KR20180091578A - Peptide for activating ampk, and uses thereof - Google Patents

Abstract

The present invention relates to an AMPK activating peptide and a use for preventing, ameliorating or treating diabetes, obesity, neuropathic disorders, and more particularly, a peptide represented by the amino acid sequence of the following general formula (I). The present invention relates to a pharmaceutical composition for the prevention and treatment of diabetes, obesity and neuropathic disorders by activating AMPK protein, which has excellent effects on side effects, and has a low molecular weight of less than 9 amino acids, have.
[Formula ^{(Ⅰ)] (CX 1 X} 2) n (X 3) 1- n (X 4) n P (CX 5) n (X 6) 1 - n P (X 7) m (1-n) (X ⁸ ) _n
N and m are each independently 0 or 1; X ¹ is S, or T; X ² is E or Q (provided that X ² = Q when X ¹ = S); X ³ is G, V, dV, VG, or VdV; X ⁴ , and X ⁵ are each independently A, G, V, or dV; X ⁶ is AG, CA, CG, CdV or WG; X ⁷ is G, or V; X ⁸ is S, T, or dT; The amino acid sequence CTQGPCGPT is excluded.

Description

AMPK-activating peptides and their uses {PEPTIDE FOR ACTIVATING AMPK, AND USES THEREOF}

The present invention relates to AMPK activating peptides and uses thereof for preventing, ameliorating or treating diabetes, obesity, neuropathic disorders.

Adenosine monophosphate-activated protein kinase (hereinafter abbreviated as 'AMPK') is an enzyme that plays an important role in cellular energy homeostasis. It has been known that activation of AMPK can prevent or treat diabetes, metabolic syndrome, obesity, cancer, neuropathic disorder (dementia) and the like, and research on AMPK activator has been continued (Non-Patent Document 1: AMPK and the biochemistry of exercise: Implications for human health and disease. Biochem J, 2009, Mar 1, 418 (2), pp. 261-275).

AMPK is an enzyme acting as an energy sensor in intracellular fat and glucose metabolism. It is activated when ATP is decreased and AMP is increased to increase enzyme activity of ATP production process, and enzyme activity of ATP consumption process . ≪ / RTI > Such AMPK is widely expressed in human organs and tissues such as heart, skeletal muscle, liver, and fat and exhibits the above-mentioned action.

When the AMPK activity of the heart is increased, the glucose uptake into the cell is increased and the process is activated (Non-Patent Document 2: Russell RR 3rd, Bergeron R, Shulman GO, and Young L. Am J Physiol. : H643-649), promotes fatty acid oxidation, increases glucose uptake in skeletal muscles, increases ATP production due to the process and fat oxidation, promotes the development of mitochondria, increases uncoupling protein (UCP ) To promote energy consumption.

It is also known that AMPK in the liver inhibits the synthesis of fatty acids and cholesterol, inhibits your biosynthesis, inhibits fatty acid synthesis in fat and promotes lipolysis (Non-Patent Document 3: Yin W, Mu J, and Bimbaum MJ, J Biol Chem, 2003, Oct, 278: 43074-43080). It has been found that the action of leptin, a typical anti-obesity hormone, is also mediated by AMPK, since studies on anti-diabetic and anti-obesity effects of AMPK have been continued since the blood glucose lowering effect of metformin was found to be mediated by AMPK. Leptin inhibits fatty acid synthesis in muscle cells and promotes fatty acid oxidation. When leptin activates AMPK, AMPK converts acetyl-CoA to malonyl-CoA (the rate of fatty acid synthesis Determining step) inhibits the activity of acetyl-CoA carboxylase (hereinafter referred to as 'ACC') to inhibit fatty acid synthesis. In addition, malonyl-CoA inhibits carnitine palmitoyltransferase (hereinafter referred to as "CPT"), which causes fatty acids to enter the mitochondria (the step of determining the rate of fatty acid oxidation) Inhibition results in decreased amounts of malonyl-CoA and activation of CPT leading to increased fatty acid oxidation in mitochondria. In addition to leptin, adiponectin, another anti-obesity hormone, is also known to be mediated by AMPK.

