Novel Peptides and Their Uses

Abstract

The present invention relates to a novel peptide and its use, and more specifically, to a peptide having a length of 12 to 20 amino acids containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 1, which has the ability to suppress the formation of abnormal protein aggregates. Among the amino acids excluding glycine, 1 to 4 amino acids are L-amino acids and the remaining amino acids are D-amino acids, and provides a novel peptide and its use.

Description

Technical Field

[0001] The present invention relates to a novel peptide and its use, and more specifically, to a novel peptide based on allatostatin and its use in the treatment of novel neurodegenerative diseases.

Background Art

[0002] Zinc is a trace element essential for cell growth and differentiation and is known as a cofactor involved in the functions and structures of proteins such as enzymes and transcription factors. Also, the homeostasis of zinc in nerve cells is known to play an important role in the survival of nerve cells. However, when zinc is deficient in nerve cells, apoptosis is induced, leading to neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (Lien, H. et al., BBRC. 268: 148 - 154, 2000). Conversely, when zinc is present in excess in nerve cells, cell damage is induced, which is known to trigger acute brain injury diseases such as ischemia and seizure (Koh et al., Science. 272: 1013 - 1016, 1996).

[0003] On the one hand, autophagy is an intracellular mechanism that decomposes organelles in a starvation situation to obtain an energy source or removes damaged organelles and abnormal or pathological protein aggregates. During autophagy, the cytoplasmic components are surrounded by a double membrane and isolated from other organelles to form an autophagosome. At this time, the water-soluble light chain I (LC3I) in the cytoplasm is converted into the form of light chain II (LC3II) attached to the autophagosome cell membrane. Subsequently, the autophagosome fuses with the lysosome to form an autolysosome, and is decomposed or recycled by various hydrolases present in the lysosome.

[0004] Recently, it has been known that protein aggregates represented by degenerative neurological diseases such as α-synuclein, β-amyloid, Tau protein, superoxide dismutase-1 (SOD-1), Huntingtin protein, and TAR DNA-binding protein 43 (TDP-43) can be removed when autophagy is promoted. In fact, when there is a defect in the function of autolysosomes, the degradation of such protein aggregates is inhibited and the autophagy flux is blocked. Eventually, there are research results showing that the absence of such autophagy leads to the accumulation of by-products of degenerative neurological diseases (Zhang et al., ABBS. 41(6):437-445, 2009; Lee et al., Cell. 141(7):1146-1158, 2010).

[0005] Previous studies have revealed the fact that supplying zinc can improve the function of lysosomes, and H 2 O 2, When treating tamoxifen, a zinc chelator TPEN that is potent in ethanol-induced lysosomal membrane permeabilization (LMP), the phenomenon of lysosomal membrane disruption is suppressed. Conversely, when the autophagic effect is decreased, there are research results indicating that if zinc is additionally supplied, the autophagic effect is promoted (Lee et al., Glia 57:1351-1361, 2009; Hwang et al., Biometals 23:997-1013, 2010; Liuzzi et al., Biol. Trace Elem. Res. 156:350-356, 2013; Kim et al., Front. Cell. Neurosci. 16:895750, 2022). Therefore, zinc is presumed to have an important impact on autophagy.

[0006] However, drugs that can treat neurodegenerative diseases by maintaining zinc homeostasis in nerve cells have not yet been developed.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention aims to solve various problems including the above-mentioned problems. By maintaining zinc homeostasis in nerve cells, abnormal or pathological protein aggregates such as amyloid-β peptide, tau protein, and superoxide dismutase, which are pathological substances of neurodegenerative diseases, are removed through autophagy or improvement of lysosomal function, thereby providing a novel peptide capable of treating neurodegenerative diseases and its use related to the treatment of neurodegenerative diseases.

Means for Solving the Problems

[0008] According to one aspect of the present invention, in a peptide having a length of 12 to 20 amino acids (a.a.) and containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 1, which has the ability to suppress the formation of abnormal protein aggregates, among the amino acids excluding glycine, 1 to 4 amino acids are L-amino acids and the remaining amino acids are D-amino acids, a novel peptide is provided.

[0009] According to another aspect of the present invention, a pharmaceutical composition for treating degenerative neurological diseases containing the peptide as an active ingredient is provided.

[0010] According to another aspect of the present invention, a method for treating an individual including the step of administering a therapeutically effective amount of the peptide to an individual suffering from a degenerative neurological disease is provided.

[0011] According to another aspect of the present invention, a method for suppressing the accumulation of pathogenic protein aggregates in the nervous system of an individual including the step of administering a therapeutically effective amount of the peptide to an individual suffering from a degenerative neurological disease is provided.

