Peptide

Abstract

To provide peptides having the ability to activate FFAR1 that is GPCR useful as a new therapeutic target for type 2 diabetes mellitus.SOLUTION: Peptides comprise (a) an amino acid sequence a represented by a specific sequence, (b) an amino acid sequence b, wherein, one to four amino acids are substituted in the amino acid sequence a, or (c) an amino acid sequence c, wherein, one to two terminal amino acids are deleted or added in the amino acid sequence a or the amino acid sequence b.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to peptides and the like.

Background Art

[0002] In recent years, GPCR called FFAR1 has attracted attention as a new therapeutic target for type 2 diabetes. FFAR1 is highly expressed in the pancreas and intestine. When FFAR1 is activated by medium- and long-chain fatty acids as ligands, insulin secretion is promoted. Since FFAR1 promotes insulin secretion only when the glucose concentration in the body is high, blood glucose levels can be controlled without the risk of hypoglycemia. However, as a problem of existing small molecule agonists, since they mimic the fatty acid skeleton, they have high lipophilicity, and side effects and toxicity due to high lipophilicity have become problems. There are many agonists that have not been able to break through clinical trials due to side effects and toxicity, and the development of agonists with low toxicity is required.

[0003] Patent Document 1 discloses a technique for regulating the activity of free fatty acid receptors such as FFAR1 by specific microorganisms.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present inventor focused on peptides as less toxic compounds. For example, peptides that can stimulate FFAR1 expressed in the intestinal tract can be expected as new health foods and health promotion supplements.

[0006] An object of the present invention is to provide a peptide having an ability to activate FFAR1.

Means for Solving the Problems

[0007] As a result of intensive research, the present inventors have found that a peptide consisting of (a) an amino acid sequence a represented by SEQ ID NO: 1, (b) an amino acid sequence b in which 1 to 4 amino acids are substituted for the amino acid sequence a, or (c) an amino acid sequence c in which 1 to 2 amino acids at the terminal are deleted or 1 to 2 amino acids are added to the terminal with respect to the amino acid sequence a or the amino acid sequence b has the ability to activate FFAR1. Based on these findings, the present inventors further conducted research and as a result, completed the present invention. That is, the present invention includes the following aspects.

[0008] Item 1. (a) An amino acid sequence a represented by SEQ ID NO: 1 (b) An amino acid sequence b in which 1 to 4 amino acids are substituted for the amino acid sequence a, or (c) An amino acid sequence c in which 1 to 2 amino acids at the terminal are deleted or 1 to 2 amino acids are added to the terminal with respect to the amino acid sequence a or the amino acid sequence b A peptide comprising the same.

[0009] Item 2. (d) An amino acid sequence d represented by SEQ ID NO: 2 (e) An amino acid sequence e in which 1 to 2 amino acids are substituted for the amino acid sequence d, or (f) An amino acid sequence f in which 1 to 2 amino acids at the terminal are deleted or 1 to 2 amino acids are added to the terminal with respect to the amino acid sequence d or the amino acid sequence e The peptide according to Item 1, comprising the same.

[0010] Item 3. The peptide according to Item 2, which consists of a partial amino acid sequence in an edible protein.

[0011] Item 4. (g) An amino acid sequence g represented by any one of SEQ ID NOs: 2 to 14, or (h) An amino acid sequence h in which 1 to 2 amino acids at the terminal are deleted or 1 to 2 amino acids are added to the terminal with respect to the amino acid sequence g The peptide according to item 2 or 3, comprising

[0012] Item 5. The peptide according to item 4, comprising the amino acid sequence g.

[0013] Item 6. The peptide according to item 5, wherein the amino acid sequence g is the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5.

[0014] Item 7. The peptide according to item 1, wherein the amino acid sequence b is an amino acid sequence in which 1 to 2 amino acids are substituted with respect to the amino acid sequence a.

[0015] Item 8. The amino acid sequence b is of the formula (1): X 1 X 2 X 3 GX 5 X 6 (1) (In the formula, X 1 is S, F, V, P, or I, X 2 is T or V, X 3 is T, V, K, I, Y, or F, X 5 is T, V, I, Y, K, or F, X 6 is Q, F, I, V, or L.) The peptide according to item 1 or 7, which is an amino acid sequence represented by or an amino acid sequence shown in any one of SEQ ID NOs: 39 to 152.

[0016] Item 9. The peptide according to item 8, wherein the amino acid sequence b is the amino acid sequence shown in any one of SEQ ID NOs: 2 and 15 to 33, or an amino acid sequence shown in any one of SEQ ID NOs: 39 to 152.

[0017] Item 10. A composition comprising the peptide according to any one of items 1 to 9.

[0018] Item 11. The composition according to item 10, for use in at least one selected from the group consisting of FFAR1 activation, promotion of insulin secretion, promotion of GLP-1 secretion, improvement of glucose metabolism, and prevention or improvement of diabetes.

[0019] Item 12. The composition according to Item 10 or 11, which is a pharmaceutical, food composition, or food additive.

[0020] Item 13. The composition according to any one of Items 10 to 12, which is in an oral dosage form.

Advantages of the Invention

[0021] According to the present invention, a peptide having an ability to activate FFAR1 can be provided.

Brief Description of the Drawings

[0022]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0023] In this specification, the expressions "comprising" and "including" include the concepts of "containing", "including", "consisting essentially of", and "consisting only of".

[0024] In this specification, conservative substitution means that an amino acid residue is substituted with an amino acid residue having a side chain of similar properties. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution technique. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine, and substitution between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also conservative substitutions.

[0025] FFAR1 (Free fatty acid receptor 1), also known as GPR40, is a class A G protein-coupled receptor that is highly expressed in the pancreas and intestine. Genes in various species are known, and the amino acid sequence of human FFAR1 is, for example, NCBI Reference Sequence: NP_005294.1, and the amino acid sequence of mouse FFAR1 is, for example, NCBI Reference Sequence: NP_918946.2.

