JP7688891B2 - peptide - Google Patents

Description

The present invention relates to peptides, etc.

In recent years, a GPCR called FFAR1 has been attracting attention as a new therapeutic target for type 2 diabetes. FFAR1 is highly expressed in the pancreas and intestine. When FFAR1 is activated by its ligand, medium- and long-chain fatty acids, it promotes insulin secretion. FFAR1 promotes insulin secretion only when the glucose concentration in the body is high, making it possible to control blood sugar levels without the risk of hypoglycemia. However, a problem with existing small molecule agonists is that they are highly lipophilic because they mimic the fatty acid skeleton, and side effects and toxicity due to this high lipophilicity are problematic. There are many agonists that have not been able to pass clinical trials due to side effects and toxicity, and there is a demand for the development of agonists with low toxicity.

Patent Document 1 discloses a technology for regulating the activity of free fatty acid receptors such as FFAR1 using specific microorganisms.

Special table 2020-532515 publication

The inventors focused on peptides as less toxic compounds. For example, peptides that can stimulate FFAR1 expressed in the intestinal tract are expected to be new health foods and health-promoting supplements.

The objective of the present invention is to provide a peptide capable of activating FFAR1.

As a result of intensive research, the present inventors have found that a peptide consisting of (a) the amino acid sequence a shown in SEQ ID NO:1, (b) the amino acid sequence b in which 1 to 4 amino acids have been substituted with respect to the amino acid sequence a, or (c) the amino acid sequence c in which 1 to 2 amino acids have been deleted from the terminal of the amino acid sequence a or the amino acid sequence b, or in which 1 to 2 amino acids have been added to the terminal of the amino acid sequence a or b, has the ability to activate FFAR1. Based on these findings, the present inventors have further researched and completed the present invention. That is, the present invention encompasses the following aspects.

Item 1. (a) an amino acid sequence a shown in SEQ ID NO: 1;
(b) an amino acid sequence b in which 1 to 4 amino acids are substituted with respect to 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 of the amino acid sequence a or b.
A peptide consisting of

Item 2. (d) an amino acid sequence d shown in SEQ ID NO: 2;
(e) an amino acid sequence e in which one or two amino acids are substituted with respect to the amino acid sequence d, or (f) an amino acid sequence f in which one or two amino acids at the terminal are deleted or one or two amino acids are added to the terminal of the amino acid sequence d or the amino acid sequence e.
Item 2. The peptide according to item 1, comprising:

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

Item 4. (g) an amino acid sequence g shown in any one of SEQ ID NOs: 2 to 14, or (h) an amino acid sequence h in which 1 to 2 amino acids are deleted from the terminal or 1 to 2 amino acids are added to the terminal of the amino acid sequence g.
Item 4. The peptide according to Item 2 or 3, comprising:

Item 5. The peptide according to Item 4, which consists of the amino acid sequence g.

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

Item 7. The peptide according to Item 1, wherein the amino acid sequence b is an amino acid sequence in which one or two amino acids are substituted with respect to the amino acid sequence a.

Item 8. The amino acid sequence b is represented by formula (1):
^{X1 X2 X3 GX5 X6} (1)
(In the formula, ^X1 is S, F, V, P, or I, ^X2 is T or V, ^X3 is T, V, K, I, Y, or F, ^X5 is T, V, I, Y, K, or F, and ^X6 is Q, F, I, V, or L.)
or an amino acid sequence shown in any one of SEQ ID NOs: 39 to 152.

Item 9. The peptide according to Item 8, wherein the amino acid sequence b is an 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.

Item 10. A composition containing the peptide described in any one of items 1 to 9.

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

Item 12. The composition according to item 10 or 11, which is a medicine, a food composition, or a food additive.

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

The present invention provides a peptide capable of activating FFAR1.