Effects of the Invention

[0012] The novel peptide of the present invention made as described above can be utilized for the development of therapeutic agents for effectively treating degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease by preventing cell death of nerve cells and regulating intracellular zinc homeostasis. Of course, the scope of the present invention is not limited by such effects.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0026] Definition of Terms:

[0027] As used herein, the term "zinc homeostasis" means the mechanism for maintaining the concentration of zinc in cells at a certain level. It is known that zinc transporters, zinc-binding proteins (metallothioneins, MTs), transcription factors (MTF1-2), etc. are involved in maintaining the concentration of zinc in cells at a certain level.

[0028] As used herein, the term "Neurodegenerative disease" refers to a disease characterized by a progressive loss of nerve structure or function due to abnormal death of nerve cells. Such neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and the like.

[0029] As used herein, the term "abnormal or pathological protein aggregates" means that proteins such as amyloid or tau are abnormally aggregated intracellularly to form insoluble fibrils. Such abnormal or pathological protein aggregates are known as neuropathological features of various intermittent or hereditary neurodegenerative diseases.

[0030] As used herein, the term "alloferon" is a natural peptide composed of 13 amino acids isolated from the larval blood of the blowfly (Calliphora vicina) infected with bacteria, and is known as a non-toxic antiviral agent developed for treating infections such as influenza virus and herpes virus (Republic of Korea Patent No. 394864).

[0031] Detailed Description of the Invention:

[0032] According to one aspect of the present invention, there is provided a novel peptide having a length of 12 to 20 a.a. and containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, which has the ability to inhibit the formation of abnormal protein aggregates, wherein among the amino acids excluding glycine, 1 to 4 amino acids are L-amino acids and the remaining amino acids are D-amino acids.

[0033] Among the amino acids of the peptide with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, one, two, three, or four amino acids excluding glycine are L-amino acids, and the remaining amino acids are D-amino acids.

[0034] More specifically, when the peptide contains the amino acid sequence shown in SEQ ID NO: 1, it may contain at least one of the following: i) At least one of the four histidines is a D-amino acid; ii) At least one of the valines at positions 3 and 11 is an L-amino acid, or both are D-amino acids; iii) The serine at position 4 is a D-amino acid; iv) The glutamine at position 8 is a D-amino acid; and v) Among i) to iv) above, two or more are D-amino acids.

[0035] When the peptide contains the amino acid sequence shown in SEQ ID NO: 2, it may contain at least one of the following: i) At least one of the four histidines is a D-amino acid; ii) At least one of the valines at positions 3 and 10 is an L-amino acid, or both are D-amino acids; iii) The serine at position 4 is a D-amino acid; and iv) At least two of i) to iii) are D-amino acids.

[0036] According to a preferred embodiment, in the peptide of the present invention, at least 3 out of 4 histidines in the L-type allopheron peptide of SEQ ID NO: 1 are replaced with D-type histidines, and at least 2 out of 2 valines, serine which is the 3rd amino acid, and glutamine which is the 8th amino acid are D-type amino acids, or all of the 4 histidines are D-type amino acids, and glutamine which is the 8th amino acid is an L-type or D-type amino acid or deleted. Desirably, the peptide according to an embodiment of the present invention can contain any one of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 14, and more desirably, can contain any one of the amino acid sequences of SEQ ID NO: 3 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12 to SEQ ID NO: 14.

[0037] As can be confirmed from FIG. 1, the inventors found that although the L-form allopheron that naturally exists in the body showed excellent activity under in vitro conditions, when administered in vivo, it has characteristics that are difficult to develop as an actual medicine due to its extremely short half-life (T1 / 2 = 51.77 minutes). Thus, the inventors hypothesized that since the allopheron peptide of the present invention not only interacts with specific proteins in vivo to exhibit physiological activity but also acts as a zinc-binding protein to supply extracellular zinc into cells as a kind of zinc homeostasis maintainer, it would be workable even if it is produced in the D-form that does not naturally exist in the body and is not an L-form peptide that is easily decomposed in vivo. As a result of manufacturing D-form allopheron and conducting the same experiments as those using L-form allopheron, it was confirmed that D-form allopheron also has the same biological activity as L-form allopheron. Moreover, as a result of additional research on whether similar functions would be exhibited even if some of the amino acids of the D-form allopheron peptide were replaced with L-form again, when one of the histidines expected to perform the function of zinc chelation was replaced with an L-form amino acid, or when the third amino acid valine and / or the fourth amino acid serine were each replaced with an L-form amino acid, or when the eighth amino acid glutamine and / or the eleventh amino acid valine were each replaced with an L-form amino acid, it was confirmed that the biological activity was equivalent to or even more excellent than that of D-form allopheron, thereby completing the present invention. Moreover, it was confirmed that in the case of a mutant in which the eighth amino acid glutamine was removed, the most excellent biological activity was exhibited.

[0038] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating degenerative neurological diseases containing the peptide as an active ingredient.