Furthermore, it has been known that AMPK improves the energy balance and thus has a neuroprotective effect in the central nervous system including the brain (Non-Patent Document 4: J Neurochem. 2009 May; 109 Suppl 1: 17-23; Non-Patent Document 5: Adv Exp Med Biol., 2016; 854: 425-30).

All of the contents of non-patent documents 1 to 5 are cited and incorporated by the background art of this specification.

 AMPK and the biochemistry of exercise: Implications for human health and disease. Biochem J, 2009, Mar 1, 418 (2), pp. 261-275.  Russell RR 3rd, Bergeron R, Shulman GO, and Young LH. Am J Physiol., 1999, Aug, 277, H643-649.  Yin W, Mu J, and Bimbaum MJ, J Biol Chem, 2003, Oct, 278: 43074-43080.  J Neurochem. 2009 May; 109 Suppl 1: 17-23.  Adv Exp Med Biol. 2016; 854: 425-30.

The inventors of the present invention have been studying a compound having an AMPK activating effect while confirming the AMPK activating effect of the osteogenic peptide and also found that a novel peptide having an AMPK activating effect having substitution, deletion or insertion of one or more amino acid residues in the osmotin peptide amino acid sequence Peptide compound and completed the present invention.

The present invention has been made to solve the problems of the prior art,

The peptide represented by the amino acid sequence of the following general formula (I).

[Formula ^{(Ⅰ)] (CX 1 X} 2) n (X 3) 1- n (X 4) n P (CX 5) n (X 6) 1 - n P (X 7) m (1-n) (X ⁸ ) _n

N and m are each independently 0 or 1; X ¹ is S, or T; X ² is E or Q (provided that X ² = Q when X ¹ = S); X ³ is G, V, dV, VG, or VdV; X ⁴ , and X ⁵ are each independently A, G, V, or dV; X ⁶ is AG, CA, CG, CdV or WG; X ⁷ is G, or V; X ⁸ is S, T, or dT; The amino acid sequence CTQGPCGPT is excluded.

The peptide of the present invention also provides a peptide represented by the amino acid sequence of the following general formula (II).

[Formula ^{(Ⅱ)] (CX 1 Q} ) n (X 3) 1- n (X 4) n P (CX 5) n (X 6) 1 - n P (V) m (1-n) (X ⁸ ) _n

N and m are each independently 0 or 1; X ¹ is S, or T; X ³ is G, or VG; X ⁴ is A, G, or dV; X ⁵ is A, G, or V; X ⁶ is AG, or CG; X ⁸ is S, or T.

In addition, the peptide of the present invention provides a peptide which is an amino acid sequence of any one of SEQ ID NOS: 2 to 20.

Also, the peptide of the present invention provides a peptide which is an amino acid sequence of any one of SEQ ID NOS: 3, 7, 11, and 14.

The peptide of the present invention also provides a peptide which is an amino acid sequence of any one of SEQ ID NOS: 2, 3, 7 to 9, 11, 14 and 20.

Also provided is a pharmaceutical composition for preventing or treating diabetes, obesity, neuropathic disorders comprising the peptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.

Also provided is a health functional food composition comprising the peptide of the present invention or an acceptable salt thereof.

In addition, there is provided a cosmetic composition comprising the peptide of the present invention or an acceptable salt thereof.

Also provided is a method for preventing or treating diabetes, obesity, neuropathic disorders, which comprises administering the peptide of the present invention or an acceptable salt thereof.

The present invention relates to a pharmaceutical composition for the prevention and treatment of diabetes, obesity and neuropathic disorders by activating AMPK protein, which has excellent effects on side effects, and has a low molecular weight of less than 9 amino acids, have.

