Novel Peptides and Their Uses

Abstract

The present invention relates to a novel peptide and its use, and more specifically, to a peptide having the ability to inhibit the formation of abnormal protein aggregates, having the ability to inhibit the formation of abnormal protein aggregates and autophagy activity against the protein aggregates, H-G-X 1 -X 2 -G-X 3 which comprises a structure of, and provides a novel peptide having a length of 4 to 10 amino acids and its use.

Description

Technical Field

[0001] The present invention relates to a novel peptide and its use. More specifically, it relates to a novel peptide capable of suppressing the formation of abnormal protein aggregates and removing the protein aggregates by autophagy, 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, and it is known to cause 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, and it is known to induce 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 to obtain an energy source in a starvation situation 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 hydrolytic enzymes present in the lysosome.

[0004] Recently, it has been known that protein aggregates represented by neurodegenerative 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, the autophagy flux is blocked, and ultimately, there are research results indicating that the absence of such autophagy leads to the accumulation of by-products of neurodegenerative 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. When treating lysosomal membrane permeabilization (LMP) induced by H2O2, tamoxifen, or ethanol with TPEN, a potent zinc chelator, the lysosomal membrane disruption phenomenon is suppressed. Conversely, when the autophagic effect is reduced, additional zinc supply can promote autophagy. Therefore, zinc is presumed to have an important impact on autophagy (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).

[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, can be removed through autophagy or improved 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, there is provided a novel peptide having a structure of the following Structural Formula 1, having the ability to suppress the formation of abnormal protein aggregates and autophagic activity against said protein aggregates, and having a length of 4 to 10 amino acids: H-G-X 1 -X 2 -G-X 3 (Structural Formula 1) (In the above formula, H is L-type or D-type histidine, G is glycine, X 1 is either absent or any D-type or L-type amino acid, X 2 is any D-type or L-type amino acid, X 3 is either absent or any D-type or L-type amino acid excluding valine).

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

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

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

Advantages 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 neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease by preventing neuronal cell death 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: As used herein, the term "zinc homeostasis" means the mechanism for maintaining the concentration of zinc in cells at a certain level, and 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.

[0027] 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.

[0028] 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.

[0029] Detailed Description of the Invention: According to one aspect of the present invention, there is provided a novel peptide having a structure of Structural Formula 1 below, having an ability to suppress the formation of abnormal protein aggregates and autophagy activity against said protein aggregates, and having a length of 4 - 10 amino acids: H - G - X 1 - X 2 - G - X 3 (Structural Formula 1) (In the above formula, H is L - type or D - type histidine, G is glycine, X 1 is either absent or any D - type or L - type amino acid, X 2 is any D - type or L - type amino acid, X 3 is either absent or any D - type or L - type amino acid excluding valine).

[0030] The peptide may contain at least one or more D - type amino acids.

[0031] In the peptide, X1 is either a non-polar amino acid or a polar amino acid. The non-polar amino acid is alanine, valine, leucine, methionine, isoleucine or proline, and the polar amino acid is glutamine or asparagine. More preferably, the X 1 is either D-valine or L-valine or D-glutamine or L-glutamine.

[0032] The peptide contains the structure of Structural Formula 1 and may have a length of 4, 5, 6, 7, 8, 9 or 10 amino acids (a.a.).

[0033] In the peptide, the X 2 is a polar amino acid, a non-polar amino acid or an aromatic amino acid. The polar amino acid is serine, cysteine, asparagine, glutamine, threonine or tyrosine, the non-polar amino acid is alanine, valine, leucine, isoleucine, methionine or proline, and the aromatic amino acid is tyrosine, tryptophan or phenylalanine. X 2 is more preferably a D-amino acid or an L-amino acid selected from the group consisting of serine, histidine, valine, threonine, cysteine and tryptophan.

[0034] In the peptide, the X 3 is a polar amino acid, a non-polar amino acid or an aromatic amino acid. The polar amino acid is serine, cysteine, asparagine, glutamine, threonine or tyrosine, the non-polar amino acid is alanine, valine, leucine, isoleucine, methionine or proline, and the aromatic amino acid is tyrosine, tryptophan or phenylalanine. The X 3 is more preferably a D-amino acid or an L-amino acid selected from the group consisting of aspartic acid, serine, asparagine, tyrosine, histidine and leucine.

[0035] Most preferably, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, or consists of the amino acid sequence.

