KR20220053745A - Composition for inhibiting Caspase comprising coprisin peptide CopA derived from Copris tripartitus - Google Patents

Abstract

The present invention relates to a reagent composition for inhibiting caspase comprising a copricin peptide CopA3 derived from Copris tripartitus. The coprisin peptide CopA3 of the present invention covalently binds to caspase to inhibit active cleavage of the caspase, thereby exhibiting excellent effects as a therapeutic agent for caspase-mediated ischemic myocardial infarction induced by caspase activity.

Description

Composition for inhibiting Caspase comprising coprisin peptide CopA derived from Copris tripartitus

It relates to a composition for inhibiting caspase comprising the coprisine peptide CopA3 derived from rhinoceros dung beetle, and more particularly, to a composition for treating caspase-mediated diseases comprising the copricin peptide CopA3, and more particularly , The present invention relates to a pharmaceutical composition for preventing or treating caspase-mediated ischemic myocardial infarction, comprising the copricin peptide CopA3.

ssa A, Lambert M, Mallet VO, Duriez M, Wassef M, Kahn A, Menaschι P, Gilgenkrantz H. Cardiac functional improvement by a human Bcl-2 transgene in a mouse model of ischemia/reperfusion injury. 2000, J Gene Med 2: 326-333), (Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94: 1621-1628, 1994), (Jeremias I, Kupatt C, Martin-Villalba A, Habazettl H, Schenkel J, Boekstegers P, Debatin KM. Involvement of CD95/Apo1/Fas in cell death after myocardial ischemia. Circulation 102: 915-920, 2000.), (Lee P, Sata M, Lefer DJ, Factor SM, Walsh K, Kitsis RN. Fas pathway is a critical mediator of cardiac myocyte death and MI during ischemia-reperfusion in vivo. Am J Physiol Heart Circ Physiol 284: H456-H463, 2003.), (Dominic P. Del Re, fundamental mechanisms of regulated cell death and implications for heart disease, 2019 99(4) 1765-1817). 즉, 허혈을 통해 비정상적으로 심근세포 자살과정이 촉진되고 이로 인해 허혈성심근경색이 초래된다고 알려져 있다. 또한 심근경색을 치료하기 위해서 건강한 심근세포를 이식하는 치료를 수행하는데, 이 과정에서의 어려움도 이식된 심근세포의 높은 사망률이라고 한다(백지석 외, Anesth pain med, 2013, 8: 175~180),(Laflamme MA, Dupras SK, et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol 2007; 25: 1015-24), (Zhang M, Methot D, Murry CE.Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 2001; 33: 907-21), (Chimenti I, Smith RR, Giacomello A, et al. Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice. Circ Res 2010; 106: 971-80). 즉, 심근세포 이식치료에서도 세포자살을 억제하는 약물개발이 주요 치료적 접근으로 알려져 있다. 이와 같은 배경으로 인해서 카스파제는 주요한 약물개발 표적분자로 소개되어 왔고, 이를 제어하는 많은 약물들이 개발 중에 있다. 예를 들어 카스파제-1을 억제하는 약물이 염증을 획기적으로 완화시키는데 도움이 된다는 보고도 있다. 본래 카스파제들은 불활성(zymogen, pro-caspases) 단백질 형태로 생산되어 세포 속에 존재하다가 상위 단백질분해효소에 의해 절단되며 그 부산물로 큰 조각(large subunit)과 작은 조각(small subunit)으로 분리된다. 절단된 두 개의 조각들은 재조립되어 이량체를 형성하며 이를 통해 활성형 카스파제가 완성된다.Caspase is a cysteine proteolytic enzyme and functions to break down substrate proteins by decomposing aspartic acid in the substrate. Caspase is known to play a key role in the apoptosis process. Twelve types of family proteins have been discovered so far, and caspases are known to be importantly involved in pyroptosis and necroptosis. Cardiomyocyte death due to acute ischemia is also closely related to apoptosis, and it is known that caspase is key in this process (Brocheriou V, Hagege AA, Oubena).