1 shows the results of the TGFα cleavage assay in Test Example 3. On the horizontal axis, Pa indicates palmitic acid, and the amino acid sequence (single letter code) indicates the sequence of the test peptide. On the horizontal axis, the test concentration for Pa and the dilution factor for the STTGTQY peptide are shown. 1 shows the results of the TGFα cleavage assay in Test Example 4. On the horizontal axis, Pa represents palmitic acid, and the amino acid sequence (single letter code) represents the sequence of the test peptide. On the horizontal axis, the test concentration of Pa is shown. The physicochemical properties of 24 types of amino acids used in calculating the feature amounts in Test Example 5 are listed below. The five overall values calculated for the physicochemical characteristics of each amino acid used in calculating the characteristic amounts in Test Example 5 and the calculation examples thereof are shown below. Shown are the amino acids at each position in the top 30 sequences predicted to have high activity in Test Example 5. The amino acid positions shown on the horizontal axis are 1, 2, 3, ... from the N-terminus of the STTGTQ peptide. 1 shows the results of the TGFα cleavage assay in Test Example 5. The horizontal axis shows the sequences of the test peptides. 1 shows the results of insulin concentration measurement in Test Example 6. The horizontal axis shows the concentration of the test substance used. ** indicates that the P value calculated by Student t-test between the two groups shown by the bar is less than 0.01. 1 shows the results of the TGFα cleavage assay in Test Example 7. The horizontal axis shows the sequences of the test peptides. 1 shows the results of GLP-1 concentration measurement in Test Example 8. The horizontal axis shows the sequence of the test peptide and the concentration used.

In this specification, the expressions "contain" and "include" include the concepts of "contain," "include," "consist essentially of," and "consist only of."

In this specification, conservative substitution means that an amino acid residue is replaced with an amino acid residue having a side chain with similar properties. For example, a conservative substitution technique involves substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine. Other conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar 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 amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

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.

1. Peptide In one aspect, the present invention relates to a peptide (sometimes referred to as the "peptide of the present invention" in the present specification) consisting of (a) the amino acid sequence a shown in SEQ ID NO: 1, (b) the amino acid sequence b in which 1 to 4 amino acids have been substituted with respect to the amino acid sequence a, or (c) the amino acid sequence c in which 1 to 2 amino acids have been deleted from the terminal of the amino acid sequence a or the amino acid sequence b, or 1 to 2 amino acids have been added to the terminal of the amino acid sequence a or b. This will be explained below.

The amino acid sequence a is represented by STTGTQ.

In the amino acid sequence b, the amino acid after substitution is not particularly limited as long as it is an amino acid that can constitute a peptide, and both natural amino acids and artificial amino acids can be used . Examples of the amino acid after substitution include hydrophobic amino acids, hydrophilic amino acids, basic amino acids, acidic amino acids, branched chain amino acids, aromatic amino acids, and sulfur-containing amino acids. More specifically, examples of the amino acid after substitution include valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, and serine.

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

The type 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 in Test Example 2 described below.

In one embodiment of the present invention, the amino acid sequence b is preferably an ^{amino acid} sequence represented by formula ( ¹ ): ^X1X2X3GX5X6 ( ¹ ) (wherein ^X1 is S, F, V, P, or I, ^X2 is T or V, ^X3 is T, V, K, I, Y, or F, ^X5 is T, V, I, Y, K, or F, and ^X6 is Q, F, I, V, or L (all amino acids are represented by one-letter code)) (amino acid sequence b1).

The amino acid sequence b1 is preferably an amino acid sequence in which one to three amino acids are substituted, more preferably two amino acids are substituted, relative to the amino acid sequence a.

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

^X2 is preferably T.

^X3 is preferably T, K, or I, more preferably T or K, and even more preferably K.

^X5 is preferably T, V, or I, and more preferably T.

^X6 is preferably F or I, and more preferably F.

As a combination of X ¹ , X ² , X ³ , X ⁵ , and X ⁶ , any combination can be adopted.

Specific examples of the amino acid sequence b1 include the amino acid sequences shown in SEQ ID NOs: 2 and 15 to 33. Among these, preferred are the amino acid sequences shown in SEQ ID NOs: 2, 15, 16, 18, 19, 20, 23 to 27, and 30, and more preferred are the amino acid sequences shown in SEQ ID NOs: 2, 16, 18, 19, 23 to 26, and 30.

In another embodiment of the present invention, the amino acid sequence b is preferably an amino acid sequence (amino acid sequence b2) shown in any one of SEQ ID NOs: 39 to 152. Among these, an amino acid sequence having a numerical value of 1.1 or more in Table 3 described below is preferably included, an amino acid sequence having a numerical value of 1.2 or more in Table 3 described below is more preferably included, an amino acid sequence having a numerical value of 1.3 or more in Table 3 described below is even more preferably included, an amino acid sequence having a numerical value of 1.4 or more in Table 3 described below is even more preferably included, an amino acid sequence having a numerical value of 1.5 or more in Table 3 described below is even more preferably included, an amino acid sequence having a numerical value of 1.6 or more in Table 3 described below is even more preferably included, an amino acid sequence having a numerical value of 1.7 or more in Table 3 described below is even more preferably included, an amino acid sequence having a numerical value of 1.8 or more in Table 3 described below is even more preferably included, an amino acid sequence having a numerical value of 1.9 or more in Table 3 described below is even more preferably included.