[0036] In order to investigate whether the generation of abnormal protein aggregates that induce autophagy is suppressed by a short peptide, the inventors designed peptides in a form in which various combinations of D-type and L-type amino acids as shown in Table 1 below were mixed, and then performed L3 puncta analysis. As a result, as confirmed from FIGS. 1A to 1D, the peptide according to an embodiment of the present invention was shown to promote autophagy by eliminating the suppression phenomenon of autophagy induced by the treatment with bafilomycin. This indicates that the peptide according to an embodiment of the present invention accumulates in nerve cells to suppress the abnormal generation of protein aggregates that induce nerve cell death, and promotes the autophagy effect and lysosomal activity against the generated abnormal protein aggregates, and thus can be used as a therapeutic agent for neurodegenerative diseases caused by abnormal protein aggregates.

[0037] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating neurodegenerative diseases containing the peptide as an active ingredient.

[0038] 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-43, p62 protein, 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 that are correlated with the accumulation of protein aggregates in the nerves. The correlation between the degenerative nerve 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).

[0039] In the composition, the peptide can prevent cell death of nerve cells, regulate intracellular zinc homeostasis, and promote lysosomal function, thereby treating the degenerative nerve disease.

[0040] 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.

[0041] As used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause common 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. Additionally, it may further contain fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives, etc.

[0042] 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 mammals. The dosage forms include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, and sterile powders.

[0043] 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, intrathecally, and also by 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.

[0044] The pharmaceutical composition according to an embodiment of the present invention is administered by general systemic administration or local administration, for example, by intramuscular injection or intravenous injection. Moreover, the peptide according to an embodiment of the present invention can also be administered orally.

[0045] The pharmaceutical composition according to an embodiment of the present invention is formulated into a suitable form together with a commonly 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].

[0046] 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 to be administered, sex, administration time and route, general health, and other drugs administered simultaneously. The therapeutically active peptide 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.

[0047] Further, the pharmaceutical composition of the present invention is administered in a therapeutically effective amount.

[0048] 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 dose level can be determined by factors including the type and severity of the individual, age, gender, drug activity, 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 at a dose of 0.1 mg / kg to 1 g / kg, and more preferably at a dose of 1 to 500 mg / kg. On the other hand, the said dose is appropriately adjusted according to the age, gender and condition of the patient.

[0049] According to another aspect of the present invention, there is provided a method of treating an individual comprising administering to the individual a composition comprising a therapeutically effective amount of said peptide, who suffers from a degenerative neurological disease.

[0050] 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 administering to the individual a therapeutically effective amount of said peptide, who suffers from a degenerative neurological disease.

[0051] 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 (IV), intraperitoneal injection (IP), or intramuscular injection (IM) is possible. In the case of local administration, intracranial administration, intracerebrospinal administration, subcutaneous injection (SC), etc. are possible.

[0052] In the method, 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-43, p62 protein, FUS protein, superoxide dismutase-1 (SOD-1), huntingtin protein or tau protein.

[0053] 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.

[0054] Examples: Devising and Synthesizing Peptides The inventors of the present invention commissioned Enigene (Republic of Korea) to synthesize a peptide having an amino acid sequence as shown in Table 1 below.

[0055]

Table 1

[0056] Experimental Example 1: Cell Culture In the present invention, H4 cell line (GL-H4) and HEK293T cell line permanently infected with GFP-LC3 plasmid were used. As the cell culture medium, Minimum Essential Medium (MEM, WellGene) supplemented with 10% fetal bovine serum (Hyclone, USA) and a mixed solution of antibiotics and antifungal agents (WellGene, Republic of Korea) was used, and the cells were cultured in a cell incubator maintained at 37 °C and 5% CO2.

[0057] Experimental Example 2: Analysis of the Autophagic Flux Blocked by Bafilomycin (A-1) In order to investigate the influence of the peptides composed of the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 15 on the autophagic flux, after changing the MEM in the H4 cell line (GL-H4) expressing LC3 linked with GFP, the autophagic flux was inhibited using the peptides (20 μM) and bafilomycin (A-1, Baf A1, 100 nM), an autophagy inhibitor, and then the effect of the peptides according to an embodiment of the present invention was investigated by fluorescence microscopy analysis.