ssa A, Lambert M, Mallet VO, Duriez M, Wassef M, Kahn A, Menaschι P, Gilgenkrantz H. Cardiac functional improvement by a human Bcl-2 transgene in a mouse model of ischemia/reperfusion injury. 2000, J Gene Med 2: 326-333), (Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94: 1621-1628, 1994), (Jeremias) I, Kupatt C, Martin-Villalba A, Habazettl H, Schenkel J, Boekstegers P, Debatin KM. Involvement of CD95/Apo1/Fas in cell death after myocardial ischemia. Circulation 102: 915-920, 2000.), (Lee P , Sata M, Lefer DJ, Factor SM, Walsh K, Kitsis RN. Fas pathway is a critical mediator of cardiac myocyte death and MI during ischemia-reperfusion in vivo. Am J Physiol Heart Circ Physiol 284: H456-H463, 2003.) , (Dominic P. Del Re, fundamental mechanisms of regulated cell death and implications for heart disease, 2019 99(4) 1765-1817). That is, it is known that myocardial cell suicide is abnormally promoted through ischemia, which leads to ischemic myocardial infarction. In addition, to treat myocardial infarction, transplantation of healthy myocardial cells is performed, and the difficulty in this process is also said to be due to the high mortality rate of the transplanted myocardial cells (Ji-Seok Baek et al., Anesth pain med, 2013, 8: 175~180). , (Laflamme MA, Dupras SK, et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol 2007; 25: 1015-24), (Zhang M, Methot D, Murry CE . Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 2001; 33: 907-21), (Chimenti I, Smith RR, Giacomello A, et al. Relative roles of direct regeneration versus paracrine effects. of human cardiosphere-derived cells transplanted into infarcted mice (Circ Res 2010; 106: 971-80). In other words, the development of drugs that inhibit apoptosis in myocardial cell transplantation is known as the main therapeutic approach. Due to this background, caspases have been introduced as major target molecules for drug development, and many drugs that control them are under development. For example, there are reports that drugs that inhibit caspase-1 can help dramatically relieve inflammation. Originally, caspases are produced in the form of inactive (zymogen, pro-caspases) proteins, exist in cells, and are cleaved by upper proteases, and are separated into large subunits and small subunits as by-products. The two cleaved fragments are reassembled to form a dimer, which completes the active caspase.

As such, caspase is recognized as a major enzyme closely related to the development of many diseases and is a major target molecule for the development of inhibitory drugs. There are also many related patents. However, most inhibitors are often toxic due to their weak selective inhibitory ability. In addition, by completely inhibiting the function of physiologically important caspase, the problem of tumorigenesis caused by the blocking of spontaneous apoptosis cannot be solved. Despite these many studies, the development of drugs that selectively bind to caspase and have fewer side effects so far is insignificant. No drug has yet entered the drug development market due to its high cytotoxicity. Therefore, there is an urgent need to develop inhibitory drugs with low side effects such as caspase-specific binding and cytotoxicity.

The inventors of the rhinoceros dung beetle ( Copris tripartitus )-derived peptide was used to study various physiological activities, and CopA3, a copricin peptide derivative isolated from Arabidopsis dung beetle, confirmed an excellent caspase inhibitory effect, and was used to treat caspase-mediated ischemic myocardial infarction The possibility of use as a therapeutic agent for Korea was confirmed.

An object of the present invention is to provide a composition for inhibiting caspase comprising a copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO: 1: LLCIALRKK-NH ₂

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating ischemic myocardial infarction, comprising the copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO: 1: LLCIALRKK-NH ₂

Another object of the present invention is to provide a food composition, feed composition, or health functional food for preventing or improving ischemic myocardial infarction, comprising the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a method for inhibiting caspase activity in vitro , comprising treating cells with the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1.

In one aspect of the present invention, the present invention provides a composition for inhibiting caspase comprising a copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1:

SEQ ID NO: 1: LLCIALRKK-NH ₂

The composition for inhibiting caspase according to the present invention preferably inhibits caspase 3 and caspase 6. More specifically, it may be a selective inhibitor for caspase 3 and caspase 6.

More specifically, it provides a reagent composition for inhibiting caspase comprising a copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1.

The copricin peptide CopA3 binds to caspase and blocks the transfer process of caspase, thereby inhibiting caspase activity, and the binding may be the binding of copricin peptide CopA3 to caspase through a disulfide bond through cysteine. there is.

In addition, the present invention provides a method of inhibiting caspase activity in vitro , comprising the step of treating cells with the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the cells are cancer cells, neurons, adipocytes, macrophages, fibroblasts, pancreatic islet cells, stem cells, hepatocytes, fibroblasts, lymphocytes, vascular endothelial cells, enamel cells, keratinocytes, osteoblasts, It may be a ciliary cell, a dendritic cell, an egg cell, a renal muscle cell, and the like. Specifically, HT29 (human colorectal adenocarcinoma), SH-SY5Y (human neuroblastoma), HepG2 (human hepatoma), HeLa (human cervical cancer), Caki (human renal carcinoma), SW13 (human adrenal carcinoma), 3T3L1 (mouse adipocytes), RAW 264.7 (mouse macrophages), NIH/3T3 (mouse fibroblasts), PC12 (rat phaeochromocytoma), 3Y1 (rat fibroblasts), etc. may be used, but the cell scope of the present invention is not limited thereto.

In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating ischemic myocardial infarction, comprising the copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO: 1: LLCIALRKK-NH ₂

In one embodiment, the present inventors revealed that the copricin peptide CopA3 has a therapeutic effect on ischemic myocardial infarction, more specifically, caspase-mediated ischemic myocardial infarction by caspase activity.

