Novel peptide and use thereof

Abstract

The present invention relates to a novel peptide and a use thereof, and more particularly, to a peptide having a length of 12 to 20 a.a. Comprising an amino acid sequence designated as SEQ ID NO: 1 or 2, in which 1 to 4 of the amino acids other than glycine are L-type amino acids and the other amino acids are D-type amino acids, which has the ability to inhibit the formation of abnormal protein aggregates.

Description

Technical Field

[0001] The present invention relates to novel peptides and uses thereof, and more particularly, to novel peptides based on alloferon and uses thereof in treating neurodegenerative diseases. Background Art

[0002] Zinc is a trace element that is essential for cell proliferation and differentiation, and is known as a cofactor in the function and structure of proteins such as enzymes and transcription factors. In addition, it is known that the homeostasis of zinc in neurons plays an important role in the survival of neurons, and it is known that zinc deficiency in neurons induces apoptosis and causes neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (Lien, H et al., BBRC. 268: 148-154, 2000). On the contrary, it is also known that excessive zinc in neurons induces cell damage, which can lead to acute brain damage, such as focal ischemia and epileptic seizures (Koh et al., Science. 272: 1013-1016, 1996).

[0003] Autophagy is the intracellular mechanism that decomposes organelles during starvation to obtain energy or remove damaged organelles and abnormal or pathological protein aggregates. During autophagy, cytoplasmic components are surrounded by double membranes and separated from other organelles to form autophagosomes. At this time, the soluble light chain I (LC3I) in the cytoplasm is converted into light chain II (LC3II), which is attached to the autophagosome membrane. Then the autophagosome merges with the lysosome to form an autolysosome, which is degraded and recycled by several hydrolases present in the lysosome.

[0004] Recently, it has been shown that protein aggregates seen in neurodegenerative diseases, such as α-synuclein, β-amyloid, tau, superoxide dismutase-1 (SOD-1), huntingtin, and TAR DNA binding protein 43 (TDP-43), can be cleared by promoting autophagy. In fact, there is evidence that defective autolysosomal function impairs the degradation of those protein aggregates, thereby blocking the flow of autophagy, and that the lack of this autophagy ultimately leads to the accumulation of neurodegenerative disease byproducts (Zhang et al., ABBS, 41(6): 437-445, 2009; Lee et al., Cell, 141(7): 1146-1158, 2010).

[0005] Previous studies have shown that the function of lysosomes can be enhanced by the supply of zinc. On the other hand, it has also been reported that when the effective zinc chelator TPEN is applied to inhibit lysosomal membrane permeabilization (LMP) induced by H2O2, tamoxifen and ethanol and thus reduce autophagy, additional zinc supply can promote autophagy (Lee et al., Glia 57: 1351-1361, 2009; Hwag et al., Biometls 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 expected to play an important role in autophagy.

[0006] However, drugs that treat neurodegenerative diseases by maintaining zinc homeostasis within neurons have not yet been developed. Summary of the invention Technical issues

[0007] The object of the present invention is to solve the above and other problems by providing novel peptides and their use in treating degenerative neurological diseases by maintaining zinc homeostasis in neurons, thereby removing abnormal or pathological protein aggregates, such as amyloid β peptide, tau protein and superoxide dismutase, which are pathological substances in degenerative neurological diseases, through autophagy or enhanced lysosomal function. Technical Solution

[0008] In one aspect of the present invention, a novel peptide with a length of 12 to 20 a.a. that is capable of inhibiting the formation of abnormal protein aggregates is provided, which comprises the amino acid sequence specified by SEQ ID NO: 1 or 2, wherein 1 to 4 of the amino acids other than glycine are L-type amino acids, and the other amino acids are D-type amino acids.

[0009] In another aspect of the present invention, a pharmaceutical composition for treating a neurodegenerative disease is provided, which comprises the above-mentioned peptide as an active ingredient.

[0010] In another aspect of the present invention, a method of treating a subject suffering from a neurodegenerative disease is provided, the method comprising administering to the subject a therapeutically effective amount of the peptide.

[0011] In another aspect of the present invention, a method for inhibiting the accumulation of pathogenic protein aggregates in the nervous system of a subject suffering from a neurodegenerative disease is provided, the method comprising the step of administering a therapeutically effective amount of the peptide to the subject. Effects of the Invention

[0012] The novel peptides of the present invention prepared as described above can be used in the development of therapeutic agents to effectively treat 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. However, the scope of the present invention is not limited by these effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a graph showing the results of measuring quantitative changes of alloferon L-type and alloferon D-type peptides by mass spectrometry to study their degradation in plasma from mice, wherein 10 μM of each alloferon peptide was treated in separated plasma and incubated at 37° C. for 0, 10, 30, 60 and 90 min.