Figure 1 shows the results of Western blot experiments on the AMPK protein activity test (Pep (#) numbers correspond to the respective sequence numbers one by one).
FIG. 2 shows the results of insulin secretion of Tolbutamide (Tolb) and Gliclazide (Glz) corresponding to the first generation, which are sulfonylurea hypoglycemic agents.
Figure 3 shows the results of insulin secretion in 3.3 mM Glucose and 16.7 mM Glucose depending on the concentration of the peptide of SEQ ID NO: 1.
4 shows the results of insulin secretion in 3.3 mM Glucose and 16.7 mM Glucose depending on the concentration of the peptide of SEQ ID NO: 2.
5 shows the results of insulin secretion in 3.3 mM Glucose and 16.7 mM Glucose depending on the concentration of the peptide of SEQ ID NO: 3.
6 shows the results of insulin secretion in 3.3 mM Glucose and 16.7 mM Glucose depending on the concentration of the peptide of SEQ ID NO: 7.
7 shows the results of insulin secretion in 3.3 mM Glucose and 16.7 mM Glucose depending on the concentration of the peptide of SEQ ID NO: 8.
8 shows the results of insulin secretion in 3.3 mM Glucose and 16.7 mM Glucose depending on the concentration of the peptide of SEQ ID NO: 9.
9 shows the results of insulin secretion in 3.3 mM Glucose and 16.7 mM Glucose depending on the concentration of the peptide of SEQ ID NO: 11.
Figure 10 shows the results of insulin secretion in 3.3 mM Glucose and 16.7 mM Glucose depending on the concentration of the peptide of SEQ ID NO: 14.
Figure 11 shows the results of insulin secretion in 3.3 mM Glucose and 16.7 mM Glucose depending on the concentration of the peptide of SEQ ID NO: 20.
12 and 13 are graphs showing insulin secretion by gluciczide in 3.3 mM Glucose and 16.7 mM Glucose and results of insulin secretion in the case of treating each peptide of SEQ ID NOS: 1 to 20 with 10 μM.

First, terms used in this specification will be described and defined.

In the present invention, the term "prevention" refers to any action that has not been diagnosed as having a disease or disease, but inhibits the occurrence of a disease or disease in an animal that tends to be susceptible to such disease or disease, do.

As used herein, the term "treatment" means (a) inhibiting the development of a disease or disease; (b) improving or benefiting from a disease or illness-induced symptom; Or (c) to remove any disease or disease.

The term " improvement " in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.

As used herein, the term "AMPK activation" means that AMPK activity is increased through direct or indirect reaction with at least one peptide, compared to AMPK activity in the absence of at least one peptide described herein. That is, the increase in AMPK activity may be the result of the peptide itself directly affecting AMPK, or AMPK activity, or interaction with other factors that affect AMPK or AMPK activity.

As used herein, the term "therapeutically effective amount" refers to an amount of a compound that is effective (eg, It means quantity.

In the present invention, the term "animal" includes a human being unless otherwise specified.

The amino acids referred to herein are abbreviated as follows according to the IUPAC-IUB nomenclature, and the suffix "d " indicates that the amino acid is a D-type amino acid.

Alanine A Arginine R

Asparagine N Aspartic acid D

Cysteine C Glutamic acid E

Glutamine Q Glycine G

Histidine H Isooru I

Louis X L Lysine K

Methionine M Phenylalanine F

Prolin P Serine S

Threonine T Tryptophan W

Tyrosine Y Valin V

Hereinafter, the present invention will be described in detail.

An aspect of the present invention is a peptide represented by the amino acid sequence of the following general formula (I).

[General Formula (I)]

_{^{(CX 1 X 2) n (}} X 3) 1-n (X 4) n P (CX 5) n (X 6) 1-n P (X 7) m (1-n) (X 8) n

N and m are each independently 0 or 1;

X ¹ is S, or T;

X ² is E or Q (provided that X ² = Q when X ¹ = S);

X ³ is G, V, dV, VG, or VdV;

X ⁴ , and X ⁵ are each independently A, G, V, or dV;

X ⁶ is AG, CA, CG, CdV or WG;

X ⁷ is G, or V;

X ⁸ is S, T, or dT; The amino acid sequence CTQGPCGPT is excluded.

Particularly, the peptide represented by the general formula (I) has AMPK activity, but in view of a more effective effect such as stronger AMPK activity and insulin secretion, the peptide represented by the amino acid sequence of the following general formula (II) desirable. A more detailed understanding of this may be made through the following Examples and Experimental Examples.

[General Formula (II)]

_{^{(CX 1 Q) n (X}} 3) 1-n (X 4) n P (CX 5) n (X 6) 1-n P (V) m (1-n) (X 8) n

N and m are each independently 0 or 1;

X ¹ is S, or T;

X ³ is G, or VG;

X ⁴ is A, G, or dV;

X ⁵ is A, G, or V;

X ⁶ is AG, or CG;

X ⁸ is S, or T.