Ischemic myocardial infarction is one of the ischemic heart diseases caused by insufficient blood supply to the heart muscle due to narrowing or blockage of the coronary arteries supplying blood to the heart. is a disease that gives

The ischemic myocardial infarction is a disease caused by sudden apoptosis of myocardial cells. Specifically, the blood vessels are damaged and the supply of nutrients and oxygen to the myocardial cells is interrupted, thereby rapidly increasing apoptosis of the myocardial cells, leading to ischemic myocardial infarction. can do it That is, caspases have a major effect on the suicide process of cardiomyocytes.

Accordingly, in an embodiment of the present invention, the copricin peptide CopA3 blocks or inhibits apoptosis in myocardial infarction by inhibiting caspase, thereby having therapeutic efficacy in ischemic myocardial infarction induced by caspase. In particular, when myocardial infarction occurs, it is known that the expression of Caspase-3 is greatly increased, and inhibition thereof can be understood as an action effect showing an excellent effect in the treatment of diseases.

The copricin peptide CopA3 of the present invention may be derived from nature, and the copricin peptide CopA3 may be derived from an insect, and preferably may be isolated from dung beetle ( Copris tripartitus ).

In addition, it can be synthesized using a known polypeptide synthesis method (genetic engineering method, chemical synthesis). The production of a peptide by a genetic engineering method can be carried out by, for example, preparing a nucleic acid encoding the polypeptide or a functional equivalent thereof according to a conventional method. The nucleic acid may be prepared by amplification by PCR using appropriate primers. Alternatively, the DNA sequence may be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer. The produced nucleic acid is operatively linked thereto and inserted into a vector containing one or more expression control sequences (eg, promoter, enhancer, etc.) for controlling the expression of the nucleic acid to produce a recombinant expression vector, After transformation into a host cell, it is cultured under an appropriate medium and conditions to recover a substantially pure polypeptide expressed from the nucleic acid from the culture. The recovery may be performed using a method known in the art (eg, chromatography). As used herein, the term 'substantially pure polypeptide' means that the polypeptide according to the present invention does not substantially contain any other proteins derived from host cells.

For the genetic engineering method for synthesizing the polypeptide of the present invention, the following documents may be referred to: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Second (1998) and Third (2000) Editions; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; and Hitzeman et al., J. Biol. Chem., 255:12073-12080, 1990.

In addition, the peptide according to the present invention can be prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press). , Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989). Peptides can also be synthesized using a C-terminal amidation reaction or an N-terminal acetylation reaction.

In addition, the peptide of the present invention includes not only a polypeptide having a native amino acid sequence, but also an amino acid sequence variant thereof having the same activity as the copricin peptide CopA3, within the scope of the present invention. A variant of the polypeptide of the present invention refers to a peptide having a different sequence by deletion, insertion, non-conservative or conservative substitution, substitution of amino acid analogs, or a combination thereof in which one or more amino acid residues in the amino acid sequence of the present invention, and caspase activity It means that it maintains the effect of suppressing the apoptotic process and inflammatory response of cells. Amino acid exchanges that do not entirely alter the activity of a molecule are known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). In the present invention, "peptide", and "protein" mean Used interchangeably and includes reference to a polymer of amino acid residues. The terms apply to natural amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding natural amino acid. The terms also apply to those polymers containing conservative amino acid substitutions such that the protein remains functional. In the present invention, the peptide may be synthesized using a gene recombination and protein expression system, preferably synthesized in vitro through a peptide synthesizer or the like.

In the present invention, "biologically active" refers to a peptide fragment having a structural function similar to that of the polypeptide of SEQ ID NO: 1, and/or a similar regulatory function, and/or a similar biochemical function, and also a peptide according to the present invention. do.

The present invention may include a variant of the sequence of SEQ ID NO: 1. Specifically, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, Sequences having at least 98%, or at least 99% sequence homology and retaining biological activity may be included within the scope of the present invention.

In the present invention, a "deletion" is defined as a change in the nucleotide or amino acid sequence that lacks one or more nucleotide or amino acid residues, respectively, compared to a wild-type polynucleotide or polypeptide.

In the present invention, "insertion" or "addition" is a change in nucleotide or amino acid sequence resulting from the addition of one or more nucleotide or amino acid residues, respectively, compared to a wild-type polynucleotide or polypeptide.

In the present invention, "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively, compared to a wild-type polynucleotide or polypeptide.

On the other hand, preferred amino acid mutation at the substitution position may be performed based on the similarity of residues in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphiphilic properties. For example, but not limited to, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; And amino acids having an uncharged polar head group having a similar hydrophilicity value include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine, threonine, phenylalanine and tyrosine; Such mutations may include conservative substitutions. A “conservative substitution” refers to a substitution in which an amino acid is substituted with another amino acid having similar properties so that those skilled in the art can predict that the secondary structure and hydropathic nature (hydropathic nature) of the polypeptide are substantially unchanged. am. In general, the following groups of amino acids exhibit conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

In some cases, the variant is phosphorylation (phosphorylation), sulfation (sulfation), acrylation (acrylation), glycosylation (glycosylation), methylation (methylation), farnesylation (farnesylation), acetylation (acetylation) and amylation (amidation) It can also be modified (modification) and the like.