[0014] Figures 2a to 2c This is a series of fluorescence micrographs of LC3-GFP dots 12 h after treatment of an H4 cell line (GL-H4) stably overexpressing GFP-LC3 protein with various variant alloferon peptides according to one embodiment of the present invention in combination with baflomycin at a concentration of 20 μM, showing that the variant alloferon peptide according to one embodiment of the present invention restored the autophagic flux blocked by baflomycin A-1. Figures 2d to 2f It is a quantitative graph showing the fluorescence intensity of LC3-GFP dots 12h after treating GL-H4 cell line with a variant alloferon peptide according to one embodiment of the present invention in combination with bafilomycin at a concentration of 20 μM. It can be seen that after bafilomycin treatment, the dots increased significantly due to the blocking of autophagy, while the number and size of the dots decreased after treatment with a variant alloferon peptide according to one embodiment of the present invention, which is the result of autophagy elimination. In Figures 2D to 2F, * indicates that the treatment of a peptide according to one embodiment of the present invention is significantly different from the treatment with bafilomycin alone (*p < 0.05, **p < 0.01, ***p < 0.001), and # indicates a significant difference compared to the treatment with bafilomycin and the previously reported D-type alloferon (DAL-1) (#p < 0.05).

[0015] Figure 3aare the results of Western blot analysis of SOD1 protein expression levels in protein samples obtained 18 hours after HEK293T cells transfected to express SOD1 were treated with 100 nM bafilomycin A1 alone or in combination with 30 μM of some variant alloferon peptides according to one embodiment of the present invention, to study the degradation of SOD1 protein aggregates caused by treatment with some variant alloferon peptides (DALS1, DAL-VS_V(-Q), DAL1 and DALV2) according to one embodiment of the present invention. Figure 3b This is a graph showing the results of quantitative analysis of the relative expression of SOD1 protein. Figure 3b In the figure, * indicates a significant difference compared with the treatment with bafilomycin alone (*p<0.05).

[0016] Figure 4a are the results of Western blot analysis of tau protein expression levels in protein samples obtained 18 hours after HEK293T cells transfected to express tau protein were treated with 100 nM bafilomycin A1 alone or 100 nM bafilomycin A1 in combination with 30 μM of some variant alloferon peptides according to one embodiment of the present invention, to study the degradation of tau protein aggregates caused by treatment with some variant alloferon peptides (DALS1, DAL-VS_V(-Q), DAL1 and DALV2) according to one embodiment of the present invention. Figure 4b This is a graph showing the results of quantitatively analyzing the relative expression of tau protein. Figure 4b In the figure, * indicates a significant difference compared with the treatment with bafilomycin alone (*p<0.05).

[0017] Figure 5 are the results of Western blot analysis of α-synuclein expression levels in protein samples obtained 18 hours after HEK293T cells transfected to express α-synuclein were treated with 100 n bafilomycin A1 alone or in combination with 30 μM of some variant alloferon peptides according to one embodiment of the present invention, to study the degradation of α-synuclein aggregates caused by treatment with some variant alloferon peptides (DALS1, DAL-VS_V(-Q), DAL1 and DALV2) according to one embodiment of the present invention.

[0018] Figure 6are the results of Western blot analysis of mutant huntingtin protein expression levels in protein samples obtained 18 hours after HEK293T cells transfected to express mutant huntingtin protein were treated with 100n bafilomycin A1 alone or in combination with 30 μM of some variant alloferon peptides according to one embodiment of the present invention, to study the degradation of mutant huntingtin protein aggregates caused by treatment with some variant alloferon peptides (DALS1, DAL-VS_V(-Q), DAL1 and DALV2) according to one embodiment of the present invention.

[0019] Figure 7 This is a graph depicting changes in body weight of Alzheimer's model animals after administering the variant alloferon peptide according to one embodiment of the present invention to the experimental animals for 25 weeks.

[0020] Figure 8 is a graph showing the ability of a variant alloferon peptide according to one embodiment of the present invention to enhance memory and learning as determined by the Morris water maze test.

[0021] Fig. 9 are a series of fluorescence micrographs (left) and graphs (right) quantifying the results of Thioflavin S staining of β-amyloid aggregate plaques in cortical (top) and hippocampal (bottom) sections from experimental animals administered with a variant alloferon peptide according to one embodiment of the present invention.

[0022] Fig.10 is a series of fluorescent micrographs (left) and graphs (right) showing quantification of β-amyloid aggregate plaques stained with Congo red in cortical (top) and hippocampal (bottom) sections from experimental animals administered a variant alloferon peptide according to one embodiment of the present invention.

[0023] FIG. 11A shows the results of Western blot analysis to determine the expression of various pathogenic aggregate-forming proteins in the cerebral cortex of experimental animals administered with a variant alloferon peptide according to one embodiment of the present invention, and FIG. 11B is a series of graphs of quantitative Western blot analysis results.

[0024] 12A shows the results of Western blot analysis to determine the expression of various pathogenic aggregate-forming proteins in the hippocampus of experimental animals administered with a variant alloferon peptide according to one embodiment of the present invention, and FIG. 12B is a series of graphs of quantitative Western blot analysis results.

[0025] Fig.13is a photograph depicting the results of Western blot analysis of spinal cord tissues of lumbar vertebrae obtained from ALS model animals which were sacrificed after the variant alloferon peptide according to one embodiment of the present invention was administered thereto. DETAILED DESCRIPTION Definition of terms:

[0026] As used herein, the term "zinc homeostasis" refers to the mechanism of maintaining a constant level of intracellular zinc, which is known to involve zinc transporters, zinc binding proteins (metallothioneins, MTs), and transcription factors (MTF1-2).

[0027] As used herein, the term "neurodegenerative disease" refers to a disease characterized by progressive loss of structure or function of nerves due to abnormal death of nerve cells. These neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD) and Huntington's disease (HD).