[0039] In the pharmaceutical composition, the neurodegenerative disease is a neurodegenerative disease having the formation of abnormal protein aggregates as a cause or pathological phenomenon, and the abnormal protein aggregates are formed by abnormal aggregation of α-synuclein, β-amyloid, TDP-34, p62, FUS protein, superoxide dismutase-1 (SOD-1), huntingtin protein or tau protein. The neurodegenerative brain diseases having the formation of abnormal protein aggregates as a cause or pathological phenomenon are specifically Alzheimer's disease (AD) associated with the accumulation of β-amyloid protein or tau protein aggregates, Parkinson's disease (PD) associated with the accumulation of α-synuclein aggregates, amyotrophic lateral sclerosis (ALS) associated with the accumulation of SOD-1 aggregates, Huntington's disease (HD) associated with the accumulation of huntingtin protein aggregates, chronic traumatic encephalopathy associated with the accumulation of tau protein or TAR DNA-binding protein 43 (TDP-43) aggregates, Lytico-bodig disease associated with the accumulation of tau protein aggregates, frontotemporal lobe degeneration associated with the accumulation of tau protein, TAR DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS) protein or p62 protein aggregates, corticobasal degeneration associated with the accumulation of tau protein aggregates, or progressive supranuclear palsy associated with the accumulation of tau protein aggregates.In addition, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, meningioangiomatosis, neuronal ceroid lipofuscinoses, etc. are also known as diseases correlated with the accumulation of protein aggregates in the nerves. The correlation between the neurodegenerative diseases and the accumulation of protein aggregates in the nerves is well described in the prior literature (Strφmland et al., J.Clin.Transl.Res. 2(1):11-26, 2016; Tutar et al., Neurodegenerative Diseases, published: May 15). th , 2013, DOI:10.5772 / 54487; Diez-Ardanuy et al., Sci.Rep., 7(1):10, 2017).

[0040] In the composition, the peptide can prevent cell death of nerve cells, regulate intracellular zinc homeostasis, and promote lysosomal function, thereby treating the neurodegenerative disease.

[0041] The pharmaceutical composition according to an embodiment of the present invention can include a pharmaceutically acceptable carrier, and may further include a pharmaceutically acceptable adjuvant, excipient or diluent in addition to the carrier.

[0042] As used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause normal gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. It may also further contain fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives, etc.

[0043] Also, the pharmaceutical composition according to an embodiment of the present invention is formulated using methods known to those skilled in the art to enable rapid release, or sustained or delayed release of the active ingredient when administered to a mammal. Dosage forms include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, and sterile powders.

[0044] The pharmaceutical composition according to an embodiment of the present invention is administered via various routes, for example, orally, parenterally, such as by suppository, transdermally, intravenously, intraperitoneally, intramuscularly, intralesionally, nasally, intraspinally, and also using an implant device for sustained release or continuous or repeated release. The number of administrations can be once a day or divided into several times within the desired range, and the administration period is also not particularly limited.

[0045] The pharmaceutical composition according to an embodiment of the present invention is administered in a general systemic or local administration manner, such as by intramuscular injection or intravenous injection. Moreover, the peptide according to an embodiment of the present invention can also be administered orally.

[0046] The pharmaceutical composition according to an embodiment of the present invention is formulated into a suitable form together with a generally used pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose, and glycols, and may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Further, the composition according to the present invention may appropriately contain a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, an antioxidant, etc., if necessary depending on the administration method and dosage form. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington’s Pharmaceutical Sciences, latest edition].

[0047] The dosage of the pharmaceutical composition according to an embodiment of the present invention for a patient varies depending on many factors including the patient's height, body surface area, age, the specific compound administered, gender, administration time and route, general health, and other drugs administered simultaneously. The therapeutically active allopheron or the polynucleotide encoding it is administered in an amount of 100 ng / kg (body weight) to 1000 mg / kg (body weight), more preferably 1 μg / kg (body weight) to 100 mg / kg (body weight), and most preferably 5 to 40 mg / kg (body weight), but the dosage is adjusted in consideration of the above factors.

[0048] Further, the pharmaceutical composition of the present invention is administered in a therapeutically effective amount.

[0049] As used herein, the term "therapeutically effective amount" means an amount sufficient for the treatment of a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined by factors including the type and severity of the individual, age, gender, activity of the drug, sensitivity to the drug, administration time, administration route and excretion ratio, treatment period, factors including co-administered drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention is administered in a volume of 0.1 mg / kg to 1 g / kg, and more preferably in a dosage of 1 to 500 mg / kg. On the other hand, the said dosage is appropriately adjusted according to the age, gender and condition of the patient.

[0050] According to another aspect of the present invention, there is provided a method of treating an individual comprising the step of administering a therapeutically effective amount of said peptide to an individual suffering from a degenerative neurological disease.

