Novel peptides and their applications

Abstract

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

Description

Technical Field

[0001] The present invention relates to a novel peptide and its use, and more specifically, to a novel peptide based on allopheron 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 neurons is known to play an important role in the survival of neurons. However, when zinc is deficient in neurons, apoptosis is induced, leading to neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (Lien, H. et al., BBRC. 268: 148 - 154, 2000). Conversely, when zinc is present in excess in neurons, cell damage is induced, which is known to trigger acute brain injury diseases such as ischemia and seizure (Koh et al., Science. 272: 1013 - 1016, 1996).

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

[0004] Recently, it has been found that protein aggregates appearing in degenerative neurological diseases, such as those of α-synuclein, β-amyloid, tau protein, superoxide dismutase-1 (SOD-1), huntingtin protein, and TAR DNA-binding protein 43 (TDP-43), can be removed if autophagy is promoted. In fact, there are studies showing that when there is a defect in the function of autolysosomes, the degradation of such protein aggregates is inhibited, blocking the autophagy flux, and ultimately leading to the accumulation of byproducts of degenerative neurological diseases (Zhang et al., ABBS. 41(6): 437-445, 2009; Lee et al., Cell. 141(7): 1146-1158, 2010).

[0005] Previous research has revealed that supplying zinc improves lysosome function, and studies have shown that treating lysosomal membrane permeabilization (LMP) induced by H2O2, tamoxifen, or ethanol with TPEN, a potent zinc chelating agent, suppresses the LMP phenomenon. Conversely, if LMP activity is reduced, additional zinc supply promotes 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). Therefore, it is presumed that zinc has an important influence on autophagy.

[0006] However, no drugs have yet been developed that can treat degenerative neurological diseases by maintaining zinc homeostasis within nerve cells. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention aims to solve a variety of problems, including the aforementioned problems, and to provide a novel peptide and its applications in the treatment of degenerative neurological diseases that can treat degenerative neurological diseases by maintaining zinc homeostasis in nerve cells, thereby removing abnormal or pathological protein aggregates such as amyloid-beta peptide, tau protein, and excess peroxide disequilibrium enzymes, which are pathological substances of degenerative neurological diseases, through autophagy or improved lysosomal function. [Means for solving the problem]

[0008] According to one aspect of the present invention, a novel peptide is provided which has the ability to suppress the formation of abnormal protein aggregates and is 12 to 20 a.a. in length, comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, wherein, excluding glycine, 1 to 4 amino acids are L-type amino acids and the remaining amino acids are D-type amino acids.

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

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

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

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

[0013] [Figure 1] Figure 1 shows the results of examining whether alloferon L-type peptide and alloferon D-type peptide are degraded in mouse plasma. Each peptide was treated with 10 μM of alloferon peptide in isolated plasma and reacted at 37°C for 0, 10, 30, 60, and 90 minutes, respectively. The resulting quantitative changes were then measured by mass spectrometry.

[0014] [Figure 2] Figures 2A to 2C show results demonstrating that the autophagy flow blocked by bafilomycin (Bafilomycin A-1) is restored by the mutant alloferon peptide according to one embodiment of the present invention. These are a series of fluorescence micrographs of LC3-GFP spots taken 12 hours after treatment of various mutant alloferon peptides according to one embodiment of the present invention in combination with bafilomycin at a concentration of 20 μM in H4 cell lines (GL-H4) in which the GFP-LC3 protein is stably overexpressed. Figures 2D to 2F are graphs showing the quantified fluorescence intensity of LC3-GFP spots after 12 hours after treatment of the GL-H4 cell line in combination with bafilomycin at a concentration of 20 μM in combination with mutant alloferon peptides according to one embodiment of the present invention. After bafilomycin treatment, it was confirmed that the autophagy was blocked and the number of spots increased significantly. However, when the mutant alloferon peptide according to one embodiment of the present invention was used for treatment, the number and size of the spots decreased, which is a result of the elimination of the autophagy. In Figures 2D to 2F, * indicates a significant difference compared to bafilomycin treatment alone (*p<0.05, **p<0.01, ***p<0.001), and # indicates a meaningful difference when the peptide according to one embodiment of the present invention is used compared to treatment with bafilomycin and conventionally reported type D alloferon (DAL-1) (#p<0.05).