[0058] As a result, the LC3 puncta shown in FIGS. 1A and 1B are phenomena represented by the LC3-GFP protein present in autophagosomes. If not degraded by lysosomes, it means that the entire autophagic process is inhibited and autophagosomes accumulate. When the GL-H4 cell line was treated with 100 nM of bafilomycin (Baf A1) to inhibit the autophagic flux, the number and size of the LC3 puncta increased significantly. However, such a phenomenon decreased as a whole, although there was a difference, when treated with the peptides (20 μM) according to an embodiment of the present invention (FIGS. 1A and 1B). Similarly, when the fluorescence intensity of the whole puncta was measured and quantified, the fluorescence intensity decreased when treated with the peptides (ZC303 to ZC317) according to an embodiment of the present invention (FIGS. 1C and 1D). This means that the autophagosomes accumulated by the blocked autophagic flux disappear due to the peptides according to an embodiment of the present invention.

[0059] Experimental Example 3: Analysis of the Effect of a Novel Peptide on the Formation of Protein Aggregates Appearing after Overexpression of SOD1 Protein Subsequently, the inventors transiently transfected the SOD1 protein into the HEK293T cell line, and then investigated whether some of the peptides (ZC303, ZC304, ZC309, ZC311) among the peptides composed of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 15 could eliminate the generated SOD1 aggregates through Western blot analysis. For this purpose, specifically, the inventors transiently transfected the EGFP-SOD1 G93A DNA into the HEK293T cell line using Lipofectamine 2000. After transfection, the cells were cultured in a cell incubator maintained at 37 °C and 5% CO2 for 30 hours, and the culture medium was replaced with minimum essential medium (MEM, Gibco, USA). Then, after pretreatment with 100 nM bafilomycin for 30 minutes, 30 μM of the peptide was treated, and the cells were cultured for 18 hours. Thereafter, Triton X-100 lysis buffer (30 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) supplemented with protease inhibitors and phosphatase inhibitors (2 μg / ml aprotinin, 2 μg / ml leupeptin, 1 μg / ml pepstatin A, 1 mM phenyl-methylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 5 mM NaF, and 10 mM Na4P2O7) was added to a 6-well culture dish at 200 μl per well to lyse the cells, and the obtained cell lysate was left at 4 °C for 30 minutes. The obtained cell lysate was quantified for protein using a BCA protein assay kit (Pierce Biotechnology, USA), then centrifuged at 17,000 xg for 20 minutes, and only the supernatant was discarded, and the cell pellet was taken to obtain a protein extract. Subsequently, 5X sample buffer (300 mM Tris, pH 6.8, 10% SDS, 50% glycerol, 0.1% bromophenol blue, 2.5% mercaptoethanol, 100 mM DTT) was mixed with the sample whose quantification was completed, and the sample was denatured at 95 °C for 5 minutes to prepare the sample.Subsequently, the proteins separated by size were electrophoresed using an 8% - 15% SDS - polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Subsequently, the membrane to which the proteins were transferred was blocked with 3% non - fat dry milk in TBST for 1 hour. The blocked membrane was reacted with an anti - GFP antibody (Santa Cruz biotechnology, USA), and an anti - Actin antibody (Sigma, USA) was used as a loading control group. Antibodies that recognize specific proteins were added to a solution of 1% BSA dissolved in TBST. For all blots, the secondary antibody was diluted at a ratio of 1:10,000 in 2% non - fat dry milk and reacted. To confirm the protein signal, enhanced chemiluminescence (iNTRoN Biotechnology, South Korea) and a bio - imaging system (MF - Chemibis, Shimadzu scientific korea corperation, South Korea) were used.

[0060] As a result, as shown in FIGS. 2A and 2B, it was confirmed that the SOD1 protein aggregates increased due to bafilomycin compared to the control group. At this time, the protein aggregates decreased due to some peptides according to an embodiment of the present invention, and when it was quantified, it was also confirmed that the accumulation of aggregates was eliminated by some peptides according to an embodiment of the present invention.

[0061] Experimental Example 4: Analysis of the Effect of a Novel Peptide on the Formation of Protein Aggregates Appearing after Overexpression of Mutant Tau Protein Subsequently, after the inventors transiently transfected mutant tau protein into the HEK293T cell line, they investigated whether mutant tau protein aggregates generated could be eliminated by some of the peptides (ZC305, ZC306, ZC307, ZC311) composed of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 15. For this purpose, EGFP-Tau P301L DNA was transiently transfected into the HEK293T cell line using Lipofectamine 2000. The analysis of protein aggregates was performed in the same manner as in Experimental Example 3 above.