The peptide represented by the amino acid sequence of the above general formula (I) can be obtained by various methods well known in the art. Specifically, it can be produced by using a gene recombination and protein expression system, synthesizing in vitro through chemical synthesis such as peptide synthesis, and cell-free protein synthesis. More specifically, it can be synthesized by a method well known in the art, for example, an automatic peptide synthesizer, and may be produced by genetic engineering techniques, but is not limited thereto. For example, a fusion gene encoding a fusion protein comprising a fusion partner and a peptide of the present invention is prepared by genetic engineering, and the host microorganism is transformed with the fusion gene, followed by expression in the form of a fusion protein in the host microorganism, The peptide of the present invention can be cleaved and separated from the fusion protein to produce a desired peptide. To this end, a DNA sequence coding for an amino acid residue which can be cleaved by, for example, a protease such as Factor Xa or enterokinase, a compound such as CNBr or hydroxylamine, is inserted between the fusion partner and the peptide gene of the present invention .

The peptide represented by the amino acid sequence of the above general formula (I) of the present invention may include a targeting sequence, a tag, an additional amino acid sequence designed for a specific purpose to increase the stability of the labeled residue, half-life or peptide, have.

Means that the peptide is directly or indirectly labeled with a marker that provides a detectable signal, such as, for example, a radioactive isotope, a fluorescent tag, or a specific binding molecule. Specific binding molecules include pairs such as biotin and streptavidin, digoxin and anti-digoxin. In the case of a specific binding element, a complementary element is normally labeled with a molecule provided for detection according to the known procedure outlined above. The marker may provide a detectable signal, either directly or indirectly.

The peptide represented by the amino acid sequence of the general formula (I) of the present invention induces AMPK activation and is useful as a therapeutic agent for obesity, diabetes, arteriosclerosis, metabolic diseases (including hypertension, hyperlipidemia and heart disease), degenerative diseases, There is an effect of preventing, ameliorating, or treating a disease including a disorder. Activation of AMPK by the peptides can be caused by binding to the adiponectin receptor.

The dose of the peptide represented by the amino acid sequence of the general formula (I) of the present invention may be determined by the specific syndrome, the severity of symptoms, the route of administration, the frequency of administration intervals, the specific compound used, the efficacy, the toxicological form, The form and presence of toxic side effects.

In one preferred embodiment, the novel peptide of the present invention may be any amino acid sequence selected from SEQ ID NOS: 2 to 20 in Table 1 below.

The present inventors confirmed that the activity and activity of AMPK in the activated mouse muscle cells of the 20 kinds of peptides were increased in a concentration-dependent manner, and the effect of activating AMPK of the 20 kinds of peptides was first identified. In addition, it was also confirmed that insulin secretion was increased in mouse muscle cells treated with 10 kinds of peptides of the above 20 kinds.

SEQ ID NOS: 3, 7, 11, and 14 peptides showed stronger AMPK activity than the other peptides, and SEQ ID NOS: 2, 3, 7 to 9, 11, 14, and 20 peptides had stronger insulin secretion effects than the other peptides Respectively. In addition, the peptide having the amino acid sequence of SEQ ID NO: 7, 9, 11, 14, and 20 shows a relatively high effect at a low concentration of 3 μM or less as compared with Gliclazide Even if absorption is not good, this means that it is possible to reach a therapeutically effective amount. A more detailed understanding of this may be made through the following Examples and Experimental Examples.

Another aspect of the present invention is a pharmaceutical composition for preventing or treating diabetes, obesity, neuropathic disorder comprising the peptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.

The peptide of the present invention can exhibit the effect of preventing or treating diabetes, obesity, neuropathic disorders through the AMPK activation effect, and it is possible that the insulin secretion amount in the mouse muscle cells treated with the peptide is increased, And the amount of secretion increased. Particularly, the peptide of any one of SEQ ID NOS: 2 to 20 may be provided as a single active ingredient as a pharmaceutical composition.

In addition, the pharmaceutical composition may further comprise one or more active ingredients that do not substantially interfere with the activity of the peptides described herein.