[0015] [Figure 3A] Figure 3A shows the results of observing whether SOD1 protein aggregates are degraded by treatment with certain mutant alloferon peptides (DALS1, DAL-VS_V(-Q), DAL1, and DALV2) according to one embodiment of the present invention. The results show Western blot analysis of protein samples obtained 18 hours after treatment of HEK293T cells trans-injected to express SOD1 with 100 nM bafilomycin A1 alone, and with 100 nM bafilomycin A1 and 30 μM of certain mutant alloferon peptides according to one embodiment of the present invention. [Figure 3B]Figure 3B is a graph showing the results of quantifying the relative expression levels. In Figure 3B, the asterisk (*) indicates a significant difference compared to bafilomycin treatment alone (*p<0.05).

[0016] [Figure 4A] Figure 4A shows the results of observing whether mutant tau protein aggregates are degraded by treatment with certain mutant alloferon peptides (DALVS, DAL-VSqV, DAL1, and DALV2) according to one embodiment of the present invention. The results show the results of Western blot analysis of the obtained protein samples after 18 hours of treatment of HEK293T cells transformed to express mutant tau protein with 100 nM bafilomycin A1 alone, and with 100 nM bafilomycin A1 and 30 μM of certain mutant alloferon peptides according to one embodiment of the present invention. [Figure 4B] Figure 4B is a graph showing the results of quantifying the relative expression levels. In Figure 3B, the asterisk (*) indicates a significant difference compared to bafilomycin treatment alone (**p<0.01).

[0017] [Figure 5] Figure 5 shows the results of observing whether α-synuclein protein aggregates are degraded by treatment with certain mutant alloferon peptides (DALV1, DALQ1, DAL1, and DALV2) according to one embodiment of the present invention. The results show the results of Western blot analysis of protein samples obtained 18 hours after treatment of HEK293T cells transformed to express α-synuclein protein with 100 nM bafilomycin A1 alone, and with 100 nM bafilomycin A1 and 30 μM of certain mutant alloferon peptides according to one embodiment of the present invention.

[0018] [Figure 6]Figure 6 shows the results of observing whether mutant huntingtin protein aggregates are decomposed by treatment with some mutant allorepheron peptides (DALH3, DALH1, DAL1, DALV2) according to an embodiment of the present invention. Western blot analysis results are shown, which measured the expression level of mutant huntingtin protein for the protein samples obtained after 18 hours of treatment with 100 nM bafilomycin A1 alone or in combination with 30 μM of some mutant allorepheron peptides according to an embodiment of the present invention in HEK293T cells transformed to express mutant huntingtin protein.

[0019] [Figure 7] Figure 7 is a graph showing the change in body weight of experimental animals when a mutant allorepheron peptide according to an embodiment of the present invention was administered to Alzheimer's model animals for 25 weeks.

[0020] [Figure 8] Figure 8 is a graph showing the ability of a mutant allorepheron peptide according to an embodiment of the present invention to improve memory and learning, which was confirmed through the Morris water maze test.

[0021] [Figure 9] Figure 9 is a series of fluorescence micrographs (left side) showing the results of staining β-amyloid aggregate plaques in cerebral cortex (upper panel) and hippocampus (lower panel) slices of experimental animals administered with a mutant allorepheron peptide according to an embodiment of the present invention with thioflavin S, and a graph (right side) showing the quantification thereof.

[0022] [Figure 10] Figure 10 is a series of fluorescence micrographs (left side) showing the results of staining β-amyloid aggregate plaques in cerebral cortex (upper panel) and hippocampus (lower panel) slices of experimental animals administered with a mutant allorepheron peptide according to an embodiment of the present invention with Congo red, and a graph (right side) showing the quantification thereof.

[0023] [Figure 11A]Figure 11A shows the results of a Western blot analysis to confirm the expression levels of various pathogenic aggregate-forming proteins in the cerebral cortex of experimental animals administered a mutant alloferon peptide according to one embodiment of the present invention. [Figure 11B] Figure 11B is a series of graphs that quantitatively show the results of the Western blot analysis.

[0024] [Figure 12A] Figure 12A shows the results of a Western blot analysis to confirm the expression levels of various pathogenic aggregate-forming proteins in the hippocampus of experimental animals administered a mutant alloferon peptide according to one embodiment of the present invention. [Figure 12B] Figure 12B is a series of graphs that quantitatively show the results of the Western blot analysis.