[0026] 1. Peptide In one aspect of the present invention, it relates to a peptide (which may also be referred to as "the peptide of the present invention" herein) consisting of (a) amino acid sequence a represented by SEQ ID NO: 1, (b) amino acid sequence b in which 1 to 4 amino acids are substituted with respect to the amino acid sequence a, or (c) amino acid sequence c in which 1 to 2 amino acids at the terminal are deleted or 1 to 2 amino acids are added at the terminal with respect to the amino acid sequence a or the amino acid sequence b. This will be described below.

[0027] The amino acid sequence a is an amino acid sequence represented by STTGTQ.

[0028] In the amino acid sequence b, the substituted amino acid is not particularly limited as long as it can form a peptide, and it can be either a natural amino acid or an artificial amino acid. Can be adopted Examples of the substituted amino acid include hydrophobic amino acids, hydrophilic amino acids, basic amino acids, acidic amino acids, branched-chain amino acids, aromatic amino acids, sulfur-containing amino acids, etc. More specifically, examples of the substituted amino acid include valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, serine, etc.

[0029] In the amino acid sequence b, the number of substituted amino acids is preferably 1 to 3, more preferably 1 to 2 or 1, in one embodiment of the present invention. In another embodiment of the present invention, the number is preferably 2 to 4, more preferably 3 to 4 or 4.

[0030] The mode of substitution in the amino acid sequence b is not particularly limited as long as the peptide of the present invention has the ability to activate FFAR1. In one embodiment of the present invention, the substitution can be a conservative substitution. The ability to activate FFAR1 is measured by the TGFα cleavage assay of Test Example 2 described below.

[0031] In one embodiment of the present invention, the amino acid sequence b is preferably the formula (1): X 1 X 2 X 3 GX 5 X 6 (1) (In the formula, X 1 is S, F, V, P, or I, X 2 is T or V, X 3 is T, V, K, I, Y, or F, X 5 is T, V, I, Y, K, or F, X 6 is Q, F, I, V, or L (all in one-letter notation of amino acids).) The amino acid sequence (amino acid sequence b1) represented by

[0032] The amino acid sequence b1 is preferably an amino acid sequence in which 1 to 3 amino acids are substituted with respect to the amino acid sequence a, and more preferably an amino acid sequence in which 2 amino acids are substituted.

[0033] X 1 is preferably S, F, P, or I, more preferably S, F, or I, and even more preferably S.

[0034] X 2 is preferably T.

[0035] X 3is preferably T, K, or I, more preferably T or K, and even more preferably K.

[0036] X 5 is preferably T, V, or I, and more preferably T.

[0037] X 6 is preferably F, or I, and more preferably F.

[0038] X 1 X 2 X 3 X 5 and X 6 As for the combination of X, X, X, X, X, and X, any combination can be adopted.

[0039] Specific examples of the amino acid sequence b1 include the amino acid sequences shown in any of SEQ ID NO: 2 and SEQ ID NOs: 15 to 33. Among these, the amino acid sequences shown in any of SEQ ID NO: 2, SEQ ID NOs: 15, 16, 18, 19, 20, 23 to 27, and 30 are preferred, and the amino acid sequences shown in any of SEQ ID NO: 2, SEQ ID NOs: 16, 18, 19, 23 to 26, and 30 are more preferred.

[0040] As the amino acid sequence b, in another aspect of the present invention, preferably, the amino acid sequence shown in any of SEQ ID NOs: 39 to 152 (amino acid sequence b2) can be mentioned. Among these, preferably, the amino acid sequence in which the numerical value in Table 3 described later is 1.1 or more can be mentioned, more preferably, the amino acid sequence in which the numerical value in Table 3 described later is 1.2 or more can be mentioned, still more preferably, the amino acid sequence in which the numerical value in Table 3 described later is 1.3 or more can be mentioned, even more preferably, the amino acid sequence in which the numerical value in Table 3 described later is 1.4 or more can be mentioned, particularly preferably, the amino acid sequence in which the numerical value in Table 3 described later is 1.5 or more can be mentioned, particularly more preferably, the amino acid sequence in which the numerical value in Table 3 described later is 1.6 or more can be mentioned, particularly still more preferably, the amino acid sequence in which the numerical value in Table 3 described later is 1.7 or more can be mentioned, particularly even more preferably, the amino acid sequence in which the numerical value in Table 3 described later is 1.8 or more can be mentioned, and most preferably, the amino acid sequence in which the numerical value in Table 3 described later is 1.9 or more can be mentioned.

[0041] In the amino acid sequence c, "the terminal 1 to 2 amino acids are deleted" means that 1 residue or 2 residues, a total of 1 to 2 residues of amino acids at the N-terminal and / or C-terminal are deleted. Specifically, it means that 1 residue at the N-terminal, 2 residues at the N-terminal, 1 residue at the C-terminal, 2 residues at the C-terminal, or 1 residue at the N-terminal and 1 residue at the C-terminal are deleted. The number of deleted amino acids is preferably 1.

[0042] In the amino acid sequence c, "1 to 2 amino acids are added to the terminal" means that 1 residue or 2 residues, a total of 1 to 2 residues of amino acids are added to the N-terminal and / or C-terminal. Specifically, it means that 1 residue is added to the N-terminal, 2 residues are added to the N-terminal, 1 residue is added to the C-terminal, 2 residues are added to the C-terminal, or 1 residue is added to the N-terminal and 1 residue is added to the C-terminal. The number of added amino acids is preferably 1.

