and Kgp.
  • One of the two types of peptides may be immobilized on a support, and the other may be labeled with a labeling substance.
  • the kit may contain reagents such as a specimen collection device, a specimen processing liquid, and a coloring substrate, and may further contain equipment necessary for the test.
  • the peptides that non-specifically bound to the beads were removed, and the peptides that did not bind to the beads were mixed with beads immobilized with gingipain (Rgp) purified from the culture supernatant of B. gingivalis (ATCC strain 33277) ( Figure 1 (b). )).
  • the supernatant was removed by centrifugation, and the peptide bound to gingipain was recovered by washing and elution (FIG. 1(c)).
  • the same operation was repeated four more times while gradually tightening the washing conditions (total of five selections). Specifically, 10mM sodium phosphate (pH 7.5), 100mM sodium chloride, and 5mM magnesium chloride were used as the selection buffer.
  • the obtained gingipain-binding peptide was cloned using a commercially available cloning kit (Zero Blunt TOPO PCR Cloning Kit, Invitrogen), and a sequence sample was prepared using the dideoxy method and the base sequence was determined using a sequencer. Based on this, the amino acid sequence of the gingipain-binding peptide was determined.
  • Rgp in the supernatant of the B. gingivalis culture was purified as follows. 1. Approximately 10 liters of the culture supernatant of P.
  • gingivalis (ATCC strain 33277) was collected, concentrated, dialyzed against 10 mM phosphate buffer (pH 7.0), and then subjected to DEAE Sepharose anion chromatography equilibrated with the same buffer. Ta. After thoroughly washing the column with the same buffer, active fractions eluted with the same buffer containing 0.1M sodium chloride were collected and concentrated. 2. The obtained active fraction was dialyzed against 10 mM acetate buffer (pH 5.5) and subjected to CM Sephadex cation chromatography equilibrated with the same buffer. Non-adsorbed fractions eluted with the same buffer were collected, concentrated, and dialyzed against 10 mM phosphate buffer (pH 7.0). 3.
  • the active fraction was subjected to Mono Q anion chromatography equilibrated with the same buffer. Active fractions eluted with a sodium chloride concentration gradient were collected and subjected to concentration dialysis. 4. The obtained enzyme fraction was subjected to affinity chromatography using Arg Sepharose. The column adsorbed fractions were eluted with lysine and they were concentrated. 5. After gel filtration of the concentrated sample using a TSK-G2000SW column (Tosoh), the active fraction was concentrated. 6. Purification into a single band was confirmed by SDS gel electrophoresis. In addition, the gingipain concentration in the culture supernatant was determined by Kadowaki et al., J. Biol. Chem. 269; 21371-8 (1994).
  • a cDNA display library consisting of a peptide having cysteine at both ends and 12 random amino acids between them (total number of amino acid residues: 14) was created, and a recombinant gingipain (Abcam "Recombinant Gingipain R1 protein (His tag)" was prepared. ), a total of seven selections were performed in the same manner as above to obtain a gingipain-binding peptide. Specifically, the second elution was performed using dithiothreitol, the third to sixth elutions were performed using biotin, and the seventh elution was performed using KYT-1, an Rgp inhibitor. Competitive elution was performed using The amino acid sequence of the resulting gingipain-binding peptide was determined in the same manner as above.
  • Candidate peptides were selected taking into consideration the characteristics of the amino acid sequence, ease of synthesis, etc., and peptide synthesis was performed by the Fmoc method (purity of the synthesized peptide by RP-HPLC ⁇ 90%).
  • alanine-substituted peptides were examined for changes in affinity for gingipain using the quartz crystal microbalance (QCM) method. Specifically, gingipain is brought into contact with a substrate on which an alanine-substituted peptide has been immobilized in advance, and the alanine-substituted peptide and gingipain are added and bonded, and then peptide "R3-1" is added.
  • the affinity of the alanine-substituted peptide for gingipain is lower than that of peptide "R3-1"
  • gingipain is transferred to peptide "R3-1" by competitive reaction (FIG. 3, upper case).
  • Figure 4 shows a summary of the results of measuring changes in affinity for seven types of alanine-substituted peptides. From the results in Figure 4, the 4 residues from the C-terminus of peptide "R3-1" play an important role in binding to gingipain, and the contribution of 1 to 4 residues from the N-terminus of the peptide to the binding activity is compared. You can see that it's not very accurate. This also suggested that the N-terminal 1 to 4 residues of peptide "R3-1" may be chemically modified without significantly affecting the interaction with gingipain.
  • a peptide analog without the N-terminal 1 to 4 residues of peptide "R3-1” was considered to reduce the cost of peptide synthesis and improve binding specificity to proteins. Therefore, we designed a peptide in which the N-terminal three residues of peptide "R3-1" were removed, and a peptide derivative in which the N-terminal three residues were chemically modified.
  • the affinity of peptides for gingipains was performed using the Biacore® system, which utilizes surface plasmon resonance (SPR) technology. Specifically, the synthesized peptide was immobilized on a sensor chip for the Biacore (R) system, and a purified culture supernatant of a wild-type strain of P. gingivalis was added thereto. In this system, when gingipain in the culture supernatant and the peptide on the chip combine, the mass of the immobilized peptide increases and the refractive index of the solvent on the sensor chip surface changes. By measuring this change, it becomes possible to monitor the state of binding in real time.
  • SPR surface plasmon resonance
  • peptide "R3-1" showed a very strong reaction to purified culture supernatant containing gingipain (Wild type, protein amount: 22 ⁇ g/mL), while The reaction to the purified culture supernatant that does not contain gingipain (-Kgp & -Rgp, protein amount: 22 ⁇ g/mL) is very weak, and the reaction to the 10 times concentrated solution (-Kgp & -Rgp, protein amount: 220 ⁇ g/mL) is somewhat weak. Although it showed a reaction, the reaction was lower than that of the culture supernatant containing gingipain. From this, it is considered that the specificity of peptide "R3-1" to gingipain is quite high.
  • Peptide "R3-1" was dissolved in dimethyl sulfoxide, and the resulting solution was diluted with distilled water (Otsuka Pharmaceutical Factory Co., Ltd., Lot. 0D96N) so that the final concentration of the peptide was 1,280 ⁇ g/mL. did. Then, after diluting with ATCC2722 medium to 160 ⁇ g/mL, 20 ⁇ g/mL, and 2.5 ⁇ g/mL, the final concentrations were 0.06 ⁇ g/mL, 0.12 ⁇ g/mL, and 0.25 ⁇ g/mL using the same medium.
  • mL 0.5 ⁇ g/mL, 1 ⁇ g/mL, 2 ⁇ g/mL, 4 ⁇ g/mL, 8 ⁇ g/mL, 16 ⁇ g/mL, 32 ⁇ g/mL, 64 ⁇ g/mL, and 128 ⁇ g/mL.
  • Diluted solutions of various concentrations were similarly prepared for gabexate methylate (manufactured by Tokyo Kasei Co., Ltd.) and KYT-1 (Peptide Institute).
  • test substance peptide, gabexate methylate (GM), or KYT-1
  • ATCC2722 medium positive control; PC
  • PC negative control
  • an Rgp solution dissolved in HEPES buffer to a concentration of 0.1 nM, 1 nM, or 10 nM was used.
  • a dimethyl sulfoxide solution (10 mM) of labeled peptide was diluted 2,000 times with HEPES buffer and added at a volume of 100 ⁇ L/well. 6) After over night reaction at 4°C, the reaction solution was discarded and each well was washed.
  • Table 6 shows the combinations of peptides used as solid-phase peptides and labeled peptides, and the absorbance measurement results.
  • the peptide according to the embodiment of the present invention is suitably used in the treatment, prevention, diagnosis, etc. of diseases related to gingipain such as periodontal disease.

Abstract

The purpose of the present invention is to provide a novel compound having an affinity and/or inhibitory function specific to gingipains. The present invention provides a peptide, or a derivative thereof, or a salt of the peptide or derivative, said peptide being selected from among peptides which include amino acid sequence (I) or (II) and in which the total number of amino acid residues is 4 to 15 [amino acid sequence (I): RX1X2RX3 (in amino acid sequence (I), X1, X2, and X3 each independently represent an arbitrary natural amino acid residue)], [amino acid sequence (II): RX4R (in amino acid sequence (II), X4 represents an arbitrary natural amino acid residue)], peptides comprising the amino acid sequence CRSVKWHHRVFGSC (SEQ ID NO: 11), and peptides comprising the amino acid sequence ITHTSRHC (SEQ ID NO: 12), wherein one or more of the following conditions (a) to (c) is satisfied. (a) The dissociation constant (KD) with respect to arginine-gingipain is not more than 1.0×10-7 M. (b) At 100 μM, the enzymatic activity of arginine-gingipain is inhibited by not less than 50%. (c) At 500 μM, the enzymatic activity of lysine-gingipain is inhibited by not less than 50%.

Description

ジンジパインに対して親和性及び／又は阻害能を有するペプチド及びその使用Peptide having affinity and/or inhibitory ability for gingipain and use thereof
　本発明は、ジンジパインに対して親和性及び／又は阻害能を有するペプチド及びその使用に関する。より具体的には、本発明は、ジンジパインに対して親和性及び／又は阻害能を有するペプチド並びに当該ペプチドを用いたジンジパイン阻害剤、ジンジバリス菌増殖抑制剤、口腔用組成物、及びジンジパインの検出方法に関する。 The present invention relates to peptides having affinity and/or inhibitory ability for gingipains and uses thereof. More specifically, the present invention provides a peptide having affinity and/or inhibitory ability for gingipain, a gingipain inhibitor, a gingivalis growth inhibitor, an oral composition, and a method for detecting gingipain using the peptide. Regarding.
　Ｐｏｒｐｈｙｒｏｍｏｎａｓ　ｇｉｎｇｉｖａｌｉｓ（ジンジバリス菌）は、歯周病の発症及び進行において重要視されている歯周病源菌であり、ジンジバリス菌が産生するプロテアーゼの１種であるジンジパインは、歯周病の重要因子として認識されている。 Porphyromonas gingivalis is a periodontal pathogen that is considered important in the onset and progression of periodontal disease, and gingipain, a type of protease produced by Porphyromonas gingivalis, is recognized as an important factor in periodontal disease. has been done.
　ジンジパインには、ペプチド切断部位特異性の異なる２種類のジンジパイン、具体的には、アルギニン残基のＣ末端側を切断するアルギニン－ジンジパイン（Ｒｇｐ）と、リジン残基のＣ末端側を切断するリジン－ジンジパイン（Ｋｇｐ）とが存在する。これらは、相互に作用しながら直接又は間接的に歯周組織を破壊する。また、ジンジパインの作用は、歯周病の進行と難治化につながるだけでなく、最近では糖尿病、動脈硬化、がん、認知症を始めとする種々の疾患の発症因子になり得るとみなされている。 There are two types of gingipains with different peptide cleavage site specificities, specifically, arginine-gingipain (Rgp), which cleaves the C-terminal side of arginine residues, and lysine-gingipains (Rgp), which cleaves the C-terminal side of lysine residues. - Gingipain (Kgp) is present. These destroy periodontal tissue directly or indirectly while interacting with each other. In addition, the effects of gingipain not only lead to the progression and intractability of periodontal disease, but have recently been considered to be a factor in the development of various diseases including diabetes, arteriosclerosis, cancer, and dementia. There is.
　そのため、ジンジパイン阻害剤は、歯周病のみならず、ジンジパインが関与する種々の疾患の治療又は予防においても有効な手段になると期待され、その開発が進められている（特許文献１～５）。また、ジンジパインに高い親和性で結合する化合物は、ジンジパインをマーカーとする歯周病診断技術等への応用が期待される。 Therefore, gingipain inhibitors are expected to be an effective means for treating or preventing not only periodontal disease but also various diseases involving gingipain, and their development is progressing (Patent Documents 1 to 5). Furthermore, compounds that bind to gingipain with high affinity are expected to be applied to periodontal disease diagnostic techniques using gingipain as a marker.
特開２０２１－３１３９９号公報JP 2021-31399 Publication 特開２０１７－５３１６９２号公報Japanese Patent Application Publication No. 2017-531692 特開２０１８－５３４３００号公報Japanese Patent Application Publication No. 2018-534300 特開２００８－１５０３２５号公報Japanese Patent Application Publication No. 2008-150325 ＷＯ２００３／０４２２３７WO2003/042237
　本発明は、ジンジパイン特異的に親和性及び／又は阻害能を有する新規物質を提供することにある。 The object of the present invention is to provide a new substance that has specific affinity and/or inhibitory ability for gingipain.
　本発明によれば、下記アミノ酸配列（Ｉ）又は（ＩＩ）を含み、総アミノ酸残基数が４～１５であるペプチド［アミノ酸配列（Ｉ）：ＲＸ１Ｘ２ＲＸ３（アミノ酸配列（Ｉ）中、Ｘ１、Ｘ２、及びＸ３はそれぞれ独立して、任意の天然アミノ酸残基を表す）］、［アミノ酸配列（ＩＩ）：ＲＸ４Ｒ（アミノ酸配列（ＩＩ）中、Ｘ４は、任意の天然アミノ酸残基を表す）］；ＣＲＳＶＫＷＨＨＲＶＦＧＳＣ（配列番号：１１）のアミノ酸配列からなるペプチド；及びＩＴＨＴＳＲＨＣ（配列番号：１２）のアミノ酸配列からなるペプチド；から選択され、下記（ａ）～（ｃ）の１つ以上を満たす、（ａ）アルギニン－ジンジパインに対する解離定数（ＫＤ）が１．０×１０－７Ｍ以下である、（ｂ）１００μＭでアルギニン－ジンジパインの酵素活性を５０％以上阻害する、（ｃ）５００μＭでリジン－ジンジパインの酵素活性を５０％以上阻害する、ペプチド若しくはその誘導体又はこれらの塩が提供される。
　１つの実施形態において、上記アミノ酸配列（Ｉ）中、Ｘ１が、Ａ、Ｒ、Ｖ、及びＥからなる群より選択され、Ｘ２が、Ａ、Ｆ、及びＫからなる群より選択され、Ｘ３が、Ｒ及びＦからなる群より選択される。
　１つの実施形態において、上記アミノ酸配列（Ｉ）中、Ｘ１が、Ｒ及びＥからなる群より選択され、Ｘ２が、Ａ及びＫからなる群より選択され、Ｘ３が、Ｒである。
　１つの実施形態において、上記アミノ酸配列（Ｉ）中、Ｘ１がＲであり、Ｘ２がＫであり、Ｘ３がＲであるか、Ｘ１がＲであり、Ｘ２がＡであり、Ｘ３がＲであるか、Ｘ１がＥであり、Ｘ２がＫであり、Ｘ３がＲである。
　１つの実施形態において、上記アミノ酸配列（ＩＩ）中、Ｘ４が、Ａ、Ｐ、及びＫからなる群より選択される。
　１つの実施形態において、上記アミノ酸配列（ＩＩ）中、Ｘ４が、Ｐである。
　１つの実施形態において、ペプチド若しくはその誘導体又はこれらの塩は、１００μＭでパパインの酵素活性を５％を超えて阻害しない。
　１つの実施形態において、上記アミノ酸配列（Ｉ）又は（ＩＩ）を含むペプチドが、配列番号：１～１０で表されるペプチドである。
　本発明の別の局面によれば、上記ペプチド若しくはその誘導体又はこれらの塩を含む、ジンジパイン阻害剤が提供される。
　本発明の別の局面によれば、上記ペプチド若しくはその誘導体又はこれらの塩を含む、ジンジバリス菌増殖抑制剤が提供される。
　本発明の別の局面によれば、上記ペプチド若しくはその誘導体又はこれらの塩を含む、口腔用組成物が提供される。
　本発明の別の局面によれば、上記ペプチド若しくはその誘導体又はこれらの塩を用いてジンジパインを免疫学的測定法により検出することを含む、ジンジパインの検出方法が提供される。
　１つの実施形態において、上記免疫学的測定法が、上記ペプチドから選択されるアミノ酸配列が異なる２種類のペプチド若しくはその誘導体又はこれらの塩を用いたサンドイッチ式免疫測定法である。
　１つの実施形態において、上記サンドイッチ式免疫測定法が、ＥＬＩＳＡ法又はイムノクロマトグラフィー測定法である。 According to the present invention, a peptide comprising the following amino acid sequence (I) or (II) and having a total number of amino acid residues of 4 to 15 [amino acid sequence (I): RX 1 X 2 RX 3 (amino acid sequence (I) [Amino acid sequence (II): RX 4 R ( In the amino acid sequence (II), X 4 is representing any natural amino acid residue); a peptide consisting of the amino acid sequence CRSVKWHHRVFGSC (SEQ ID NO: 11); and a peptide consisting of the amino acid sequence ITHTSRHC (SEQ ID NO: 12); and the following (a) to ( satisfies one or more of c); (a) the dissociation constant (K D ) for arginine-gingipain is 1.0×10 −7 M or less; (b) the enzymatic activity of arginine-gingipain is 50% or more at 100 μM (c) provides a peptide, a derivative thereof, or a salt thereof, which inhibits the enzymatic activity of lysine-gingipain by 50% or more at 500 μM.