[0028] As used herein, the term "abnormal or pathological protein aggregates" refers to abnormal aggregation of proteins such as amyloid or tau in cells to form insoluble fibrils. These abnormal or pathological protein aggregates are known to be neuropathological hallmarks of various intermittent or hereditary neurodegenerative diseases.

[0029] As used herein, the term "alloferon" refers to a natural peptide consisting of 13 amino acids isolated from the blood of larvae of bacteria-infected red-headed blowflies (Calliphora vicina). Alloferon is known to be a non-toxic antiviral agent developed for the treatment of infections caused by influenza virus, herpes virus, etc. (Korean Patent No. 394864). DETAILED DESCRIPTION OF THE INVENTION

[0030] In one aspect of the present invention, a novel peptide with a length of 12 to 20 a.a. that is capable of inhibiting the formation of abnormal protein aggregates is provided, which comprises the amino acid sequence specified by SEQ ID NO: 1 or 2, wherein 1 to 4 of the amino acids other than glycine are L-type amino acids, and the other amino acids are D-type amino acids.

[0031] The peptide may comprise amino acids specified by SEQ ID NO: 1 or 2, wherein one, two, three or four amino acids other than glycine are L-form amino acids, and the other amino acids are D-form amino acids.

[0032] More specifically, if the peptide comprises the amino acid sequence designated as SEQ ID NO: 1, it may comprise 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 third and eleventh amino acids, valine, is an L-amino acid, or both are D-amino acids; iii) the fourth amino acid, serine, is a D-amino acid; iv) the eighth amino acid glutamine is a D-amino acid; and v) At least two of the above i to iv are D-amino acids.

[0033] In case the peptide comprises the amino acid sequence designated as SEQ ID NO: 2, it may comprise 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 third and tenth amino acids, valine, is an L-amino acid, or both are D-amino acids; iii) all fourth amino acids, serine, are D-amino acids; and iv) at least two of i to iii are D-amino acids.

[0034] According to a preferred embodiment, the peptide of the present invention is an L-type alloferon peptide of SEQ ID NO: 1, wherein at least three of the four histidines are substituted with D-type histidines, and in addition, at least two of the following amino acids are L-type or D-type amino acids or are missing: two valines, the third amino acid serine, and the eighth amino acid glutamine. Preferably, the peptide according to one embodiment of the present invention may comprise an amino acid sequence of any one of SEQ ID NOs: 2 to 14, and more preferably an amino acid sequence of any one of SEQ ID NOs: 3 to 5, 7, 8, 10, and 12 to 14.

[0035] like Figure 1 As shown in , the present inventors have found that the naturally occurring L-form of alloferon in vivo exhibits excellent activity under in vitro conditions, but has an extremely short half-life (T 1 / 2=51.77min), which makes it difficult to develop into a practical drug. Therefore, based on the fact that the alloferon peptide of the present invention not only exhibits biological activity by interacting with certain proteins in the body, but also acts as a zinc binding protein, and thus acts as a zinc homeostasis maintainer by transporting zinc from the outside of the cell to the cell, the inventors assume that the D-type peptide that does not naturally exist in the body can replace the L-type peptide that is easily degraded in the body. The inventors synthesized D-type alloferon and conducted the same experiment as using L-type alloferon, and proved that D-type alloferon exhibits the same biological activity as L-type alloferon. In addition, as a result of further research on whether D-type alloferon in which some amino acids are replaced by L-type amino acids will show similar functionality, it was found that replacing any of the histidines expected to act as zinc chelators in D-type alloferon with L-type amino acids, or the third amino acid valine and / or the fourth amino acid serine, or the eighth amino acid glutamine and / or the eleventh amino acid valine, respectively, with L-type amino acids, produces biological activities equivalent to or even better than D-type alloferon. Furthermore, the biological activity of the variant in which the eighth amino acid glutamine was removed was found to be optimal.

[0036] In another aspect of the present invention, a pharmaceutical composition for treating a neurodegenerative disease is provided, which comprises the above-mentioned peptide as an active ingredient.

[0037] In the pharmaceutical composition, the neurodegenerative disease may be a neurodegenerative disease having abnormal protein aggregate formation as its etiology or pathology, wherein the abnormal protein aggregate is formed by abnormal aggregation of α-synuclein, β-amyloid protein, TDP-34, p62, FUS protein, superoxide dismutase-1 (SOD-1), huntingtin protein or tau protein. The neurodegenerative disease may specifically be Alzheimer's disease (AD), which is associated with the accumulation of β-amyloid protein or tau protein aggregates; Parkinson's disease (PD), which is associated with the accumulation of α-synuclein aggregates; amyotrophic lateral sclerosis (ALS), which is associated with the accumulation of SOD-1 aggregates; Huntington's disease (HD), which is associated with the accumulation of huntingtin protein aggregates; chronic traumatic encephalopathy, which is associated with the accumulation of tau protein or TAR DNA-binding protein 43 (TDP-43) aggregates; Lytico-Bodig disease, which is associated with the accumulation of tau protein aggregates; frontotemporal lobar degeneration, which is associated with the accumulation of tau protein, TAR DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS) or p62 protein aggregates; corticobasal degeneration, which is associated with the accumulation of tau protein aggregates; progressive supranuclear palsy, which is associated with the accumulation of tau protein aggregates. In addition, diseases such as Creuzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, meningioangiomatosis, and neuronal ceroid lipofuscinosis are known to be associated with the accumulation of protein aggregates in nerves. These neurodegenerative diseases and their association with the accumulation of protein aggregates in nerves are described in detail in the prior art (Stromland et al., J. Clin. Transl. Res. 2(1): 11-26, 2016; Tutar et al., Neurodegenerative Diseases, published: May 15, 2013, DOI: 10.5772 / 54487; Diez-Ardanuy et al., Sci. Rep: 7(1): 10, 2017).