[0043] In the amino acid sequence c, the added amino acid is not particularly limited as long as it is an amino acid that can form a peptide, and either natural amino acids or artificial amino acids Can be adoptedExamples of the amino acids after substitution include hydrophobic amino acids, hydrophilic amino acids, basic amino acids, acidic amino acids, branched-chain amino acids, aromatic amino acids, sulfur-containing amino acids, and the like. More specifically, examples of the amino acids after substitution include valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, serine, and the like.

[0044] The modes of deletion and addition in the amino acid sequence c are not particularly limited as long as the peptide of the present invention has the ability to activate FFAR1. The ability to activate FFAR1 is measured by the TGFα cleavage assay in Test Example 2 described below.

[0045] A preferred embodiment of the peptide (amino acid sequence b or c) of the present invention is (d) the amino acid sequence d represented by SEQ ID NO: 2, (e) the amino acid sequence e in which 1 to 2 amino acids are substituted with respect to the amino acid sequence d, or (f) the amino acid sequence d or the amino acid sequence e, in which 1 to 2 amino acids at the terminal are deleted or 1 to 2 amino acids are added to the terminal, and the peptide (peptide A of the present invention) is exemplified.

[0046] The amino acid sequence d is an amino acid sequence represented by STKGTF.

[0047] In the amino acid sequence e, the amino acids after substitution and the mode of substitution are the same as those in the amino acid sequence b.

[0048] Regarding the definition of the amino acid sequence f, the added amino acids, and the modes of deletion and addition, they are the same as those in the amino acid sequence c.

[0049] As the peptide A of the present invention, preferably, (g) an amino acid sequence g shown in any of SEQ ID NOs: 2 to 14, or (h) an amino acid sequence h in which 1 to 2 amino acids at the terminal are deleted or 1 to 2 amino acids are added to the amino acid sequence g, a peptide (peptide A1 of the present invention) is exemplified.

[0050] As the amino acid sequence g, preferably, an amino acid sequence shown in any of SEQ ID NOs: 2 to 5 is exemplified.

[0051] Regarding the definition of the amino acid sequence h, additional amino acids, and the modes of deletion and addition, they are the same as those of the amino acid sequence c.

[0052] The peptide A1 of the present invention is preferably a peptide consisting of the amino acid sequence g.

[0053] The peptide of the present invention (particularly, the peptide A of the present invention) is preferably a peptide consisting of a partial amino acid sequence in an edible protein. The edible protein is not particularly limited as long as it is a protein rich in food materials. Specifically, for example, milk proteins (such as casein, sodium caseinate, MPC (Milk Protein Concentrate), α-casein, β-casein, κ-casein, lactalbumin, etc., and their degradation products, etc.), soybean proteins (such as glycinin, β-conglycinin, convicilin, histone, etc.), cereal (such as rice, wheat, etc.) proteins (such as gluten, gliadin, glutelin, gluten storage protein, etc.), livestock meat proteins (such as muscle structure protein, myosin, actin, etc.), fish meat proteins (such as muscle fiber protein, actomyosin, myosin, actin, etc.), chicken egg proteins (such as ovalbumin, yolk lipoprotein, etc.), pig skin proteins (such as gelatin, etc.) and the like can be mentioned.

[0054] When the ability of the peptide of the present invention to activate FFAR1 is set to 1 with respect to the ability of the non-chemically modified peptide consisting of amino acid sequence a, it is, for example, 0.1 or more, preferably 0.2 or more, more preferably 0.5 or more, still more preferably 0.7 or more, even more preferably 1.0 or more, particularly preferably 1.5 or more, particularly more preferably 2.0 or more, and particularly even more preferably 3.0 or more. The upper limit of the activation ability is not particularly limited and is, for example, 20, 10, 8, 7, 6, or 5.

[0055] The peptide of the present invention is preferably an isolated, concentrated, or purified peptide.

[0056] The peptide of the present invention also includes those in which the terminal amino acid residues are chemically modified as long as the ability to activate FFAR1 is not significantly reduced.

[0057] The peptide of the present invention includes those in which the C-terminus is a carboxyl group (-COOH), carboxylate (-COO - )), amide (-CONH 2 ), or ester (-COOR), etc.

[0058] Here, as R in the ester, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 alkyl group; for example, C such as cyclopentyl, cyclohexyl, etc. 3-8 cycloalkyl group; for example, C such as phenyl, α-naphthyl, etc. 6-12 aryl group; for example, phenyl-C such as benzyl, phenethyl, etc. 1-2 alkyl group; α-naphthyl-C such as α-naphthylmethyl, etc. 1-2 alkyl group such as C 7-14 aralkyl group; pivaloyloxymethyl group, etc. are used.

[0059] Furthermore, in the peptide of the present invention, the amino group on the main chain of the N-terminal amino acid residue is a protecting group (for example, C such as formyl group, acetyl group, etc. 1-6 alkanoyl such as C 1-6Those protected with an acyl group etc., including myristoylation, pyroglutamylation, methylation, etc.

[0060] The peptide of the present invention includes those in which amino acid residues other than the termini are chemically modified, as long as the ability to activate FFAR1 is not significantly reduced. Preferably, however, the peptide of the present invention does not include those in which amino acid residues other than the termini are chemically modified. Examples of such chemical modifications include amidation, esterification, etc. of carboxyl groups; protection of amino groups with protecting groups, etc. Esterification and protecting groups are the same as those for the terminal chemical modifications described above.

[0061] The peptide of the present invention also includes forms as salts with acids or bases. The salts are not particularly limited, and either acidic salts or basic salts can be employed. For example, examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, p-toluenesulfonate, etc.; and amino acid salts such as aspartate, glutamate, etc. Examples of basic salts include alkali metal salts such as sodium salt, potassium salt, etc.; and alkaline earth metal salts such as calcium salt, magnesium salt, etc.

[0062] The peptide of the present invention also includes forms as solvates. The solvent is not particularly limited, and examples include water, ethanol, glycerol, acetic acid, etc.