In one embodiment, in the amino acid sequence (I), X 1 is selected from the group consisting of A, R, V, and E, and X 2 is selected from the group consisting of A, F, and K, X 3 is selected from the group consisting of R and F.
In one embodiment, in the above amino acid sequence (I), X 1 is selected from the group consisting of R and E, X 2 is selected from the group consisting of A and K, and X 3 is R.
In one embodiment, in the amino acid sequence (I), X 1 is R, X 2 is K, and X 3 is R, or X 1 is R, and X 2 is A, X 3 is R, or X 1 is E, X 2 is K, and X 3 is R.
In one embodiment, in the above amino acid sequence (II), X 4 is selected from the group consisting of A, P, and K.
In one embodiment, X 4 in the above amino acid sequence (II) is P.
In one embodiment, the peptide or derivative thereof or salt thereof does not inhibit the enzymatic activity of papain by more than 5% at 100 μM.
In one embodiment, the peptide comprising the above amino acid sequence (I) or (II) is a peptide represented by SEQ ID NOs: 1 to 10.
According to another aspect of the present invention, there is provided a gingipain inhibitor comprising the above-mentioned peptide, a derivative thereof, or a salt thereof.
According to another aspect of the present invention, there is provided a growth inhibitor of P. gingivalis, which comprises the above-mentioned peptide, a derivative thereof, or a salt thereof.
According to another aspect of the present invention, there is provided an oral composition comprising the above-mentioned peptide, a derivative thereof, or a salt thereof.
According to another aspect of the present invention, there is provided a method for detecting gingipain, which comprises detecting gingipain by an immunoassay method using the above-mentioned peptide, a derivative thereof, or a salt thereof.
In one embodiment, the immunoassay is a sandwich immunoassay using two types of peptides selected from the peptides with different amino acid sequences, derivatives thereof, or salts thereof.
In one embodiment, the sandwich immunoassay method is an ELISA method or an immunochromatography assay method.
　本発明の実施形態によれば、ジンジパインに対して親和性及び／又は阻害能を有する新規ペプチドが提供される。 According to an embodiment of the present invention, a novel peptide having affinity and/or inhibitory ability for gingipain is provided.
ジンジパイン結合ペプチドのセレクションプロセスを示す図である。FIG. 2 is a diagram showing the selection process of gingipain-binding peptides. ペプチド「Ｒ３－１」のアラニン置換ペプチドを示す図である。FIG. 2 is a diagram showing an alanine-substituted peptide of peptide “R3-1”. ＱＣＭを用いた競合実験を説明する概略図である。It is a schematic diagram explaining a competition experiment using QCM. アラニン置換ペプチドの親和性変化を示すグラフである。It is a graph showing affinity changes of alanine-substituted peptides. ペプチド「Ｒ３－１」の特異性評価結果を示す図である。FIG. 3 is a diagram showing the results of specificity evaluation of peptide “R3-1”. ペプチド「Ｋ７２９４」」及び「Ｒ５－７」の特異性評価結果を示す図である。FIG. 3 is a diagram showing the results of specificity evaluation of peptides “K7294” and “R5-7”. ジンジバリス菌の増殖抑制能の評価効果を示す図である。FIG. 3 is a diagram showing the evaluation effect of the ability to inhibit the growth of P. gingivalis bacteria.
　以下、本発明の好ましい実施形態について説明するが、本発明はこれらの実施形態には限定されない。なお、本明細書中で、数値範囲を表す「～」は、その上限及び下限の数値を含む。 Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments. In addition, in this specification, "~" representing a numerical range includes the numerical value of the upper limit and the lower limit.
　本明細書において、天然アミノ酸とは、アラニン（Ａ）、ロイシン（Ｌ）、アルギニン（Ｒ）、リジン（Ｋ）、アスパラギン（Ｎ）、メチオニン（Ｍ）、アスパラギン酸（Ｄ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、プロリン（Ｐ）、グルタミン（Ｑ）、セリン（Ｓ）、グルタミン酸（Ｅ）、トレオニン（Ｔ）、グリシン（Ｇ）、トリプトファン（Ｗ）、ヒスチジン（Ｈ）、チロシン（Ｙ）、イソロイシン（Ｉ）、及びバリン（Ｖ）を意味する。 In this specification, natural amino acids include alanine (A), leucine (L), arginine (R), lysine (K), asparagine (N), methionine (M), aspartic acid (D), and phenylalanine (F). , cysteine (C), proline (P), glutamine (Q), serine (S), glutamic acid (E), threonine (T), glycine (G), tryptophan (W), histidine (H), tyrosine (Y) , isoleucine (I), and valine (V).
Ａ．ペプチド
　本発明の実施形態によれば、
・下記アミノ酸配列（Ｉ）又は（ＩＩ）を含み、総アミノ酸残基数が４～１５であるペプチド
　　　［アミノ酸配列（Ｉ）：ＲＸ１Ｘ２ＲＸ３
（アミノ酸配列（Ｉ）中、Ｘ１、Ｘ２、及びＸ３はそれぞれ独立して、任意の天然アミノ酸残基を表す）］、
　　　［アミノ酸配列（ＩＩ）：ＲＸ４Ｒ
（アミノ酸配列（ＩＩ）中、Ｘ４は、任意の天然アミノ酸残基を表す）］；
・ＣＲＳＶＫＷＨＨＲＶＦＧＳＣ（配列番号：１１）のアミノ酸配列からなるペプチド（ペプチド「Ｋ７２２６」）；及び
・ＩＴＨＴＳＲＨＣ（配列番号：１２）のアミノ酸配列からなるペプチド（ペプチド「Ｒ５－７」）；
　から選択され、
　下記（ａ）～（ｃ）の１つ以上を満たし、好ましくは２つ以上を満たす、ペプチド若しくはその誘導体又はこれらの塩が提供される。
（ａ）Ｒｇｐに対する解離定数（ＫＤ）が１．０×１０－７Ｍ以下であり、好ましくは１．０×１０－８Ｍ以下であり、より好ましくは１．０×１０－９Ｍ以下である。
（ｂ）１００μＭでＲｇｐの酵素活性を５０％以上阻害する。換言すれば、Ｒｇｐに対するＩＣ５０が、１００μＭ以下であり、好ましくは６０μＭ以下であり、より好ましくは４０μＭ以下である。
（ｃ）５００μＭでＫｇｐの酵素活性を５０％以上阻害する。換言すれば、Ｋｇｐに対するＩＣ５０が、５００μＭ以下であり、好ましくは４００μＭ以下であり、より好ましくは３００μＭ以下である。 A. Peptide According to an embodiment of the invention:
- A peptide containing the following amino acid sequence (I) or (II) and having a total number of amino acid residues of 4 to 15 [Amino acid sequence (I): RX 1 X 2 RX 3
(In the amino acid sequence (I), X 1 , X 2 , and X 3 each independently represent any natural amino acid residue)],
[Amino acid sequence (II): RX 4 R
(In the amino acid sequence (II), X 4 represents any natural amino acid residue)];
- A peptide (peptide "K7226") consisting of the amino acid sequence of CRSVKWHHRVFGSC (SEQ ID NO: 11); and - A peptide (peptide "R5-7") consisting of the amino acid sequence of ITHTSRHC (SEQ ID NO: 12);
selected from
A peptide, a derivative thereof, or a salt thereof that satisfies one or more of the following (a) to (c), preferably two or more, is provided.
(a) The dissociation constant (K D ) for Rgp is 1.0×10 −7 M or less, preferably 1.0×10 −8 M or less, more preferably 1.0×10 −9 M or less It is.
(b) Inhibits Rgp enzyme activity by 50% or more at 100 μM. In other words, the IC 50 for Rgp is 100 μM or less, preferably 60 μM or less, and more preferably 40 μM or less.
(c) Kgp enzyme activity is inhibited by 50% or more at 500 μM. In other words, the IC 50 for Kgp is 500 μM or less, preferably 400 μM or less, more preferably 300 μM or less.
　１つの実施形態において、上記ペプチドは、１００μＭでジンジパインと同じシステインプロテアーゼに分類されるパパインの酵素活性を５％を超えて阻害しない。 In one embodiment, the peptide does not inhibit the enzymatic activity of papain, which is classified as a cysteine protease like gingipain, by more than 5% at 100 μM.
　配列番号：１１のアミノ酸配列からなるペプチド（ペプチド「Ｋ７２２６」）は、両末端のＣがジスルフィド結合により環状となっていてもよい。 The peptide (peptide "K7226") consisting of the amino acid sequence of SEQ ID NO: 11 may have a cyclic form at both terminal Cs due to disulfide bonds.
　ペプチドのアミノ酸配列中におけるＲの数は、代表的には１以上、例えば１、２、３又は４であり、５以上であってもよい。 The number of R's in the amino acid sequence of the peptide is typically 1 or more, for example 1, 2, 3, or 4, and may be 5 or more.
　アミノ酸配列（Ｉ）中、Ｘ１は、例えばアラニン残基（Ａ）、アルギニン残基（Ｒ）、バリン残基（Ｖ）、及びグルタミン酸残基（Ｅ）からなる群より選択され、好ましくはＲ及びＥからなる群より選択される。 In the amino acid sequence (I), X1 is selected from the group consisting of, for example, an alanine residue (A), an arginine residue (R), a valine residue (V), and a glutamic acid residue (E), preferably R and E.
　アミノ酸配列（Ｉ）中、Ｘ２は、例えばアラニン残基（Ａ）、フェニルアラニン残基（Ｆ）、及びリジン残基（Ｋ）からなる群より選択され、好ましくはＡ及びＫからなる群より選択される。 In the amino acid sequence (I), X2 is selected from the group consisting of, for example, alanine residue (A), phenylalanine residue (F), and lysine residue (K), preferably selected from the group consisting of A and K. be done.
　アミノ酸配列（Ｉ）中、Ｘ３は、例えばアルギニン残基（Ｒ）及びフェニルアラニン残基（Ｆ）からなる群より選択され、好ましくはＲである。 In the amino acid sequence (I), X 3 is selected, for example, from the group consisting of arginine residue (R) and phenylalanine residue (F), and is preferably R.
　１つの実施形態において、アミノ酸配列（Ｉ）中、Ｘ１がＲであり、Ｘ２がＫであり、Ｘ３がＲである。
　１つの実施形態において、アミノ酸配列（Ｉ）中、Ｘ１がＲであり、Ｘ２がＡであり、Ｘ３がＲである。
　１つの実施形態において、アミノ酸配列（Ｉ）中、Ｘ１がＥであり、Ｘ２がＫであり、Ｘ３がＲである。 In one embodiment, in amino acid sequence (I), X 1 is R, X 2 is K, and X 3 is R.
In one embodiment, in amino acid sequence (I), X 1 is R, X 2 is A, and X 3 is R.
In one embodiment, in amino acid sequence (I), X 1 is E, X 2 is K, and X 3 is R.
　アミノ酸配列（Ｉ）を含むペプチドの総アミノ酸残基数は、代表的には５～１５であり、好ましくは５～１４であり、例えば５～１２であり得る。 The total number of amino acid residues of the peptide containing the amino acid sequence (I) is typically 5 to 15, preferably 5 to 14, and may be, for example, 5 to 12.
　アミノ酸配列（Ｉ）を含むペプチドの総アミノ酸残基が６以上である場合、アミノ酸配列（Ｉ）のＮ末端側に付加されるアミノ酸残基の数は、Ｃ末端側に付加されるアミノ酸残基の数以上であり得る。アミノ酸配列（Ｉ）のＮ末端側に付加されるアミノ酸残基の数は、代表的には１～１０であり、例えば１～８であり得る。アミノ酸配列（Ｉ）のＣ末端側に付加されるアミノ酸残基の数は、代表的には１～６であり、例えば１～５であり得る。 When the total number of amino acid residues in the peptide containing the amino acid sequence (I) is 6 or more, the number of amino acid residues added to the N-terminus of the amino acid sequence (I) is equal to the number of amino acid residues added to the C-terminus. can be greater than or equal to the number of The number of amino acid residues added to the N-terminal side of the amino acid sequence (I) is typically 1 to 10, and may be 1 to 8, for example. The number of amino acid residues added to the C-terminal side of the amino acid sequence (I) is typically 1 to 6, and may be 1 to 5, for example.
　アミノ酸配列（Ｉ）を含むペプチドの具体例を表１に示す。表中、下線はアミノ酸配列（Ｉ）に対応する配列を示す。
Figure JPOXMLDOC01-appb-T000001
 Specific examples of peptides containing the amino acid sequence (I) are shown in Table 1. In the table, the underline indicates the sequence corresponding to amino acid sequence (I).
Figure JPOXMLDOC01-appb-T000001
　アミノ酸配列（ＩＩ）中、Ｘ４は、例えばアラニン残基（Ａ）、プロリン残基（Ｐ）、及びリジン残基（Ｋ）からなる群より選択され、好ましくはＰである。 In the amino acid sequence (II), X 4 is selected from the group consisting of, for example, an alanine residue (A), a proline residue (P), and a lysine residue (K), and is preferably P.
　アミノ酸配列（ＩＩ）を含むペプチドの総アミノ酸残基数は、代表的には４～１５であり、好ましくは５～１４であり、例えば５～１０である。 The total number of amino acid residues of the peptide containing the amino acid sequence (II) is typically 4 to 15, preferably 5 to 14, for example 5 to 10.