[0025] [Figure 13] Figure 13 is a photograph showing the results of a Western blot analysis performed on spinal cord tissue obtained from the lumbar region after subcutaneous administration of a mutant alloferon peptide according to one embodiment of the present invention to an ALS model animal according to a predetermined schedule, and after the experimental animals were sacrificed following the completion of the experiment. [Modes for carrying out the invention]

[0026] Definitions of terms:

[0027] As used herein, the term "zinc homeostasis" refers to the mechanism that maintains the concentration of zinc within a cell at a constant level. It is known that zinc transporters, zinc-binding proteins (metallothioneins, MTs), and transcription factors (MTF1-2) are involved in maintaining the concentration of zinc within a cell at a constant level.

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

[0029] As used herein, the term “abnormal or pathological protein aggregates” refers to abnormal aggregation of proteins such as amyloid or tau within cells, forming insoluble fibrills. Such abnormal or pathological protein aggregates are known as neuropathological features of a variety of intermittent or hereditary degenerative neurological disorders.

[0030] As used herein, the term "alloferon" refers to a 13-amino acid-based natural peptide isolated from the blood of bacterial-infected Calliphora vicina insect larvae, and is known as a non-toxic antiviral agent developed for the treatment of infections such as influenza virus and herpes virus (Korean Patent No. 394864).

[0031] Detailed description of the invention:

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

[0033] The peptide in question has an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, in which one, two, three, or four amino acids excluding glycine are L-type amino acids, and the remaining amino acids are D-type amino acids.

[0034] More specifically, if the peptide contains the amino acid sequence shown in SEQ ID NO: 1, it may contain at least one of the following: i) At least one of the four histidines is a D-type amino acid; ii) At least one of the valine amino acids at the 3rd and 11th positions is an L-type amino acid, or both are D-type amino acids; iii) The fourth amino acid, serine, is a D-type amino acid; iv) The eighth amino acid, glutamine, is a D-type amino acid; and v) Two or more of the above i through iv are D-type amino acids.

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

[0036] In a preferred embodiment, the peptide of the present invention is the L-type allopherone peptide of SEQ ID NO: 1, in which at least three of the four histidines are replaced with D-type histidines, and at least two of the two valine, the third amino acid serine, and the eighth amino acid glutamine are D-type amino acids, or all four histidines are D-type amino acids, and the eighth amino acid glutamine is either an L-type or D-type amino acid or deleted. Preferably, the peptide according to one embodiment of the present invention may contain any one amino acid sequence from SEQ ID NOs: 2 to 14, and more preferably, it may contain any one amino acid sequence from SEQ ID NOs: 3 to 5, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 10, and SEQ ID NOs: 12 to 14.

[0037] As can be seen from Figure 1, the inventors confirmed that while L-type alloferon, which naturally exists in the body, showed excellent activity under test tube conditions, it has characteristics that make it difficult to develop as an actual pharmaceutical product when administered in vivo, such as an extremely short half-life (T1 / 2 = 51.77 minutes). Based on this, the inventors hypothesized that since the alloferon peptide of the present invention not only exhibits physiological activity by interacting with specific proteins in the body, but also acts as a zinc-binding protein, supplying extracellular zinc into cells and acting as a type of zinc homeostasis maintainer, it would still be functional even if it were manufactured as the D-type, which does not naturally exist in the body and is not the L-type peptide that is easily degraded in the body. As a result of manufacturing D-type alloferon and conducting the same experiments as those using L-type alloferon, they confirmed that D-type alloferon also has the same biological activity as L-type alloferon. Furthermore, the inventors conducted additional research to determine whether substituting some of the amino acids in the D-type alloferon peptide back into the L-type would result in similar functionality. As a result, they confirmed that substituting one of the histidine amino acids, which is expected to perform zinc chelation, with an L-type amino acid, or substituting the third amino acid (valine) and / or the fourth amino acid (serine) with L-type amino acids, or substituting the eighth amino acid (glutamine) and / or the eleventh amino acid (valine) with L-type amino acids, results in biological activity equivalent to or even superior to that of D-type alloferon, thus completing the present invention. Moreover, they were able to confirm that the most superior biological activity was observed in the mutant in which the eighth amino acid (glutamine) was removed.