[0051] According to another aspect of the present invention, there is provided a method of suppressing the accumulation of pathogenic protein aggregates in the nervous system of an individual comprising the step of administering a therapeutically effective amount of said peptide to an individual suffering from a degenerative neurological disease.

[0052] In the said method, the said composition is administered by oral or parenteral administration. In the case of parenteral administration, it can be administered through any route of systemic administration or local administration. In the case of systemic administration, intravenous injection, intraperitoneal injection, or intramuscular injection is possible. In the case of local administration, intracranial administration, intracerebrospinal administration, subcutaneous injection, etc. are possible.

[0053] As used herein, the term "therapeutically effective amount" means a dosage that exhibits a palliative, inhibitory, ameliorative and / or curative effect on the symptoms of the disease to be treated.

[0054] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, and can be embodied in various different forms. The following examples are provided to complete the disclosure of the present invention and to fully inform those skilled in the art of the scope of the invention.

[0055] Example 1: Peptide Preparation 1-1: Synthesis of L-Type Peptide The L-type peptide (SEQ ID NO: 16, LAL) of allomone (SEQ ID NO: 1) used in the present invention was synthesized by commissioning Peptron Inc. (Republic of Korea).

[0056] 1-2: Synthesis of D-Type Peptide Instead of the L-type allomone used in the present invention, a D-type allomone peptide (SEQ ID NO: 15, DAL1) in which all amino acids except glycine are replaced with D-type amino acids was also synthesized by commissioning Peptron Inc. (Republic of Korea) and Enigen Inc. (Republic of Korea).

[0057] Experimental Example 1: Analysis of the Stability of Blood Plasma in Vitro The inventors considered that the mechanism of action of allopheron peptides acts through the endocytosis influx process via a multi-ligand receptor rather than physiological activity, and that the zinc-binding ability acts as an important mechanism. They investigated the stability in blood of the D-type allopheron of Examples 1-2, in which all amino acids except glycine of allopheron were replaced with D-type amino acids, and the L-type allopheron of Example 1-1. For comparison of the stability of allopheron L-type peptide and D-type peptide in mouse serum, in vitro serum stability analysis was performed. For this purpose, after obtaining 500-600 μl of blood through orbital blood collection in ICR rats, centrifugation was carried out at 2000 xg for 20 minutes at 4°C using a centrifuge, and then only the supernatant was taken to obtain serum. After adding 10 μM each of allopheron L-type and D-type peptides to 100 μl of serum, it was left at 37°C for 0, 5, 10, 30, 60, and 90 minutes. Next, 500 μl of cold methanol was added to precipitate the protein, and after freeze-drying the protein, liquid chromatography and protein mass spectrometry were carried out. As a result, it was found that the allopheron L-type peptide was decomposed from an early time in serum, while the D-type peptide was not decomposed over time and remained stable (Figure 1).

[0058] 1-3: Design and Synthesis of Mutant Alloferon Peptide From the results of Experimental Example 1 above, the inventors investigated whether a mutant peptide in an L-type / D-type hybrid form, in which some amino acids are L-type amino acids, exhibits equivalent biological activity instead of the D-type peptide (DAL1) in which all amino acids except glycine in allopheron were replaced with D-type amino acids. For this purpose, 12 mutant allopheron peptides in which 1 to 4 amino acids are L-type amino acids were synthesized (Table 1).

[0059]

Table 1

[0060] The mutant peptides consisted of peptides in which one of the four histidines predicted to be involved in zinc chelation had been reduced to the L-type (DALH1, DALH2, DALH3, DALH4), peptides in which at least one of the third, fourth, and eleventh amino acids had been reduced to the L-type amino acid (DALVS, DALV2, DALV1, DALS1), peptides in which the eighth amino acid, glutamine, had been reduced to the L-type amino acid (DALQ1), and peptides in which at least three of the two valines, glutamines, and serine amino acids had been reduced to the L-type amino acids (DALVSQV, DALVSqV, and DALVS_V).

[0061] Peptide synthesis was outsourced to Peptron Corporation (Republic of Korea) and Anygen (Republic of Korea).

[0062] Experimental Example 2: Cell Culture In the present invention, H4 cell line (GL-H4) and HEK293T cell line permanently infected with GFP-LC3 plasmid were used. The cell culture medium was Minimum Essential Medium (MEM, WellGene) supplemented with 10% fetal bovine serum (Hyclone, USA) and a mixed solution of antibiotics and antimycotics (WellGene, Republic of Korea), and incubated at 37°C and 5% CO. 2 The cells were cultured in a cell incubator where the conditions were maintained.

[0063] Experimental Example 3: Analysis of the Autophagic Flux Blocked by Bafilomycin (A-1) In order to investigate the effect of peptides consisting of the amino acid sequences shown in SEQ ID NO: 3 to SEQ ID NO: 16 on cell cycle function, the inventors inhibited autophagy in an H4 cell line (GL-H4) expressing LC3 linked to GFP using the peptides (20 μM) and / or the autophagy inhibitor bafilomycin (A-1, Baf A1, 100 nM), and then investigated the effect of a mutant alloferon peptide according to one embodiment of the present invention by fluorescence microscopy.