　アミノ酸配列（ＩＩ）を含むペプチドの総アミノ酸残基が４以上である場合、アミノ酸配列（ＩＩ）のＮ末端側に付加されるアミノ酸残基の数は、Ｃ末端側に付加されるアミノ酸残基の数以上であり得る。アミノ酸配列（ＩＩ）のＮ末端側に付加されるアミノ酸残基の数は、代表的には１～１２であり、例えば１～８であり得る。アミノ酸配列（ＩＩ）のＣ末端側に付加されるアミノ酸残基の数は、代表的には１～４であり、例えば１～３であり得る。１つの実施形態において、アミノ酸配列（ＩＩ）を含むペプチドは、ＬＲＸ４Ｒで表されるアミノ酸配列を含み、例えばＫＬＲＸ４Ｒ又はＩＬＲＸ４Ｒで表されるアミノ酸配列を含むペプチドであり得る。 When the total number of amino acid residues of the peptide containing the amino acid sequence (II) is 4 or more, the number of amino acid residues added to the N-terminal side of the amino acid sequence (II) is equal to the number of amino acid residues added to the C-terminal side. can be greater than or equal to the number of The number of amino acid residues added to the N-terminal side of the amino acid sequence (II) is typically 1 to 12, and may be 1 to 8, for example. The number of amino acid residues added to the C-terminal side of the amino acid sequence (II) is typically 1 to 4, and may be 1 to 3, for example. In one embodiment, the peptide comprising the amino acid sequence (II) comprises the amino acid sequence represented by LRX 4 R, and may be a peptide comprising the amino acid sequence represented by KLRX 4 R or ILRX 4 R, for example.
　アミノ酸配列（ＩＩ）を含むペプチドの具体例を表２に示す。表中、下線はアミノ酸配列（ＩＩ）に対応する配列を示す。
Figure JPOXMLDOC01-appb-T000002
 Specific examples of peptides containing the amino acid sequence (II) are shown in Table 2. In the table, the underline indicates the sequence corresponding to amino acid sequence (II).
Figure JPOXMLDOC01-appb-T000002
　上記ペプチドは、固相合成法、段階的伸長法、液相合成法等の化学合成法によって合成することができる。なかでも、固相合成法が好ましい。固相合成法としては、例えば、Ｆｍｏｃ合成法、Ｂｏｃ合成法等が挙げられる。 The above peptide can be synthesized by chemical synthesis methods such as solid phase synthesis, stepwise extension, and liquid phase synthesis. Among these, solid phase synthesis is preferred. Examples of the solid phase synthesis method include Fmoc synthesis method and Boc synthesis method.
　上記ペプチドは、ジンジパインに対して所定の親和性及び／又は阻害能を有することから、ジンジパインが関連する疾患に対する治療薬、予防薬、又は診断薬に用いることができる。よって、本発明の別の局面によれば、上記ペプチドを有効成分として含む、ジンジパインが関連する疾患に対する治療薬、予防薬、又は診断薬が提供され得る。当該治療薬、予防薬、又は診断薬におけるペプチドの配合量は、その機能を発揮し得る有効量であればよく、用途、剤形等に応じて適切に設定され得る。ジンジパインが関連する疾患としては、例えば、歯周病、２型糖尿病、循環器疾患、肺炎、リウマチ関節炎、アルツハイマー型認知症、早産、低体重児出産等が挙げられる。 Since the above-mentioned peptide has a predetermined affinity and/or inhibitory ability for gingipain, it can be used as a therapeutic agent, prophylactic agent, or diagnostic agent for diseases related to gingipain. Therefore, according to another aspect of the present invention, a therapeutic, prophylactic, or diagnostic agent for diseases associated with gingipain can be provided, which contains the above-mentioned peptide as an active ingredient. The amount of the peptide in the therapeutic, prophylactic, or diagnostic agent may be an effective amount capable of exerting its function, and may be appropriately set depending on the intended use, dosage form, and the like. Examples of diseases related to gingipains include periodontal disease, type 2 diabetes, cardiovascular disease, pneumonia, rheumatoid arthritis, Alzheimer's disease, premature birth, and low birth weight.
　上記ペプチドは、本発明の効果が得られる限りにおいて、誘導体又は塩の形態であってもよい。よって、本明細書において、特段の記載がない場合、ペプチドは、当該ペプチドの誘導体あるいは当該ペプチド又はその誘導体の塩であり得る。 The above peptide may be in the form of a derivative or salt as long as the effects of the present invention can be obtained. Therefore, in this specification, unless otherwise specified, a peptide may be a derivative of the peptide or a salt of the peptide or its derivative.
　ペプチドの誘導体としては、ペプチドのＮ末端アミノ基、Ｃ末端カルボキシル基、側鎖のカルボキシル基、アミノ基、グアニジノ基、ヒドロキシル基、チオール基等の官能基が各種置換基により置換されたものが挙げられる。置換基としては、特に限定されず、例えば、アルキル基、アシル基、水酸基、アミノ基、アルキルアミノ基、ニトロ基、アミド基、スルホニル基、ハロゲン、及び各種保護基が挙げられる。これらの置換基は、フッ素等のハロゲンでさらに置換されていてもよい。また、置換は、蛍光標識、ビオチン標識等の標識の導入であってもよい。 Examples of peptide derivatives include those in which functional groups such as the N-terminal amino group, C-terminal carboxyl group, side chain carboxyl group, amino group, guanidino group, hydroxyl group, and thiol group are substituted with various substituents. It will be done. Substituents are not particularly limited, and include, for example, alkyl groups, acyl groups, hydroxyl groups, amino groups, alkylamino groups, nitro groups, amide groups, sulfonyl groups, halogens, and various protective groups. These substituents may be further substituted with halogen such as fluorine. Further, the substitution may be the introduction of a label such as a fluorescent label or a biotin label.
　ペプチドの塩は、好ましくは薬理的に許容される塩である。薬理的に許容される塩としては、酸付加塩及び塩基付加塩が挙げられる。酸付加塩としては、例えば、無機酸塩、有機酸塩等が挙げられる。無機酸塩としては、例えば、塩酸塩、臭化水素酸塩、硫酸塩、ヨウ化水素酸塩、硝酸塩、リン酸塩等が挙げられる。有機酸塩としては、クエン酸塩、シュウ酸塩、酢酸塩、ギ酸塩、プロピオン酸塩、安息香酸塩、トリフルオロ酢酸塩、マレイン酸塩、酒石酸塩、メタンスルホン酸塩、ベンゼンスルホン酸塩、パラトルエンスルホン酸塩等が挙げられる。塩基付加塩としては、無機塩基塩、有機塩基塩等が挙げられる。無機塩基塩としては、例えば、ナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩、アンモニウム塩等が挙げられる。有機塩基塩としては、例えば、トリエチルアンモニウム塩、トリエタノールアンモニウム塩、ピリジニウム塩、ジイソプロピルアンモニウム塩等の有機塩基塩等が挙げられる。 The peptide salt is preferably a pharmacologically acceptable salt. Pharmaceutically acceptable salts include acid addition salts and base addition salts. Examples of acid addition salts include inorganic acid salts and organic acid salts. Examples of inorganic acid salts include hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, and the like. Examples of organic acid salts include citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, Examples include paratoluenesulfonate. Examples of base addition salts include inorganic base salts and organic base salts. Examples of inorganic base salts include sodium salts, potassium salts, calcium salts, magnesium salts, and ammonium salts. Examples of the organic base salt include organic base salts such as triethylammonium salt, triethanolammonium salt, pyridinium salt, and diisopropylammonium salt.
Ｂ．ジンジパイン阻害剤
　Ａ項に記載のペプチドは、ジンジパイン阻害効果を発揮し得る。よって、本発明の別の局面によれば、Ａ項に記載のペプチドを含む、ジンジパイン阻害剤が提供される。阻害対象のジンジパインは、Ｒｇｐ及びＫｇｐのいずれか一方又は両方であり得る。ペプチドとしては、阻害対象のジンジパインに対するＩＣ５０が例えば５００μＭ以下、好ましくは３００μＭ以下、より好ましくは１００μＭ以下であるペプチドが好ましく用いられ得る。 B. Gingipain Inhibitor The peptide described in Section A can exhibit a gingipain inhibitory effect. Therefore, according to another aspect of the present invention, there is provided a gingipain inhibitor comprising the peptide described in Section A. The gingipain to be inhibited may be one or both of Rgp and Kgp. As the peptide, a peptide having an IC 50 of, for example, 500 μM or less, preferably 300 μM or less, and more preferably 100 μM or less against the gingipain to be inhibited can be preferably used.
　ジンジパイン阻害剤は、当該ペプチドのみから構成されるものであってもよく、他の成分を含むものであってもよい。他の成分としては、薬理的に許容される担体、当該ペプチド以外のジンジパイン阻害剤（例えば、メシル酸ガベキサート、ロイペプチン、アンチパイン、ＫＹＴ－０１、Ｅ－６４、ＫＹＴ－３６、ＫＹＴ－４１、ヒノキチオール、銅クロロフィリン金属塩）等が挙げられる。 The gingipain inhibitor may be composed only of the peptide, or may contain other components. Other ingredients include a pharmacologically acceptable carrier, gingipain inhibitors other than the peptide (for example, gabexate mesylate, leupeptin, antipain, KYT-01, E-64, KYT-36, KYT-41, hinokitiol). , copper chlorophyllin metal salt), etc.
　ジンジパイン阻害剤におけるペプチドの含有量は、当該ペプチドによるジンジパイン阻害効果が得られる有効量であればよく、例えば０．１重量％～１００重量％、好ましくは１重量％～１００重量％であり得る。 The content of the peptide in the gingipain inhibitor may be any effective amount that provides the gingipain inhibitory effect of the peptide, and may be, for example, 0.1% to 100% by weight, preferably 1% to 100% by weight.
　ジンジパイン阻害剤は、ジンジパインが関連する疾患に対する治療薬、予防薬、又は診断薬に用いることができる。ジンジパインが関連する疾患としては、Ａ項に記載の通りである。 A gingipain inhibitor can be used as a therapeutic, preventive, or diagnostic agent for diseases related to gingipain. Diseases related to gingipain are as described in Section A.
Ｃ．ジンジバリス菌増殖抑制剤
　Ａ項に記載のペプチドは、ジンジバリス菌増殖抑制効果を発揮し得る。よって、本発明の別の局面によれば、Ａ項に記載のペプチドを含む、ジンジバリス菌増殖抑制剤が提供される。ジンジバリス菌増殖抑制剤は、当該ペプチドのみから構成されるものであってもよく、他の成分を含むものであってもよい。他の成分としては、薬理的に許容される担体、当該ペプチド以外のジンジバリス菌増殖抑制剤（例えば、ミノサイクリン等の抗生物質）等が挙げられる。 C. Gingivalis bacteria growth inhibitor The peptide described in section A can exhibit the effect of inhibiting the growth of Gingivalis bacteria. Therefore, according to another aspect of the present invention, there is provided a growth inhibitor of P. gingivalis, which comprises the peptide described in Section A. The P. gingivalis growth inhibitor may be composed only of the peptide, or may contain other components. Other components include a pharmacologically acceptable carrier, a growth inhibitor of G. gingivalis bacteria other than the peptide (for example, an antibiotic such as minocycline), and the like.
　ジンジバリス菌増殖抑制剤におけるペプチドの含有量は、当該ペプチドによるジンジバリス菌増殖抑制効果が得られる有効量であればよく、例えば０．１重量％～１００重量％、好ましくは１重量％～１００重量％であり得る。 The content of the peptide in the P. gingivalis growth inhibitor may be any effective amount that provides the peptide with the effect of inhibiting the growth of P. gingivalis, for example, 0.1% to 100% by weight, preferably 1% to 100% by weight. It can be.
Ｄ．口腔用組成物
　本発明の別の局面によれば、Ａ項に記載のペプチドを含む口腔用組成物が提供される。口腔用組成物は、当該ペプチドを含むことに起因して、ジンジパイン活性の阻害効果及び／又はジンジバリス菌の増殖抑制効果を発揮し得る。 D. Oral Composition According to another aspect of the present invention, there is provided an oral composition comprising the peptide described in Section A. Due to the inclusion of the peptide, the oral composition can exhibit an inhibitory effect on gingipain activity and/or an inhibitory effect on the growth of Gingivalis bacteria.
　口腔用組成物におけるペプチドの配合量は、ジンジパイン活性の阻害効果及び／又はジンジバリス菌の増殖抑制効果が得られる有効量であればよい。口腔用組成物におけるペプチド等の配合量は、組成物の剤形によって変動し得るが、例えば０．１重量％～８０重量％、好ましくは０．５重量％～５０重量％、より好ましくは１重量％～５０重量％である。 The amount of peptide to be blended in the oral composition may be an effective amount that achieves the effect of inhibiting gingipain activity and/or the effect of inhibiting the growth of gingivalis bacteria. The amount of the peptide etc. blended in the oral composition may vary depending on the dosage form of the composition, but for example, 0.1% to 80% by weight, preferably 0.5% to 50% by weight, more preferably 1% by weight. % to 50% by weight.
　口腔用組成物は、例えば、練り歯磨剤、潤製歯磨剤、粉歯磨剤、液状歯磨剤、液体歯磨剤等の歯磨剤、洗口剤（マウスウォッシュ）、口中清涼剤、口腔用スプレー剤、口腔用塗布剤、又は嗽剤等であり得る。また例えば、口腔用組成物は、例えば、タブレット、ガム、グミ、飴、又は飲料とすることができる。 Oral compositions include, for example, dentifrices such as toothpastes, moisturizing dentifrices, powdered dentifrices, liquid dentifrices, and liquid dentifrices, mouthwashes, mouth fresheners, oral sprays, It may be an oral ointment, a gargle, or the like. Also, for example, the oral composition can be, for example, a tablet, gum, gummy, candy, or drink.
　口腔用組成物には、剤形、用途等に応じて、上記ペプチドに加えて、任意の適切な成分を本発明の効果を妨げない範囲で配合することができる。例えば、歯磨剤では、研磨剤、粘稠剤、粘結剤、界面活性剤、香料、甘味料、着色剤、防腐剤、有効成分等が配合され得る。 In addition to the above-mentioned peptide, any appropriate components may be added to the oral composition according to the dosage form, use, etc., within a range that does not impede the effects of the present invention. For example, dentifrices may contain abrasives, thickeners, binders, surfactants, fragrances, sweeteners, colorants, preservatives, active ingredients, and the like.
　研磨剤としては、シリカゲル、沈降シリカ、アルミノシリケート、ジルコノシリケート等のシリカ系研磨剤、第２リン酸カルシウム２水和物及び無水和物、第３リン酸カルシウム、第４リン酸カルシウム、ピロリン酸カルシウム、炭酸カルシウム、水酸化アルミニウム、アルミナ、炭酸マグネシウム、第３リン酸マグネシウム、ゼオライト、ハイドロキシアパタイト、合成樹脂系研磨剤等が挙げられる。研磨剤の配合量は、剤形により調整され、練歯磨では、例えば２重量％～４０重量％、好ましくは１０重量％～３０重量％であり、液体歯磨では０重量％であり得る。 Examples of abrasives include silica-based abrasives such as silica gel, precipitated silica, aluminosilicate, and zirconosilicate, dibasic calcium phosphate dihydrate and anhydrate, tribasic calcium phosphate, quaternary calcium phosphate, calcium pyrophosphate, calcium carbonate, and water. Examples include aluminum oxide, alumina, magnesium carbonate, tertiary magnesium phosphate, zeolite, hydroxyapatite, and synthetic resin abrasives. The amount of abrasive is adjusted depending on the dosage form, and is, for example, 2% to 40% by weight, preferably 10% to 30% by weight in toothpaste, and may be 0% by weight in liquid dentifrice.
　粘稠剤としては、ソルビット、キシリット、マルチット、ラクチット等の糖アルコール、グリセリン、プロピレングリコール、ポリエチレングリコール等の多価アルコールが挙げられる。配合量は通常、５重量％～５０重量％％、好ましくは２０重量％～４５重量％である。 Examples of the thickening agent include sugar alcohols such as sorbitol, xylitol, maltitol, and lactitol, and polyhydric alcohols such as glycerin, propylene glycol, and polyethylene glycol. The blending amount is usually 5% to 50% by weight, preferably 20% to 45% by weight.