In addition, the sequence according to the present invention may further include a cell penetrating peptide sequence for intracellular penetration. The cell-penetrating peptide is a kind of signal peptide and is a peptide sequence for the purpose of delivering a substance into a cell. It mainly consists of a peptide sequence of about 7-30 amino acids, and any sequence having a signal peptide that can be delivered into a cell may be included in the present invention.

For example, the cell-penetrating peptide mainly contains basic amino acid residues such as lysine/arginine, and thus serves to penetrate the fused proteins into the cell membrane and into the cell. The cell-penetrating peptide is HIV-1 Tat protein, Drosophila antennapedia homeodomain (phenetratin), HSV VP22 transcriptional regulatory protein, vFGF-derived MTS peptide, PTD-5, transpotan or Pep-1 peptide Sequences derived from, but are not limited thereto. As such, various CPPs identified/synthesized from viruses or cationic peptides (eg, TAT48-60, penetratin (pAntp) 43-58, polyarginine, Pep-1, transpotan, etc.) are biologically active substances. has an activity capable of internalization of cells to mediate the movement of drug carriers.

The copricin peptide CopA3 according to the present invention inhibits caspase activity, and preferably, the copricin peptide CopA3 binds directly to caspase to inhibit cleavage of the active form of caspase, thereby inhibiting caspase activity. can do.

In addition, the bond may be a disulfide bond of the copricin peptide CopA3 through cysteine.

As used herein, "treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject or the performance of a procedure or modality in a subject for the purpose of obtaining the benefit of treatment of a disease or health-related condition. Prevention", "preventing" and the like refer to an approach for preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or condition. Also delaying the onset or recurrence of a disease or condition, or the occurrence or recurrence of a disease or condition. It refers to delaying relapse.

As used herein, “individual”, “subject” and “patient” refer to a human or non-human, such as a primate, mammal, or vertebrate. In certain embodiments, the subject is a human.

In the present invention, "therapeutic benefit" or "therapeutically effective" refers to anything that promotes or enhances the health of a subject in connection with the medical treatment of such condition. This includes, but is not limited to, a decrease in the frequency or severity of signs or symptoms of a disease.

The copricin peptide CopA3 contained in the composition according to the present invention may be used by itself or in the form of a pharmaceutically acceptable salt. In the present invention, 'pharmaceutically acceptable' means that it is physiologically acceptable, does not inhibit the action of the active ingredient when administered to humans, and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions. . The salt is preferably an acid addition salt formed with a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid can be used as the free acid. The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid.

The copricin peptide CopA3 according to the present invention may be added to the pharmaceutical composition of the present invention in an amount of 0.001 to 95% by weight. The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient to the copricin peptide CopA3 of the present invention, for example, starch, calcium carbonate, sucrose. Or it is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and generally dosages range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection. The pharmaceutical composition of the present invention is suitable for parenteral administration, including intravenous or body cavity administration.

The present invention provides the use of the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1 in the manufacture of a medicament for the treatment of ischemic myocardial infarction.

The present invention provides a method for treating ischemic myocardial infarction, comprising administering a therapeutically effective amount of the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1 to a subject in need thereof.

In addition, the present invention provides a food composition for preventing or improving ischemic myocardial infarction, comprising the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1.

According to the present invention, there is provided a health functional food for preventing or improving ischemic myocardial infarction comprising the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1.

The food composition of the present invention may contain conventional food additives, and the suitability as the "food additive" is determined according to the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety, unless otherwise specified. It is judged according to the standards and standards related to the item.

The term "health functional food" means a food manufactured and processed using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act, and "functionality" refers to the structure and function of the human body. It refers to ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological effects.

The items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; Mixed preparations such as sodium L-glutamate preparation, noodle-added alkali agent, preservative agent, and tar color agent can be mentioned.

The food composition of the present invention may contain 0.01 to 95% by weight of copricin peptide CopA3, preferably 1 to 80% by weight, based on the total weight of the composition.

In addition, the food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, and the like.

For example, the health functional food in tablet form is granulated with a mixture of the copricin peptide CopA3 of the present invention, excipients, binders, disintegrants, and other additives in a conventional manner, and then a lubricant is added and compression molded. Alternatively, the mixture may be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent, etc., if necessary, and may be coated with a suitable skinning agent if necessary.

Among health functional foods in the form of capsules, hard capsules can be prepared by filling conventional hard capsules with a mixture of the copricin peptide CopA3 of the present invention and additives such as excipients, or their granules or coated granules, and soft capsules The formulation can be prepared by filling a mixture with additives such as copricin peptide CopA3 and excipients in a capsule base such as gelatin. The soft capsule formulation may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The health functional food in the form of a ring can be prepared by molding a mixture of the copricin peptide CopA3 of the present invention, excipients, binders, disintegrants, etc. by an appropriate method. The gown may be made of talc or a suitable material.