In the amino acid sequence c, "one or two amino acids at the terminus have been deleted" means that one or two residues at the N-terminus and/or C-terminus have been deleted, for a total of one or two amino acid residues. Specifically, this means that one residue at the N-terminus, two residues at the N-terminus, one residue at the C-terminus, two residues at the C-terminus, or one residue at the N-terminus and one residue at the C-terminus have been deleted. The number of deleted amino acids is preferably one.

In the amino acid sequence c, "one or two amino acids have been added to the termini" means that one or two amino acid residues have been added to the N-terminus and/or C-terminus, for a total of one or two amino acid residues. Specifically, this means that one residue has been added to the N-terminus, two residues to the N-terminus, one residue to the C-terminus, two residues to the C-terminus, or one residue to the N-terminus and one residue to the C-terminus. The number of added amino acids is preferably one.

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

The manner of deletion and addition in amino acid sequence c is not particularly limited, so 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.

A preferred embodiment of the peptide of the present invention (amino acid sequence b or c) is (d) the amino acid sequence d shown in SEQ ID NO: 2, (e) the amino acid sequence e in which 1 to 2 amino acids have been substituted with respect to the amino acid sequence d, or (f) the amino acid sequence f in which 1 to 2 amino acids have been deleted from the terminal or 1 to 2 amino acids have been added to the terminal of the amino acid sequence d or the amino acid sequence e (peptide A of the present invention).

The amino acid sequence d is represented by STKGTF.

In amino acid sequence e, the amino acids after substitution and the manner of substitution are the same as those in amino acid sequence b.

The definition of amino acid sequence f, the additional amino acids, and the deletion and addition patterns are the same as those of amino acid sequence c.

Peptide A of the present invention preferably includes (g) an amino acid sequence g shown in any one of SEQ ID NOs: 2 to 14, or (h) a peptide (peptide A1 of the present invention) consisting of an amino acid sequence h in which 1 to 2 amino acids are deleted from the terminal of the amino acid sequence g or 1 to 2 amino acids are added to the terminal of the amino acid sequence g.

The amino acid sequence g is preferably an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5.

The definition of amino acid sequence h, the additional amino acids, and the modes of deletion and addition are the same as those of amino acid sequence c.

Peptide A1 of the present invention is preferably a peptide consisting of the amino acid sequence g.

The peptide of the present invention (particularly, peptide A of the present invention) is preferably a peptide consisting of a partial amino acid sequence in an edible protein. There are no particular limitations on the edible protein as long as it is a protein that is abundant in food materials, and specific examples include milk proteins (e.g., casein, sodium caseinate, MPC (Milk Protein Concentrate), α-casein, β-casein, κ-casein, lactalbumin, and hydrolysates thereof), soy proteins (e.g., glycinin, β-conglycinin, convicilin, histone, and the like), cereal (e.g., rice, wheat, and the like) proteins (e.g., gluten, gluadin, glutelin, glutenin storage protein, and the like), livestock meat proteins (e.g., muscle structural proteins, myosin, actin, and the like), fish meat proteins (e.g., muscle fiber proteins, actomyosin, myosin, actin, and the like), chicken egg proteins (e.g., ovalbumin, egg yolk lipoprotein, and the like), and pig skin proteins (e.g., gelatin, and the like).

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

The peptides of the present invention are preferably isolated, enriched, or purified peptides.

The peptides of the present invention also include those in which the terminal amino acid residues have been chemically modified, so long as the FFAR1 activation ability is not significantly reduced.

The peptides of the present invention also include those in which the C-terminus is a carboxyl group (--COOH), a carboxylate (--COO ^- ), an amide (--CONH ₂ ), or an ester (--COOR).

Here, examples of R in the ester include _C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl; _C3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; _C6-12 aryl groups such as phenyl and α-naphthyl; C1-2 phenyl- _C1-2 alkyl groups such as benzyl and phenethyl; _C7-14 aralkyl groups such as α-naphthyl- _C1-2 alkyl groups such as α-naphthylmethyl; and pivaloyloxymethyl groups.