[0038] In the pharmaceutical composition, the peptide can treat neurodegenerative diseases by preventing neuronal cell death, regulating intracellular zinc homeostasis, and promoting lysosomal function.

[0039] The pharmaceutical composition according to one embodiment of the present invention may include a pharmaceutically acceptable carrier, and may further include other pharmaceutically acceptable adjuvants, excipients or diluents in addition to the carrier.

[0040] As used herein, the term "pharmaceutically acceptable" refers to physiologically acceptable and does not usually cause allergic reactions when applied to people, such as gastrointestinal disorders, dizziness or similar reactions. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, deflocculants, lubricants, wetting agents, spices, emulsifiers and preservatives may be further included.

[0041] In addition, the pharmaceutical composition according to one embodiment of the present invention can be formulated using methods known in the art to adjust the rapid release, sustained release or delayed release of the active ingredient after administration to a mammal. Preparations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions and sterile powder forms.

[0042] The pharmaceutical composition according to one embodiment of the present invention can be administered by various routes, such as orally, or parenterally, such as as a suppository, percutaneously, intravenously, intraperitoneally, intramuscularly, locally at the lesion site, intranasally or intrathecally. It can also be administered by using an implant device for sustained release, continuous release or repeated release. The frequency of administration can be once or multiple times per day in any desired range, and the duration of administration is not particularly limited.

[0043] The pharmaceutical composition according to one embodiment of the present invention can be administered by conventional systemic administration or local administration, such as by intramuscular or intravenous injection. In addition, the peptide according to one embodiment of the present invention can also be administered orally.

[0044] The pharmaceutical composition according to one embodiment of the present invention can be formulated in any suitable form together with any commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, water, suitable oil, saline, carriers for parenteral administration such as glucose aqueous solution and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants, such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl paraben or propyl paraben and chlorobutanol. Depending on the method or preparation used, the pharmaceutical composition according to the present invention may also include suspending agents, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorbents, surfactants, diluents, excipients, pH regulators, analgesics, buffers and antioxidants. Pharmaceutically acceptable carriers and preparations suitable for the present invention, including those illustrated above, are described in detail in the document [Remington's Pharmaceutical Sciences, latest edition].

[0045] The dosage of the pharmaceutical composition according to one embodiment of the present invention to the patient depends on many factors, including the patient's height, body surface area, age, specific compound administered, sex, time and route of administration, overall health status and other drugs administered simultaneously. The therapeutically active alloferon or polynucleotide encoding it can be administered in an amount of 100 ng to 1000 mg per body weight (kg), more preferably in an amount of 1 μg to 100 mg per body weight (kg), and most preferably in an amount of 5 to 40 mg per body weight (kg), and the dosage can be adjusted considering the above factors.

[0046] Furthermore, the pharmaceutical composition of the present invention is administered in a therapeutically effective amount.

[0047] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a condition at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the following and other factors known in the medical field: individual type, severity, age, sex, activity of the drug, sensitivity to the drug, administration time, route of administration and excretion level, duration of treatment, concomitant medication. The pharmaceutical composition of the present invention can be administered at a dosage of 0.1 mg / kg to 1 g / kg, more preferably at a dosage of 1 mg / kg to 500 mg / kg. At the same time, the dosage can be appropriately adjusted according to the patient's age, sex and condition.

[0048] In another aspect of the present invention, a method of treating a subject suffering from a neurodegenerative disease is provided, the method comprising administering to the subject a therapeutically effective amount of the peptide.

[0049] In another aspect of the present invention, a method for inhibiting the accumulation of pathogenic protein aggregates in the nervous system of a subject suffering from a neurodegenerative disease is provided, the method comprising administering a therapeutically effective amount of the peptide to the individual.

[0050] In any of the above methods, the composition can be administered by oral or parenteral administration as described above, and in the case of parenteral administration, it can be administered by systemic or local administration. In the case of systemic administration, it can be administered by intravenous injection, intraperitoneal injection or intramuscular injection. On the other hand, in the case of local administration, it can be administered by intracranial administration, intracerebrospinal administration or subcutaneous injection.