[0062] As a result, as shown in FIG. 3A, it was confirmed that the mutant tau protein aggregates increased by bafilomycin compared to the control group. At this time, the protein aggregates decreased by some of the peptides according to an embodiment of the present invention, and when it was quantified, it was also confirmed that the accumulation of aggregates significantly decreased by some of the peptides according to an embodiment of the present invention.

[0063] Experimental Example 5: Analysis of the Effect of a Novel Peptide on the Formation of Protein Aggregates Appearing after Overexpression of α-Synuclein Protein Subsequently, after the inventors transiently transfected α-synuclein protein into the HEK293T cell line, they investigated whether α-synuclein protein aggregates generated could be eliminated by some of the peptides (ZC308, ZC310, ZC311, ZC312) composed of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 15. For this purpose, EGFP-α-synuclein A53T DNA was transiently transfected into the HEK293T cell line using Lipofectamine 2000. The analysis of protein aggregates was performed in the same manner as in Experimental Example 3 above.

[0064] As a result, as shown in FIG. 4, it was confirmed that the α-synuclein protein aggregates increased by bafilomycin compared to the control group. At this time, it was confirmed that the protein aggregates decreased by some of the peptides according to an embodiment of the present invention.

[0065] Experimental Example 6: Analysis of the Effect of a Novel Peptide on the Formation of Protein Aggregates Appearing after Overexpression of Mutant Huntingtin Protein Subsequently, after transiently transducing the mutant huntingtin protein into the HEK293T cell line, the inventors investigated whether the generated mutant huntingtin protein aggregates could be eliminated by some of the peptides (ZC311, ZC314, ZC315, ZC316) composed of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 15. For this purpose, GFP-mHttQ74 DNA was transiently transduced into the HEK293T cell line using Lipofectamine 2000. The analysis of protein aggregates was performed in the same manner as in Experimental Example 3 above.

[0066] As a result, as shown in FIG. 5, it was confirmed that the mutant huntingtin protein aggregates increased by bafilomycin compared to the control group, and at this time, it was confirmed that the protein aggregates decreased by some of the peptides according to an embodiment of the present invention.

[0067] Experimental Example 7: Alzheimer's Model Animal Experiment 7-1: Analysis of Body Weight Changes From the results of Experimental Examples 4 to 6 above, the inventors conducted animal experiments using disease model animals to confirm whether the peptide according to an embodiment of the present invention can be used as a therapeutic agent for various neurodegenerative diseases.

[0068] First, in order to confirm whether it shows toxicity during in vivo administration, the peptide (ZC311) produced from Example 9 of the present invention, 20 mg / kg, was intraperitoneally (IP) injected into a dementia model animal (5XFAD) once a day, 5 days a week (Monday to Friday) for 25 weeks, and the body weight change was confirmed. As control groups, wild-type mice administered only physiological saline and Alzheimer's model rats administered only physiological saline were used.

[0069] As a result, as shown in FIG. 6, a constant increase in body weight was shown during the 25-week injection period, and no difference was shown between the experimental groups.

[0070] 7-2: Analysis of the Effect on the Accumulation of β-Amyloid Aggregates in the Brain As in the results of Experimental Examples 4 to 5 described above, in order to confirm whether the peptide according to an embodiment of the present invention can suppress the accumulation of β-amyloid aggregates in the brain, which is a pathological phenomenon of Alzheimer's disease, even under actual in-vivo conditions, after sacrificing the experimental animals that had completed 25 weeks of peptide administration, brain tissue sections were obtained and stained for β-amyloid aggregates using Thioflavin S and Congo red, which have selective affinity for β-amyloid plaques.

[0071] Specifically, for Thioflavin S staining, the brains of the amyloidosis model rats were excised, placed in O.C.T compound (Sakura finetec, USA), freeze-dried, and then brain sections were obtained by 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, stained in 1% Thiflavin S solution (MilliporeSigma, USA) for 15 minutes. Subsequently, after washing in 80% ethanol for 1 minute, then washing again in 70% ethanol for 1 minute, they were washed twice with distilled water, and β-amyloid plaques were observed through a fluorescence microscope.