Furthermore, the peptide of the present invention also includes those in which the amino group on the main chain of the N-terminal amino acid residue is protected with a protecting group (e.g., a _C1-6 acyl group, such as a formyl group, or a _C1-6 alkanoyl group such as an acetyl group), myristoylated, pyroglutamylated, methylated, etc.

The peptides of the present invention include those in which amino acid residues other than the terminal ones are chemically modified, so long as the FFAR1 activation ability is not significantly reduced. However, preferably, the peptides of the present invention do not include those in which amino acid residues other than the terminal ones are chemically modified. In this case, examples of chemical modifications include amidation and esterification of carboxyl groups; protection of amino groups with protecting groups, etc. The esterification and protecting groups are the same as those for the chemical modification of the terminals described above.

The peptide of the present invention also includes salt forms with acids or bases. There are no particular limitations on the salt, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

The peptides of the present invention also include solvate forms. The solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, and acetic acid.

The peptide of the present invention can be produced by various methods. For example, the peptide of the present invention can be produced by solid-phase synthesis. In addition, 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 hydrolysis of the protein.

The enzyme is not particularly limited as long as it has proteolytic activity, but examples include aspartic acid proteases, serine proteases, cysteine proteases, metalloproteases, and threonine proteases.

Examples of aspartic acid proteases include pepsin, renin, cathepsin D, cathepsin E, napsin, β-secretase, γ-secretase, signal peptide peptidase, HIV protease, HTLV protease, NS3A protease, plasmepsin, saspase, and chymosin.

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

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

Metalloproteases include, for example, ADAM, matrix metalloproteases, thermolysin, neprilysin, carboxypeptidase, endothelin-converting enzyme, KELL antigen, bone morphogenetic protein-1, meprin, serralysin, PAPP, mitochondrial processing protease, insulin-degrading enzyme, aminopeptidase, and prenyl protease.

Examples of threonine proteases include proteasomes and gamma glutamyl transferase.

As an enzyme, one type can be used alone, or two or more types can be used in combination.

The combination of edible protein and 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 that is produced when the amino acid sequence of the edible protein is decomposed with an enzyme from the amino acid sequence of the edible protein. In this way, it is possible to obtain an edible protein/enzyme combination that gives the amino acid sequence of the peptide of the present invention. The amino acid sequence of the edible protein and the cleavage specificity of the enzyme can be easily determined according to publicly known information.

A method including a step of decomposing a protein with an enzyme can produce an enzymatic decomposition product of a protein. Specifically, the decomposition can be carried out, 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, the presence or absence of additives and their types, etc. can be appropriately set depending on the types of protein and enzyme.

After the step of hydrolyzing the protein with an enzyme, it is preferable to further carry out a step of purifying the peptide of the present invention.

The purification method is not particularly limited as long as it is a method that can concentrate the peptide of the present invention (i.e., can increase the concentration of the peptide of the present invention in the entire peptide). Examples of the purification method include purification with silica gel, purification with a 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 (for example, by using a carrier carrying FFAR1 or cells expressing FFAR1). As the purification method, one type alone can be adopted, or two or more types can be adopted in combination.

2. Uses In one aspect, the present invention relates to a composition containing the peptide of the present invention (sometimes referred to herein as the "composition of the present invention").

The composition of the present invention can be used for at least one selected from the group consisting of FFAR1 activation, insulin secretion promotion, GLP-1 secretion promotion, glucose metabolism improvement, and prevention or amelioration 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 diabetes prevention or amelioration agent, each of which contains the peptide of the present invention.

The peptides of the present invention can be used alone or in combination of two or more types.

The composition of the present invention may also contain a peptide other than the peptide of the present invention as a peptide. 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, even more preferably 70% by mass or more, even more preferably 80% by mass or more, particularly preferably 90% by mass or more, and particularly preferably 95% by mass or more, based on 100% by mass of the peptide.

The composition of the present invention can be used in various fields, for example, as a food additive, a food composition (including health enhancers and nutritional supplements (supplements, etc.)), a medicine, etc.