The health functional food in the form of granules can be prepared in a granular form by a suitable method of a mixture of the copricin peptide CopA3 of the present invention, excipients, binders, disintegrants, etc., and may contain flavoring agents, flavoring agents, etc. as necessary. For health functional foods in granular form, use No. 12 (1680 μm), No. 14 (1410 μm), and No. 45 (350 μm) sieves to pass all the sieves in the next particle size test, and the remaining amount in No. 14 sieves 5.0% or less and passing through No. 45 may be less than 15.0% of the total amount.

The term definitions for the excipients, binders, disintegrants, lubricants, flavoring agents, flavoring agents, etc. are described in documents known in the art and include those having the same or similar functions (Explanation of the Korean Pharmacopoeia, Moonseongsa, Korea) Association of Colleges of Pharmacy, 5th ed., p33-48, 1989).

There is no particular limitation on the type of the food. Examples of foods to which the copricin peptide CopA3 of the present invention can be added include beverages, gums, vitamin complexes, drinks, and the like, and include all health functional foods in a conventional sense.

The food composition may contain food supplement additives, and the food products include, for example, beverages, gum, tea, vitamin complexes, health supplements, etc., capsules, tablets, powders, granules, liquids, pills, slices, pastes. It can be used in the form of phases, syrups, gels, beverages, jellies or vines.

In the present invention, the content of copricin peptide CopA3 may be contained in an amount of 0.01 to 15% by weight based on the total weight of the health functional food, and in the case of a health beverage composition, 0.02 to 10 g, preferably based on 100 g may be contained in an amount of 0.3 to 1 g.

In addition, the food supplement additives include food additives conventional in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. In addition, the health drink composition is an essential ingredient in the indicated ratio, and there is no particular limitation on the ingredients added other than the copricin peptide CopA3, and it may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. . Examples of the natural carbohydrate include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; polysaccharides such as dextrins, cyclodextrins; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. there is.

In addition to the above, food compositions or health functional foods containing the copricin peptide CopA3 are various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.) , pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

In addition, the present invention provides a feed composition for preventing or improving ischemic myocardial infarction comprising the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1.

In the present invention, the feed may be a feed for fish, birds or mammals, and preferably, it is suitable for breeding as its wild habits are acclimatized, as defined in Article 2 No. 1 of the Livestock Act and Article 2 of the Enforcement Rule of the same Act. and may be feed for livestock or aquatic organisms that can contribute to the increase of farm household income. The livestock may include cattle, horses, mules, donkeys, goats, goats, sheep, deer, pigs, rabbits, poultry, etc., and the poultry includes chickens, turkeys, ducks, ostriches, geese, quails, etc., preferably chickens, If it is suitable for breeding and obtaining livestock products, it is not limited thereto. The above "livestock products" are meat, milk, eggs, honey and their processed products, raw hides (including raw fur), raw wool, and other livestock products, which are defined by Article 2, 3 of the Livestock Act, as determined by the Ordinance of the Ministry of Agriculture and Forestry. means that In addition, it may be a dog, a cat, etc., including companion animals, but is not limited thereto.

The term "feed" in the present invention means any natural or artificial diet, meal, etc., or a component of said meal, intended for or suitable for being eaten, consumed, and digested by an animal. In one aspect, the composition for feed containing the copricin peptide CopA3 of the present invention may include a concentrated feed, roughage and/or special feed.

In the enriched feed, seed fruits containing grains such as wheat, oats, and corn, bran containing rice bran, bran, barley bran, etc. Fish-soluble, meat powder, which is a concentrated product of fish meal, fish waste, and fresh liquids obtained from fish meal, fish waste, and residual starch, which is the main component of starch residue, which is the remainder of starch residue obtained by subtracting starch from sweet potatoes and potatoes. , animal feed such as dried whey, yeast, chlorella, seaweed, etc., which are dried whey, which is the remainder of the production of casein from milk, cheese, and casein from skim milk.

For roughage, raw grass feed such as wild grasses, grasses, and green cuttings, turnips for feed, beets for feed, root vegetables such as ruther beargers, a type of turnip, raw herbs, green crops, grains, etc. are placed in a silo. There are silage, which is stored feed fermented with lactic acid, wild grass, hay dried after cutting grass, straw for breeding crops, and leaves from legumes. Special feed includes mineral feed such as oyster shells and rock salt, urea feed such as urea or its derivative diureide isobutane, and supplementation of ingredients that are likely to be lacking when only natural feed ingredients are mixed, or added to formulated feed to increase feed storage. There are feed additives, which are substances added in trace amounts.

The copricin peptide CopA3 of the present invention inhibits caspase activity by covalently binding to caspase and inhibiting cleavage of the active type of caspase, and in particular, inhibits the activity of caspase-3 and caspase-6 to induce apoptosis and cell death. It has the effect of inhibiting pyroptosis and necrosis. Due to this effect, it can be used as a preventive or therapeutic agent for ischemic myocardial infarction caused by caspase activity, as a health food supplement, and as a feed composition.