　粘結剤としては、カルボキシメチルセルロースナトリウム、メチルセルロース、ヒドロキシエチルセルロース等のセルロース誘導体、キサンタンガム、アラビアガム等のガム類、カラゲナン、ポリビニルアルコール、ポリアクリル酸ナトリウム等の有機粘結剤、ゲル化性シリカ、ゲル化性アルミニウムシリカ、ビーガム、ラポナイト等の無機粘結剤が挙げられる。配合量は、通常、練歯磨では０．１重量％～５重量％、液体歯磨や洗口剤では０重量％～５重量％である。 Examples of binders include cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxyethylcellulose, gums such as xanthan gum and gum arabic, organic binders such as carrageenan, polyvinyl alcohol, and sodium polyacrylate, gelling silica, and gel. Inorganic binders such as oxidizable aluminum silica, vegum, and laponite can be mentioned. The blending amount is usually 0.1% to 5% by weight in toothpastes, and 0% to 5% by weight in liquid toothpastes and mouthwashes.
　界面活性剤としては、アニオン性界面活性剤、ノニオン性界面活性剤、カチオン性界面活性剤、両性界面活性剤を配合できる。 As the surfactant, anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants can be blended.
　アニオン性界面活性剤としては、ラウリル硫酸ナトリウム等のアルキル硫酸塩、Ｎ－ラウロイルサルコシンナトリウム、Ｎ－ミリストイルサルコシンナトリウム等のＮ－アシルサルコシン酸塩、Ｎ－パルミトイルグルタミン酸ナトリウム等のＮ－アシルグルタミン酸塩、Ｎ－メチル－Ｎ－アシルタウリンナトリウム、Ｎ－メチル－Ｎ－アシルアラニンナトリウム、α－オレフィンスルホン酸ナトリウム等が挙げられる。 Examples of anionic surfactants include alkyl sulfates such as sodium lauryl sulfate, N-acyl sarcosinates such as sodium N-lauroyl sarcosine and sodium N-myristoyl sarcosine, N-acyl glutamates such as sodium N-palmitoyl glutamate, Examples include sodium N-methyl-N-acyl taurate, sodium N-methyl-N-acylalanine, and sodium α-olefin sulfonate.
　ノニオン性界面活性剤としては、ショ糖脂肪酸エステル、マルトース脂肪酸エステル等の糖脂肪酸エステル、マルチトール脂肪酸エステル、ラクチトール脂肪酸エステル等の糖アルコール脂肪酸エステル、ソルビタン脂肪酸エステル、グリセリン脂肪酸エステル、モノラウリン酸ヘキサグリセリル、モノミリスチン酸ヘキサグリセリル、モノラウリン酸デカグリセリル、モノミリスチン酸デカグリセリル等のポリグリセリン脂肪酸エステル、ポリオキシエチレンソルビタンモノラウレート、ポリオキシエチレンソルビタンモノステアレート等のポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレン硬化ヒマシ油等のポリオキシエチレン脂肪酸エステル、ポリオキシエチレンラウリルエーテル等のポリオキシエチレン高級アルコールエーテル、ラウリン酸ジエタノールアミド等の脂肪酸アルカノールアミド、ポリオキシエチレンポリオキシプロピレン共重合体、ポリオキシエチレンポリオキシプロピレン脂肪酸エステル等が挙げられる。 Examples of nonionic surfactants include sugar fatty acid esters such as sucrose fatty acid ester and maltose fatty acid ester, sugar alcohol fatty acid esters such as maltitol fatty acid ester and lactitol fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, hexaglyceryl monolaurate, Polyglycerin fatty acid esters such as hexaglyceryl monomyristate, decaglyceryl monolaurate, decaglyceryl monomyristate, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, polyoxyethylene Polyoxyethylene fatty acid esters such as hydrogenated castor oil, polyoxyethylene higher alcohol ethers such as polyoxyethylene lauryl ether, fatty acid alkanolamides such as lauric acid diethanolamide, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene polyoxy Examples include propylene fatty acid ester.
　カチオン性界面活性剤としては、アルキルアンモニウム、アルキルベンジルアンモニウム塩等、両性界面活性剤としては、アルキルベタイン、脂肪酸アミドプロピルベタイン、アルキルイミダゾリニウムベタイン等のベタイン系等が挙げられる。 Examples of cationic surfactants include alkylammonium and alkylbenzylammonium salts, and examples of amphoteric surfactants include betaine-based surfactants such as alkyl betaines, fatty acid amidopropyl betaines, and alkylimidazolinium betaines.
　口腔用組成物における界面活性剤の配合量は、例えば０．００１重量％～１０重量％、好ましくは０．１重量％～５重量％である。 The blending amount of the surfactant in the oral composition is, for example, 0.001% to 10% by weight, preferably 0.1% to 5% by weight.
　香料としては、ペパーミント油、スペアミント油、アニス油、ユーカリ油、ウィンターグリーン油、カシア油、クローブ油、タイム油、セージ油、レモン油、オレンジ油、ハッカ油、カルダモン油、コリアンダー油、マンダリン油、ライム油、ラベンダー油、ローズマリー油、ローレル油、カモミル油、キャラウェイ油、マジョラム油、ベイ油、レモングラス油、オリガナム油、パインニードル油、ネロリ油、ローズ油、ジャスミン油、グレープフルーツ油、スウィーティー油、柚油、イリスコンクリート、アブソリュートペパーミント、アブソリュートローズ、オレンジフラワー等の天然香料、及び、これら天然香料の加工処理（前溜部カット、後溜部カット、分留、液液抽出、エッセンス化、粉末香料化等）した香料、及び、メントール、カルボン、アネトール、シネオール、サリチル酸メチル、シンナミックアルデヒド、オイゲノール、３－ｌ－メントキシプロパン－１，２－ジオール、チモール、リナロール、リナリールアセテート、リモネン、メントン、メンチルアセテート、Ｎ－置換－パラメンタン－３－カルボキサミド、ピネン、オクチルアルデヒド、シトラール、プレゴン、カルビールアセテート、アニスアルデヒド、エチルアセテート、エチルブチレート、アリルシクロヘキサンプロピオネート、メチルアンスラニレート、エチルメチルフェニルグリシデート、バニリン、ウンデカラクトン、ヘキサナール、ブタノール、イソアミルアルコール、ヘキセノール、ジメチルサルファイド、シクロテン、フルフラール、トリメチルピラジン、エチルラクテート、エチルチオアセテート等の単品香料、更に、ストロベリーフレーバー、アップルフレーバー、バナナフレーバー、パイナップルフレーバー、グレープフレーバー、マンゴーフレーバー、バターフレーバー、ミルクフレーバー、フルーツミックスフレーバー、トロピカルフルーツフレーバー等の調合香料等、口腔用組成物に用いられる公知の香料素材を組み合わせて使用することができる。 Fragrances include peppermint oil, spearmint oil, anise oil, eucalyptus oil, wintergreen oil, cassia oil, clove oil, thyme oil, sage oil, lemon oil, orange oil, peppermint oil, cardamom oil, coriander oil, mandarin oil, Lime oil, lavender oil, rosemary oil, laurel oil, chamomile oil, caraway oil, marjoram oil, bay oil, lemongrass oil, origanum oil, pine needle oil, neroli oil, rose oil, jasmine oil, grapefruit oil, sweetie oil Natural fragrances such as oil, yuzu oil, iris concrete, absolute peppermint, absolute rose, orange flower, etc., and processing of these natural fragrances (front distillation cut, rear distillation cut, fractional distillation, liquid-liquid extraction, essence formation, powdered fragrances, etc.), and menthol, carvone, anethole, cineole, methyl salicylate, cinnamic aldehyde, eugenol, 3-l-menthoxypropane-1,2-diol, thymol, linalool, linaryl acetate, limonene , menthone, menthyl acetate, N-substituted-paramenthane-3-carboxamide, pinene, octylaldehyde, citral, pulegone, carbyl acetate, anisaldehyde, ethyl acetate, ethyl butyrate, allylcyclohexanepropionate, methyl anthranilate, Individual fragrances such as ethylmethylphenylglycidate, vanillin, undecalactone, hexanal, butanol, isoamyl alcohol, hexenol, dimethyl sulfide, cyclotene, furfural, trimethylpyrazine, ethyl lactate, ethylthioacetate, strawberry flavor, apple flavor, Known flavoring materials used in oral compositions can be used in combination, such as blended flavorings such as banana flavor, pineapple flavor, grape flavor, mango flavor, butter flavor, milk flavor, mixed fruit flavor, and tropical fruit flavor. .
　香料素材は、口腔用組成物中に０．０００００１重量％～１重量％使用することが好ましい。また、上記香料素材を使用した賦香用香料は、口腔用組成物中に０．１重量％～２重量％使用することが好ましい。 The fragrance material is preferably used in an amount of 0.000001% to 1% by weight in the oral composition. Further, it is preferable that the perfume for flavoring using the above-mentioned perfume material is used in the oral composition in an amount of 0.1% to 2% by weight.
　甘味料としては、サッカリンナトリウム、ステビオサイド、パラメトキシシンナミックアルデヒド、ペリラルチン等が挙げられる。着色剤としては、青色１号、黄色４号、二酸化チタン等が挙げられる。 Examples of sweeteners include saccharin sodium, stevioside, paramethoxycinnamic aldehyde, perilartine, and the like. Examples of the coloring agent include Blue No. 1, Yellow No. 4, and titanium dioxide.
　防腐剤としては、パラオキシ安息香酸エステル、安息香酸ナトリウム等の安息香酸又はその塩等が挙げられる。 Examples of the preservative include benzoic acid or its salts such as paraoxybenzoic acid ester and sodium benzoate.
　口腔用組成物には、上記ペプチドに加えて、イソプロピルメチルフェノール等の非イオン性殺菌剤、塩化セチルピリジニウム、塩酸クロルヘキシジン、塩化ベンザルコニウム、塩化ベンゼトニウム等のカチオン性殺菌剤、トラネキサム酸、イプシロンアミノカプロン酸、アラントイン、グリチルレチン酸、グリチルリチン酸等の抗炎症剤、デキストラナーゼ、ムタナーゼ、アミラーゼ、プロテアーゼ等の酵素、フッ化ナトリウム、モノフルオロリン酸ナトリウム等のフッ化物、正リン酸のカリウム塩、ナトリウム塩等の水溶性リン酸化合物、グルコン酸銅、銅クロロフィリンナトリウム等の銅化合物、塩化ナトリウム、硝酸カリウム、乳酸アルミニウム、塩化亜鉛、クエン酸亜鉛、塩化ストロンチウム等の無機塩類、酢酸トコフェロール等のビタミン類、ゼオライト、アズレン、ジヒドロコレステロール、クロロフィル、トウキ軟エキス、タイム、オウゴン、チョウジ、ハマメリス等の植物抽出物、歯石防止剤、歯垢防止剤等を配合してもよい。これらの成分は、本発明の効果を妨げない範囲で有効量配合することができる。 In addition to the above-mentioned peptides, the oral composition contains nonionic disinfectants such as isopropylmethylphenol, cationic disinfectants such as cetylpyridinium chloride, chlorhexidine hydrochloride, benzalkonium chloride, and benzethonium chloride, tranexamic acid, and epsilon aminocaprone. Acid, anti-inflammatory agents such as allantoin, glycyrrhetinic acid, glycyrrhizic acid, enzymes such as dextranase, mutanase, amylase, protease, fluoride such as sodium fluoride, sodium monofluorophosphate, potassium salt of orthophosphoric acid, sodium Water-soluble phosphoric acid compounds such as salts, copper compounds such as copper gluconate and sodium copper chlorophyllin, inorganic salts such as sodium chloride, potassium nitrate, aluminum lactate, zinc chloride, zinc citrate, and strontium chloride, vitamins such as tocopherol acetate, Zeolite, azulene, dihydrocholesterol, chlorophyll, Japanese angelica soft extract, plant extracts such as thyme, scutellariae, clove, and hamamelis, anti-tartar agents, anti-plaque agents, etc. may be blended. These components can be blended in an effective amount within a range that does not impede the effects of the present invention.
Ｅ．ジンジパインの検出方法
　本発明の別の局面によれば、Ａ項に記載のペプチドを用いてジンジパインを免疫学的測定法により検出することを含む、ジンジパインの検出方法が提供される。検出対象のジンジパインは、Ｒｇｐ及びＫｇｐのいずれか一方又は両方であり得る。検出対象のジンジパインに対するペプチドの解離定数は、好ましくは１．０×１０－８Ｍ以下、より好ましくは１．０×１０－９Ｍ以下である。 E. Method for Detecting Gingipains According to another aspect of the present invention, there is provided a method for detecting gingipains, which comprises detecting gingipains by an immunoassay using the peptide described in Section A. The gingipain to be detected may be one or both of Rgp and Kgp. The dissociation constant of the peptide for gingipain to be detected is preferably 1.0×10 −8 M or less, more preferably 1.0×10 −9 M or less.
　免疫学的測定法としては、イムノブロッティング法、酵素免疫測定法（ＥＩＡ、ＥＬＩＳＡ）、放射線免疫測定法（ＲＩＡ）、蛍光抗体法、イムノクロマトグラフィー法等が挙げられる。なかでも、ＥＬＩＳＡ法又はイムノクロマトグラフィー法が好ましい。 Examples of immunoassay methods include immunoblotting, enzyme-linked immunosorbent assay (EIA, ELISA), radiation immunoassay (RIA), fluorescent antibody method, and immunochromatography. Among these, ELISA method or immunochromatography method is preferred.
　免疫学的測定法は、好ましくはサンドイッチ式免疫学的測定法であり、例えばサンドイッチＥＬＩＳＡ又はサンドイッチイムノクロマトグラフィー法であり得る。サンドイッチ式免疫学的測定法は、支持体に捕捉抗体を固定化し、標識抗体と捕捉抗体とがサンドイッチ状に被検物質に対して結合することにより被検物質を定性分析又は定量分析する方法である。 The immunoassay method is preferably a sandwich immunoassay method, and can be, for example, a sandwich ELISA or a sandwich immunochromatography method. Sandwich immunoassay is a method for qualitative or quantitative analysis of a test substance by immobilizing a capture antibody on a support and binding the labeled antibody and capture antibody to the test substance in a sandwich-like manner. be.
　サンドイッチ式免疫学的測定法においては、Ａ項に記載のペプチドであって、アミノ酸配列が異なる２種類のペプチドＡ及びＢ（結果として、ペプチドＡ及びＢはそれぞれ、検出対象のジンジパインの異なる部位を認識する）を捕捉抗体及び標識抗体として用いることができる。 In the sandwich immunoassay, two types of peptides A and B, which are the peptides described in Section A and have different amino acid sequences, are used (as a result, peptides A and B each target different sites of the gingipain to be detected). can be used as a capture antibody and a labeled antibody.