The administration (or ingestion) form of the composition of the present invention is not particularly limited as long as the desired effect is obtained, and it can be administered to mammals, including humans, by either oral administration or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, topical administration). The preferred administration form is oral administration. Dosage forms for oral and parenteral administration and methods for their preparation are well known to those skilled in the art, and can be prepared in accordance with conventional methods by mixing the active ingredient (the peptide of the present invention) with a pharma- ceutically acceptable carrier, etc.

The form of the composition of the present invention is not particularly limited, and may be any form commonly used for each application, depending on the application.

When the composition of the present invention is used as a food additive, medicine, health enhancer, nutritional supplement (supplement, etc.), the form of the composition may be, for example, a tablet (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), a pill, a granule, a fine granule, a powder, a hard capsule, a soft capsule, a dry syrup, a liquid (including drinks, suspensions, syrups), a jelly, etc. In the case of medicines in particular, other than these, for example, an injectable preparation (for example, drip injection, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection), an external preparation (for example, ointment, cataplasm, lotion, cream, gel), a suppository, an inhalant, an eye preparation, an eye ointment, a nasal drop, an ear drop, a liposome, etc.

When the composition of the present invention is used as a food composition, the form of the composition may be liquid, gel or solid food, for example, ramen, hamburgers, fried foods, juice, soft drinks, tea, soup, beverages such as soy milk, edible oils and fats such as salad oil and butter, dressings, yogurt, jellies, puddings, furikake, powdered milk for infants, cake mixes, powdered or liquid dairy products, bread, cookies, etc.

The composition of the present invention may further contain other components as necessary. The other components are not particularly limited as long as they are components that can be blended into food additives, food compositions, medicines, health enhancers, nutritional supplements (supplements, etc.), and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, flavorings, chelating agents, etc.

The content of the active ingredient in the composition of the present invention depends on the application, mode of use, state of the subject of application, 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.

The amount of the composition of the present invention to be applied (e.g., administered, ingested, inoculated, etc.) is not particularly limited as long as it is an effective amount that exerts its effect, and is generally 0.01 to 1000 mg/kg body weight per day in terms of dry weight of the peptide of the present invention. The lower limit of the application amount 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 application amount is preferably applied once or more times a day (e.g., 1 to 3 times), and can be increased or decreased as appropriate depending on age, pathology, and symptoms.

The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

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

<Test Example 1-1. Preparation of activated membrane>
Twenty-one sheets of cellulose filter paper FILTER PAPER 542 HARDENED ASHLESS 24.0 cm (1542-185, Whatman, England) (hereafter referred to as membrane) were cut to a size of 10 cm x 15 cm, placed in a polypropylene Tupperware, immersed in 150 mL of DMF (N,N-Dimethylformamide) (A00185, Kanto Chemical Co., Ltd., Tokyo), and shaken for a day. Next, an activation solution (0.5M β-Ala 32 mL, 1-Methylimidazole 1.6 mL, DIPCI 2 mL, DMF 150 mL) was prepared using Fmoc-β-Ala-OH (K00410, Watanabe Chemical Industry Co., Ltd., Hiroshima), 1-Methylimidazole, redistilled, 99+% (336092-100ML, SIGMA-ALDRICH, USA), and DIPCI (N,N`-Diisopropylcarbodiimide) (A00011, Watanabe Chemical Industry Co., Ltd., Hiroshima). 21 membranes were immersed in this solution and shaken for a day, after which 1.6 mL of 1-Methylimidazole and 2 mL of DIPCI were added and shaken for another day. The membranes were immersed in the activation solution, both the front and back, two or three times a day using tweezers. 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 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 to color the _NH2 groups blue. After confirming that the membrane was colored blue, the remaining 20 pieces were washed three times each with 50 mL of DMF and 50 mL of methanol (139-01827, Wako Pure Chemical Industries, Ltd., Osaka), air-dried, and then vacuum-packed and stored at 4°C.