1 is a result of analyzing caspase protein expression level change and electrophoretic phase change by copricin peptide CopA3 treatment according to an embodiment of the present invention.
Figure 2 is the result of confirming the binding relationship between the copricin peptide CopA3 and the caspase protein according to an embodiment of the present invention through electrophoretic phase change and surface plasmon resonance (SPR, Surface Plasmon Resonance) analysis.
3 shows the results of analyzing the effect of inhibiting caspase enzyme activity by binding the copricin peptide CopA3 to caspase protein and blocking the caspase protein cleavage process according to an embodiment of the present invention.
4 shows the results of analyzing the caspase enzyme activity inhibitory effect of the copricin peptide CopA3 in various cells according to an embodiment of the present invention.

Hereinafter, preferred examples are presented to help the understanding of the present invention, but the following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, It goes without saying that such variations and modifications fall within the scope of the appended claims.

Example One: copricin peptide derivative CopA3 Produce ( copricin peptide derivative CopA3 synthesis)

CopA3, a copricin peptide derivative having the amino acid sequence of SEQ ID NO: 1 (LLCIALRKK-NH ₂ ) was requested by AnyGen (Gwang-ju, Korea) and synthesized with a purity of 98% or more, and was used by dissolving in 0.01% acetic acid.

Example 2: copricin peptide CopA3 by caspase changes in protein expression and electrophoresis Through phase change analysis copricin peptide CopA3 and caspase Confirmation of protein binding

To determine whether the copricin peptide CopA3 derived from Arabidopsis binds to caspase, a key enzyme for apoptosis, human colonic epithelial cells (HT29) were cultured and then treated with CopA3 to express caspase protein. The amount change and the electrophoretic phase change were confirmed, and the results are shown in FIG. 1 .

As can be seen in FIG. 1A , it was confirmed that the treatment with the copricin peptide CopA3 caused a significant shift in the SDS-PAGE electrophoresis phase shift of the caspase protein in a concentration-dependent manner. Although most caspases were shifted by the treatment of copricin peptide CopA3, it was confirmed that, in particular, caspase-3 and caspase-6 proteins were shifted the most. And other caspases were confirmed to be shifted only in the high concentration copricin peptide CopA3. Therefore, it was confirmed that among the caspases, caspase-3 and caspase-6 showed the most sensitive molecular weight change upon stimulation of the copricin peptide CopA3. In addition, the shift phenomenon in the SDS-PAGE electrophoresis phase transfer process shows the possibility that the molecular weight increased due to the binding of the CopA3 peptide and caspase.

In addition, after synthesizing the recombinant proteins GST-caspase-3 and GST-caspase-6 in bacteria, an in vitro binding test was performed with the copricin peptide CopA3 of the present invention, and the results are shown in B, C, and D of FIG. .

First, there was no change in the electrophoretic phase shift of the GST protein even when the copricin peptide CopA3 was treated, indicating that CopA3 does not bind to the GST protein (FIG. 1B). However, when the copricin peptide CopA3 was mixed with the GST-caspase-3 protein for 30 minutes and then subjected to SDS-PAGE electrophoresis, it was confirmed that the GST-caspase-3 protein was shifted upward ( FIG. 3C ). The phenomenon of protein shift on SDS-PAGE electrophoresis means an increase in molecular weight, and this result indicates that caspase-3 protein binds with CopA3 to increase molecular weight, and thus the electrophoretic phase shift is different. The same phenomenon was also observed when CopA3 and GST-caspase-6 protein were reacted (FIG. 1D). The binding of caspase and CopA3 did not dissociate even during SDS-PAGE electrophoresis, which leads to protein denaturation, indicating that the two substances strongly interact with each other through a covalent bond.

In addition, it was also confirmed that CopA3 binds to the large subunit and small subunit, which are fragments of cleaved caspase, respectively, and the results are as shown in FIG. When converted, the caspase protein is cleaved into two fragments, one called the large subunit and the other called the small subunit).

As can be seen in FIG. 1E , when the purified large subunit and small subunit of caspase-3 were reacted with CopA3 and then electrophoresed, the large subunit and small subunit proteins were shifted upward in the electrophoretic phase. This result appears because CopA3 binds to increase the molecular weight of each. In addition, as can be seen in FIG. 1F , CopA3 treatment induced changes in the large subunit and small subunit mobile phases of caspase-6.

These results indicate that the copricin peptide CopA3 of the present invention binds to caspase-3 and caspase-6 proteins, which are apoptosis inducing enzymes in cells, and CopA3 binds to both the cleaved fragment proteins (large subunit and small subunit).

Example 3: copricin peptide with CopA3 caspase Analysis of protein binding relationships

In order to confirm the binding relationship between the copricin peptide CopA3 and the caspase protein of the present invention, amino acids that are essential in binding to caspase among amino acids constituting the copricin peptide CopA3 were identified, and the results are shown in FIG. 2 . .