　例えば、サンドイッチＥＬＩＳＡ法においては、捕捉抗体（一次抗体）として未標識のペプチドＡを９６ウェルマイクロタイタープレート、ポリスチレンビーズ等の担体に固相化し、又はビオチン標識したペプチドＡをアビジンコートされた９６ウェルマイクロタイタープレートに固相化し、標識抗体（二次抗体）として、西洋ワサビペルオキシダーゼ（ＨＲＰ)、アルカリフォスファターゼ（ＡＬＰ)等の酵素、ＦＩＴＣ等の蛍光物質、ビオチン等の標識物質で標識したペプチドＢを用いることができる。標識抗体をビオチンで標識し、標識したアビジン又はストレプトアビジンをビオチンに結合させて発色させてもよい。ジンジパインと捕捉抗体又は標識抗体との結合はそれぞれ、例えば０℃～３７℃、好ましくは４℃～２５℃の温度条件で、例えば３０分～２４時間、好ましくは６０分～１２０分の反応時間で行うことができる。 For example, in the sandwich ELISA method, unlabeled peptide A is immobilized as a capture antibody (primary antibody) on a carrier such as a 96-well microtiter plate or polystyrene beads, or biotin-labeled peptide A is immobilized on a 96-well avidin-coated well. Peptide B is immobilized on a microtiter plate and labeled with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), a fluorescent substance such as FITC, or a labeling substance such as biotin as a labeled antibody (secondary antibody). Can be used. The labeled antibody may be labeled with biotin, and the labeled avidin or streptavidin may be bound to the biotin to develop color. The binding of gingipain and the capture antibody or the labeled antibody is carried out under temperature conditions of, for example, 0°C to 37°C, preferably 4°C to 25°C, and for a reaction time of, for example, 30 minutes to 24 hours, preferably 60 minutes to 120 minutes. It can be carried out.
　上記サンドイッチ式免疫学的測定法において、ペプチドを支持体に固相化する場合、支持体とペプチドとの間にスタンドペプチドを介在させることにより、支持体の影響を緩和して、ジンジパインの測定精度を向上することができる。 In the sandwich immunoassay method described above, when the peptide is immobilized on the support, a stand peptide is interposed between the support and the peptide to alleviate the influence of the support and improve the measurement accuracy of gingipain. can be improved.
Ｆ．ジンジパインを検出又は測定するためのキット
　本発明の別の局面によれば、少なくとも、Ａ項に記載のペプチドであって、アミノ酸配列が異なる２種類のペプチド（結果として、２種類のペプチドはそれぞれ、ジンジパインの異なる部位を認識する）を含むジンジパインを検出又は測定するためのキットが提供される。検出又は測定対象のジンジパインは、Ｒｇｐ及びＫｇｐのいずれか一方又は両方であり得る。２種類のペプチドの一方は、支持体に固相化され、他方は標識物質で標識されていてもよい。 F. Kit for detecting or measuring gingipains According to another aspect of the present invention, at least two types of peptides having different amino acid sequences among the peptides described in Section A (as a result, the two types of peptides each have A kit for detecting or measuring gingipain is provided. The gingipain to be detected or measured may be one or both of Rgp and Kgp. One of the two types of peptides may be immobilized on a support, and the other may be labeled with a labeling substance.
　キットは、検体採取器具、検体処理液、発色基質等の試薬を含んでいてもよく、さらに、試験に必要な器具等を含んでいてもよい。 The kit may contain reagents such as a specimen collection device, a specimen processing liquid, and a coloring substrate, and may further contain equipment necessary for the test.
　以下、実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.
１．ジンジパイン結合ペプチドのセレクション
　ＷＯ２００６／０４１１９４に記載のリンカーを利用したｉｎ　ｖｉｔｒｏ　Ｖｉｒｕｓ法によって、図１に示すスキームでジンジパインに親和性を有するペプチドをセレクションした。
　具体的には、ＷＯ２００６／０４１１９４の記載に従って作製したランダム８アミノ酸からなるｃＤＮＡディスプレイライブラリをターゲット固定化用ビーズ（ストレプトアビジン修飾磁気ビーズ）と混合し（図１（ａ））、ターゲット固定化用ビーズと非特異的に結合したペプチドを取り除き、当該ビーズと結合しなかったペプチドをジンジバリス菌（ＡＴＣＣ　３３２７７株）培養上清から精製したジンジパイン（Ｒｇｐ）を固定化したビーズと混合した（図１（ｂ））。次いで、遠心分離を行って上清を除去し、洗浄及び溶出を行うことにより、ジンジパインと結合したペプチドを回収した（図１（ｃ））。
　徐々に洗浄条件を厳しくしながら同様の操作をさらに４回繰り返した（計５回のセレクション）。具体的には、セレクション緩衝液として、１０ｍＭリン酸ナトリウム（ｐＨ７．５）、１００ｍＭ塩化ナトリウム、５ｍＭ塩化マグネシウムを使用した。１回目セレクションの洗浄には、１０ｍＭリン酸ナトリウム（ｐＨ７．５）、１Ｍ塩化ナトリウム、２～３回目セレクションの洗浄には、１０ｍＭリン酸ナトリウム（ｐＨ７．５）、２Ｍ塩化ナトリウム、４回目セレクションの洗浄には、１０ｍＭリン酸ナトリウム（ｐＨ７．５）、３Ｍ塩化ナトリウム、５回目セレクションの洗浄には、１０ｍＭリン酸ナトリウム（ｐＨ７．５）、３Ｍ塩化ナトリウム、０．１％ＳＤＳを用いた。
　市販のクローニングキット（Ｚｅｒｏ　Ｂｌｕｎｔ　ＴＯＰＯ　ＰＣＲ　Ｃｌｏｎｉｎｇ　Ｋｉｔ，　Ｉｎｖｉｔｒｏｇｅｎ）を用いて、得られたジンジパイン結合ペプチドのクローニングを行い、ダイデオキシ法によるシーケンスサンプル調製、及びシーケンサーによる塩基配列の決定を行い、塩基配列に基づいてジンジパイン結合ペプチドのアミノ酸配列を決定した。
　なお、ジンジバリス菌培養液の上清中のＲｇｐの精製は、以下の通り行った。
１．ジンジバリス菌（ＡＴＣＣ　３３２７７株）培養上清約１０リットルを集め、濃縮、さらに１０ｍＭ　リン酸緩衝液（ｐＨ７．０）に対して透析した後、同緩衝液で平衡化したＤＥＡＥセファロース陰イオンクロマトグラフィーにかけた。同緩衝液でカラムを十分洗浄した後、０．１Ｍ塩化ナトリウムを含む同緩衝液で溶出される活性画分を集め、濃縮した。
２．得られた活性画分を１０ｍＭ酢酸緩衝液（ｐＨ５．５）に対して透析し、同緩衝液で平衡化したＣＭセファデックス陽イオンクロマトグラフィーにかけた。同緩衝液で溶出される非吸着画分を集め、濃縮、１０ｍＭ　リン酸緩衝液（ｐＨ７．０）に対して透析した。
３．活性画分を同緩衝液で平衡化したＭｏｎｏ　Ｑ陰イオンクロマトグラフィーにかけた。塩化ナトリウムの濃度勾配によって溶出される活性画分を集め、濃縮透析を行った。
４．得られた酵素画分をＡｒｇセファロースのアフィニティークロマトにかけた。カラム吸着画分をリジンで溶出し、それらを濃縮した。
５．濃縮試料をＴＳＫ―Ｇ２０００ＳＷカラム（東ソー）でゲルろ過した後、活性画分を濃縮した。
６．ＳＤＳゲル電気泳動で、単一バンドに精製されていることを確認した。
　また、培養上清中のジンジパイン濃度は、門脇ら、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２６９；２１３７１－８（１９９４）記載の方法によって決定した。 1. Selection of gingipain-binding peptides Peptides having affinity for gingipain were selected according to the scheme shown in FIG. 1 by an in vitro virus method using a linker described in WO2006/041194.
Specifically, a cDNA display library consisting of random 8 amino acids prepared according to the description in WO2006/041194 is mixed with target immobilization beads (streptavidin-modified magnetic beads) (Figure 1(a)), and the target immobilization beads are mixed with target immobilization beads (streptavidin-modified magnetic beads). The peptides that non-specifically bound to the beads were removed, and the peptides that did not bind to the beads were mixed with beads immobilized with gingipain (Rgp) purified from the culture supernatant of B. gingivalis (ATCC strain 33277) (Figure 1 (b). )). Next, the supernatant was removed by centrifugation, and the peptide bound to gingipain was recovered by washing and elution (FIG. 1(c)).
The same operation was repeated four more times while gradually tightening the washing conditions (total of five selections). Specifically, 10mM sodium phosphate (pH 7.5), 100mM sodium chloride, and 5mM magnesium chloride were used as the selection buffer. For the first selection wash, 10mM sodium phosphate (pH 7.5), 1M sodium chloride; for the second and third selection washes, 10mM sodium phosphate (pH 7.5), 2M sodium chloride; for the fourth selection, For washing, 10 mM sodium phosphate (pH 7.5), 3M sodium chloride were used, and for the fifth selection wash, 10 mM sodium phosphate (pH 7.5), 3M sodium chloride, and 0.1% SDS were used.
The obtained gingipain-binding peptide was cloned using a commercially available cloning kit (Zero Blunt TOPO PCR Cloning Kit, Invitrogen), and a sequence sample was prepared using the dideoxy method and the base sequence was determined using a sequencer. Based on this, the amino acid sequence of the gingipain-binding peptide was determined.
Note that Rgp in the supernatant of the B. gingivalis culture was purified as follows.
1. Approximately 10 liters of the culture supernatant of P. gingivalis (ATCC strain 33277) was collected, concentrated, dialyzed against 10 mM phosphate buffer (pH 7.0), and then subjected to DEAE Sepharose anion chromatography equilibrated with the same buffer. Ta. After thoroughly washing the column with the same buffer, active fractions eluted with the same buffer containing 0.1M sodium chloride were collected and concentrated.
2. The obtained active fraction was dialyzed against 10 mM acetate buffer (pH 5.5) and subjected to CM Sephadex cation chromatography equilibrated with the same buffer. Non-adsorbed fractions eluted with the same buffer were collected, concentrated, and dialyzed against 10 mM phosphate buffer (pH 7.0).
3. The active fraction was subjected to Mono Q anion chromatography equilibrated with the same buffer. Active fractions eluted with a sodium chloride concentration gradient were collected and subjected to concentration dialysis.
4. The obtained enzyme fraction was subjected to affinity chromatography using Arg Sepharose. The column adsorbed fractions were eluted with lysine and they were concentrated.
5. After gel filtration of the concentrated sample using a TSK-G2000SW column (Tosoh), the active fraction was concentrated.
6. Purification into a single band was confirmed by SDS gel electrophoresis.
In addition, the gingipain concentration in the culture supernatant was determined by Kadowaki et al., J. Biol. Chem. 269; 21371-8 (1994).
　また、両端にシステインを有し、その間にランダム１２アミノ酸を含んだペプチド（総アミノ酸残基数１４）からなるｃＤＮＡディスプレイライブラリを作製し、組み換えジンジパイン（アブカム社「Ｒｅｃｏｍｂｉｎａｎｔ　Ｇｉｎｇｉｐａｉｎ　Ｒ１　ｐｒｏｔｅｉｎ　（Ｈｉｓ　ｔａｇ）」）を用い、上記と同様にして計７回のセレクションを行ってジンジパイン結合ペプチドを得た。具体的には、２回目までの溶出は、ジチオトレイトールを用いて行い、３回目～６回目の溶出は、ビオチンを用いて行い、７回目の溶出は、Ｒｇｐ阻害剤であるＫＹＴ－１を用いた競合溶出を行った。
　得られたジンジパイン結合ペプチドのアミノ酸配列を上記と同様にして決定した。 In addition, a cDNA display library consisting of a peptide having cysteine at both ends and 12 random amino acids between them (total number of amino acid residues: 14) was created, and a recombinant gingipain (Abcam "Recombinant Gingipain R1 protein (His tag)") was prepared. ), a total of seven selections were performed in the same manner as above to obtain a gingipain-binding peptide. Specifically, the second elution was performed using dithiothreitol, the third to sixth elutions were performed using biotin, and the seventh elution was performed using KYT-1, an Rgp inhibitor. Competitive elution was performed using
The amino acid sequence of the resulting gingipain-binding peptide was determined in the same manner as above.
　アミノ酸配列の特徴、合成の容易さ等を考慮して、候補ペプチドを選択し、Ｆｍｏｃ法によりペプチド合成を行った（ＲＰ－ＨＰＬＣによる合成ペプチドの純度≧９０％）。 Candidate peptides were selected taking into consideration the characteristics of the amino acid sequence, ease of synthesis, etc., and peptide synthesis was performed by the Fmoc method (purity of the synthesized peptide by RP-HPLC ≧90%).
２．ジンジパイン結合ペプチドの分子設計
　合成ペプチドに対して、ジンジパインに対する親和性評価を行い、評価結果が良好であったペプチドに関してさらなる高機能化を目的として分子設計を行った。以下に分子設計の一例を説明する。また、ジンジパインに対する親和性評価については後述する。 2. Molecular design of gingipain-binding peptide Synthetic peptides were evaluated for their affinity for gingipain, and those peptides with good evaluation results were subjected to molecular design with the aim of further increasing their functionality. An example of molecular design will be explained below. Furthermore, evaluation of affinity for gingipain will be described later.
　高いジンジパイン親和性が確認されたペプチド「Ｒ３－１」（配列番号：１０）について、ジンジパインとの結合に重要な部位の調査を行った。具体的には、ペプチド側鎖の影響を検討するために、ペプチドの各アミノ酸残基をアラニンに置換したアナログを合成した（ただし、グリシン残基は側鎖に水素原子しか持たないため、グリシン残基をアラニンに置換したアナログは合成せず、活性評価も行わなかった）。合成したアラニン置換ペプチドを図２に示す（図中、「Ａｈｘ」は、疎水性スペーサーとしての６－アミノヘキサン酸を表す）。 Regarding the peptide "R3-1" (SEQ ID NO: 10), which has been confirmed to have high affinity for gingipain, we investigated the important site for binding to gingipain. Specifically, in order to examine the influence of peptide side chains, we synthesized analogs in which each amino acid residue of the peptide was replaced with alanine (however, since glycine residues only have hydrogen atoms in their side chains, Analogs in which the groups were replaced with alanine were not synthesized nor were their activity evaluated). The synthesized alanine-substituted peptide is shown in FIG. 2 (in the figure, "Ahx" represents 6-aminohexanoic acid as a hydrophobic spacer).
　これらのアラニン置換ペプチドに対し、水晶振動子マイクロバランス（ＱＣＭ）法を用いて、ジンジパインに対する親和性の変化を調べた。具体的には、予めアラニン置換ペプチドを固定した基板に対してジンジパインを接触させて、アラニン置換ペプチドとジンジパインとを添加して結合させておき、その後、ペプチド「Ｒ３－１」を添加する。ここで、アラニン置換ペプチドのジンジパイン親和性がペプチド「Ｒ３－１」よりも低下していた場合、競合反応によってジンジパインがペプチド「Ｒ３－１」に移る（図３、上のケース）。一方、濃度にもよるが、アラニン置換ペプチドのジンジパイン親和性がペプチド「Ｒ３－１」以上であった場合、基本的にはジンジパインに結合したままになると考えられる（図３、下のケース）。よって、親和性が低下していれば、基板上の重量が変化することとなり、その変化を読み取ることで親和性の変化として評価することが可能となる。 These alanine-substituted peptides were examined for changes in affinity for gingipain using the quartz crystal microbalance (QCM) method. Specifically, gingipain is brought into contact with a substrate on which an alanine-substituted peptide has been immobilized in advance, and the alanine-substituted peptide and gingipain are added and bonded, and then peptide "R3-1" is added. Here, if the affinity of the alanine-substituted peptide for gingipain is lower than that of peptide "R3-1", gingipain is transferred to peptide "R3-1" by competitive reaction (FIG. 3, upper case). On the other hand, although it depends on the concentration, if the affinity of the alanine-substituted peptide to gingipain is higher than that of peptide "R3-1", it is thought that it basically remains bound to gingipain (FIG. 3, bottom case). Therefore, if the affinity decreases, the weight on the substrate will change, and by reading this change, it becomes possible to evaluate it as a change in affinity.