<Test Example 1-2. Peptide synthesis>
The peptide array was prepared by Fmoc solid-phase synthesis. Fmoc-amino acids (all from Watanabe Chemical Industry Co., Ltd.) were dissolved in NMP (N-methyl-2-pyrrolidone) (133-15115, Wako Pure Chemical Industries Co., Ltd., Osaka) to prepare a 0.5 M amino acid solution, which was then mixed with DIPCI (N,N'-Diisopropylcarbodiimide) and HOBt (1-hydrozybenzotriazole) (A00015, Watanabe Chemical Industry Co., Ltd., Hiroshima) to a final concentration of 1:2:2. The amino acids were spotted onto an activated membrane using a peptide synthesizer (ASP222, IntavisAG, Koln, Germany) (1 spot=1.1 μl × 3 times, with 0.8 μl × 3 times for the first residue only). Fmoc-photo-linker (sc-294977A, SANTA CRUZ, USA) was synthesized as a linker at the first residue. After synthesis of the first residue, the membrane was washed three times with DMF to remove unreacted activated amino acids. Then, to block the unreacted amino groups, 5% acetic anhydride (011-00276, Wako Pure Chemical Industries, Osaka)/DMF 50 ml was reacted for 15 min × 2 times to acetylate the unreacted amino groups on the activated membrane. After that, the membrane was washed three times with DMF and immersed in 20% piperidine/DMF for 1 h to deprotect the Fmoc group. The above procedure was repeated to synthesize any peptide by peptide elongation reaction. The synthesis reaction was confirmed by using a 1% BPB (bromophenol blue) (021-02911, Wako Pure Chemical Industries, Ltd., Osaka)/DMF solution, adding 100 μl of 1% BPB/DMF solution and 100 μl of acetic acid (017-00256, Wako Pure Chemical Industries, Ltd., Osaka) to the methanol solution in which the membrane was immersed, and coloring the NH2 group blue.

After synthesizing a 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, a deprotection reagent was prepared by mixing TFA (trifluoroacetic acid) (A00025, Watanabe Chemical Industry Co., Ltd., Hiroshima), m-cresol (034-04646, Watanabe Chemical Industry Co., Ltd., Osaka), EDT (1,2-Ethanedithiol) (A00057, Watanabe Chemical Industry Co., Ltd., Hiroshima), and thioanisole (T0191, Tokyo Chemical Industry Co., Ltd., Tokyo) in a ratio of 40:1:3:6 to prepare 50 ml, and the membrane was immersed in this solution for 2.5 h. After deprotection, the product was washed three times each with diethyl ether (051-01157, Wako Pure Chemical Industries, Osaka) and methanol, and then washed several times with diethyl ether and methanol until the odor disappeared. It was then dried with a hair dryer and used in the experiment.

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

Test Example 2. TGFα cleavage assay The TGFα cleavage assay in the test example described below was carried out as follows.

<Test Example 2-1. Cell culture>
HEK293 cells were cultured in a T75 flask (658170, Greiner Bio One, AT) at 37℃ in a _CO2 incubator under 5% _CO2 and 95% air. DMEM containing 10% FBS (biosera, NUAILLE, France) and 1% Penicillin-Streptomycin (PS) (15140122, Wako Pure Chemical Industries, Osaka) was used as the medium. For subculture, the medium was removed from the cells that had become subconfluent (80%-90%), and the cells were washed twice with PBS, detached by trypsinization, and reseeded. The number of cells was measured using a hemocytometer, and the cells were reseeded so that the initial cell number was 1.0× ¹⁰⁶ cells/well. The cells used in the assay were seeded in a 100 mm dish (664160, Greiner Bio One, AT) at an appropriate cell concentration so that they were subconfluent at the start of the assay.

<Test Example 2-2. Preparation of plasmid dissolution buffer>
The plasmids used were pCAGGS/AP-TGFα and pCAGGS/FFAR1. Tris-EDTA buffer (TE buffer: 10 mM Tris-HCl buffer, 1 mM EDTA) was used as a buffer to dissolve the plasmids. 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 diluting 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 purified water, adjusting the pH to 8.0 with NaOH (193-13775, Wako Pure Chemical Industries, Osaka), and diluting to 500 mL.

<Test Example 2-3. Preparation of pNPP solution and dissolution buffer>
p-Nitrophenyl phosphate disodium salt hexahydrate (pNPP; 4264-83-9, Life Technologies, Tokyo) 1 g was dissolved in 2.692 mL of pure water (1 M solution). 2xAP buffer (2 M Tris-HCl buffer 60 mL, 4 M NaCl (191-01665, Wako Pure Chemical Industries, Osaka) 10 mL, 1 M MgCl ₂ ·6H ₂ O (132-00175, Wako Pure Chemical Industries, Osaka) 10 mL, pure water 920 mL) was prepared as a dissolution buffer for pNPP solution. The 10 mM solution was diluted 100 times with 2xAP buffer to prepare pNPP solution.