Such neuropathic disorders may be mediated by beta amyloid. Examples of such disorders include Huntington's disease and spinal cord cerebellar atrophy, Alzheimer's disease, Parkinson's disease, hypertensive neurological syndrome, dystonia, olivary cerebral atrophy, amyotrophic lateral sclerosis, Cerebral ischemia, seizure, cerebral ischemia, hypoxia, multiple infarct dementia, brain trauma or injury, spinal cord injury, AIDS-dementia complex, viral or bacterial meningitis; General central nervous system (CNS) infections such as viruses, bacteria or parasites; Or anemia withdrawal syndrome, eating behavior, schizophrenia, chronic anxiety, depressive disorder, growth or anemia, and / or an infectious disease, such as Alzheimer ' s disease, Alzheimer ' s disease, polio, Lyme disease (Borrelia burgdorferi infection) and malaria, brain localized cancer, Tourette ' s syndrome, And other di-glutamine diseases such as aging brain disorders, diabetes mellitus, and complications thereof.

The peptides described herein can be administered in therapeutically effective amounts. The therapeutically effective amount may vary depending on a number of factors such as the severity of the disease to be treated, age and relative health, the potency of the compound employed, the route and form of administration, and other factors well known to those of ordinary skill in the art. The drug may be administered at least once a day, for example once or twice a day.

The composition of the present invention may be formulated into oral or parenteral formulations according to the route of administration by the ordinarily skilled artisan. In the case of formulation, it can be prepared by using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

The composition thus prepared can be formulated into oral formulations such as powders, granules, tablets, pills, capsules, suspensions, extracts, emulsions, syrups and aerosols according to conventional pharmaceutical manufacturing processes. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The peptides may be in the form of pharmaceutically acceptable salts, chelates, non-covalent complexes and tautomers, and such compounds may exist in crystalline form, including polymorphs and / or clathrates.

"Pharmaceutically acceptable salts" refer to salts with inorganic acids such as hydrochlorates, phosphates, diphosphates, hydrobromates, sulphates, sulfinates, , Nitrates and similar salts; Or salts with organic acids such as malate, maleate, fumarate, tartrate, succinate, citrate, acetate, Lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate, salicylate, and the like. , Stearates, alkanoates, and similar salts; Alternatively, acetate (acetate), HOOC- (CH ₂₎ n-COOH include (where, n is from 0 to and 4), and the like salts, but not limited to this.

Yet another aspect of the present invention is a health functional food composition comprising the peptide of the present invention or an acceptable salt thereof.

The health functional food composition may be for preventing or ameliorating diabetes, obesity, neuropathic disorders. More specifically, the peptides of the present invention or salts thereof may be added to health functional foods for the purpose of preventing or ameliorating diabetes, obesity, neuropathic disorders. When the peptide of the present invention or a salt thereof is used as an additive for health functional food, the peptide or its salt may be added as it is or may be used in combination with other health functional food or health functional food ingredients and may be suitably used according to a conventional method . The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

There is no particular limitation on the kind of the health functional food of the present invention. Examples of the health functional food to which the peptide or its salt can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, , A tea, a drink, an alcoholic beverage, and a vitamin complex, and may include all the health functional foods in the conventional sense, and foods used as feeds for animals.

In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

Yet another aspect of the present invention is a cosmetic composition comprising the peptide of the present invention or an acceptable salt thereof.

In particular, the cosmetic composition of the present invention can be more preferably used for prevention or improvement of obesity. That is, the peptide of the present invention or a salt thereof can exhibit effects such as inhibition of fatty acid synthesis in fat or promotion of fat dissolution through activation of AMPK, and thus can be used in a slimming cosmetic composition.

The cosmetic composition of the present invention can be produced in the form of a general emulsified formulation and a solubilized formulation. Examples of the emulsified formulations include nutritive lotions, creams, essences, and the like, and the solubilization formulations include softening longevity. Suitable formulations include, but are not limited to, solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) A cream, a skin, a lotion, a powder, an ointment, a spray, or a conical stick. It may also be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, perfumes, surfactants, water, ionic or nonionic emulsifiers, Auxiliaries such as ionic sequestering agents, chelating agents, preservatives, vitamins, blocking agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients conventionally used in cosmetic compositions ≪ / RTI >

Another aspect of the present invention is a method for preventing or treating diabetes, obesity, neuropathic disorder characterized by comprising administering the peptide of the present invention or an acceptable salt thereof to an animal.