Previously, in Example 2, it was confirmed that caspase and the coprisine peptide CopA3 interact by a covalent bond, and the fact that the coprisine peptide CopA3 is the only amino acid cysteine capable of forming a covalent bond was confirmed. Based on this, we prepared mutant CopA3 (CopA3-CS mutant) by substituting a serine amino acid for cysteine present in CopA3. And after treating this mutant CopA3, to check whether the electrophoretic change of caspase is maintained, recombinant proteins GST-caspase-3 and GST-caspase-6 were mixed with CopA3 and CopA3-CS mutant, respectively, and then electrophoresed. The migration phase shift change was tracked, and the result is as shown in A of FIG. 2 .

As can be seen in FIG. 2A , the copricin peptide CopA3 was shown to strongly induce an electrophoretic shift of caspase-3 and caspase-6, but CopA3-CS mutant treatment was performed with caspase-3 and caspase-6 It did not cause a shift in the electrophoretic phase of the protein. These results show that the cysteine amino acid present in the copricin peptide CopA3 plays a key role in binding to caspase.

In addition, the binding of the copricin peptide CopA3 to caspase was re-verified by a surface plasmon resonance (SPR) method. Specifically, for the SPR measurement experiment, a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, and 5 mM dithiothreitol (DTT) was used, and the experiment was performed at a temperature of 25 °C. . First, the separated and purified recombinant proteins GST-caspase-3 and GST-caspase-6 were attached to CM5 sensor chips, and then the solution was flowed at a rate of 10 ml/min. At this time, CopA3 and CopA3-CS mutant were treated at different concentrations, and then the solution was flowed on the CM5 sensor chip, and through this, the binding force between CopA3 and caspase protein was quantified, and the result is shown in FIG. 2B.

2900 mM)과 GST-capase-6에 결합하지 못하는 것으로 나타났으며, CopA3를 GST단백질에 처치했을 때에도 유의한 결합력을 보이지 않는 것으로 나타났다(K _D

243.56 mM). As can be seen in FIG. 2B , it was confirmed that CopA3 bound to caspase-3 with a 22.9 K _D value, and it was also confirmed that CopA3 binds to caspase-6 with a 801 K _D value. These results show that CopA3 strongly binds to GST-caspase-3 and GST-caspase-6, and in particular, CopA3 shows a higher binding affinity to caspase-6 than caspase-3. On the other hand, CopA3-CS mutant treatment was GST-caspase-3 ( K _D

2900 mM) and GST-capase-6 did not bind, and CopA3 did not show significant binding force even when GST protein was treated ( K _D

243.56 mM).

These results indicate that the thiol group of cysteine present on the copricin peptide CopA3 strongly binds to each other through a disulfide bond with the thiol group of cysteine present on caspase. It was confirmed that the number of amino acids was higher in caspase-3 (caspase-3: 8, caspase-6: 10).

Example 4: copricin peptide with CopA3 caspase by protein binding caspase Analysis of the effect of blocking the active cleavage process

Caspase enzyme proteins must be cleaved by other proteases to be activated. Accordingly, it was confirmed whether this active cleavage process is blocked when the copricin peptide CopA3 binds to caspase. In this regard, caspase-8 is known to cleave and activate caspase-3, and caspase-3 is known to cleave and activate caspase-6. Under the above conditions, cleavage of the copricin peptide CopA3 of the present invention is blocked. In order to confirm the efficacy, GST-caspase-3 was cleaved by adding active caspase-8 to GST-caspase-3, and the results are as shown in FIG. 3 .

As can be seen in FIG. 3A , active caspase-8 cleaves the GST-caspase-3 protein to form a fragment protein, and this fragment is a large subunit fragment, which is active caspase-3. As can be seen in FIG. 3C , it was found that when DEVD, which is a substrate, was added to the cleaved caspase-3, an active type of caspase that cleaves the substrate is formed. However, GST-caspase-3 was first reacted with the copricin peptide CopA3 for 30 minutes to induce binding, and then, when active caspase-8 was added, cleavage was blocked (FIG. 3A), and the enzymatic activity of caspase was significantly reduced. It was shown to block (FIG. 3C).

In addition, this inhibitory effect of CopA3 was also confirmed in caspase-6. Active caspase-3 cleaves GST-caspase-6 to form an active fragment (FIG. 3B), and it was confirmed that the cleaved caspase-6 activity of cleaving the substrate DEID is enhanced (FIG. 3D) ). On the other hand, when GST-caspase-6 was first bound to CopA3, cleavage blocking (FIG. 3C) and caspase activity were significantly reduced (FIG. 3D) even if active caspase-3 was added.

These results indicate that when the copricin peptide CopA3 binds to caspase having an inactive structure, it strongly blocks subsequent cleavage of the active protein.

Example 5: variety in the cell copricin peptide with CopA3 caspase by protein binding caspase Analysis of the effect of blocking the active cleavage process

Previously, in Example 4, it was confirmed that the copricin peptide CopA3 of the present invention strongly binds to the caspase enzyme, thereby inhibiting the active type of caspase-protein cleavage. Therefore, it was checked whether this phenomenon appeared in various cells as it is, and the value as a drug applicable to various diseases was evaluated.