[0064] As a result, the LC3 spots shown in Figures 2A to 2C are a phenomenon caused by the LC3-GFP protein present in the autophagosome, and if they are not degraded by lysosomes, the entire process of autophagic action is inhibited and autophagic action is accumulated. When the GL-H4 cell line was treated with 100 nM bafilomycin (Baf A1) to inhibit the autophagy process, the number and size of the LC3 spots increased significantly, but this phenomenon was reduced by treatment with the mutant alloferon peptide (20 μM) according to one embodiment of the present invention (Figures 2A to 2C). When the fluorescence intensity of the entire spots was measured and quantified, the fluorescence intensity was also reduced by treatment with the mutant alloferon peptide according to one embodiment of the present invention (Figures 2D to 2F). This means that the autophagic action accumulated due to the blocked autophagic action is eliminated by the mutant alloferon peptide according to one embodiment of the present invention. In particular, as shown in Figures 2E and 2F, when at least one to three of the two amino acids, valine, serine, and glutamine, of the mutant alloferon peptides according to one embodiment of the present invention are reduced to L-amino acids (DALV1, DALS1, DALV2, DALVS, and DALVSq), or when all amino acids except histidine are reduced to L-amino acids (DALVSQV), and when glutamine is removed from DALVSQV (DALVS_V), they exhibited superior autopredator elimination activity compared to the D-alloferon peptide (DAL1) in which all amino acids are replaced with D-amino acids. It was confirmed that the autopredator elimination activity was most excellent when at least three of the four amino acids were L-amino acids.

[0065] Experimental Example 4: Analysis of the Effect of Mutant Alloferon Peptide on the Formation of Protein Aggregates Expressed after Overexpression of SOD1 Protein Next, the present inventors investigated whether some mutant peptides (DALS1, DALVS_V, DAL1, DALV2) among the peptides consisting of the amino acid sequences shown in SEQ ID NO: 3 to SEQ ID NO: 16, could resolve the generated SOD1 aggregates after transiently transducing SOD1 protein into 293T cell line. For this purpose, EGFP-SOD1 G93A DNA was transiently transduced into HEK293T cell line using Lipofectamine 2000. After transfection, the cells were incubated at 37°C, 5% CO for 30 hours. 2 The cells were cultured in a cell culture vessel where the conditions were maintained, and the culture medium was replaced with a minimum essential medium (Minimum Essential Medium, MEM, Gibco, USA). After pretreatment with 100 nM bafilomycin for 30 minutes, the cells were treated with 30 μM mutant alloferon peptide and cultured for 18 hours. After that, the cells were cultured in the presence of protein hydrolase inhibitors and phosphohydrolase inhibitors (2 μg / ml aprotinin, 2 μg / ml leupeptin, 1 μg / ml pepstatin A, 1 mM phenyl-methylsulfonyl fluoride (PMSF), 1 mM Na 3 VO 4 , 5 mM NaF, and 10 mM Na 4 P 2 O 7) was added to a 6-well culture dish, and 200 μl of Triton X-100 lysis buffer (30 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) was added per well to lyse the cells, and the obtained cell lysate was left at 4°C for 30 minutes. The obtained cell lysate was subjected to protein quantification using a BCA protein assay kit (Pierce Biotechnology, USA), centrifuged at 17,000xg for 20 minutes, and the supernatant was discarded and the cell debris was removed to obtain a protein extract. Next, 5X sample buffer (300 mM Tris, pH 6.8, 10% SDS, 50% glycerol, 0.1% BPB, 2.5% mercaptoethanol, 100 mM DTT) was mixed with the quantified sample, and the sample was denatured at 95°C for 5 minutes to prepare the sample. Proteins were then electrophoresed using 8%-15% SDS-polyacrylamide gels and separated by size, then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane with the transferred proteins was then blocked for 1 hour with 3% nonfat dry milk in TBST. The blocked membrane was reacted with an anti-GFP antibody (Santa Cruz biotechnology, USA), and anti-Actin antibody (Sigma, USA) was used as a loading control. Antibodies that recognize specific proteins were added to a solution of 1% BSA in TBST. Secondary antibodies were diluted 1:10,000 in 2% nonfat dry milk for all blots, and enhanced chemiluminescence (enhanced chemiluminescense, iNTRoN Biotechnology, South Korea) and a biological imaging system (MF-Chemibis, Shimadzu Scientific Korea Corperation, South Korea) were used to confirm protein signals.