[0063] The peptide of the present invention can be produced by various methods. The peptide of the present invention can be produced, for example, by solid-phase synthesis. Also, when the peptide of the present invention is a peptide consisting of a partial amino acid sequence in an edible protein, it can be obtained by enzymatic digestion of the protein.

[0064] The enzyme is not particularly limited as long as it has proteolytic activity. Examples include aspartic proteases, serine proteases, cysteine proteases, metalloproteases, threonine proteases, and the like.

[0065] Examples of aspartic proteases include pepsin, renin, cathepsin D, cathepsin E, napsin, β-secretase, γ-secretase, signal peptide peptidase, HIV protease, HTLV protease, NS3A protease, plasmepsin, suspaase, chymosin, and the like.

[0066] Examples of serine proteases include dipeptidyl peptidase 4, trypsin, chymotrypsin, plasmin, thrombin, factors of the blood coagulation - fibrinolysis system and complement system and their regulatory systems such as factor Xa, neutrophil elastase, subtilisin, furin, PACE4, PC2, PC7, kekunisin, cucumisin, lantibiotic peptidase, thermitase, acrosin, kallikrein, urokinase, granzyme, tryptase, kimerase, cathepsin A, prolyl aminopeptidase, P-type signal peptidase, prostate-specific antigen, HCMV protease, V8 protease, protease K, and the like.

[0067] Examples of cysteine proteases include cathepsins such as cathepsin B, cathepsin H, cathepsin L, cathepsin S, cathepsin K, legumain, angiotensin-converting enzyme, bleomycin hydrolase, calpain, caspase, ER-60, papain, coronavirus 3CL protease, falcipain, TEV protease, HRV3C protease, and the like.

[0068] Examples of metalloproteases include ADAM, matrix metalloprotease, thermolysin, neprilysin, carboxypeptidase, endothelin converting enzyme, KELL antigen, bone morphogenetic factor-1, meprin, serralysin, PAPP, mitochondrial processing protease, insulin degrading enzyme, aminopeptidase, prenyl protease, and the like.

[0069] Examples of threonine proteases include proteasome, γ-glutamyltransferase, and the like.

[0070] As the enzyme, one kind alone can be employed, or two or more kinds can be employed in combination.

[0071] The combination of the edible protein and the enzyme can be determined, for example, as follows. That is, it can be determined by a method of searching for the amino acid sequence of the peptide of the present invention generated when decomposed by an enzyme from the amino acid sequence of the edible protein. Thereby, it is possible to obtain a combination of an edible protein / enzyme from which the amino acid sequence of the peptide of the present invention can be obtained. The amino acid sequence of the edible protein and the cleavage specificity of the enzyme can be easily determined according to known information.

[0072] A protein enzymatic digest can be obtained by a method including a step of decomposing a protein with an enzyme. Specifically, the decomposition can be performed, for example, by incubating a reaction solution containing the protein and the enzyme. The composition of the reaction solution, reaction temperature, reaction time, protein concentration, enzyme concentration, presence or absence of additives and their types, etc. can be appropriately set according to the types of the protein and the enzyme.

[0073] After the step of decomposing the protein with an enzyme, it is preferable to further perform a step of purifying the peptide of the present invention.

[0074] The purification method is not particularly limited as long as it can concentrate the peptide of the present invention (i.e., increase the concentration of the peptide of the present invention in the whole peptide). Examples of the purification method include purification by silica gel, purification by synthetic adsorption resin, normal-phase partition chromatography, reverse-phase partition chromatography, anion-exchange chromatography, desalting by electrodialysis, molecular weight fractionation by ultrafiltration membrane, size exclusion chromatography, affinity chromatography, etc. The peptide of the present invention can also be purified by utilizing FFAR1 binding ability (for example, using a carrier carrying FFAR1 or cells expressing FFAR1). As the purification method, one kind alone can be employed, or two or more kinds can be combined and employed.

[0075] 2. Use In one aspect, the present invention relates to a composition (which may also be referred to as "the composition of the present invention" herein) containing the peptide of the present invention.

[0076] The composition of the present invention can be used for at least one selected from the group consisting of FFAR1 activation, promotion of insulin secretion, promotion of GLP-1 secretion, improvement of glucose metabolism, and prevention or improvement of diabetes. In one aspect, the present invention relates to an FFAR1 activator, an insulin secretion promoter, a GLP-1 secretion promoter, a glucose metabolism improver, and a preventive or therapeutic agent for diabetes, each containing the peptide of the present invention.

[0077] As the peptide of the present invention, one kind alone can be employed, or two or more kinds can be combined and employed.

[0078] The composition of the present invention includes, as a peptide, cases where it contains peptides other than the peptide of the present invention. The content of the peptide of the present invention in the composition of the present invention is, for example, 10% by mass or more, preferably 30% by mass or more, more preferably 50% by mass or more, still more preferably 70% by mass or more, even more preferably 80% by mass or more, particularly preferably 90% by mass or more, especially preferably 95% by mass or more, based on 100% by mass of the peptide.

[0079] The composition of the present invention can be used in various fields, for example, as a food additive, a food composition (including a health promoter, a nutritional supplement (such as a supplement)), a medicine, and the like.

[0080] The administration (or ingestion) form of the composition of the present invention is not particularly limited as long as the desired effect can be obtained, and it can be administered to mammals including humans by any administration route of oral administration and parenteral administration (for example, intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, topical administration). The preferred administration form is oral administration. Dosage forms for oral administration and parenteral administration and methods for manufacturing them are well known to those skilled in the art, and the active ingredient (the peptide of the present invention) can be manufactured according to a conventional method by mixing it with a pharmaceutically acceptable carrier and the like.

[0081] The form of the composition of the present invention is not particularly limited, and depending on the use, it can take the form usually used in each use.