[0072] Congo red staining for amyloid confirmation was performed as follows. Brain sections obtained in coronal section were attached to glass slides coated with 0.1% poly-L-lysine. 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 with PBS solution, the operation of dipping into and then taking out from an alkaline alcohol solution containing 0.01% sodium hydroxide was quickly performed 10 times. Then, after washing in PBS solution for 3 minutes, washing in 95% ethanol for 3 minutes, and washing in 100% ethanol for 3 minutes for dehydration, amyloid senile plaques were observed through a fluorescence microscope. The number of stained senile plaques and amyloid per brain section was counted and quantified using a statistical processing program (Graph prism).

[0073] As a result, as confirmed from FIGS. 7 and 8, in the case of Alzheimer's model animals administered with only PBS, a considerable number of β-amyloid plaques were confirmed, but in the case of Alzheimer's model animals administered with the peptide according to an embodiment of the present invention, it was found that the accumulation of β-amyloid aggregates was suppressed at a significant level.

[0074] 7-3: Analysis of the Effect on the Expression of Various Aggregating Proteins in the Brain The present inventors investigated the expression levels of pathogenic aggregate-forming proteins such as tau protein, p62, and LC3 other than β-amyloid in brain tissues through Western blot analysis based on the results of Experimental Example 7-2.

[0075] Specifically, after sacrificing the experimental animals that had completed the experiment, the cerebral cortex and hippocampal tissues were obtained. After lysing the tissues, Western blot analysis was performed in the same manner as described in Experimental Example 3 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), respectively.

[0076] As a result, as confirmed from FIGS. 9A to 10B, not only the cerebral cortex but also all six proteins in the hippocampus showed an increased pattern in Alzheimer's disease model mice compared to normal mice in the control group, while in the experimental animals administered with the peptide (ZC311) according to an embodiment of the present invention, the expression of the aggregate-forming protein was significantly lower in both the cerebral cortex and the hippocampus. This is a result indicating that the peptide according to an embodiment of the present invention can suppress the formation of pathogenic protein aggregates in the brain even after in vivo administration.

[0077] Experimental Example 8: Confirmation of the Therapeutic Effect of Amyotrophic Lateral Sclerosis 8-1: Rotarod Test In order to confirm whether the peptide according to an embodiment of the present invention has a therapeutic effect on amyotrophic lateral sclerosis (ALS) in an actual living body, the present inventors repeatedly administered the peptide (ZC311) according to an embodiment of the present invention to amyotrophic lateral sclerosis model animals (G93A-SOD1) by subcutaneous (SC) injection at 40 mg / kg 5 days a week (Monday to Friday) from 112 days old to 200 days old.

[0078] Subsequently, in order to evaluate the motor function of the experimental animals, a rotarod test was performed twice a week at intervals of up to 3 to 4 days. The rotarod test was performed using a rotarod treadmill (Cat. No. DJ-345, Dae Jong, Seoul, Korea) at a fixed speed of 15 rpm for 300 seconds in 3 repetitions to measure the time for the target animals (n = 11, 8, 10) to fall from the rod, and the highest score was used with 300 seconds as the full score.

[0079] As a result, as confirmed from FIG. 11, the ALS model mice began to show ataxia after 116 days, and showed a complete ataxic state at about 150 days, but it was confirmed that the ataxia phenomenon was delayed in the experimental animals administered with the peptide (ZC311) according to an embodiment of the present invention.

[0080] 8-2: Investigation of Body Weight Changes The inventors measured the change in body weight over time while conducting the behavioral analysis of Experimental Example 8-1.

[0081] As a result, as confirmed from FIG. 12, the body weight of the ALS model animals gradually decreased over time, and such a phenomenon remained unchanged even when the peptide according to the examples of the present invention was administered.

[0082] 8-3: Investigation of Survival Rate Amyotrophic lateral sclerosis is a fatal disease that eventually leads to death as the disease progresses after onset. In fact, actual ALS animal models also ultimately die 100% after the onset, but the inventors attempted to investigate the effect of the peptide according to one embodiment of the present invention on the survival rate in ALS model animals.

[0083] As a result of measuring the survival rate over time of the animals during the progress of the experiment of Experimental Example 8-1, as confirmed from FIG. 13, the ALS model mice began to die from about 140 days after the onset, and by about 178 days after the onset, all the experimental animals had died (50% survival day: 162 days). On the other hand, the ALS model mice administered with the peptide according to one embodiment of the present invention began to die at the same time as the control group, but it was confirmed that the time point of death was delayed (50% survival day: 183 days). Such results indicate that the peptide according to one embodiment of the present invention delays ataxia, which is a major symptom of ALS, and death caused thereby.