Specifically, HT29 (human colorectal adenocarcinoma), SH-SY5Y (human neuroblastoma), HepG2 (human hepatoma), HeLa (human cervical cancer), Caki (human renal carcinoma), SW13 (human adrenal carcinoma), 3T3L1 (mouse adipocytes) , RAW 264.7 (mouse macrophages), NIH/3T3 (mouse fibroblasts), PC12 (rat phaeochromocytoma), and 3Y1 (rat fibroblasts) cells were used. All cells were prepared with the same number of cells (10 ⁵ cells/well), and then treated with CopA3 at a concentration of 50 mg/ml, proteins were separated, and then SDS-PAGE electrophoresis (15% gels) was performed to electrophoresis of caspases. Changes in migration phase were confirmed, and the results are as shown in FIG. 4 .

First, as can be seen in FIG. 4A , when CopA3 was treated, the electrophoretic phase shift of caspase-3 and caspase-6 proteins was found to be upward in the entire human cell line used in the experiment.

In addition, as can be seen in FIG. 4B , shifts of caspase-3 and caspase-6 due to CopA3 treatment were observed in all mouse and rat cell lines. However, in consideration of the characteristics of the peptide drug, when the appropriate concentration of 50 mg/ml CopA3 was treated, the shift of other caspases did not appear.

As a result, it was confirmed that the main target molecules regulated by the copricin peptide CopA3 of the present invention were caspase-3 and caspase-6 among caspases.

From the above results, the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1 according to the present invention has an effect of inhibiting the activity of caspase by binding to caspase protein, in particular, of caspase-3 and caspase-6 As it was confirmed that there is an effect of inhibiting activity, it was confirmed that there is an effect of inhibiting apoptosis, apoptosis, and necrosis. It was confirmed that the copricin peptide CopA3 of the present invention is effective in inhibiting ischemic myocardial infarction mediated by caspase. .

<110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) Daejin University Center for Educational Industrial Cooperation <120> Composition for inhibiting Caspase comprising coprisin peptide CopA derived from Copris tripartitus <130> nsh1141 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CopA3 <400> 1 Leu Leu Cys Ile Ala Leu Arg Lys Lys 1 5

Claims (14)

A reagent composition for inhibiting caspase comprising a copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1.
SEQ ID NO: 1: LLCIALRKK-NH ₂ According to claim 1,
The copricin peptide CopA3 binds to caspase and blocks the transfer process of caspase, thereby inhibiting caspase activity. According to claim 1,
The binding is that the coprisine peptide CopA3 binds to caspase through a disulfide bond through cysteine, caspase inhibitory reagent composition. According to claim 1,
The caspase inhibition is to inhibit caspase-3 and caspase-6, a reagent composition for inhibiting caspase. A method of inhibiting caspase activity in vitro , comprising the step of treating cells with the copricin peptide CopA3 having the amino acid sequence of SEQ ID NO: 1.
SEQ ID NO: 1: LLCIALRKK-NH ₂ 6. The method of claim 5,
The cells include cancer cells, nerve cells, adipocytes, macrophages, fibroblasts, pancreatic islet cells, stem cells, hepatocytes, fibroblasts, lymphocytes, vascular endothelial cells, enamel cells, keratinocytes, osteoblasts, ciliate cells, dendritic cells, Which is selected from the group consisting of egg cells and renal muscle cells. A pharmaceutical composition for preventing or treating ischemic myocardial infarction comprising a copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1.
SEQ ID NO: 1: LLCIALRKK-NH ₂ 8. The method of claim 7,
The copricin peptide CopA3 is Arabidopsis dung beetle ( Copris tripartitus ), which is isolated from, ischemic myocardial infarction preventing or treating pharmaceutical composition. 8. The method of claim 7,
The copricin peptide CopA3 is a pharmaceutical composition for preventing or treating ischemic myocardial infarction, which inhibits caspase activity. 8. The method of claim 7,
The copricin peptide CopA3 is a pharmaceutical composition for preventing or treating ischemic myocardial infarction, which inhibits the activity of caspase by directly binding to caspase and inhibiting cleavage of the active form of caspase. 8. The method of claim 7,
The ischemic myocardial infarction is a caspase-mediated ischemic myocardial infarction induced by caspase activity, a pharmaceutical composition for preventing or treating ischemic myocardial infarction. A food composition for preventing or improving ischemic myocardial infarction comprising a copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1.
SEQ ID NO: 1: LLCIALRKK-NH ₂ A feed composition for preventing or improving ischemic myocardial infarction comprising a copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1.
SEQ ID NO: 1: LLCIALRKK-NH ₂ A health functional food for preventing or improving ischemic myocardial infarction comprising a copricin peptide CopA3 (9-mer disulfide dimer) having the amino acid sequence of SEQ ID NO: 1.
SEQ ID NO: 1: LLCIALRKK-NH ₂

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