[0082] As forms of the composition of the present invention, when the use is a food additive, medicine, health promoter, nutritional supplement (such as a supplement), etc., for example, tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquid preparations (including drinks, suspensions, syrups), jelly preparations, etc. can be mentioned. Particularly in the case of medicine, in addition to these, for example, injection preparations (such as intravenous drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (such as ointments, poultices, lotions, creams, gels), suppositories, inhalants, eye drops, eye ointments, nasal drops, ear drops, liposome preparations, etc. can be mentioned.

[0083] As forms of the composition of the present invention, when the use is a food composition, it includes liquid, gel-like or solid foods, such as ramen, hamburgers, fried foods, juices, soft drinks, tea, soups, soy milk and other beverages, edible oils such as salad oil and butter, dressings, yogurt, jelly, pudding, furikake, powdered or liquid milk products for infants, cake mixes, bread, cookies, etc.

[0084] The composition of the present invention may further contain other components as necessary. The other components are not particularly limited as long as they can be formulated in food additives, food compositions, medicines, health promoters, nutritional supplements (such as supplements), etc., and examples include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, fragrances, chelating agents, etc.

[0085] The content of the active ingredient in the composition of the present invention depends on the use, usage mode, state of the application target, etc., and is not limited, but can be, for example, 0.0001 to 100% by mass, preferably 0.001 to 50% by mass. The lower limit of the content is, for example, 0.01% by mass, 0.1% by mass, 1% by mass, 5% by mass, 10% by mass, 20% by mass, 30% by mass, 40% by mass, 50% by mass, 60% by mass, 70% by mass, 80% by mass, or 90% by mass.

[0086] The dosage of the composition of the present invention (for example, administration, ingestion, inoculation, etc.) is not particularly limited as long as it is an effective amount that exhibits its effect, and is usually, as the dry weight of the peptide of the present invention, generally 0.01 to 1000 mg / kg body weight per day. The lower limit of the dosage is, for example, 0.1 mg / kg body weight, 1 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, 20 mg / kg body weight, 50 mg / kg body weight, 100 mg / kg body weight, 200 mg / kg body weight, or 500 mg / kg body weight. The above dosage is preferably applied once or more a day (for example, 1 to 3 times), and can be appropriately increased or decreased according to age, disease state, and symptoms.

Examples

[0087] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited by these examples.

[0088] Test Example 1. Synthesis of Peptide Array The peptide array used in the test examples described below was synthesized as follows.

[0089] <Test Example 1-1. Preparation of Activated Membrane> 21 sheets of cellulose filter paper FILTER PAPER 542 HARDENED ASHLESS 24.0 cm (1542 - 185, Whatman, England) (hereinafter referred to as membrane) were cut into 10 cm × 15 cm, placed in a polypropylene tapper, and immersed in 150 mL of DMF (N,N - Dimethylformamide) (A00185, Kanto Chemical Co., Ltd., Tokyo) and shaken for one day. Next, using Fmoc - β - Ala - OH (K00410, Watanabe Chemical Industries, Ltd., Hiroshima), 1 - Methylimidazole, redistilled, 99 + % (336092 - 100ML, SIGMA - ALDRICH, USA), DIPCI (N,N` - Diisopropylcarbodiimide) (A00011, Watanabe Chemical Co., Ltd., Hiroshima), an activation solution (0.5M β - Ala 32 mL, 1 - Methylimidazole 1.6 ml, DIPCI 2 mL, DMF 150 mL) was prepared. The 21 membranes were immersed in this solution and shaken for one day. Then, 1.6 ml of 1 - Methylimidazole and 2 mL of DIPCI were added and shaken for another day. Using tweezers, each membrane was immersed on both sides one by one 2 or 3 times a day to ensure that the activation solution thoroughly soaked the membranes. The synthesis reaction was confirmed using one membrane. Using a 1% BPB (Bromophenol Blue) (021 - 02911, Wako Pure Chemical Industries, Ltd., Osaka) / DMF solution, 100 μl of the 1% BPB / DMF solution and 100 μl of acetic acid (017 - 00256, Wako Pure Chemical Industries, Ltd., Osaka) were added to the methanol solution in which the membrane was immersed, and the NH 2 group was colored blue. After confirming that the color turned blue, the remaining 20 sheets were each washed 3 times with 50 mL of DMF and 50 mL of methanol (139 - 01827, Wako Pure Chemical Industries, Ltd., Osaka), air - dried, then placed in a vacuum pack and stored at 4°C.

[0090] <Test Example 1 - 2. Peptide Synthesis> The peptide array was prepared using the Fmoc solid-phase synthesis method. Fmoc-amino acids (all from Watanabe Chemical Industries, Ltd.) were dissolved in a 0.5 M amino acid solution in NMP (N-methyl-2-pyrrolidone) (133-15115, Wako Pure Chemical Industries, Ltd., Osaka) to which DIPCI (N,N`-Diisoprppylcarbodiimide) and HOBt (1-hydrozybenzotriazole) (A00015, Watanabe Chemical Industries, Ltd., Hiroshima) were mixed so that the final concentrations were 1:2:2. This amino acid was spotted onto the activated membrane using a peptide synthesizer (ASP222, Intavis AG, Koln, Germany) (1 spot = 1.1 μl × 3 times, only 0.8 μl × 3 times for the first residue). For the first residue, Fmoc-photo-linker (sc-294977A, SANTA CRUZ, USA) was synthesized as a linker. After synthesizing one residue, the membrane was washed three times with DMF to remove unreacted activated amino acids. Then, to block unreacted amino groups, 50 ml of 5% acetic anhydride (011-00276, Wako Pure Chemical Industries, Ltd., Osaka) / DMF was reacted twice for 15 min each to acetylate the unreacted amino groups on the activated membrane. Thereafter, the membrane was washed three times with DMF, immersed in 20% piperidine / DMF for 1 h to deprotect the Fmoc group. By repeating the above operations to perform the peptide elongation reaction, an arbitrary peptide was synthesized. The confirmation of the synthesis reaction was performed by adding 100 μl of 1% BPB (bromophenol blue) (021-02911, Wako Pure Chemical Industries, Ltd., Osaka) / DMF solution and 100 μl of acetic acid (017-00256, Wako Pure Chemical Industries, Ltd., Osaka) to a methanol solution in which the membrane was immersed, and coloring the NH2 group blue.