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

[0039] In the above pharmaceutical composition, the degenerative neurological disease is a degenerative neurological disease having the formation of abnormal protein aggregates as an etiological or pathological phenomenon, and the abnormal protein aggregates are formed by the abnormal aggregation of α-synuclein, β-amyloid, TDP-34, p62, FUS protein, superperoxide disequilibrium-1 (SOD-1), huntingtin protein, or tau protein. Degenerative brain diseases having the formation of the aforementioned abnormal protein aggregates as an etiology or pathological phenomenon include, 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, and 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. This includes degeneration, 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, and neuronal ceroid lipofuscinoses are also known to be diseases that correlate with the accumulation of protein aggregates in nerves. The correlation between the aforementioned degenerative neurological diseases and the accumulation of protein aggregates in nerves has been well described in previous literature (Strömland et al., J.Clin.Transl.Res.2(1):11-26, 2016; Tutar et al., Neurodegenerative Diseases, published: May 15). th ,2013,DOI:10.5772 / 54487;Diez-Ardanuy et al.,Sci.Rep.,7(1):10,2017).

[0040] In the above composition, the peptide can treat the degenerative neurological disease by preventing cell death in nerve cells, regulating intracellular zinc homeostasis, and promoting lysosomal function.

[0041] A pharmaceutical composition according to one embodiment of the present invention may contain a pharmaceutically acceptable carrier, and may further contain pharmaceutically acceptable auxiliaries, excipients, or diluents in addition to the carrier.

[0042] As used herein, the term "pharmaceutically acceptable" means a composition that is physiologically acceptable and, when administered to humans, does not cause typical gastrointestinal disturbances, allergic reactions such as dizziness, or similar reactions. Examples of 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. They may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

[0043] Furthermore, a pharmaceutical composition according to one embodiment of the present invention may be formulated using methods known to those skilled in the art to enable rapid release, or sustained or delayed release of the active ingredient upon administration to a mammal. The dosage forms include powder, granules, tablets, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injection solution, and sterile powder.

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

[0045] A pharmaceutical composition according to one embodiment of the present invention is administered by general systemic or topical methods, such as intramuscular or intravenous injection. Furthermore, the peptide according to one embodiment of the present invention can also be administered orally.

[0046] A pharmaceutical composition according to one embodiment of the present invention is formulated into a suitable dosage form with a commonly used pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include water, suitable oils, saline solutions, parenteral carriers such as aqueous glucose and glycol, 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. Furthermore, the composition according to the present invention may appropriately contain suspending agents, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, antioxidants, etc., as needed depending on the method of administration and dosage form. Suitable pharmaceutically acceptable carriers and formulations for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].

[0047] The dosage of a pharmaceutical composition according to one embodiment of the present invention for a patient varies depending on many factors, including the patient's height, body surface area, age, the specific compound administered, sex, time and route of administration, general health, and other drugs administered concurrently. The therapeutically active alloferone or the polynucleotide encoding it is administered in amounts of 100 ng / kg to 1000 mg / kg, more preferably 1 μg / kg to 100 mg / kg, and most preferably 5 to 40 mg / kg, but the dosage is adjusted considering the aforementioned factors.

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

[0049] As used herein, the term "therapeutably effective dose" means an amount sufficient to treat a disease in a reasonable benefit-to-risk ratio applicable to medical treatment. The effective dose level may be determined by factors including the individual's species and severity, age, sex, drug activity, sensitivity to the drug, administration time, route of administration and elimination ratio, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical compositions of the present invention are administered in doses of 0.1 mg / kg to 1 g / kg, and more preferably, in doses of 1 to 500 mg / kg. On the other hand, the dose is appropriately adjusted according to the patient's age, sex, and condition.

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

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

[0052] In the above method, the composition is administered orally or parenterally, as described above. In the case of parenteral administration, it can be administered by any route, whether systemic or local. In the case of systemic administration, it can be administered by intravenous injection, intraperitoneal injection, or intramuscular injection. In the case of local administration, it can be administered by intracerebral administration, intracerebral administration, subcutaneous injection, etc.

[0053] As used herein, the term "therapeutic dose" means a dose that produces a mitigating, suppressing, improving, and / or curative effect on the symptoms of the disease being treated.

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

[0055] Example 1: Peptide preparation 1-1: Synthesis of L-type peptides The L-type peptide (SEQ ID NO: 16, LAL) of alloferon (SEQ ID NO: 1) used in this invention was synthesized by Peptron Corporation (South Korea).

[0056] 1-2: Synthesis of D-type peptides In addition to the L-type alloferon used in this invention, a D-type alloferon peptide (SEQ ID NO: 15, DAL1), in which all amino acids except glycine are replaced with D-type amino acids, was also synthesized by commissioning Peptron Corporation (South Korea) and Eniegen (South Korea).