In the present invention, the animal is an animal such as a cow, horse, elephant, pig, rat, human, deer, squirrel, rabbit, tiger, lion, wolf, goat, leopard, bear, whale, seal, , Eagles, swans, thunder ducks, penguins, pigeons, ostriches, emu, and the like. However, the subject to which the composition of the present invention can be administered is not limited by the above examples.

The subject to be treated may vary depending upon the particular syndrome to be treated, the severity of the symptoms, the route of administration, the frequency of administration intervals, the particular compound employed, the efficacy, the toxicological form, the pharmacokinetic profile of the peptide and the presence of toxic side effects. Preferably in an amount of 0.001 to 1000 ug / g in the case of a rat, more preferably in an amount of 0.1 to 500 ug / g, more preferably in an amount of 0.2 to 10 ug / g There will be. However, such dosage can be determined by a person skilled in the art according to various factors such as the kind, volume, characteristic, weight and the like of a cell or an individual to be administered.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the following Examples and Experimental Examples are intended to illustrate the present invention in more detail and are not intended to limit the scope of the present invention.

Example

Amino acids were chemically synthesized through methods known in the art to obtain peptides of SEQ ID NOS: 1-20.

Experimental Example

Experimental Example  One. AMPK  Activation increase measurement experiment

The effect of each peptide on AMPK activation was evaluated based on phosphorylation of AMPK protein during peptide treatment.

Cell culture and drug treatment

Mouse muscle cells C2C12 were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, Grand Island, NY), 100 U / ml penicillin G, 100 μg / ml streptomycin sulfate, 0.25 μg / ml amphotericin The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 50 μM amphotericin B, and 2 μM mercaptoethanol. To induce the differentiation of C2C12 cells, 10% fetal bovine serum was replaced with 2% horse serum when the cells reached 70% confluence. After 4 days of culture, the cells were differentiated into polynuclear myotubes. The cells were treated with the normal medium to give a control, and the peptides of the present invention were added to the medium of the experimental group in amounts of 5 and 10 μM, respectively. All drug treated groups were incubated in vitro for 24 hours.

Western Blot (Western blot) analysis

After the cells were washed with phosphate buffered saline (PBS), phosphate buffered saline (PBS) was added and the cells were removed from the culture dish using a cell scraper. The cells were centrifuged at 13,200 rpm for 10 minutes The cells were separated and separated. The protease inhibitor cocktail and phosphatase inhibitor cocktail were added to lysis buffer (pH 7.2) consisting of 320 mM sucrose, 200 mM HEPES, and 1 mM EDTA, and the cell membrane was disrupted using an ultrasonic machine at 4 ° C. After crushing, 20 μg of each sample was quantitated by using BCA Protein Assay kit (BCA Protein Assay kit). The samples were mixed with a sample buffer and subjected to 10% SDS PAGE at 90 volts for 1 hour at room temperature Followed by electrophoresis to perform protein separation. Gel separated proteins were transferred to polyvinylidene fluoride microporous membrane at 15 ° C over 12 hours at 4 ° C using a blocking solution containing 25 mM Tris, 192 mM glycine, and 10% methanol as a PVDF microporous membrane. The transferred PVDF microporous membrane was incubated with 5% non-fat dry milk or 5% bovine serum albumin (TBS-T buffer, 40 mM Tris-HCl pH 7.4, 25 mM NaCl, 0.1% Tween®20) bovine serum albumin) was added and reacted for 1 hour on a shaker to inhibit non-specific binding with the antibody. Thereafter, the cells were reacted on a shaker for 2 hours at room temperature using an antibody in a blocking solution, washed 5 times with TBS-T buffer for 5 minutes each, and then reacted with HRP-conjugated 2 The secondary antibody was added to the blocking solution and reacted for 2 hours under the same conditions as the primary antibody. The result of AMPK activity by the peptide of the present invention through the expression pattern of each protein after being washed five times with TBS-T buffer for 5 minutes and then exposed to a film using an ECL system in a dark room is shown in Table 2 and FIG.