[0051] As used herein, the term "therapeutically effective amount" refers to an amount that produces a relieving, suppressing, improving and / or curing effect on the symptoms of the condition being treated. Mode for carrying out the invention

[0052] The present invention will now be described in more detail with reference to the following examples. However, the present invention is not limited to the embodiments disclosed herein, but can be implemented in many different forms, and the following embodiments are provided to complete the disclosure of the present invention and give those of ordinary skill in the art a complete concept of the scope of the present invention. Example 1: Preparation of peptides 1-1: Synthesis of L-type peptide

[0053] The L-type peptide (SEQ ID NO: 16, LAL) of alloferon (SEQ ID NO: 1) used in the present invention was synthesized by Corp. Peptron (Korea). 1-2: Synthesis of D-peptide

[0054] The D-form alloferon peptide (SEQ ID NO: 15, DAL1) in which all amino acids except glycine were substituted with D-form amino acids instead of L-form alloferon used in the present invention was also synthesized by Corp. Peptron (Korea) and Anigen (Korea). Experimental Example 1: In vitro plasma stability assay

[0055] Because the inventors believe that the mechanism of action of alloferon peptides is through endocytosis via multi-ligand receptors rather than physiological activity, and zinc binding ability is an important mechanism, they studied the blood stability of D-type alloferon of Example 1-2 (wherein all amino acids except glycine are replaced by D-type amino acids) and L-type alloferon of Example 1-1. In vitro plasma stability assays were performed to compare the stability of L-type and D-type peptides of alloferon in the plasma of mice. For this purpose, 500 to 600 μl of blood was obtained from ICR mice by orbital blood sampling, which was then centrifuged at 2000 × g at 4 ° C for 20 min using a centrifuge, and the supernatant was collected to obtain plasma. After adding 10 μM alloferon L-type and D-type peptides to 100 μL of plasma, the plasma was incubated at 37 ° C for 0, 5, 10, 30, 60 and 90 min. Then, 500 μl of cold methanol was added to precipitate the protein, and the protein was freeze-dried and subjected to liquid chromatography and protein spectrum analysis. The results showed that the alloferon L-type peptide was degraded early in plasma, while the D-type peptide remained stable over time without degradation ( Figure 1 ). 1-3: Design and synthesis of variant alloferon peptides

[0056] From the results of the above-mentioned Experimental Example 1, the present inventors investigated whether a variant alloferon peptide in the form of an L-type / D-type hybrid in which some amino acids are L-type amino acids, rather than a D-type peptide (DAL1) in which all amino acids except glycine are substituted with D-type amino acids, would exhibit equivalent biological activity. To this end, 12 variant alloferon peptides were synthesized in which one to four amino acids were L-type amino acids (Table 1). [Table 1] A peptide according to one embodiment of the present invention

[0057] The variant peptides include products in which one of the four histidines believed to be involved in zinc chelation is restored to the L-type (DALH1, DALH2, DALH3, DALH4), products in which at least one of the third, fourth and eleventh amino acids is restored to the L-type amino acid (DALVS, DALV2, DALV1 and DALS1), a product in which the eighth amino acid glutamine is restored to the L-type amino acid (DALQ1), and products in which at least three of the two valine, glutamine and serine are restored to the L-type amino acids (DALVSQV, DALVSqV and DALVS_V).

[0058] The synthesis of peptides was performed by Corp. Peptron (Korea) and Anigen (Korea). Experimental Example 2: Cell culture

[0059] H4 cell line (GL-H4) and HEK293T cell line permanently transfected with GFP-LC3 plasmid were used in the present invention. The cell culture medium was the minimum essential medium (MEM, WellGene) supplemented with 10% fetal bovine serum (Hyclone, USA) and antibiotic and antimycotic mixture (WellGene, South Korea), and cultured in a cell culture machine at 37°C and 5% CO2. Experimental Example 3: Analysis of autophagic flux blocked by bafilomycin A-1

[0060] In order to study the effect of peptides comprising the amino acid sequences designated as SEQ ID NOs: 3 to 16 on autophagic flux, the present inventors treated the H4 cell line (GL-H4) expressing GFP-linked LC3 with the peptides (20 μM) and / or the autophagy inhibitor bafilomycin A-1 (Baf A1, 100 nM) to inhibit autophagy, and then studied the effect of the variant alloferon peptide according to one embodiment of the present invention by fluorescence microscopy analysis.

[0061] As a result, Figures 2a to 2c The LC3 dots shown in the figure are caused by the LC3-GFP protein present in the autophagosome, which inhibits the entire autophagic process if not degraded by the lysosome, indicating the accumulation of autophagosomes. When the GL-H4 cell line was treated with 100 nM of bafilomycin (Baf A1) to inhibit the autophagic flow, there was a significant increase in the number and size of LC3 dots, which decreased after treatment with the variant alloferon peptide (20 μM) according to one embodiment of the present invention ( Figures 2a to 2c When quantification was performed by measuring the fluorescence intensity of all spots, the fluorescence intensity was similarly reduced after treatment with the variant alloferon peptide according to one embodiment of the present invention ( Figures 2d to 2f ). This indicates that the autophagosomes accumulated due to the blockage of autophagy are cleared by the variant alloferon peptide according to one embodiment of the present invention. In particular, Figure 2e and 2fAs shown in, compared with the D-type alloferon peptide (DAL1) in which all amino acids are replaced by D-type amino acids, among the variant alloferon peptides according to one embodiment of the present invention, those produced by restoring at least one to three amino acids of two valines, serine and glutamine to L-type amino acids (DALV1, DALS1, DALV2, DALVS and DALVSq) or those produced by restoring all amino acids except histidine to L-type (DALVSQV), and those produced by removing glutamine from DALVSQV (DALVS_V) exhibited excellent autophagy clearance activity. When at least three of the four amino acids were L-type amino acids, the autophagy killing activity was found to be optimal. Experimental Example 4: Effect of variant alloferon peptide on protein aggregate formation after overexpression of SOD1 protein analyze