[0091] After synthesizing any peptide on the membrane, the membrane after the peptide elongation reaction was immersed in 20% piperidine / DMF for 1 h to deprotect the Fmoc group. Next, to remove the protecting groups attached to each amino acid side chain, the deprotection reagent was prepared by mixing trifluoroacetic acid (TFA) (A00025, Watanabe Chemical Industries, Ltd., Hiroshima), m-cresol (034-04646, Watanabe Chemical Industries, Ltd., Osaka), EDT (1,2-Ethanedithiol) (A00057, Watanabe Chemical Industries, Ltd., Hiroshima), and thioanisole (T0191, Tokyo Chemical Industry Co., Ltd., Tokyo) at a ratio of 40:1:3:6 to make 50 ml, and the membrane was immersed for 2.5 h. After deprotection, the membrane was washed three times each with diethyl ether (051-01157, Wako Pure Chemical Industries, Ltd., Osaka) and methanol, and then washed multiple times with diethyl ether and methanol until the odor disappeared. Then, it was dried with a dryer and used in the experiment.

[0092] <Test Example 1-3. Preparation of Peptide Solution> The peptide array synthesized by the operation of Test Example 1-2 was irradiated with UV at 365 nm for 3 h using a transilluminator (DT-20LCP, Atto, Tokyo). The photolinker was cleaved by UV irradiation to release the peptide. Each spot on the peptide array was punched out into a 96-well filter (MSRLN0410, Multiscreen HTS Vacuum Manifold, Merck Millipore, Germany) in groups of two spots. An appropriate HBSS (14025076, Biotechnology, Tokyo) buffer was added for each experiment to dissolve the peptide, and the peptide was eluted at 37°C for 1 h. Then, each peptide solution was collected in a 96-well plate by vacuum filtration.

[0093] Test Example 2. TGFα Cleavage Assay The TGFα cleavage assay performed in the test examples described below was carried out as follows.

[0094] <Test Example 2-1. Cell Culture> HEK293 cells were cultured at 37°C, 5% CO2 , CO under 95% Air 2 In an incubator, cells were cultured in a T75 flask for cell culture (658170, Greiner Bio One, AT). DMEM containing 10% FBS (biosera, NUAILLE, France) and 1% Penicillin - Streptomycin (PS) (15140122, Wako Pure Chemical Industries, Osaka) was used as the medium. For sub - culturing, the medium of cells in a sub - confluent state (80% - 90%) was removed, the cells were washed twice with PBS, detached by trypsin treatment, and then re - seeded. The cell count was measured using a hemocytometer, and the cells were re - seeded so that the initial cell count was 1.0×10 6 cells / well. The cells used for the assay were seeded in a 100 mm dish (664160, Greiner Bio One, AT) at an appropriate cell concentration so that they were sub - confluent at the start of the assay.

[0095] <Test Example 2 - 2. Preparation of Plasmid Lysis Buffer> As plasmids, pCAGGS / AP - TGFα and pCAGGS / FFAR1 were used. Tris - EDTA buffer (TE buffer: 10 mM Tris - HCl buffer, 1 mM EDTA) was used as the buffer for dissolving the plasmid. The Tris - HCl buffer was prepared by dissolving 60.55 g of tris (hydroxymethyl) aminomethane (207 - 06275, Wako Pure Chemical Industries, Ltd., Osaka) in 400 mL of water, adjusting the pH to 8.0 with hydrochloric acid (080 - 01066, Wako Pure Chemical Industries, Ltd., Osaka), and making up to 500 mL. The EDTA solution was prepared by dissolving 90.06 g of disodium ethylenediaminetetraacetate dihydrate (345 - 01865, Dojindo Laboratories, Kumamoto) in 400 mL of pure water, adjusting the pH to 8.0 with NaOH (193 - 13775, Wako Pure Chemical Industries, Ltd., Osaka), and making up to 500 mL.

[0096] <Test Example 2 - 3. Preparation of pNPP Solution and Lysis Buffer> 1 g of disodium p-nitrophenyl phosphate hexahydrate (pNPP; 4264-83-9, Life Technologies, Tokyo) was dissolved in 2.692 mL of pure water (1 M solution). 2×AP buffer (60 mL of 2 M Tris-HCl buffer, 10 mL of 4 M NaCl (191-01665, Wako Pure Chemical Industries, Ltd., Osaka), 10 mL of 1 M MgCl 2 ·6H 2 O (132-00175, Wako Pure Chemical Industries, Ltd., Osaka), 920 mL of pure water) was prepared as the dissolution buffer for the pNPP solution. A 10 mM solution diluted 100-fold with 2×AP buffer was used as the pNPP solution.

[0097] <Test Example 2-4. Preparation of Ligand> HBSS buffer, HBSS (2% DMSO) was prepared as a negative control. Palmitic acid (165-00102, Wako Pure Chemical Industries, Ltd., Osaka), an existing FFAR1 agonist, was dissolved in HBSS (2% DMSO) to prepare a positive control. The peptide solution was prepared by eluting 72 μL each with HBSS buffer (5 mM HEPES).

[0098] <Test Example 2-5. Cell Assay Using Peptide Array> HEK293 cells cultured by the method of Test Example 2-1 were seeded into 12 wells of a 24-well plate so that the initial cell number was 1.0×10 5 cells, and incubated at 37°C, 5% CO 2 , 95% Air in a CO 2 incubator for 24 h.