From Table 2 and FIG. 1, it can be seen that the peptide of the present invention has an AMPK activity. More specifically, in the case of the peptides represented by SEQ ID NOS: 11 and 14, when the peptide was treated at a concentration of 5 μM, it was confirmed that the AMPK activation effect was superior to other peptides described in the present specification. 11 and 14 showed that the effect of AMPK activation was excellent when treated with 10 μM of the peptide. Further, in the case of treatment with 10 μM peptide, threonine (T) located at the second amino acid of the peptide shown in SEQ ID NO: 1 is substituted with serine (S), glycine (G) located at the fourth or seventh amino acid is substituted with alanine (A), it was confirmed that AMPK activation effect was very high. Particularly, it was confirmed that the effect of AMPK activity was excellent even when glycine (G) located at the 7th amino acid of SEQ ID NO: 1 was substituted with valine (V). Glycine (G), a neutral, weak hydrophobic amino acid, (V), which is an unusual and surprising discovery. Specifically, in the case of the fourth glycine (G) of the peptide shown in SEQ ID NO: 1, a strong AMPK activity was exhibited only when it was substituted with D-valine (dV) This is presumably due to structural specificity.

Experimental Example  2. AMPK  Experiment to measure increase of insulin secretion by activation

In order to measure the increase of the insulin secretion of the peptide according to the present invention, the following experiment was conducted using Insulin Secretion Assay kit: ALPCO (80-INSMSH-E01).

The mouse muscle cells C2C12 differentiated into polynuclear myotubes were treated with 10 μM of the peptide of the present invention in the experimental group and Gliclazide: Sigma (G2167-5G) having the following formula was treated in place of the peptide of the present invention as a control (control) , And all of the drug treated groups were cultured in vitro for 24 hours.

The cells were treated with KHBB solution (1 mM KH2PO4, 24 mM NaHCO3, 115 mM NaCl, 5 mM KCI, 1 mM MgSO4.7H2O, 2.5 mM CaCl2.2H2O, 0.25% BSA) and incubated with INS-1 Cells Growth Medium supplement RPMI-1640 Medium with 10 mM Hepes, 2 mM L-glutamine, 1 mM sodium-pyruvate, 0.05 mM 2-mercaptoethanol) was used.

 Gliclazide: Sigma (G2167-5G) is a sulfonylurea drug that is used for the treatment of diabetes due to a slight side effect as a non-insulin dependent diabetes mellitus. It binds to sulfonylurea receptor in pancreatic beta cells and stimulates pancreatic beta cells to secrete insulin It decreases the blood sugar, and has superior drug effect among the same series of drugs and its side effects are also slight.

In addition, glyclazide (Glz) was higher in insulin content per protein when 20 iM gliclazide (Glz) and 200 iM tolbutamide (Tolb) were administered to INS-1 rat pancreatic a-cells (Molecular Pharmacology January 2013, 83 (1), pp. 191-205) (see FIG. 2).

FIGS. 3 to 11 are graphs showing the effect of insulin secretion on 3.3 mM glucose and 16.7 mM glucose according to the respective concentrations of the peptides of the present invention in the control group and SEQ ID NOs: 1, 2, 3, 7, 8, 9, 11, 14, .

3 to 11, it was confirmed that the peptide of the present invention was superior to the control group in the insulin secretion ability of 16.7 mM Glucose, and the peptide of SEQ ID Nos. 1, 2, 7, 9, 11, 14 and 20 It was confirmed that the peptide of the present invention exhibited excellent activity at low concentration.

In addition, the results of insulin secretion activity test according to the peptide treatment of 10 μM of the present invention are shown in FIG. 12 and FIG. 13, and the results are summarized in Table 3 below.

12 and 13, the control group treated with Gliclazide: Sigma (G2167-5G), which is currently being used as an anti-diabetic drug, is about 1.8 ng / ml / h in 3.3 mM glucose and about 2.4 ng / ml in 16.7 mM glucose / h insulin, the insulin secretion amount in the group treated with the peptide of the present invention according to the present invention is similar to that of the control group in the 3.3 mM glucose, and the insulin secretion amount in the 16.7 mM glucose is in the range of 1 to 5 and 7 The results are shown in FIGS. 1, 2, 3, 7, 8, 9, 11, 14, and 20, which show a significantly increased amount of insulin secretion relative to that of the control group in comparison with the insulin secretion amount of 16.7 mM Glucose treated in the control group And the effect of increasing the insulin secretion amount was excellent.