[0062] The inventors then investigated whether some of the variant peptides (DALS1, DALVS_V, DAL1, DALV2) comprising the amino acid sequences designated as SEQ ID NOs: 3 to 16 could eliminate SOD1 aggregates formed after transient transfection of SOD1 protein into 293T cell lines. To this end, EGFP-SOD1 G93A DNA was transiently transfected into HEK293T cell lines using Lipofectamine 2000. After transfection, the cell lines were incubated for 30 h in a cell culture machine at 37 ° C and 5% CO2, and the culture medium was replaced with minimum essential medium (MEM, Gibco, USA). Subsequently, the cells were pretreated with 100 nM bafilomycin for 30 min, and then treated with μM variant alloferon peptides and incubated for 18 hours. After this, protease inhibitors and phosphatase inhibitors (2 μg / ml aprotinin, 2 μg / ml leupeptin, 1 μg / ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 5 mM NaF and 10 mM Na4P2O7) were placed in 6-well culture dishes with 200 μl per well to lyse the cells. The obtained cell lysate was first placed at 4°C for 30 min. Then, protein was quantified using a BCA protein assay kit (Pierce Biotechnology, USA) and centrifuged at 17,000 × g for 20 min, wherein the supernatant was discarded and only the cell debris was taken to obtain the protein. Then, 5× 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 quantitative sample, denatured at 95°C for 5 min, and prepared for analysis. Then, 8% to 15% SDS-polyacrylamide gel was used for electrophoresis, and the proteins separated by their size were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes of the transferred proteins were then blocked with 3% skim milk powder in TBST for 1 h. The blocked membranes were reacted with anti-GFP antibodies (Santa Cruz Biotechnology, USA), while anti-actin antibodies (Sigma, USA) were used as loading controls. Antibodies that recognize specific proteins were added to a solution of 1% BSA dissolved in TBST. For all blots, the secondary antibody was diluted and incubated at a ratio of 1:10,000 in 2% skim milk. Protein signals were detected using enhanced chemiluminescence (iNTRoN Biotechnology, South Korea) and a bioimaging system (MF-Chemibis, Shimadzu ScientifcKorea Corporation, South Korea).

[0063] As a result, Figure 3aAs shown in , it can be seen that bafilomycin increased SOD1 protein aggregates compared to the control. Here, some variant alloferon peptides according to one embodiment of the present invention reduced protein aggregates, and quantitatively demonstrated that some variant alloferon peptides according to one embodiment of the present invention eliminated aggregate accumulation. In particular, as Figure 3b As shown in, among the variant alloferon peptides according to one embodiment of the present invention, those produced by restoring the fourth and / or eleventh amino acid to L-type amino acids (DALS1, DALV2) and those produced by restoring at least three of two valines, glutamine and serine to L-type amino acids (DALVS_V) exhibited better protein aggregate decomposition ability compared to the D-type alloferon peptide (DAL1) in which all amino acids were substituted with D-type amino acids. Experimental Example 5: Effect of variant alloferon peptide on protein aggregate formation after overexpression of mutant tau protein Analysis of the sound

[0064] The present inventors then investigated whether some of the variant peptides (DALVS, DALVSqV, DAL1 and DALV2) comprising the amino acid sequences designated as SEQ ID NOs: 3 to 16 can eliminate mutant tau protein aggregates formed after transient transfection of mutant tau protein into HEK293T cell line. For this purpose, EGFP-Tau P301L DNA was transiently transfected into HEK293T cell line using Lipofecta mine 2000. Protein aggregate analysis was performed in the same manner as in Experimental Example 4 above.

[0065] As a result, Figure 4a As shown in , it can be seen that bafilomycin increased mutant tau protein aggregates compared to the control. Quantification also demonstrated that some variant alloferon peptides according to one embodiment of the present invention reduced protein aggregates, such as Figure 4b as shown in . Experimental Example 6: Effect of variant alloferon peptide on protein aggregate formation after α-synuclein overexpression Analysis of the sound

[0066] The present inventors then investigated whether some of the variant peptides (DALV1, DALQ1, DAL1, DALV2) comprising the amino acid sequences designated as SEQ ID NOs: 3 to 16 can eliminate α-synuclein aggregates formed after transient transfection of α-synuclein into HEK293T cell lines. For this purpose, EGFP-α-synuclein A53T DNA was transiently transfected into HEK293T cell lines using Lipofectamine 2000. Protein aggregate analysis was performed in the same manner as in Experimental Example 4 above.

[0067] As a result, in Figure 5 As can be seen in Figure 1, bafilomycin increased α-synuclein aggregates compared to the control. It was also demonstrated that some variant alloferon peptides according to one embodiment of the present invention reduced protein aggregates. In this case, DALQ1 and DALV2 also showed better protein aggregate decomposition ability compared to the D-type alloferon peptide (DAL-1) in which all amino acids are replaced by D-type amino acids. Experimental Example 7: Effect of variant alloferon peptide on protein aggregate formation after overexpression of mutant huntingtin protein Impact Analysis

[0068] The present inventors then investigated whether some of the variant peptides (DALH3, DALH1, DAL1 and DALV2) comprising the amino acid sequences designated as SEQ ID NOs: 3 to 16 can eliminate mutant huntingtin protein aggregates formed after transient transfection of 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 above.