[0057] Experimental Example 1: Analysis of plasma stability in a test tube The inventors hypothesized that the mechanism of action of alloferon peptide is not physiological activity but rather through endocytosis via multi-ligand receptors, with zinc binding ability playing a crucial role. Therefore, they investigated the blood stability of D-type alloferon from Examples 1-2, in which all amino acids except glycine are replaced with D-type amino acids, and L-type alloferon from Example 1-1. To compare the stability of L-type and D-type alloferon peptides in mouse plasma, in vitro plasma stability analysis was performed. For this purpose, 500-600 μl of blood was obtained from ICR rats via orbital blood collection. The blood was then centrifuged at 4°C at 2000xg for 20 minutes, and only the supernatant was collected to obtain plasma. 10 μM alloferon L-type and D-type peptides were added to 100 μl of plasma, and the mixtures were left to stand at 37°C for 0, 5, 10, 30, 60, and 90 minutes. Next, 500 μl of cold methanol was added to precipitate the proteins, and after lyophilization, liquid chromatography and protein mass spectrometry were performed. As a result, it was found that alloferon L-type peptide was degraded in the plasma from an early stage, while D-type peptide remained stable and undegraded over time (Figure 1).

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

[0059] [Table 1]

[0060] The aforementioned mutant peptides consisted of those in which one of the four histidines presumed to be associated with zinc chelation was reduced to the L-type (DALH1, DALH2, DALH3, DALH4), those in which at least one of the 3rd, 4th, and 11th amino acids was reduced to the L-type amino acid (DALVS, DALV2, DALV1, DALS1), those in which the 8th amino acid, glutamine, was reduced to the L-type amino acid (DALQ1), and those in which at least three of the two valine, glutamine, and serine amino acids were reduced to the L-type amino acid (DALVSQV, DALVSqV, and DALVS_V).

[0061] Peptide synthesis was outsourced to Peptron Corporation (South Korea) and Eniegen (South Korea).

[0062] Experimental Example 2: Cell Culture In this invention, H4 cell lines (GL-H4) and HEK293T cell lines permanently infected with the GFP-LC3 plasmid were used. As the cell culture medium, Minimum Essential Medium (MEM, WellGene) was used, supplemented with 10% fetal bovine serum (Hyclone, USA) and a mixed solution of antibiotics and antifungals (WellGene, South Korea). The cells were cultured in a cell incubator maintained at 37°C and 5% CO2.

[0063] Experimental Example 3: Analysis of self-predation flow blocked by bafilomycin (A-1) To investigate the effect of peptides composed of amino acid sequences shown in SEQ ID NOs: 3 to 16 on the cell flow, the inventors suppressed autophagy in H4 cell lines (GL-H4) expressing GFP-linked LC3 using the peptide (20 μM) and / or the autophagy inhibitor bafilomycin (A-1, Baf A1, 100 nM). Then, they investigated the effect of a mutant allopheron peptide according to one embodiment of the present invention by fluorescence microscopy analysis.

[0064] As a result, the LC3 spots shown in Figures 2A to 2C are a phenomenon caused by the LC3-GFP protein present in the autopredators, and if they are not degraded by lysosomes, it means that the entire autopredation process is suppressed and autopredators accumulate. When the GL-H4 cell line was treated with 100 nM bafilomycin (Baf A1) to suppress the autopredation flow, the number and size of LC3 spots increased significantly, but this phenomenon decreased when treated with the mutant alloferon peptide (20 μM) according to one embodiment of the present invention (Figures 2A to 2C). Similarly, when the fluorescence intensity of the overall spots was measured and quantified, the fluorescence intensity decreased when treated with the mutant alloferon peptide according to one embodiment of the present invention (Figures 2D to 2F). This means that the autopredators accumulated by the blocked autopredation process are eliminated by the mutant alloferon peptide according to one embodiment of the present invention. In particular, as shown in Figures 2E and 2F, among the mutant allopheron peptides according to one embodiment of the present invention, when at least 1 to 3 of the two valine, serine, and glutamine amino acids were reduced to L-type amino acids (DALV1, DALS1, DALV2, DALVS, and DALVSq), or when all amino acids except histidine were reduced to L-type (DALVSQV), and when glutamine was removed from DALVSQV (DALVS_V), the self-predator elimination activity was even better than that of the D-type allopheron peptide (DAL1) in which all amino acids were substituted with D-type amino acids. The self-predator elimination activity was confirmed to be best when at least 3 or more of the four amino acids were L-type amino acids.