[0066] As a result, as shown in Figure 3A, it was confirmed that SOD1 protein aggregates were increased by bafilomycin compared to the control group, and in this case, some mutant alloferon peptides according to an embodiment of the present invention reduced protein aggregates, and when the protein aggregates were quantified, it was confirmed that some mutant alloferon peptides according to an embodiment of the present invention eliminated the accumulation of aggregates. In particular, as shown in Figure 3B, among the mutant alloferon peptides according to an embodiment of the present invention, those in which the 4th and / or 11th amino acids were reduced to L-amino acids (DALS1, DALV2) and at least three amino acids out of two valines, glutamines, and serine were reduced to L-amino acids (DALVS_V) showed a better ability to eliminate protein aggregates than the D-alloferon peptide (DAL1) in which all amino acids were replaced with D-amino acids.

[0067] Experimental Example 5: Analysis of the Effect of Mutant Alloferon Peptide on the Formation of Protein Aggregates Expressed after Overexpression of Mutant Tau Protein Next, the present inventors investigated whether some mutant peptides (DALVS, DALVSqV, DAL1, DALV2) among the peptides consisting of the amino acid sequences shown in SEQ ID NO: 3 to SEQ ID NO: 16 can resolve the generated mutant tau protein aggregates after transiently transfecting the mutant tau protein into HEK293T cell line. For this purpose, EGFP-Tau P301L DNA was transiently transfected into HEK293T cell line using Lipofectamine 2000. Protein aggregate analysis was performed in the same manner as in Experimental Example 4.

[0068] As a result, as shown in Figure 4A, it was confirmed that mutant tau protein aggregates were increased by bafilomycin compared to the control group, and in this case, protein aggregates were reduced by some mutant alloferon peptides according to one embodiment of the present invention, and when this was quantified, it was similarly confirmed that the accumulation of aggregates was significantly reduced by some mutant alloferon peptides according to one embodiment of the present invention, as shown in Figure 4B.

[0069] Experimental Example 6: Analysis of the Effect of Mutant Alloferon Peptide on the Formation of Protein Aggregates Expressed after Overexpression of α-Synuclein Protein Next, the present inventors investigated whether some mutant peptides (DALV1, DALQ1, DAL1, DALV2) among the peptides consisting of the amino acid sequences shown in SEQ ID NO: 3 to SEQ ID NO: 16 can resolve the α-synuclein protein aggregates that have formed after transiently transfecting the α-synuclein protein into HEK293T cell line. To this end, EGFP-α-synuclein A53T DNA was transiently transfected into HEK293T cell line using Lipofectamine 2000. Protein aggregate analysis was performed in the same manner as in Experimental Example 4.

[0070] As a result, as shown in Figure 5, it was confirmed that alpha-synuclein protein aggregates were increased by bafilomycin compared to the control group, and that protein aggregates were reduced by some mutant alloferon peptides according to an embodiment of the present invention. In this case, DALQ1 and DALV2 also showed a better ability to dissolve protein aggregates than the D-alloferon peptide (DAL-1) in which all amino acids are replaced with D-amino acids.

[0071] Experimental Example 7: Analysis of the Effect of Mutant Alloferon Peptide on the Formation of Protein Aggregates Expressed after Overexpression of Mutant Huntingtin Protein Next, the present inventors investigated whether some mutant peptides (DALH3, DALH1, DAL1, DALV2) among the peptides consisting of the amino acid sequences shown in SEQ ID NO: 3 to SEQ ID NO: 16 can resolve the generated mutant huntingtin protein aggregates after transiently transfecting the mutant huntingtin protein into HEK293T cell line. For this purpose, GFP-mHttQ74 DNA was transiently transfected into HEK293T cell line using Lipofectamine 2000. Protein aggregate analysis was performed in the same manner as in Experimental Example 4.

[0072] As a result, as shown in Figure 6, it was confirmed that mutant huntingtin protein aggregates were increased by bafilomycin compared to the control group, and that protein aggregates were reduced by some mutant alloferon peptides according to one embodiment of the present invention. In this case, DALH1 and DALV2 showed a better ability to dissolve protein aggregates than the D-alloferon peptide (DAL1) in which all amino acids are replaced with D-amino acids.

[0073] Experimental Example 8: Animal Experiment Related to Alzheimer's Disease Experiment 8-1: Body Weight Change by Administration of Mutant Alloferon Peptide Based on the results of Experimental Examples 4 to 7, the inventors conducted animal experiments using disease model animals to determine whether the mutant alloferon peptide according to one embodiment of the present invention can be used as a therapeutic agent for various degenerative neurological diseases.

[0074] First, to confirm whether or not toxicity is observed when administered in vivo, 20 mg / kg of the mutant alloferon peptide (DALV2) according to one embodiment of the present invention was administered intraperitoneally (IP) to a dementia model animal (5XFAD) once a day, five days a week (Monday to Friday), for 25 weeks, and weight changes were observed. As a comparison group, DAL, in which all amino acids are D-type, was used.