[0069] As a result, in Figure 6 As can be seen in the results, bafilomycin increased mutant huntingtin protein aggregates compared to the control, and some of the variant alloferon peptides according to one embodiment of the present invention reduced protein aggregates. In this case, DALH1 and DALV2 also showed better protein aggregate decomposition ability compared to the D-type alloferon peptide (DAL1) in which all amino acids were substituted with D-type amino acids. Experimental Example 8: Animal Experiment on Alzheimer's Disease 8-1: Body weight changes after administration of variant alloferon peptide

[0070] The present inventors conducted animal experiments using disease model animals to determine whether the variant alloferon peptide from the results of the above-mentioned Experimental Experiments 4 to 7 according to one embodiment of the present invention can be used as a therapeutic agent for various neurodegenerative diseases.

[0071] First, in order to determine whether it is toxic when administered in vivo, dementia model animals (5XFAD) were administered 20 mg / kg of a variant alloferon peptide (DALV2) according to one embodiment of the present invention by intraperitoneal (IP) injection once a day, five days a week (Monday to Friday) for 25 weeks to study changes in body weight. DAL in which all amino acids are D-type was used as a control.

[0072] As a result, Figure 7As shown in , body weight gain was consistent over the 25-week injection period with no differences between treatment groups. 8-2: Morris water maze test

[0073] Since the inventors observed from the results of the above-mentioned Experimental Examples 4 to 7 that the variant alloferon peptide according to one embodiment of the present invention decomposes abnormal protein aggregates, they intraperitoneally administered the variant alloferon peptide according to one embodiment of the present invention to Alzheimer's model mice (5XFAD) at a concentration of 20 mg / kg once a day, 5 days a week (Monday to Friday) for 25 weeks, and observed whether the memory of the experimental animals was improved from day 1 to day 6 after 25 weeks of administration by Morris water maze test.

[0074] As a result, Figure 8 As shown in , the peptide according to one embodiment of the present invention significantly restored the memory and learning ability of Alzheimer's model animals. 8-3: Analysis of the inhibition of β-amyloid aggregate accumulation in brain tissue

[0075] In order to determine whether the improvement in the behavioral index observed in the above Experimental Example 8-2 was due to the inhibition of the accumulation of β-amyloid aggregates in the brain, the present inventors sacrificed Figure 8 Brain tissue sections were obtained after Morris water maze testing of the animals shown in. These sections were stained with Thioflavin S, which has a selective affinity for β-amyloid plaques, in order to visualize β-amyloid aggregates.

[0076] Specifically, for Thioflavin S staining, the brain of the dementia model mouse was harvested and freeze-dried in OCT compound (Sakurafintec, USA), and brain slices were obtained by coronal sectioning and fixed on a slide coated with 0.1% poly-L-lysine. The obtained brain slices were washed in 70% ethanol for 1 min, then washed again in 80% ethanol for 1 min, and stained in 1% Thioflavin S solution (MilliporeSigma, USA) for 15 min. After this, it was washed in 80% ethanol for 1 min, washed again in 70% ethanol for 1 min, and then washed twice in distilled water. Finally, β-amyloid senile plaques were observed by fluorescence microscopy.

[0077] Congo red staining was performed as follows for identification of amyloid. The brain slices obtained by coronal sectioning were fixed on a slide coated with 0.1% poly-L-lysine. The brain slices obtained were washed in phosphate buffered saline (PBS) solution for 2 min, and then stained in 0.5% Congo red solution (Thermo Fisher, USA) for 20 min. After washing the Congo red solution in PBS solution, the slices were quickly immersed and taken out 10 times in an alkaline alcohol solution containing 0.01% sodium hydroxide. The slices were washed in PBS solution for 3 min, washed in 95% ethanol for 3 min, and then washed in 100% ethanol for 3 min to dehydrate. Finally, amyloid was observed under a fluorescent microscope, and the number of stained senile plaques and amyloid of each brain slice was counted and quantified with a statistical program (Graph Prism).

[0078] As a result, Fig. 9 and 10 As shown in , in the Alzheimer's model animals, a significant number of β-amyloid protein plaques were identified, but neither the control DAL1 nor the variant alloferon peptide according to one embodiment of the present invention significantly inhibited the accumulation of β-amyloid protein aggregates. 8-4: Analysis of the effects on the expression of different aggregate-forming proteins in the brain

[0079] The present inventors performed Western blot analysis to investigate the expression of other pathogenic aggregate-forming proteins, such as tau protein, p62, and LC3, in brain tissues in addition to β-amyloid protein from the results of the above-mentioned Experimental Examples 8-2 and 8-3.

[0080] Specifically, cortical and hippocampal tissues were obtained from experimental animals sacrificed after behavioral experiments, and the tissues were lysed and analyzed by western blot using antibodies that specifically bind to phosphorylated tau S214 (P-Tau S214), phosphorylated tau T205 (P-Tau T205), unphosphorylated tau, p62, LC3 and β-amyloid protein (APP), respectively.