　７種類のアラニン置換ペプチドに対して親和性の変化を測定した結果をまとめたものを図４に示す。図４の結果から、ペプチド「Ｒ３－１」のＣ末端から４残基はジンジパインとの結合に重要な役割を示し、ペプチドのＮ末端から１～４残基は結合活性への寄与度が比較的低いことがわかる。また、このことから、ペプチド「Ｒ３－１」のＮ末端１～４残基はジンジパインとの相互作用に大きな影響を与えずに化学修飾が可能である可能性が示唆された。また、ペプチド「Ｒ３－１」のＮ末端１～４残基を有さないペプチドアナログはペプチド合成のコスト削減や、タンパク質への結合特異性の向上が考えられた。そこで、ペプチド「Ｒ３－１」のＮ末端３残基を除いたペプチド、Ｎ末端３残基に化学修飾を施したペプチド誘導体等を設計した。 Figure 4 shows a summary of the results of measuring changes in affinity for seven types of alanine-substituted peptides. From the results in Figure 4, the 4 residues from the C-terminus of peptide "R3-1" play an important role in binding to gingipain, and the contribution of 1 to 4 residues from the N-terminus of the peptide to the binding activity is compared. You can see that it's not very accurate. This also suggested that the N-terminal 1 to 4 residues of peptide "R3-1" may be chemically modified without significantly affecting the interaction with gingipain. Furthermore, a peptide analog without the N-terminal 1 to 4 residues of peptide "R3-1" was considered to reduce the cost of peptide synthesis and improve binding specificity to proteins. Therefore, we designed a peptide in which the N-terminal three residues of peptide "R3-1" were removed, and a peptide derivative in which the N-terminal three residues were chemically modified.
　上記１．に記載のペプチドセレクション及び２．に記載の分子設計によって、種々のペプチド（ペプチド誘導体を含む）を得た。得られたペプチドの配列及び構造を表３に示す。
Figure JPOXMLDOC01-appb-T000003
 Above 1. Peptide selection and 2. Various peptides (including peptide derivatives) were obtained by the molecular design described in . The sequences and structures of the obtained peptides are shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
３．ジンジパインに対する親和性評価
　ペプチドのジンジパインに対する親和性は、表面プラズモン共鳴（ＳＰＲ）技術を利用したＢｉａｃｏｒｅ（Ｒ）システムを用いて行った。具体的には、合成したペプチドをＢｉａｃｏｒｅ（Ｒ）システム用のセンサーチップ上に固定化し、そこにジンジバリス菌野生株培養上清の精製液を添加した。当該システムにおいては、培養上清中のジンジパインとチップ上のペプチドとが結合した場合には、固定化されているペプチドの質量が増加し、センサーチップ表面の溶媒の屈折率に変化が生じるため、その変化を測定することによって、結合の様子をリアルタイムにモニターすることが可能となる。
　培養上清添加時のセンサグラムカーブと添加終了後のセンサグラムカーブから結合速度定数（ｋａ）、解離速度定数（ｋｄ）を求め、これらに基づいて解離定数（ＫＤ＝ｋｄ／ｋａ）を算出した。結果を表４に示す。 3. Affinity evaluation for gingipains The affinity of peptides for gingipains was performed using the Biacore® system, which utilizes surface plasmon resonance (SPR) technology. Specifically, the synthesized peptide was immobilized on a sensor chip for the Biacore (R) system, and a purified culture supernatant of a wild-type strain of P. gingivalis was added thereto. In this system, when gingipain in the culture supernatant and the peptide on the chip combine, the mass of the immobilized peptide increases and the refractive index of the solvent on the sensor chip surface changes. By measuring this change, it becomes possible to monitor the state of binding in real time.
Obtain the association rate constant (ka) and dissociation rate constant (kd) from the sensorgram curve at the time of addition of the culture supernatant and the sensorgram curve after the addition, and calculate the dissociation constant (K D = kd/ka) based on these. did. The results are shown in Table 4.
４．特異性評価
　ペプチド「Ｒ３－１」に関して、特異性評価を実施した。
　具体的には、Ｂｉａｃｏｒｅ（Ｒ）システムを用いて、各ペプチドを固定化したセンサーチップ上にジンジバリス菌野生株（ＡＴＣＣ　３３２７７株）培養上清の精製液（Ｗｉｌｄ　ｔｙｐｅ、タンパク量：２２μｇ／ｍＬ）とジンジパイン遺伝子をノックアウトしたジンジバリス菌変異株培養上清の精製液（－Ｋｇｐ＆－Ｒｇｐ、タンパク量：２２μｇ／ｍＬ）及び当該精製液の１０倍濃縮液（－Ｋｇｐ＆－Ｒｇｐ、タンパク量：２２０μｇ／ｍＬ）を添加した。 4. Specificity Evaluation Specificity evaluation was performed regarding peptide “R3-1”.
Specifically, using the Biacore (R) system, a purified solution (Wild type, protein amount: 22 μg/mL) of the culture supernatant of B. gingivalis wild strain (ATCC 33277 strain) was placed on a sensor chip on which each peptide was immobilized. A purified solution of a culture supernatant of a mutant strain of M. gingivalis with the gingipain gene knocked out (-Kgp & -Rgp, protein amount: 22 μg/mL) and a 10-fold concentrated solution of the purified solution (-Kgp & -Rgp, protein amount: 220 μg/mL ) was added.
　その結果、図５に示すように、ペプチド「Ｒ３－１」は、ジンジパインを含む精製培養上清（Ｗｉｌｄ　ｔｙｐｅ、タンパク量：２２μｇ／ｍＬ）に対しては非常に強い反応を示した一方で、ジンジパインを含まない精製培養上清（－Ｋｇｐ＆－Ｒｇｐ、タンパク量：２２μｇ／ｍＬ）に対する反応は非常に弱く、１０倍濃縮液（－Ｋｇｐ＆－Ｒｇｐ、タンパク量：２２０μｇ／ｍＬ）に対しては多少反応を示すものの、ジンジパインを含む培養上清よりも低い反応であった。このことから、ペプチド「Ｒ３－１」のジンジパインに対する特異性はかなり高いと考えられる。 As a result, as shown in Figure 5, peptide "R3-1" showed a very strong reaction to purified culture supernatant containing gingipain (Wild type, protein amount: 22 μg/mL), while The reaction to the purified culture supernatant that does not contain gingipain (-Kgp & -Rgp, protein amount: 22 μg/mL) is very weak, and the reaction to the 10 times concentrated solution (-Kgp & -Rgp, protein amount: 220 μg/mL) is somewhat weak. Although it showed a reaction, the reaction was lower than that of the culture supernatant containing gingipain. From this, it is considered that the specificity of peptide "R3-1" to gingipain is quite high.
　さらに、ペプチド「Ｋ７２９４」及び「Ｒ５－７」についても同様の特異性評価を行った。その結果、図６に示すように、これらは共にジンジパインを含む培養上清（Ｗｉｌｄ　ｔｙｐｅ、タンパク量：２２μｇ／ｍＬ）に対してはペプチド「Ｒ３－１」と同様に非常に強い反応を示した一方で、ジンジパインを含まない培養上清（－Ｋｇｐ＆－Ｒｇｐ、タンパク量：２２μｇ／ｍＬ）及び１０倍濃縮液（－Ｋｇｐ＆－Ｒｇｐ、タンパク量：２２０μｇ／ｍＬ）に対してはペプチド「Ｒ３－１」よりも低い反応を示した。このことから、ペプチド「Ｋ７２９４」及び「Ｒ５－７」は、「Ｒ３－１」よりもジンジパインに対する特異性が高いことが示唆される。 Furthermore, similar specificity evaluations were performed for peptides "K7294" and "R5-7". As a result, as shown in Figure 6, both of these showed a very strong reaction to the culture supernatant containing gingipain (Wild type, protein amount: 22 μg/mL), similar to the peptide "R3-1". On the other hand, the peptide "R3-1 ” showed a lower response. This suggests that peptides "K7294" and "R5-7" have higher specificity for gingipain than "R3-1".
５．Ｒｇｐ活性の阻害能評価
　以下の方法でペプチドのＲｇｐ活性の阻害能を評価した。
１）９０μＬの１Ｍ　Ｌ－Ｃｙｓｔｅｉｎｅ水溶液に１０μＬの４Ｍ　ＮａＯＨ水溶液を添加して中和した（０．９Ｍ　Ｃｙｓｔｅｉｎｅ）。
２）ＧｉｎｇｉｓＲＥＸ（ＧＥＮＯＶＩＳ社製Ａｒｇ－ジンジパイン、１μＭ）をＮｕｃｌｅａｓｅ　ｆｒｅｅ　Ｗａｔｅｒ（ＮＦＷ）（Ａｍｂｉｏｎ社製）で４０倍に希釈した（２５ｎＭ　ＧｉｎｇｉｓＲＥＸ）。
３）９６ｗｅｌｌ　ＮＢＳ　ｂｌａｃｋ　ｐｌａｔｅ（Ｃｏｒｎｉｎｇ　＃３９９１）の各ウェルに、１０μＬの１Ｍ　Ｔｒｉｓ－ＨＣｌ（ｐＨ７．５）、１．１１μＬの０．９Ｍ　Ｃｙｓｔｅｉｎｅ、及び７１．８９μＬのＮＦＷを添加及び混和し、次いで、２μＬの２５ｎＭ　ＧｉｎｇｉｓＲＥＸを添加した（計８５μＬ／ウェル）。
４）溶媒としてジメチルスルホキシドを用いて種々の濃度に調整したペプチド溶液又は溶媒のみを５μＬ／ウェルとなるようにウェルに添加し、プレートシェーカーで約５００ｒｐｍ、１０秒間攪拌した。
５）ＮＦＷを用いて１ｍＭに希釈したＺ－Ｐｈｅ－Ａｒｇ－ＭＣＡ（ＥＰＴＩＤＥ　ＩＮＳＴＩＴＵＴＥ社製　＃３０９５－Ｖ）を各ウェルに１０μＬずつ添加し、プレートシェーカーで約５００ｒｐｍ、１０秒間攪拌した。
６）速やかにマルチスペクトロマイクロプレートリーダーＶａｒｉｏｓｋａｎ　Ｆｌａｓｈ（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ，＃５２５００４０）にて３７℃下で蛍光値（Ｅｘ：３８０ｎｍ，Ｅｍ：４６０ｎｍ）を３０秒間隔で２０分間測定した。
７）ウェル毎に、測定した値５分間あたりの傾きを取り、最大値を用いて溶媒のみを添加したウェルに対する比率を算出し、活性比率とした。
８）１から活性比率を減算し、阻害率とした。
　　５０％阻害濃度（ＩＣ５０）を表４に示す。 5. Evaluation of ability to inhibit Rgp activity The ability of the peptide to inhibit Rgp activity was evaluated by the following method.
1) 10 μL of 4M NaOH aqueous solution was added to 90 μL of 1M L-Cysteine aqueous solution to neutralize it (0.9M Cysteine).
2) GingisREX (Arg-gingipain, manufactured by GENOVIS, 1 μM) was diluted 40 times with Nuclease free Water (NFW) (manufactured by Ambion) (25 nM GingisREX).
3) Add and mix 10 μL of 1M Tris-HCl (pH 7.5), 1.11 μL of 0.9 M Cysteine, and 71.89 μL of NFW to each well of a 96-well NBS black plate (Corning #3991), and then mix. , 2 μL of 25 nM GingisREX was added (total 85 μL/well).
4) Peptide solutions adjusted to various concentrations using dimethyl sulfoxide as a solvent or only the solvent were added to the wells at 5 μL/well and stirred for 10 seconds at about 500 rpm using a plate shaker.
5) 10 μL of Z-Phe-Arg-MCA (manufactured by EPTIDE INSTITUTE, #3095-V) diluted to 1 mM using NFW was added to each well, and the mixture was stirred with a plate shaker at about 500 rpm for 10 seconds.
6) Fluorescence values (Ex: 380 nm, Em: 460 nm) were immediately measured at 37° C. for 20 minutes at 30 second intervals using a multi-spectro microplate reader Varioskan Flash (Thermo Scientific, #5250040).
7) For each well, the slope of the measured value per 5 minutes was taken, and using the maximum value, the ratio to the well to which only the solvent was added was calculated, and this was taken as the activity ratio.
8) The activity ratio was subtracted from 1 to obtain the inhibition rate.
The 50% inhibitory concentrations (IC 50 ) are shown in Table 4.
６．Ｋｇｐ活性の阻害能評価
　以下の方法でペプチドのＫｇｐ活性の阻害能を評価した。
１）９０μＬの１Ｍ　Ｌ－Ｃｙｓｔｅｉｎｅ水溶液に１０μＬの４Ｍ　ＮａＯＨ水溶液を添加して中和した（０．９Ｍ　Ｃｙｓｔｅｉｎｅ）。
２）９６ｗｅｌｌ　ＮＢＳ　ｂｌａｃｋ　ｐｌａｔｅ（Ｃｏｒｎｉｎｇ　＃３９９１）の各ウェルに、１０μＬの１Ｍ　Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）、０．２２２μＬの０．９Ｍ　Ｃｙｓｔｅｉｎｅ、及び７２．７７８μＬのＮＦＷを添加及び混和し、次いで、２μＬのＧｉｎｇｉｓＫＨＡＮ（ＧＥＮＯＶＩＳ社製Ｌｙｓ－ジンジパイン、１０Ｕ／μＬ）を添加した（計８５μＬ／ウェル）。
３）溶媒としてジメチルスルホキシドを用いて種々の濃度に調整したペプチド溶液又は溶媒のみを５μＬ／ウェルとなるようにウェルに添加し、プレートシェーカーで約５００ｒｐｍ、１０秒間攪拌した。
４）ＮＦＷを用いて１ｍＭに希釈したＺ－Ｈｉｓ－Ｇｌｕ－Ｌｙｓ－ＭＣＡ（ＥＰＴＩＤＥ　ＩＮＳＴＩＴＵＴＥ社製　＃３２１５－Ｖ）を各ウェルに１０μＬずつ添加し、プレートシェーカーで約５００ｒｐｍ、１０秒間攪拌した。
５）速やかにマルチスペクトロマイクロプレートリーダーＶａｒｉｏｓｋａｎ　Ｆｌａｓｈ（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ，＃５２５００４０）にて３７℃下で蛍光値（Ｅｘ：３８０ｎｍ，Ｅｍ：４６０ｎｍ）を３０秒間隔で２０分間測定した。
６）ウェル毎に、測定した値５分間あたりの傾きを取り、最大値を用いて溶媒のみを添加したウェルに対する比率を算出し、活性比率とした。
７）１から活性比率を減算し、阻害率とした。
　５０％阻害濃度（ＩＣ５０）を表４に示す。 6. Evaluation of ability to inhibit Kgp activity The ability of the peptide to inhibit Kgp activity was evaluated by the following method.
1) 10 μL of 4M NaOH aqueous solution was added to 90 μL of 1M L-Cysteine aqueous solution to neutralize it (0.9M Cysteine).
2) Add and mix 10 μL of 1M Tris-HCl (pH 8.0), 0.222 μL of 0.9 M Cysteine, and 72.778 μL of NFW to each well of a 96-well NBS black plate (Corning #3991), and then , 2 μL of GingisKHAN (Lys-gingipain manufactured by GENOVIS, 10 U/μL) was added (total of 85 μL/well).