[0099] Next, the plasmid solutions of pCAGGS / AP-TGFα and pCAGGS / FFAR1 prepared in Test Example 2-2, the transfection reagent PEI (49553-93-7, Polyscience, Inc, USA), and Opti-MEM (31985-070, Life Technologies, Tokyo) were mixed in Eppendorf tubes A and B as shown in the following table (Table 1). After tapping and flashing the mixed Eppendorf tubes, they were allowed to stand for 5 min. Then, the Eppendorf tubes A and B were mixed at a ratio of 1:1, and after tapping and flashing, they were allowed to stand for 20 min. Then, the FFAR1 plasmid solution was added to a 24-well plate seeded with HEK293 cells at 50 μL / well. Then, it was incubated in an incubator at 37 °C, 5% CO 2 2, and 95% Air under CO 2 for 24 h.

[0100] Next, the transfected HEK293 cells were washed twice with PBS, detached by trypsin treatment, and collected into centrifuge tubes containing DMEM according to the type of plasmid. After centrifugation at 1000 rpm for 5 min, HBSS was added and allowed to stand for 10 min. Then, it was centrifuged again at 1000 rpm for 5 min, and HBSS was removed to remove AP-TGFα cleaved by trypsin treatment. The HEK293 cells were diluted with HBSS so that they could be seeded in a 96-well plate at 80 μL / well, and the HEK293 cells were reseeded in the 96-well plate. Then, it was incubated in an incubator at 37 °C, 5% CO 2 2, and 95% Air under CO 2 for 1 h to adhere the cells. 20 μL each of the ligand and peptide solutions prepared in Test Example 2-4 were added, and it was incubated at 37 °C, 5% CO 2 2, and 95% Air under CO 2It was incubated in an incubator. Then, 80 μL of the supernatant containing the cleaved and released AP-TGFα was transferred to a new 96-well plate in 80-μL aliquots. After removing 20 μL of the solution from each remaining well, the pNPP solution prepared in Test Example 2-3 was added to each well of each 96-well plate at 80 μL / well. The absorbance at 405 nm was measured using an absorbance meter immediately after addition and 30 min after addition. The cleavage rate of TGFα was calculated by the following formula.

[0101] ΔOD405 = (OD405 after 30 min of reaction) − (OD405 immediately after pNPP addition) Cleavage rate of AP-TGFα = ((ΔOD405 in the culture supernatant)) / ((ΔOD405 in the culture supernatant)+(ΔOD405 in the cell plate)).

[0102]

Table 1

[0103] Test Example 3. Screening of FFAR1-Binding Peptide Using a 7-mer peptide library (phage library, library size: 10 9 )), screening for FFAR1-activating peptides was performed by the phage display method. Briefly, by combining negative panning and positive panning, phage peptides that did not bind to HEK293 cells and specifically bound to HEK293 cells in which FFAR1 was forcibly expressed were obtained. Specifically, phages were incubated with HEK293 cells (1×10 7Incubated with cells / 1 mL PBS (1% BSA), and the supernatant of phages that did not bind to HEK293 cells was collected (negative panning). Subsequently, the collected phages were incubated with HEK293 cells that overexpressed FFAR1, and the phages that bound to FFAR1-expressing HEK293 cells were collected (positive panning). Two rounds of negative panning and one round of positive panning were defined as one cycle. After performing three and four cycles of panning, sequence analysis was carried out to obtain peptides with high binding affinity to FFAR1.

[0104] As a result, a peptide consisting of STTGTQY (SEQ ID NO: 34) (STTGTQY peptide) was obtained as a peptide obtained by both sequence analysis after round 3 and sequence analysis after round 4.

[0105] Subsequently, the STTGTQY peptide was synthesized by resin synthesis, and a TGFα cleavage assay was performed at a high concentration to measure the ability of the peptide to activate FFAR1. Specifically, it was carried out according to Test Example 1-2 and Test Example 2. As a test substance for the TGFα cleavage assay, in addition to the above peptide, palmitic acid (Pa), which is a ligand of FFAR1, was also used.

[0106] The results are shown in Figure 1. It was found that the STTGTQY peptide activates FFAR1 in a concentration-dependent manner.

[0107] Test Example 4. Examination of Short Residue Formation of STTGTQY Peptide A peptide array of peptides in which 1 to 2 amino acids at the ends of the STTGTQY peptide (1 to 2 residues of amino acids at the N-terminus and / or C-terminus, a total of 1 to 2 residues of amino acids), STTGTQ (SEQ ID NO: 1), TTGTQY (SEQ ID NO: 35), STTGT (SEQ ID NO: 36), TGTQY (SEQ ID NO: 37), TTGTQ (SEQ ID NO: 38) was synthesized according to Test Example 1, and a TGFα cleavage assay was performed according to Test Example 2 using this. As a test substance for the TGFα cleavage assay, in addition to the above peptide, palmitic acid (Pa), which is a ligand of FFAR1, was also used.

[0108] The results are shown in Figure 2. The STTGTQ peptide showed higher FFAR1 activation ability than the STTGTQY peptide. In addition, other truncated peptides also showed a certain degree of FFAR1 activation ability.

[0109] Test Example 5. Prediction of Highly Active Peptide by Machine Learning An alanine substitution comprehensive peptide array consisting of 114 peptides (Table 2: SEQ ID NOs: 39 to 152) in which one amino acid of the STTGTQ peptide was substituted with 19 other natural amino acids was prepared according to Test Example 1, and the FFAR1 activation ability of each peptide was evaluated by the TGFα cleavage assay according to Test Example 2. By using the calibration curve of Pa for the TGFα cleavage rate, it was converted into the Pa concentration, and the value obtained by dividing the Pa-converted concentration of each peptide by the Pa-converted concentration of the original sequence (STTGTQ peptide) was used as the activity value. The results are shown in Table 3.