[0075] As a result, as shown in FIG. 7, the body weight increase was constant over the 25-week injection period, and no difference was observed between the experimental groups.

[0076] 8-2: Morris Water Maze Test The inventors observed through the results of Experimental Examples 4 to 7 that the mutant alloferon peptide according to one embodiment of the present invention decomposes abnormal protein aggregates, and therefore intraperitoneally administered the mutant alloferon peptide according to one embodiment of the present invention at a concentration of 20 mg / kg once a day, 5 days a week (Monday to Friday) for 25 weeks to Alzheimer's model rats (5XFAD), and observed whether there was any improvement in the memory of the experimental animals from days 1 to 6 after 25 weeks of administration using the Morris water maze test.

[0077] As a result, as confirmed from FIG. 8, the peptide according to one embodiment of the present invention was shown to significantly restore the memory and learning functions of Alzheimer's model animals.

[0078] 8-3: Analysis of the Presence or Absence of Inhibition of the Accumulation of β-Amyloid Aggregates in Brain Tissue In order to confirm whether the improvement of the behavioral index in Experimental Example 8-2 described above is a phenomenon due to the suppression of the accumulation of β-amyloid aggregates in the brain, after sacrificing the experimental animals that had completed the Morris water maze test shown in FIG. 8, brain tissue sections were obtained and stained for β-amyloid aggregates using Thioflavin S, which has a selective affinity for β-amyloid plaques.

[0079] Specifically, for Thioflavin S staining, the brains of the amyloidosis model rats were removed, placed in O.C.T compound (Sakura finetec, USA), freeze-dried, and then brain sections were obtained in coronal section and attached to glass slides coated with 0.1% poly-L-lysine. The obtained brain sections were washed in 70% ethanol for 1 minute, then washed again in 80% ethanol for 1 minute, and stained in 1% Thiflavin S solution (MilliporeSigma, USA) for 15 minutes. Then, after washing in 80% ethanol for 1 minute and then washing again in 70% ethanol for 1 minute, they were washed twice with distilled water, and β-amyloid senile plaques were observed through a fluorescence microscope.

[0080] Congo red staining to confirm amyloid was performed as follows. Brain sections obtained by coronal sectioning were attached to 0.1% poly-L-lysine-coated glass slides. The obtained brain sections were washed in PBS (phosphate-buffered saline) solution for 2 minutes and then stained in 0.5% Congo red solution (Thermo Fisher, USA) for 20 minutes. After washing the Congo red solution in PBS solution, the sections were quickly immersed in an alkaline alcohol solution containing 0.01% sodium hydroxide and then removed 10 times. The sections were then washed in PBS solution for 3 minutes, washed in 95% ethanol for 3 minutes, and washed in 100% ethanol for 3 minutes for dehydration, and amyloid was observed under a fluorescent microscope. The number of stained senile plaques and amyloid per brain section was counted and quantified using a statistical processing program (Graph Prism).

[0081] As a result, as can be seen from Figures 9 and 10, a considerable number of β-amyloid plaques were observed in Alzheimer's model animals, but it was found that the comparison group DAL1 and the mutant alloferon peptide according to one embodiment of the present invention suppressed the accumulation of β-amyloid aggregates to a significant level.

[0082] 8-4: Analysis of the Effect on the Expression of Various Aggregative Proteins in the Brain Based on the results of Experimental Examples 8-2 and 8-3, the present inventors investigated the expression levels of pathological aggregate-forming proteins such as tau protein, p62, and LC3 other than β-amyloid in brain tissues by Western blot analysis.

[0083] Specifically, after sacrificing the experimental animals after the behavioral experiment, the cerebral cortex and hippocampus tissues were obtained and lysed, and then Western blot analysis was performed using antibodies that specifically bind to phosphorylated Tau S214 (P-Tau S214), phosphorylated Tau T205 (P-Tau T205), non-phosphorylated Tau, p62, LC3 and β-amyloid (APP).

[0084] As a result, as can be seen from FIG. 11A to FIG. 12B, all of the six proteins in the cerebral cortex and hippocampus were increased in the Alzheimer's disease model mice compared to the control group of normal mice, while the expression of the aggregate-forming proteins was significantly lower in the cerebral cortex and hippocampus in both experimental animals administered with the comparison group DAL1 or the peptide according to an embodiment of the present invention (DALV2). In the case of the Western blot analysis of the cerebral cortex, DALV2 showed a more excellent ability to dissolve protein aggregates than the D-type alloferron peptide (DAL1) in which all amino acids are replaced with D-type amino acids. In the Western blot analysis of the hippocampus, the reduction in the amount of β-amyloid was not significant in the case of DAL1, while DALV2 showed a significant reduction. This result indicates that the peptide according to an embodiment of the present invention can inhibit the formation of pathological protein aggregates in the brain even after in vivo administration, and indicates the excellent inhibitory effect of DALV2.