[0081] As a result, as shown in Figures 11A to 12B, compared with normal mice as controls, in Alzheimer's disease model mice, all six proteins in the cerebral cortex and hippocampus increased, while animals treated with the control DAL1 or a peptide (DALV2) according to one embodiment of the present invention showed significantly lower expression of the above-mentioned aggregate-forming proteins in the cortex and hippocampus. In the cortical protein blot analysis, DALV2 showed better protein aggregate decomposition ability compared with the D-type alloferon peptide (DAL1) in which all amino acids were replaced by D-type amino acids. Protein blot analysis of hippocampal slices showed that DALV2 significantly reduced the amount of β-amyloid protein, while DAL1 did not show significance in reducing β-amyloid protein levels. These results show that the peptide according to one embodiment of the present invention can inhibit the formation of pathogenic protein aggregates in the brain after in vivo administration, and DALV2 has excellent inhibition. Experimental Example 9: Analysis of the Effect on Amyotrophic Lateral Sclerosis Model Animals

[0082] In order to determine whether the variant alloferon peptide according to one embodiment of the present invention has a therapeutic effect on amyotrophic lateral sclerosis (ALS) in vivo, the present inventors administered a peptide (DALV2) according to one embodiment of the present invention to an amyotrophic lateral sclerosis model animal (SOD1G93A) aged 112 to 180 days. The peptide was administered at a dose of 40 mg / kg by subcutaneous (SC) injection repeated five days a week (Monday to Friday). After the experiment, the animals were sacrificed and spinal cord sections from the lumbar region were obtained and subjected to Western blot analysis using an anti-SOD-1 antibody.

[0083] As a result, Fig.13 As shown in , significantly lower levels of SOD-1 aggregates were detected in ALS model animals treated with a variant alloferon peptide according to one embodiment of the present invention. This indicates that a variant alloferon peptide according to one embodiment of the present invention inhibits the accumulation of pathogenic protein aggregates in the nervous system even in vivo.

[0084] The present invention has been described with reference to the above-mentioned embodiments, but these are only exemplary, and those skilled in the art will understand that various modifications and other equivalent effective embodiments can be obtained from them. Therefore, the true scope of the technical protection of the present invention should be determined by the technical concept of the attached claims of this patent. Industrial Applicability

[0085] By inhibiting abnormal hyperphosphorylation of pathogenic proteins and the resulting aggregate formation in the central nervous system and removing parasitic protein aggregates, the peptide according to one embodiment of the present invention can be developed as a therapeutic agent for various neurodegenerative diseases caused by pathogenic protein aggregates, such as Alzheimer's disease and amyotrophic lateral sclerosis.

Claims

1. A novel peptide having a length of 12 to 20 a.a. that can inhibit the formation of abnormal protein aggregates, the novel peptide comprising the amino acid sequence specified by SEQ ID NO: 1 or 2, wherein 1 to 4 of the amino acids other than glycine are L-type amino acids, and the other amino acids are D-type amino acids.

2. The peptide according to claim 1, wherein in the case of comprising the amino acid sequence designated as SEQ ID NO: 1, the peptide comprises 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 third and eleventh amino acids, valine, is an L-amino acid, or both are D-amino acids; iii) the fourth amino acid, serine, is a D-amino acid; iv) the eighth amino acid glutamine is a D-amino acid; and v) At least two of the above i to iv are D-amino acids.

3. The peptide according to claim 1, wherein in the case of comprising the amino acid sequence designated as SEQ ID NO: 2, the peptide comprises 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 third and tenth amino acids, valine, is an L-amino acid, or both are D-amino acids; iii) the fourth amino acid, serine, is a D-amino acid; and iv) at least two of i to iii are D-amino acids.

4. The peptide according to claim 1, wherein 1) at least three of the four histidines in the L-alloferon peptide designated as SEQ ID NO: 1 are substituted with D-histidines, and at least two of the two valines, the third amino acid serine, and the eighth amino acid glutamine are D-amino acids; or 2) All four histidines in the L-alloferon peptide designated as SEQ ID NO: 1 are D amino acids, and the eighth amino acid glutamine is an L or D amino acid or is missing. 5 . The peptide according to claim 1 , comprising an amino acid sequence designated as any one of the amino acid sequences designated as SEQ ID NOs: 3 to 14. 6 . The peptide according to claim 1 , comprising the amino acid sequence of any one of SEQ ID NOs: 3 to 5, 7, 8, 10, and 12 to 14.

7. A pharmaceutical composition for treating a neurodegenerative disease, comprising the peptide according to any one of claims 1 to 6 as an active ingredient.

8. The pharmaceutical composition according to claim 7, wherein the neurodegenerative disease is a neurodegenerative disease having the formation of abnormal protein aggregates as its etiology or pathology.

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

10. The pharmaceutical composition according to claim 8, wherein the neurodegenerative disease is Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), chronic traumatic encephalopathy (CTE), Lytico-bodig disease (LBD), frontotemporal lobar degeneration (FTD), corticobasal degeneration or progressive supranuclear palsy.

11. A method of treating a subject suffering from a neurodegenerative disease, the method comprising administering to the subject a therapeutically effective amount of the peptide of any one of claims 1 to 6.

12. A method for inhibiting the accumulation of pathogenic protein aggregates in the nervous system of a subject suffering from a neurodegenerative disease, the method comprising administering to the subject a therapeutically effective amount of the peptide of any one of claims 1 to 6.

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