[0110] [Table 2]

[0111] [Table 3]

[0112] Next, discriminant analysis was performed using all 120 feature quantities to associate the activity of the peptide with the feature quantities. As the feature quantities, 120 values calculated by multiplying the physicochemical properties of 24 types of amino acids (Figure 3) by five comprehensive values (Figure 4) calculated for the physicochemical characteristics of each amino acid were used.

[0113] Using the obtained training data (Table 3) and the above 120 feature quantities, logistic regression analysis was performed to predict the high-activity sequences from among the peptides with two-residue substitutions of the STTGTQ peptide. In the prediction, hydrophobicity indices such as LogP and Hydropathy index, and charge indices such as pK2 were largely used. The amino acids at each position in the top 30 sequences predicted to be highly active are shown in Figure 5. From Figure 5, formula (1): X 1 X 2 X3 GX 5 X 6 (1) (In the formula, X 1 is S, F, V, P, or I, X 2 is T or V, X 3 is T, V, K, I, Y, or F, X 5 is T, V, I, Y, K, or F, X 6 is Q, F, I, V, or L.) It can be seen that the amino acid sequence represented by (1) is a consensus sequence having a certain level of activity.

[0114] Twenty peptides (Table 4: SEQ ID NOs: 2 and 15 to 33) that satisfy the consensus sequence were prepared according to Test Example 1, and the ability of each peptide to activate FFAR1 was evaluated by the TGFα cleavage assay according to Test Example 2. By using the calibration curve of Pa for the TGFα cleavage rate, it was converted into the Pa concentration, and the value obtained by dividing the Pa-converted concentration of each peptide by the Pa-converted concentration of the original sequence (STTGTQ peptide) was used as the activity value.

[0115]

Table 4

[0116] Typical results are shown in FIG. 6. It was found that by satisfying the consensus sequence, a high ability to activate FFAR1 was exhibited.

[0117] Test Example 6. Evaluation of Insulin Secretion-Promoting Ability A test substance was added to MIN6 cells, and after 1 hour, the supernatant was collected, and the insulin concentration in the supernatant was quantified by ELISA. The test substances are as follows. ·Negative control: Krebs-Ringer-bicarbonate-HEPES (KRBH) buffer (0.2% fatty acid-free BSA, 25 mM Glucose) ·Positive control: 50 μM GW9508 (0.2% fatty acid-free BSA, 25 mM Glucose) · Test peptide: STKGTF peptide.

[0118] Specifically, it was carried out according to Reference 1 (Chen et al., Br. J. Pharmacol., 2020). The insulin concentration when using each of the positive control and the test peptide was divided by the insulin concentration when using the negative control to calculate the activity ratio.

[0119] The results are shown in Figure 7. It was confirmed that the STKGTF peptide has the ability to promote insulin secretion.

[0120] Test Example 7. Search and Evaluation of Peptides Derived from Edible Proteins From the amino acid sequences of 710 proteins in the Edible Protein Database (URL: http: / / www.uwm.edu.pl / biochemia / index.php / en / biopep), 6-mer amino acid sequences were extracted while shifting residues one by one (total 98,387 kinds), and from these, 2-residue substitution sequences were extracted from the STKGTF peptide (Table 5: total 12 kinds (SEQ ID NOs: 3 to 14)). For the peptides of these 12 amino acid sequences, peptide arrays were synthesized according to Test Example 1, and using these, TGFα cleavage assays were carried out according to Test Example 2.

[0121]

Table 5

[0122] The results are shown in Figure 8. Peptides with 2-residue substitution sequences from the STKGTF peptide, which is derived from an edible protein, also showed FFAR1 activation ability above a certain level.

[0123] Test Example 8. Evaluation of GLP-1 Secretion-Promoting Ability For the STKGTF peptide and two peptides (VQKGTF, SILGTF) shown to have higher FFAR1 activation ability than the peptide in Test Example 7, it was verified whether GLP-1 secretion was promoted via FFAR1 expressed in enteroendocrine cells. Specifically, the test peptide was added to GLUTag cells (an enteroendocrine cell line), and after 2 hours, the supernatant was collected, and the GLP-1 concentration in the supernatant was quantified by ELISA. The GLP-1 concentration when each test peptide was used was divided by the GLP-1 concentration when the negative control (the same as in Test Example 6) was used to calculate the activity ratio.

[0124] The results are shown in Figure 9. It was confirmed that all of the test peptides had the ability to promote GLP-1 secretion.

Claims

**Claim 1**: A peptide comprising an amino acid sequence shown in any of SEQ ID NOs: 3 to 14. **Claim 2**: A peptide comprising an amino acid sequence shown in any of SEQ ID NOs: 3 to 5. **Claim 3**: A peptide comprising an amino acid sequence shown in any of SEQ ID NOs: 1, 2, 16, 18, 19, 23 to 25, 40, 41, 49 to 53, 57, 60, 67 to 69, 71, 72, 75 to 77, 81, 85 to 89, 91, 93 to 95, 97, 124, 132, 136, 138, 140, 142 to 147, and 152. **Claim 4** A composition comprising the peptide according to any one of Claims 1 to 3. **Claim 5** **Claim 6**: The composition according to Claim 4, for use in at least one selected from the group consisting of FFAR1 activation, promotion of insulin secretion, promotion of GLP-1 secretion, improvement of glucose metabolism, and prevention or improvement of diabetes. **Claim 7** **Claim 8**: The composition according to Claim 4 or 5, which is a pharmaceutical, a food composition, or a food additive. **Claim 9** **Claim 10**: The composition according to any one of Claims 4 to 6, which is in an oral dosage form.

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