3) Peptide solutions adjusted to various concentrations using dimethyl sulfoxide as a solvent or only the solvent were added to the wells at 5 μL/well, and stirred at about 500 rpm for 10 seconds using a plate shaker.
4) 10 μL of Z-His-Glu-Lys-MCA (manufactured by EPTIDE INSTITUTE, #3215-V) diluted to 1 mM with NFW was added to each well, and the mixture was stirred with a plate shaker at about 500 rpm for 10 seconds.
5) Fluorescence values (Ex: 380 nm, Em: 460 nm) were immediately measured at 37° C. for 20 minutes at 30 second intervals using a multi-spectro microplate reader Varioskan Flash (Thermo Scientific, #5250040).
6) For each well, the slope of the measured value over 5 minutes was taken, and using the maximum value, the ratio to the well to which only the solvent was added was calculated, and this was taken as the activity ratio.
7) The activity ratio was subtracted from 1 to obtain the inhibition rate.
The 50% inhibitory concentrations (IC 50 ) are shown in Table 4.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
７．パパイン活性の阻害能評価
　以下の方法でペプチドのシステインプロテアーゼ（パパイン）活性の阻害能を評価した。
１）９０μＬの１Ｍ　Ｌ－Ｃｙｓｔｅｉｎｅ水溶液に１０μＬの４Ｍ　ＮａＯＨ水溶液を添加して中和した。（０．９Ｍ　Ｃｙｓｔｅｉｎｅ）
２）パパイン（ＳＩＧＭＡ社製＃Ｐ４７６２）をＮｕｃｌｅａｓｅ　ｆｒｅｅ　Ｗａｔｅｒ（ＮＦＷ）（Ａｍｂｉｏｎ社製）で溶解した。（５μｇ／ｍＬパパイン）
３）９６ｗｅｌｌ　ＮＢＳ　ｂｌａｃｋ　ｐｌａｔｅ（Ｃｏｒｎｉｎｇ　＃３９９１）の各ウェルに５０μＬの１００ｍＭ　ＨＥＰＥＳ（ｐＨ７．０）、４μＬの５０ｍＭ　ＥＤＴＡ、１．１１μＬの０．９Ｍ　Ｃｙｓｔｅｉｎｅ、及び２７．８９μＬのＮＦＷを添加及び混和し、次いで、２μＬの５μｇ／ｍＬパパインを添加した（計８５μＬ／ウェル）。
４）溶媒としてジメチルスルホキシドを用いて種々の濃度に調整したペプチド溶液又は溶媒のみを５μＬ／ウェルとなるようにウェルに添加し、プレートシェーカーで約５００ｒｐｍ、１０秒間攪拌した。
５）ＮＦＷを用いて１ｍＭに希釈したＺ－Ｐｈｅ－Ａｒｇ－ＭＣＡ（ＥＰＴＩＤＥ　ＩＮＳＴＩＴＵＴＥ社製　＃３０９５－Ｖ）を各ウェルに１０μＬずつ添加し、プレートシェーカーで約５００ｒｐｍ、１０秒間攪拌した。
６）速やかにマルチスペクトロマイクロプレートリーダーＶａｒｉｏｓｋａｎ　Ｆｌａｓｈ（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ，　＃５２５００４０）にて３７℃下で蛍光値（Ｅｘ：３８０ｎｍ，Ｅｍ：４６０ｎｍ）を３０秒間隔で２０分間測定した。
７）ウェル毎に、測定した値５分間あたりの傾きを取り、最大値を用いて溶媒のみを添加したウェルに対する比率を算出し、活性比率とした。
８）１から活性比率を減算し、阻害率とした。
　その結果、ペプチド「Ｒ３－１」、「Ｒ５－７」、「Ｒ５－３５」、「Ｋ７４１９」、及び「Ｋ７４５５」はいずれも、１００μＭにおいて阻害率が検出限界（約１％）未満であり、有意な阻害活性が確認されなかった。対して、システインプロテアーゼの阻害剤として知られるＥ－６４のパパインに対する阻害活性を同様に調べた結果、そのＩＣ５０値は７ｎＭであった。
　また、ペプチド「Ｒ３－１」、「Ｋ７４１９」、及び「Ｋ７４５５」の１００μＭにおける他の酵素に対する阻害活性を常法に従い評価した例として、セリンプロテアーゼであるトロンビン、アスパラギン酸プロテアーゼであるカテプシンＤに対しては、いずれの阻害率も検出限界（約１％）未満であり、有意な阻害活性が確認されなかった。 7. Evaluation of ability to inhibit papain activity The ability of the peptide to inhibit cysteine protease (papain) activity was evaluated by the following method.
1) 10 μL of 4M NaOH aqueous solution was added to 90 μL of 1M L-Cysteine aqueous solution to neutralize it. (0.9M Cysteine)
2) Papain (#P4762 manufactured by SIGMA) was dissolved in Nuclease free Water (NFW) (manufactured by Ambion). (5μg/mL papain)
3) Add 50 μL of 100 mM HEPES (pH 7.0), 4 μL of 50 mM EDTA, 1.11 μL of 0.9 M Cysteine, and 27.89 μL of NFW to each well of a 96-well NBS black plate (Corning #3991) and mix. , then 2 μL of 5 μg/mL papain was added (85 μL/well total).
4) Peptide solutions adjusted to various concentrations using dimethyl sulfoxide as a solvent or only the solvent were added to the wells at 5 μL/well and stirred for 10 seconds at about 500 rpm using a plate shaker.
5) 10 μL of Z-Phe-Arg-MCA (manufactured by EPTIDE INSTITUTE, #3095-V) diluted to 1 mM using NFW was added to each well, and the mixture was stirred with a plate shaker at about 500 rpm for 10 seconds.
6) Fluorescence values (Ex: 380 nm, Em: 460 nm) were immediately measured at 37° C. for 20 minutes at 30 second intervals using a multi-spectro microplate reader Varioskan Flash (Thermo Scientific, #5250040).
7) For each well, the slope of the measured value per 5 minutes was taken, and using the maximum value, the ratio to the well to which only the solvent was added was calculated, and this was taken as the activity ratio.
8) The activity ratio was subtracted from 1 to obtain the inhibition rate.
As a result, the inhibition rate of peptides "R3-1", "R5-7", "R5-35", "K7419", and "K7455" was below the detection limit (about 1%) at 100 μM, No significant inhibitory activity was confirmed. On the other hand, the inhibitory activity of E-64, which is known as a cysteine protease inhibitor, against papain was similarly investigated and its IC 50 value was 7 nM.
In addition, as an example of evaluating the inhibitory activity of peptides "R3-1", "K7419", and "K7455" against other enzymes at 100 μM according to a conventional method, All inhibition rates were below the detection limit (approximately 1%), and no significant inhibitory activity was confirmed.
８．ジンジバリス菌増殖抑制能評価
　理化学研究所バイオリソース研究センター　Ｊａｐａｎ　Ｃｏｌｌｅｃｔｉｏｎ　ｏｆ　Ｍｉｃｒｏｏｒｇａｎｉｓｍｓ（ＪＣＭ）から凍結乾燥状態で入手したジンジバリス菌（ＪＣＭ１２２５７）をＡＴＣＣ　Ｍｅｄｉｕｍ　２７２２　Ｓｕｐｐｌｅｍｅｎｔｅｄ　Ｔｒｙｐｔｉｃ　Ｓｏｙ　Ｂｒｏｔｈ（以下ＡＴＣＣ２７２２培地）で３７℃、嫌気条件下で２日間での継代培養後、６２０ｎｍで吸光度を測定し、培地のみ（ブランク）の吸光度を引いた値がマックファーランド懸濁標準液０．５と同程度（小数点第３位を四捨五入）になるようにして、菌液を調製した。 8. Evaluation of ability to suppress the growth of Tris gingivalis bacteria The RIKEN BioResource Research Center Japan Collection of Microorganisms (JCM) obtained lyophilized bacteria Gingivalis bacteria (JCM12257) using ATCC Medium 2722 Supplemented Tryptic Soy. Broth (hereinafter referred to as ATCC2722 medium) at 37°C under anaerobic conditions. After subculturing for 2 days under ), a bacterial solution was prepared.
　ペプチド「Ｒ３－１」をジメチルスルホキシドに溶解し、得られた溶液を、蒸留水（株式会社大塚製薬工場、Ｌｏｔ．０Ｄ９６Ｎ）を用いてペプチドの終濃度が１，２８０μｇ／ｍＬになるように希釈した。その後、１６０μｇ／ｍＬ、２０μｇ／ｍＬ及び２．５μｇ／ｍＬになるようにＡＴＣＣ２７２２培地で希釈した後、同培地を用いて終濃度が０．０６μｇ／ｍＬ、０．１２μｇ／ｍＬ、０．２５μｇ／ｍＬ、０．５μｇ／ｍＬ、１μｇ／ｍＬ、２μｇ／ｍＬ、４μｇ／ｍＬ、８μｇ／ｍＬ、１６μｇ／ｍＬ、３２μｇ／ｍＬ、６４μｇ／ｍＬ及び１２８μｇ／ｍＬになるように希釈した。メチル酸ガベキサート（東京化成社製）とＫＹＴ－１（ペプチド研究所）についても同様にして、種々の濃度の希釈液を調製した。 Peptide "R3-1" was dissolved in dimethyl sulfoxide, and the resulting solution was diluted with distilled water (Otsuka Pharmaceutical Factory Co., Ltd., Lot. 0D96N) so that the final concentration of the peptide was 1,280 μg/mL. did. Then, after diluting with ATCC2722 medium to 160 μg/mL, 20 μg/mL, and 2.5 μg/mL, the final concentrations were 0.06 μg/mL, 0.12 μg/mL, and 0.25 μg/mL using the same medium. mL, 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 4 μg/mL, 8 μg/mL, 16 μg/mL, 32 μg/mL, 64 μg/mL, and 128 μg/mL. Diluted solutions of various concentrations were similarly prepared for gabexate methylate (manufactured by Tokyo Kasei Co., Ltd.) and KYT-1 (Peptide Institute).
　９６穴Ｕ字型マイクロプレートに、各濃度の被験物質（ペプチド、メチル酸ガベキサート（ＧＭ）、又はＫＹＴ－１）及びＡＴＣＣ２７２２培地（陽性対照；ＰＣ）１００μＬを添加し、その後１０μＬの菌液を添加した。陰性対照（ＮＣ）として、菌液の代わりにＡＴＣＣ２７２２培地１０μＬを添加した。その後、嫌気条件下３７℃で２日間培養した。 Add each concentration of the test substance (peptide, gabexate methylate (GM), or KYT-1) and 100 μL of ATCC2722 medium (positive control; PC) to a 96-well U-shaped microplate, then add 10 μL of bacterial solution. did. As a negative control (NC), 10 μL of ATCC2722 medium was added instead of the bacterial solution. Thereafter, the cells were cultured for 2 days at 37°C under anaerobic conditions.
　マイクロプレートミキサーで振とう後、各ウェルの波長６２０ｎｍにおける吸光度を測定（ｉＭａｒｋマイクロプレートリーダー、バイオ・ラッド　ラボラトリーズ株式会社）し、ＯＤ値とした。濃度反応曲線を作製し、当該曲線に基づいて下記の計算式の値を満たす検体濃度を求めて５０％増殖阻害濃度（ＧＩ５０）とした。結果を表５及び図７に示す。
　　　ＧＩ５０に相当するＯＤ値＝（ＰＣのＯＤ値－ＮＣのＯＤ値）×０．５＋ＮＣのＯＤ値 After shaking with a microplate mixer, the absorbance of each well at a wavelength of 620 nm was measured (iMark microplate reader, Bio-Rad Laboratories, Inc.) and determined as the OD value. A concentration-response curve was prepared, and based on the curve, a sample concentration satisfying the value of the following calculation formula was determined and defined as a 50% growth inhibition concentration (GI 50 ). The results are shown in Table 5 and FIG. 7.
OD value equivalent to GI 50 = (OD value of PC - OD value of NC) x 0.5 + OD value of NC
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
９．ペプチドを用いたジンジパインのＥＬＩＳＡ測定
１）アビジンプレート（住友ベークライト株式会社「＃ＢＳ－Ｘ７６０３」）に１５０ｍＭ　ＮａＣｌ含有１０ｍＭ　ＨＥＰＥＳ　ｂｕｆｆｅｒ（ｐＨ７．０）で希釈した固相ペプチド溶液（１μｇ／ｍＬ）を１００μＬ／ｗｅｌｌの容量で添加した。
２）４℃でｏｖｅｒ　ｎｉｇｈｔ反応後、反応液を廃棄して各ｗｅｌｌを洗浄した。洗浄液として０．０５％　Ｔｗｅｅｎ含有ＰＢＳ（ｐＨ７．４）を用い、３００μＬ／ｗｅｌｌの容量で３回洗浄した。
３）検体を１００μＬ／ｗｅｌｌの容量で添加した。なお、検体としては、０．１ｎＭ、１ｎＭ、又は１０ｎＭとなるようにＨＥＰＥＳ　ｂｕｆｆｅｒに溶解したＲｇｐ溶液を用いた。
４）４℃でｏｖｅｒ　ｎｉｇｈｔ反応後、反応液を廃棄して各ｗｅｌｌを洗浄した。洗浄液として０．０５％　Ｔｗｅｅｎ含有ＰＢＳ（ｐＨ７．４）を用い、３００μＬ／ｗｅｌｌの容量で３回洗浄した。
５）標識ペプチドのジメチルスルホキシド溶液（１０ｍＭ）をＨＥＰＥＳ　ｂｕｆｆｅｒで２，０００倍希釈し、１００μＬ／ｗｅｌｌの容量で添加した。
６）４℃でｏｖｅｒ　ｎｉｇｈｔ反応後、反応液を廃棄して各ｗｅｌｌを洗浄した。洗浄液として０．０５％　Ｔｗｅｅｎ含有ＰＢＳ（ｐＨ７．４）を用い、３００μＬ／ｗｅｌｌの容量で３回洗浄した。
７）ＨＲＰ標識ストレプトアビジンをＨＥＰＥＳ　ｂｕｆｆｅｒで１０，０００倍希釈し、１００μＬ／ｗｅｌｌで添加した。
８）４℃でｏｖｅｒ　ｎｉｇｈｔ反応後、反応液を廃棄して各ｗｅｌｌを洗浄した。洗浄液として０．０５％　Ｔｗｅｅｎ含有ＰＢＳ（ｐＨ７．４）を用い、３００μＬ／ｗｅｌｌの容量で３回洗浄した。
９）洗浄液を廃棄後、ＴＭＢ基質（ＳｅｒａＣａｒｅ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅｓ社）を１００μＬ／ｗｅｌｌで添加し、室温で２０分間反応させた。
１０）２Ｎ硫酸を１００μＬ／ｗｅｌｌの容量で添加し、直ちにプレートリーダー（プラクティカル社製）で４５０ｎｍにおける吸光度を測定した。 9. ELISA measurement of gingipain using peptides 1) Add 100 μL of solid-phase peptide solution (1 μg/mL) diluted with 10 mM HEPES buffer (pH 7.0) containing 150 mM NaCl to an avidin plate (Sumitomo Bakelite Co., Ltd. “#BS-X7603”). It was added at a volume of /well.
2) After over night reaction at 4°C, the reaction solution was discarded and each well was washed. Washing was performed three times at a volume of 300 μL/well using PBS containing 0.05% Tween (pH 7.4) as a washing solution.
3) The specimen was added in a volume of 100 μL/well. As the specimen, an Rgp solution dissolved in HEPES buffer to a concentration of 0.1 nM, 1 nM, or 10 nM was used.