[0084] The present invention has been described with reference to the foregoing examples and experimental examples, but this is merely illustrative, and those skilled in the art will understand that various modifications and equivalent other examples and experimental examples are possible therefrom. Therefore, the true technical protection scope of the present invention must be determined by the technical idea of the claims.

Industrial Applicability

[0085] The peptide according to an embodiment of the present invention suppresses abnormal hyperphosphorylation of pathogenic proteins and the formation of aggregates thereby in the central nervous system, and removes pre-existing protein aggregates, and thus can be developed as a therapeutic agent for various neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis caused by pathogenic protein aggregates.

Claims

1. A novel peptide having the ability to inhibit the formation of abnormal protein aggregates and autophagy activity against said protein aggregates, comprising the structure of Structural Formula 1 below and having a length of 4 - 10 a.a.: H-G-X １ -X ２ -G-X ３ (Structural formula 1) (In the above formula, H is L-type or D-type histidine, G is glycine, X １ is either absent or any D-type or L-type amino acid, X ２ is any D-type or L-type amino acid, X ３ is either absent or any D-type or L-type amino acid excluding valine).

2. The peptide according to Claim 1, comprising at least one or more D - type amino acids.

3. Said X １ is a non-polar amino acid or a polar amino acid, the peptide according to claim 1.

4. The peptide according to Claim 3, wherein the non - polar amino acid is alanine, valine, leucine, methionine, isoleucine, or proline.

5. The peptide according to Claim 3, wherein the polar amino acid is glutamine or asparagine.

6. Said X １ is the peptide according to claim 3, which is absent, or is D- or L-valine or glutamine.

7. Said X ２ The peptide according to claim 1, wherein said X is a polar amino acid, a nonpolar amino acid or an aromatic amino acid.

8. The peptide according to Claim 7, wherein the polar amino acid is serine, cysteine, asparagine, glutamine, threonine, or tyrosine.

9. The peptide according to Claim 7, wherein the non - polar amino acid is alanine, valine, leucine, isoleucine, methionine, or proline.

10. The peptide according to Claim 7, wherein the aromatic amino acid is tyrosine, tryptophan, or phenylalanine.

11. Said X ２ is a D-type or L-type amino acid selected from the group consisting of serine, histidine, valine, threonine, cysteine, and tryptophan, the peptide according to claim 1.

12. Said X ３ is not, a polar amino acid, a nonpolar amino acid, or an aromatic amino acid, the peptide according to claim 1.

13. The peptide according to Claim 12, wherein the polar amino acid is serine, cysteine, asparagine, glutamine, threonine, or tyrosine.

14. The peptide according to Claim 12, wherein the non - polar amino acid is alanine, valine, leucine, isoleucine, methionine, or proline.

15. The peptide according to Claim 12, wherein the aromatic amino acid is tyrosine, tryptophan, or phenylalanine.

16. Said X ３ is a D- or L-amino acid selected from the group consisting of aspartic acid, serine, asparagine, tyrosine, histidine, and leucine, the peptide according to claim 12.

17. The peptide according to Claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

15.

18. A pharmaceutical composition for treating degenerative neurological diseases, comprising the peptide according to any one of Claims 1 to 17 as an active ingredient.

19. The pharmaceutical composition according to Claim 18, wherein the degenerative neurological disease is a degenerative neurological disease having the formation of abnormal protein aggregates as an etiology or pathological phenomenon.

20. The pharmaceutical composition according to claim 19, wherein the abnormal protein aggregate is formed by abnormal aggregation of α-synuclein, β-amyloid, TDP-43, p62 protein, FUS protein, superoxide dismutase-1 (SOD-1), huntingtin protein or tau protein.

21. The pharmaceutical composition according to claim 19, wherein the degenerative brain disease having the formation of the abnormal protein aggregate as an etiology or pathological phenomenon is 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, or progressive supranuclear palsy.

22. A method of treating an individual, comprising administering a therapeutically effective amount of the peptide according to any one of claims 1 to 17 to the individual suffering from a degenerative nerve disease.

23. A method of suppressing the accumulation of pathogenic protein aggregates in the nervous system of an individual, comprising administering a therapeutically effective amount of the peptide according to any one of claims 1 to 17 to the individual suffering from a degenerative nerve disease.

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