[0085] Experimental Example 9: Analysis of the Effect on Amyotrophic Lateral Sclerosis Model Animals In order to confirm whether the mutant alloferon peptide according to one embodiment of the present invention has a therapeutic effect against amyotrophic lateral sclerosis (ALS) in vivo, the present inventors conducted a study on an animal model for amyotrophic lateral sclerosis (SOD1 G93A The peptide (DALV2) according to one embodiment of the present invention was repeatedly administered subcutaneously (SC) at 40 mg / kg, 5 days a week (Monday to Friday) from 112 days to 180 days of age. After the experiment, the experimental animals were sacrificed, and spinal cord slices were obtained from the lumbar spinal cord and subjected to Western blot analysis using anti-SOD-1 antibody.

[0086] As a result, as can be seen from Figure 13, a significantly lower level of SOD-1 aggregates was detected in the ALS model animals administered with the mutant alloferon peptide according to one embodiment of the present invention, indicating that the mutant alloferon peptide according to one embodiment of the present invention suppresses the accumulation of pathogenic protein aggregates in the nervous system in vivo.

[0087] Although the present invention has been described with reference to the above-mentioned embodiment, it is understood that the embodiment is merely illustrative and that various modifications and equivalent embodiments are possible. Therefore, the true technical scope of the present invention should be determined by the technical spirit of the claims. [Industrial Applicability]

[0088] The peptide according to one embodiment of the present invention can be developed as a therapeutic agent for various degenerative neurological diseases caused by pathogenic protein aggregates, such as Alzheimer's disease and amyotrophic lateral sclerosis, by suppressing abnormal hyperphosphorylation of pathogenic proteins in the central nervous system and the resulting formation of aggregates and removing pre-existing protein aggregates.

Claims

1. A novel peptide having a length of 12 to 20 a.a. and containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, which has the ability to suppress the formation of abnormal protein aggregates. Among the amino acids excluding glycine, 1 to 4 amino acids are L-amino acids, and the remaining amino acids are D-amino acids.

2. The peptide according to claim 1, when the peptide contains the amino acid sequence represented by SEQ ID NO: 1, comprising at least one of the following: i) At least one of the four histidines is a D-amino acid; ii) At least one of the valines at positions 3 and 11 is an L-amino acid or both are D-amino acids; iii) The serine at position 4 is a D-amino acid; iv) The glutamine at position 8 is a D-amino acid; and v) Among i) to iv) above, two or more are D-amino acids.

3. The peptide according to claim 1, when the peptide contains the amino acid sequence represented by SEQ ID NO: 2, comprising at least one of the following: i) At least one of the four histidines is a D-amino acid: ii) At least one of the valines at positions 3 and 10 is an L-amino acid or both are D-amino acids; iii) The serine at position 4 is a D-amino acid; and iv) At least two or more of i) to iii) are D-amino acids.

4. 1) In the L-allopheron peptide of SEQ ID NO: 1, at least 3 of the four histidines are replaced with D-histidines, and at least 2 of the two valines, the serine at position 3, and the glutamine at position 8 are D-amino acids, or 2) In the L-allopheron peptide of SEQ ID NO: 1, all four histidines are D-amino acids, and the glutamine at position 8 is an L-type or D-type amino acid or has been deleted. The peptide according to claim 1.

5. The peptide according to claim 1, containing any one of the amino acid sequences represented by SEQ ID NO: 3 to SEQ ID NO:

14.

6. The peptide according to claim 1, containing any one of the amino acid sequences of SEQ ID NO: 3 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12 to SEQ ID NO:

14.

7. A pharmaceutical composition for treating degenerative neurological diseases, comprising, as an active ingredient, the peptide according to any one of Claims 1 to 6.

8. The pharmaceutical composition according to Claim 7, wherein the degenerative neurological disease is a degenerative neurological disease having the formation of abnormal protein aggregates as an etiology or pathological phenomenon.

9. The pharmaceutical composition according to Claim 8, wherein the abnormal protein aggregates are formed by abnormal aggregation of α-synuclein, β-amyloid, TDP-34, p62, FUS protein, superoxide dismutase-1 (SOD-1), huntingtin protein or tau protein.

10. The degenerative brain diseases having the formation of abnormal protein aggregates as an etiology or pathological phenomenon are specifically Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), chronic traumatic encephalopathy, corticobasal degeneration, frontotemporal lobar degeneration, progressive supranuclear palsy, etc. The pharmaceutical composition according to Claim 8.

11. A method for treating an individual, comprising the step of administering a therapeutically effective amount of the peptide according to any one of Claims 1 to 6 to the individual suffering from a degenerative neurological disease.

12. A method for suppressing the accumulation of pathogenic protein aggregates in the nervous system of an individual, comprising the step of administering a therapeutically effective amount of the peptide according to any one of Claims 1 to 6 to the individual suffering from a degenerative neurological disease.

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