Test Example 2-4. Preparation of Ligand
HBSS buffer and HBSS (2% DMSO) were prepared as negative controls. Palmitic acid (165-00102, Wako Pure Chemical Industries, Osaka, Japan), an existing FFAR1 agonist, was dissolved in HBSS (2% DMSO) as a positive control. Peptide solutions were prepared by eluting with 72 μL of HBSS buffer (5 mM HEPES) at each time point.

<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 for 24 hours in a CO ₂ incubator at 37° C. under 5% CO ₂ and 95% air.

Next, the pCAGGS/AP-TGFα and pCAGGS/FFAR1 plasmid solutions prepared in Test Example 2-2, transfection reagents 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 table below (Table 1). The mixed Eppendorf tubes were tapped, flushed, and then left to stand for 5 min. Then, Eppendorf tubes A and B were mixed 1:1, tapped, flushed, and then left to stand for 20 min. Then, 50 μL/well of the FFAR1 plasmid solution was added to the 24-well plate seeded with HEK293 cells. The plates were then incubated for 24 h at 37°C in a _CO2 incubator under 5% _CO2 and 95% air.

Next, the transfected HEK293 cells were washed twice with PBS, detached by trypsinization, and collected in a centrifuge tube containing DMEM according to the type of plasmid. After centrifugation at 1000 rpm for 5 min, HBSS was added and left to stand for 10 min. Then, the cells were centrifuged again at 1000 rpm for 5 min, and the HBSS was removed to remove AP-TGFα cleaved by trypsinization. The HEK293 cells were diluted with HBSS so that they could be seeded on a 96-well plate at 80 μL/well, and the HEK293 cells were reseeded on the 96-well plate. The cells were then incubated for 1 h at 37°C in a _CO2 incubator under 5% _CO2 and 95% air to allow the cells to adhere. 20 μL each of the ligand and peptide solutions prepared in Test Example 2-4 was added, and the cells were incubated for 1 h at 37°C in a _CO2 incubator under 5% _CO2 and 95% air. Then, 80 μL of the supernatant containing the cleaved and released AP-TGFα was transferred to a new 96-well plate. After removing the remaining 20 μL of solution from each well, 80 μL/well of the pNPP solution prepared in Test Example 2-3 was added to all wells of each 96-well plate. The absorbance at 405 nm was measured using an absorbance meter immediately after the addition and 30 min after the addition. The cleavage rate of TGFα was calculated using the following formula.

ΔOD405 = (OD405 after 30 min of reaction) - (OD405 immediately after adding pNPP)
Cleavage rate of AP-TGFα=((ΔOD405 in the culture medium supernatant))/((ΔOD405 in the culture medium supernatant)+(ΔOD405 in the cell plate)).

Test Example 3. Screening of FFAR1-binding peptides
A 7-mer peptide library (phage library, library size: 10 ⁹ ) was used to screen for FFAR1-activating peptides by phage display. In summary, by combining negative and positive panning, phage peptides that do not bind to HEK293 cells but specifically bind to HEK293 cells in which FFAR1 is forcibly expressed were obtained. Specifically, phages were incubated with HEK293 cells (1×10 ⁷ cells/1 mL PBS (1% BSA)) in liquid phase, and the supernatant of phages that did not bind to HEK293 cells was collected (negative panning). The collected phages were then incubated with HEK293 cells in which FFAR1 was forcibly expressed, and phages that bound to FFAR1-expressing HEK293 cells were collected (positive panning). After three and four rounds of panning, consisting of two rounds of negative panning and one round of positive panning, sequence analysis was performed to obtain peptides that bind highly to FFAR1.

As a result, a peptide consisting of STTGTQY (sequence number 34) (STTGTQY peptide) was obtained from both the sequence analysis after round 3 and the sequence analysis after round 4.

Next, the STTGTQY peptide was resin-synthesized and a TGFα cleavage assay was performed at high concentration to measure the FFAR1 activation ability of the peptide. Specifically, the procedure was similar to Test Examples 1-2 and 2. In addition to the above peptides, palmitic acid (Pa), a ligand for FFAR1, was also used as a test substance for the TGFα cleavage assay.