4) After over night reaction at 4°C, the reaction solution was discarded and each well was washed. Washing was performed three times at a volume of 300 μL/well using PBS containing 0.05% Tween (pH 7.4) as a washing solution.
5) A dimethyl sulfoxide solution (10 mM) of labeled peptide was diluted 2,000 times with HEPES buffer and added at a volume of 100 μL/well.
6) After over night reaction at 4°C, the reaction solution was discarded and each well was washed. Washing was performed three times at a volume of 300 μL/well using PBS containing 0.05% Tween (pH 7.4) as a washing solution.
7) HRP-labeled streptavidin was diluted 10,000 times with HEPES buffer and added at 100 μL/well.
8) After over night reaction at 4°C, the reaction solution was discarded and each well was washed. Washing was performed three times at a volume of 300 μL/well using PBS containing 0.05% Tween (pH 7.4) as a washing solution.
9) After discarding the washing solution, TMB substrate (SeraCare Life Sciences) was added at 100 μL/well and reacted at room temperature for 20 minutes.
10) 2N sulfuric acid was added at a volume of 100 μL/well, and the absorbance at 450 nm was immediately measured using a plate reader (manufactured by Practical).
　固相ペプチド及び標識ペプチドとして用いたペプチドの組合せ並びに吸光度の測定結果を表６に示す。
Figure JPOXMLDOC01-appb-T000006
 Table 6 shows the combinations of peptides used as solid-phase peptides and labeled peptides, and the absorbance measurement results.
Figure JPOXMLDOC01-appb-T000006
　表６に示される通り、吸光度はジンジパインの濃度依存的に増大しており、このことから、配列が異なる２種類のジンジパイン結合ペプチドを用いたサンドイッチＥＬＩＳＡ法によってＲｇｐを検出できることがわかる。 As shown in Table 6, the absorbance increased depending on the concentration of gingipain, which indicates that Rgp can be detected by the sandwich ELISA method using two types of gingipain-binding peptides with different sequences.
１０．ジンジバリス菌に対するペプチドの最小発育阻止濃度（ＭＩＣ）の測定
　ジンジバリス菌（ＪＣＭ１２２５７）をＧＡＭブイヨン「ニッスイ」（日水製薬社製）を用いて調製した寒天平板培地で３７℃、４８時間、嫌気培養した。得られた新鮮な培養菌体をＡＴＣＣ２７２２培地に分散し、培地のみ（ブランク）を引いた値がマックファーランド懸濁標準液１．０と同程度になるよう調製した。これをさらに滅菌生理食塩水にて１０倍希釈した菌液（約１×１０７ｃｆｕ／ｍＬ）を接種菌液とした。 10. Measurement of Minimum Inhibitory Concentration (MIC) of Peptide against Gingivalis Bacteria Gingivalis bacteria (JCM12257) was cultured anaerobically at 37°C for 48 hours in an agar plate medium prepared using GAM broth "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.). . The obtained freshly cultured bacterial cells were dispersed in ATCC 2722 medium and adjusted so that the value obtained by subtracting only the medium (blank) was approximately the same as the McFarland suspension standard solution 1.0. This was further diluted 10 times with sterile physiological saline and a bacterial solution (approximately 1×10 7 cfu/mL) was used as an inoculum solution.
　ペプチドとして「Ｋ７３９３」、「Ｋ７４１９」、「Ｋ７４５５」、「Ｋ７５２８」、「ＫＹＴ－４１」、及び「Ｒ３－１」を用い、それらのＤＭＳＯ溶液をＡＴＣＣ２７２２培地で倍々希釈し、試験液とした。ペプチド濃度が２００μＭから０．２μＭになるよう調製し、試験液２００μＬを９６ウェルプレートの各ウェルに分注した後、菌液を１０μＬずつ接種した（最終菌量　約１×１０５ｃｆｕ／ｗｅｌｌ）。陰性対照として、菌液の代わりにＡＴＣＣ２７２２培地１０μＬを添加した。陽性対照として、ペプチド未添加で菌液入りのＡＴＣＣ２７２２培地１０μＬを添加した。３７℃、４８時間、嫌気培養した後、肉眼でウェルを観察し、次の判定基準に従って、発育陽性又は発育陰性を判定した。発育陰性と判定された最小濃度をＭＩＣ値とした。結果を表７に示す。
＜判定基準＞
発育陽性：試験液に混濁が認められる、直径１ｍｍ以上の沈殿が認められる、又は沈殿の直径が１ｍｍ以下であっても沈殿（沈殿塊）が２個以上認められる。
発育陰性：試験液に混濁が認められない、沈殿が認められない、又は直径が１ｍｍ以下の沈殿（沈殿塊）が１個のみ認められる。 "K7393", "K7419", "K7455", "K7528", "KYT-41", and "R3-1" were used as peptides, and their DMSO solutions were diluted several times with ATCC2722 medium to prepare test solutions. After adjusting the peptide concentration from 200 μM to 0.2 μM and dispensing 200 μL of the test solution into each well of a 96-well plate, 10 μL of the bacterial solution was inoculated (final amount of bacteria: approximately 1×10 5 cfu/well). . As a negative control, 10 μL of ATCC2722 medium was added instead of the bacterial solution. As a positive control, 10 μL of ATCC2722 medium containing bacterial solution without addition of peptide was added. After anaerobic culture at 37° C. for 48 hours, the wells were visually observed and growth positive or growth negative was determined according to the following criteria. The minimum concentration at which growth was determined to be negative was defined as the MIC value. The results are shown in Table 7.
<Judgment criteria>
Positive growth: Turbidity is observed in the test solution, a precipitate with a diameter of 1 mm or more is observed, or 2 or more precipitates (precipitate lumps) are observed even if the diameter of the precipitate is 1 mm or less.
Negative growth: No turbidity or precipitate is observed in the test solution, or only one precipitate (precipitate mass) with a diameter of 1 mm or less is observed.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　表７に示される通り、評価したペプチド全てに関して少なくとも４８時間のジンジバリス菌に対する静菌効果が確認された。 As shown in Table 7, all of the evaluated peptides were confirmed to have a bacteriostatic effect on B. gingivalis for at least 48 hours.
　本発明の実施形態によるペプチドは、歯周病等のジンジパインが関連する疾患の治療、予防、及び診断等において好適に用いられる。 The peptide according to the embodiment of the present invention is suitably used in the treatment, prevention, diagnosis, etc. of diseases related to gingipain such as periodontal disease.

Claims (14)

  1. 　下記アミノ酸配列（Ｉ）又は（ＩＩ）を含み、総アミノ酸残基数が４～１５であるペプチド
    　　　［アミノ酸配列（Ｉ）：ＲＸ１Ｘ２ＲＸ３
    （アミノ酸配列（Ｉ）中、Ｘ１、Ｘ２、及びＸ３はそれぞれ独立して、任意の天然アミノ酸残基を表す）］、
    　　　［アミノ酸配列（ＩＩ）：ＲＸ４Ｒ
    （アミノ酸配列（ＩＩ）中、Ｘ４は、任意の天然アミノ酸残基を表す）］；
    　ＣＲＳＶＫＷＨＨＲＶＦＧＳＣ（配列番号：１１）のアミノ酸配列からなるペプチド；及び
    　ＩＴＨＴＳＲＨＣ（配列番号：１２）のアミノ酸配列からなるペプチド；から選択され、
    　下記（ａ）～（ｃ）の１つ以上を満たす、
    　（ａ）アルギニン－ジンジパインに対する解離定数（ＫＤ）が１．０×１０－７Ｍ以下である、
    　（ｂ）１００μＭでアルギニン－ジンジパインの酵素活性を５０％以上阻害する、
    　（ｃ）５００μＭでリジン－ジンジパインの酵素活性を５０％以上阻害する、
    　ペプチド若しくはその誘導体又はこれらの塩。     A peptide containing the following amino acid sequence (I) or (II) and having a total number of amino acid residues of 4 to 15 [Amino acid sequence (I): RX 1 X 2 RX 3
    (In the amino acid sequence (I), X 1 , X 2 , and X 3 each independently represent any natural amino acid residue)],
    [Amino acid sequence (II): RX 4 R
    (In the amino acid sequence (II), X 4 represents any natural amino acid residue)];
    a peptide consisting of the amino acid sequence CRSVKWHHRVFGSC (SEQ ID NO: 11); and a peptide consisting of the amino acid sequence ITHTSRHC (SEQ ID NO: 12);
    Satisfies one or more of the following (a) to (c),
    (a) the dissociation constant (K D ) for arginine-gingipain is 1.0×10 −7 M or less;
    (b) inhibiting the enzyme activity of arginine-gingipain by 50% or more at 100 μM;
    (c) inhibiting the enzyme activity of lysine-gingipain by 50% or more at 500 μM;
    Peptides or derivatives thereof or salts thereof.
  2. 　前記アミノ酸配列（Ｉ）中、
    　Ｘ１が、Ａ、Ｒ、Ｖ、及びＥからなる群より選択され、
    　Ｘ２が、Ａ、Ｆ、及びＫからなる群より選択され、
    　Ｘ３が、Ｒ及びＦからなる群より選択される、
    　請求項１に記載のペプチド若しくはその誘導体又はこれらの塩。     In the amino acid sequence (I),
    X 1 is selected from the group consisting of A, R, V, and E;
    X 2 is selected from the group consisting of A, F, and K;
    X 3 is selected from the group consisting of R and F;
    The peptide according to claim 1, a derivative thereof, or a salt thereof.
  3. 　前記アミノ酸配列（Ｉ）中、
    　Ｘ１が、Ｒ及びＥからなる群より選択され、
    　Ｘ２が、Ａ及びＫからなる群より選択され、
    　Ｘ３が、Ｒである、
    　請求項１に記載のペプチド若しくはその誘導体又はこれらの塩。     In the amino acid sequence (I),
    X 1 is selected from the group consisting of R and E;
    X 2 is selected from the group consisting of A and K;
    X 3 is R,
    The peptide according to claim 1, a derivative thereof, or a salt thereof.
  4. 　前記アミノ酸配列（Ｉ）中、
    　Ｘ１がＲであり、Ｘ２がＫであり、Ｘ３がＲであるか、
    　Ｘ１がＲであり、Ｘ２がＡであり、Ｘ３がＲであるか、
    　Ｘ１がＥであり、Ｘ２がＫであり、Ｘ３がＲである、
    　請求項１に記載のペプチド若しくはその誘導体又はこれらの塩。     In the amino acid sequence (I),
    X 1 is R, X 2 is K, and X 3 is R,
    X 1 is R, X 2 is A, and X 3 is R,
    X 1 is E, X 2 is K, and X 3 is R,
    The peptide according to claim 1, a derivative thereof, or a salt thereof.
  5. 　前記アミノ酸配列（ＩＩ）中、Ｘ４が、Ａ、Ｐ、及びＫからなる群より選択される、
    　請求項１に記載のペプチド若しくはその誘導体又はこれらの塩。     In the amino acid sequence (II), X 4 is selected from the group consisting of A, P, and K.
    The peptide according to claim 1, a derivative thereof, or a salt thereof.
  6. 　前記アミノ酸配列（ＩＩ）中、Ｘ４が、Ｐである、
    　請求項１に記載のペプチド若しくはその誘導体又はこれらの塩。     In the amino acid sequence (II), X 4 is P;
    The peptide according to claim 1, a derivative thereof, or a salt thereof.
  7. 　１００μＭでパパインの酵素活性を５％を超えて阻害しない、請求項１に記載のペプチド若しくはその誘導体又はこれらの塩。 The peptide or a derivative thereof, or a salt thereof according to claim 1, which does not inhibit the enzymatic activity of papain by more than 5% at 100 μM.
  8. 　前記アミノ酸配列（Ｉ）又は（ＩＩ）を含むペプチドが、配列番号：１～１０で表されるペプチドである、請求項１に記載のペプチド若しくはその誘導体又はこれらの塩。 The peptide or a derivative thereof, or a salt thereof according to claim 1, wherein the peptide comprising the amino acid sequence (I) or (II) is a peptide represented by SEQ ID NOs: 1 to 10.
  9. 　請求項１から８のいずれか一項に記載のペプチド若しくはその誘導体又はこれらの塩を含む、ジンジパイン阻害剤。 A gingipain inhibitor comprising the peptide according to any one of claims 1 to 8, a derivative thereof, or a salt thereof.
  10. 　請求項１から８のいずれか一項に記載のペプチド若しくはその誘導体又はこれらの塩を含む、ジンジバリス菌増殖抑制剤。 gingivalis growth inhibitor, comprising the peptide according to any one of claims 1 to 8, a derivative thereof, or a salt thereof.
  11. 　請求項１から８のいずれか一項に記載のペプチド若しくはその誘導体又はこれらの塩を含む、口腔用組成物。 An oral composition comprising the peptide according to any one of claims 1 to 8, a derivative thereof, or a salt thereof.
  12. 　請求項１から８のいずれか一項に記載のペプチド若しくはその誘導体又はこれらの塩を用いてジンジパインを免疫学的測定法により検出することを含む、ジンジパインの検出方法。 A method for detecting gingipain, which comprises detecting gingipain by an immunoassay using the peptide according to any one of claims 1 to 8, a derivative thereof, or a salt thereof.
  13. 　前記免疫学的測定法が、請求項１から８のいずれか一項に記載のペプチドから選択されるアミノ酸配列が異なる２種類のペプチド若しくはその誘導体又はこれらの塩を用いたサンドイッチ式免疫測定法である、請求項１２に記載のジンジパインの検出方法。 The immunoassay method is a sandwich immunoassay method using two types of peptides having different amino acid sequences selected from the peptides according to any one of claims 1 to 8, derivatives thereof, or salts thereof. The method for detecting gingipain according to claim 12.
  14. 　前記サンドイッチ式免疫測定法が、ＥＬＩＳＡ法又はイムノクロマトグラフィー測定法である、請求項１３に記載の検出方法。
          14. The detection method according to claim 13, wherein the sandwich immunoassay is an ELISA method or an immunochromatography assay.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055742A1 (en) * 1998-04-29 1999-11-04 Marchant Roger E Surfactants that mimic the glycocalyx
JP2012232923A (en) * 2011-04-28 2012-11-29 Sunrise Kogyo Kk Short peptide
JP2015529221A (en) * 2012-09-07 2015-10-05 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ Peptides and their use
JP2017520569A (en) * 2013-06-26 2017-07-27 ザイジェン インフラメーション エルティーディー Novel use of JNK inhibitor molecules for the treatment of various diseases
US20190209646A1 (en) * 2016-09-29 2019-07-11 Meharry Medical College Bacterial inhibitors
CN110305193A (en) * 2019-07-18 2019-10-08 河南科技大学 A kind of resisting porphyromonas gingivalis polypeptide and application

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055742A1 (en) * 1998-04-29 1999-11-04 Marchant Roger E Surfactants that mimic the glycocalyx
JP2012232923A (en) * 2011-04-28 2012-11-29 Sunrise Kogyo Kk Short peptide
JP2015529221A (en) * 2012-09-07 2015-10-05 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ Peptides and their use
JP2017520569A (en) * 2013-06-26 2017-07-27 ザイジェン インフラメーション エルティーディー Novel use of JNK inhibitor molecules for the treatment of various diseases
US20190209646A1 (en) * 2016-09-29 2019-07-11 Meharry Medical College Bacterial inhibitors
CN110305193A (en) * 2019-07-18 2019-10-08 河南科技大学 A kind of resisting porphyromonas gingivalis polypeptide and application

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