For reference, the AMPK activation effect was relatively weak compared to other peptides (see Table 2), but very good peptides (SEQ ID NOS: 2, 8, 9, and 20) were found in the effect of increasing insulin secretion, In addition to antidiabetic effects due to activation, it is presumed that insulin secretion is increased through direct or indirect reaction with other factors such as enzymes that promote insulin secretion.

<110> KOREA PHARMA CO., LTD. <120> PEPTIDE FOR ACTIVATING AMPK AND USES THEREOF <130> JSP2016-0080 <160> 20 <170> KoPatentin 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 1 Cys Thr Gln Gly Pro Cys Gly Pro Thr   1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 2 Gly Pro Cys Gly Pro   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 3 Cys Ser Gln Gly Pro Cys Gly Pro Thr   1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 4 Cys Ser Gln Gly Pro Cys Gly Pro Ser   1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 5 Cys Thr Gln Val Pro Cys Gly Pro Thr   1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 6 Cys Thr Glu Gly Pro Cys Gly Pro Thr   1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 7 Cys Thr Gln Gly Pro Cys Ala Pro Thr   1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 8 Cys Thr Gln Gly Pro Cys Val Pro Thr   1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 9 Cys Thr Gln Ala Pro Cys Ala Pro Ser   1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 10 Val Pro Cys Gly Pro   1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 11 Gly Pro Ala Gly Pro   1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 12 Gly Pro Cys Ala Pro   1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 13 Gly Pro Trp Gly Pro   1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide, which is the fourth amino acid is D-form <400> 14 Cys Thr Gln Val Pro Cys Gly Pro Thr   1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptides which first and fourth amino acids are D-form <400> 15 Val Pro Cys Val Pro   1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptides which are seventh and ninth amino acids are D-form <400> 16 Cys Thr Gln Gly Pro Cys Val Pro Thr   1 5 <210> 17 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 17 Gly Pro Cys Gly Pro Gly   1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 18 Val Gly Pro Cys Gly Pro Gly   1 5 <210> 19 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide, which is the second and fifth amino acids are D-form <400> 19 Val Val Pro Cys Val Pro   1 5 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 20 Val Gly Pro Cys Gly Pro Val   1 5

Claims (9)

A peptide represented by the amino acid sequence of the following general formula (I).
[General Formula (I)]
_{^{(CX 1 X 2) n (}} X 3) 1-n (X 4) n P (CX 5) n (X 6) 1-n P (X 7) m (1-n) (X 8) n
N and m are each independently 0 or 1;
X ¹ is S, or T;
X ² is E or Q (provided that X ² = Q when X ¹ = S);
X ³ is G, V, dV, VG, or VdV;
X ⁴ , and X ⁵ are each independently A, G, V, or dV;
X ⁶ is AG, CA, CG, CdV or WG;
X ⁷ is G, or V;
X ⁸ is S, T, or dT; The amino acid sequence CTQGPCGPT is excluded. The peptide according to claim 1, which is represented by the amino acid sequence of the following general formula (II).
[General Formula (II)]
_{^{(CX 1 Q) n (X}} 3) 1-n (X 4) n P (CX 5) n (X 6) 1-n P (V) m (1-n) (X 8) n
N and m are each independently 0 or 1;
X ¹ is S, or T;
X ³ is G, or VG;
X ⁴ is A, G, or dV;
X ⁵ is A, G, or V;
X ⁶ is AG, or CG;
X ⁸ is S, or T. The peptide according to claim 1, which is an amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 20. The peptide of claim 1, which is an amino acid sequence of any one of SEQ ID NOS: 3, 7, 11, and 14. The peptide of claim 1, which is an amino acid sequence of any one of SEQ ID NOS: 2, 3, 7 to 9, 11, 14, A pharmaceutical composition for preventing or treating diabetes, obesity, neuropathic disorders comprising the peptide of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof as an active ingredient. A health functional food composition comprising a peptide according to any one of claims 1 to 5 or an acceptable salt thereof. A cosmetic composition comprising the peptide of any one of claims 1 to 5 or an acceptable salt thereof. A method for the prophylaxis or treatment of diabetes, obesity, neuropathic disorders, comprising administering a peptide or an acceptable salt thereof of any one of claims 1 to 5 to an animal other than a human.

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