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

Test Example 4. Examination of shortening residues of STTGTQY peptide
A peptide array of peptides (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)) in which 1 to 2 amino acids (1 or 2 residues at the N-terminus and/or C-terminus, a total of 1 to 2 amino acid residues) were deleted from the terminal of the STTGTQY peptide was synthesized according to Test Example 1, and a TGFα cleavage assay was performed using this according to Test Example 2. In addition to the above peptides, palmitic acid (Pa), a ligand of FFAR1, was also used as a test substance for the TGFα cleavage assay.

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

Test Example 5. Prediction of highly active peptides using machine learning
A peptide array covering all single amino acid substitutions 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. The TGFα cleavage rate was converted to Pa concentration using a Pa calibration curve, and the activity value was calculated by dividing the Pa concentration of each peptide by the Pa concentration of the original sequence (STTGTQ peptide). The results are shown in Table 3.

Next, a discriminant analysis was performed using all 120 feature values to correlate the peptide activity with the feature values. The feature values used were 120 values calculated by multiplying the physicochemical properties of 24 types of amino acids (Figure 3) by the five overall values calculated for the physicochemical properties of each amino acid (Figure 4).

Using the obtained teacher data (Table 3) and the above 120 feature values, logistic regression analysis was performed to predict highly active sequences from 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, it can be seen that the amino acid sequence represented by formula (1): X ¹ X ² X ³ GX ⁵ X ⁶ (1) (wherein 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, and X ⁶ is Q, F, I, V, or L) is a consensus sequence with a certain level of activity.

Twenty peptides satisfying the consensus sequence (Table 4: SEQ ID NOs: 2 and 15-33) were 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. The TGFα cleavage rate was converted to Pa concentration using a Pa calibration curve, and the activity value was calculated by dividing the Pa concentration of each peptide by the Pa concentration of the original sequence (STTGTQ peptide).

Representative results are shown in Figure 6. It was found that fulfilling the consensus sequence resulted in high FFAR1 activation ability.

Test Example 6. Evaluation of insulin secretion promoting activity
Test substances were added to MIN6 cells, and after 1 hour, the supernatant was collected and the insulin concentration in the supernatant was quantified by ELISA.
Negative control: Krebs-Ringer-bicarbonate-HEPES (KRBH) buffer (0.2% fatty acid-free BSA, 25 mM Glucose)
・Positive control: 50μM GW9508 (0.2% fatty acid-free BSA, 25mM Glucose)
Test peptide: STKGTF peptide.

Specifically, the experiment was performed according to Reference 1 (Chen et al., Br. J. Pharmacol., 2020). The activity ratio was calculated by dividing the insulin concentration when the positive control and test peptide were used by the insulin concentration when the negative control was used.

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

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 by shifting the residues one by one (total of 98,387), and from these, 2-residue replacement sequences were extracted from the STKGTF peptide (Table 5: total of 12 types (SEQ ID NOs: 3 to 14)). For these 12 types of amino acid sequence peptides, a peptide array was synthesized according to Test Example 1, and a TGFα cleavage assay was performed using this according to Test Example 2.

The results are shown in Figure 8. A peptide derived from an edible protein, which has a two-residue substitution sequence from the STKGTF peptide, also showed a certain level of FFAR1 activation ability.

Test Example 8. Evaluation of GLP-1 secretion promoting ability
STKGTF peptide and two peptides (VQKGTF, SILGTF) that were shown to have a higher FFAR1 activation ability than STKGTF peptide in Test Example 7 were examined to see whether they promote GLP-1 secretion via FFAR1 expressed by enteroendocrine cells. Specifically, the test peptides were added to GLUTag cells (enteroendocrine cell line), and after 2 hours, the supernatant was collected and the GLP-1 concentration in the supernatant was quantified by ELISA. The activity ratio was calculated by dividing the GLP-1 concentration when each test peptide was used by the GLP-1 concentration when the negative control (same as Test Example 6) was used.

The results are shown in Figure 9. All of the test peptides were confirmed to have the ability to promote GLP-1 secretion.

Claims (7)

A peptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 14 . A peptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 5. A peptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 16, 18, 19, 23-25, 40, 41, 49-53, 57, 60, 67-69, 71, 72, 75-77, 81, 85-89, 91, 93-95, 97, 124, 132, 136, 138, 140, 142-147, and 152. A composition comprising the peptide according to any one of claims 1 to 3 . 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 amelioration of diabetes. The composition according to claim 4 or 5 , which is a medicine, a food composition, or a food additive. The composition according to any one of claims 4 to 6 , which is in an oral dosage form.

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