[0065] Experimental Example 4: Analysis of the effect of mutant alloferon peptide on the formation of protein aggregates that appear after SOD1 protein overexpression. Subsequently, the inventors investigated whether certain mutant peptides (DALS1, DALVS_V, DAL1, DALV2) composed of amino acid sequences shown in SEQ ID NOs. 3 to 16 could resolve the resulting SOD1 aggregates after transient transduction of SOD1 protein into the 293T cell line. For this purpose, EGFP-SOD1 G93A DNA was transiently transdone into the HEK293T cell line using Lipofectamine 2000. After transduction, the cells were cultured for 30 hours in a cell incubator maintained at 37°C and 5% CO2, and the culture medium was replaced with Minimum Essential Medium (MEM, Gibco, USA). Afterward, the cells were pre-treated with 100 nM bafilomycin for 30 minutes, then treated with 30 μM mutant alloferon peptide, and cultured for 18 hours. Subsequently, Triton X-100 lysis buffer (30 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA), to which protein hydrolase inhibitors and phosphate hydrolase 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) were added, and 200 μl was placed per well in a 6-well culture dish to lyse the cells. The obtained cell lysates were left at 4°C for 30 minutes. After quantification of the obtained cell lysates using the BCA protein assay kit (Pierce Biotechnology, USA), the cells were centrifuged at 17,000xg for 20 minutes, the supernatant was discarded, and the cell residue was collected to obtain the protein extract. Next, the 5X sample buffer (300 mM Tris, pH 6.8, 10% SDS, 50% Glycerol, 0.1% BPB, 2.5% Mercaptoethanol, 100 mM DTT) was mixed with the quantified sample, and the sample was prepared by denaturing it at 95°C for 5 minutes.Subsequently, proteins separated by size by electrophoresis using 8%-15% SDS-polyacrylamide gel were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes with transferred proteins were then blocked for 1 hour with 3% skim milk powder added to TBST. The blocked membranes were reacted with an anti-GFP antibody (Santa Cruz Biotechnology, USA), and an anti-Actin antibody (Sigma, USA) was used as a loading control. Antibodies recognizing specific proteins were added to a solution of 1% BSA dissolved in TBST. For each blot, a secondary antibody was diluted in 2% skim milk powder at a ratio of 1:10,000 and reacted. Enhanced chemiluminescence (iNTRoN Biotechnology, South Korea) and a bioimaging system (MF-Chemibis, Shimadzu Scientific Korea Corporation, South Korea) were used to confirm the protein signal.

[0066] As a result, as shown in Figure 3A, it was confirmed that bafilomycin increased SOD1 protein aggregates compared to the control group. In this case, some mutant alloferon peptides according to one embodiment of the present invention reduced the protein aggregates, and when these were quantified, it was similarly confirmed that some mutant alloferon peptides according to one embodiment of the present invention resolved the accumulation of aggregates. In particular, as shown in Figure 3B, among the mutant alloferon peptides according to one embodiment of the present invention, those in which the 4th and / or 11th amino acid was reduced to an L-type amino acid (DALS1, DALV2), and those in which at least three of the two valine, glutamine, and serine amino acids were reduced to L-type amino acids (DALVS_V), showed even better protein aggregate resolution ability compared to the D-type alloferon peptide (DAL1) in which all amino acids were substituted with D-type amino acids.

[0067] Experimental Example 5: Analysis of the effect of mutant alloferon peptide on the formation of protein aggregates that appear after overexpression of mutant tau protein. Subsequently, the inventors temporarily transduced mutant tau protein into the HEK293T cell line and then investigated whether some mutant peptides (DALVS, DALVSqV, DAL1, DALV2) composed of amino acid sequences shown in SEQ ID NOs. 3 to 16 could resolve the resulting mutant tau protein aggregates. For this purpose, EGFP-Tau P301L DNA was temporarily transduced into the HEK293T cell line using Lipofectamine 2000. The protein aggregates were analyzed using the same method as in Experimental Example 4.

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

[0069] Experimental Example 6: Analysis of the effect of mutant alloferon peptide on the formation of protein aggregates that appear after overexpression of α-synuclein protein. Subsequently, the inventors investigated whether some mutant peptides (DALV1, DALQ1, DAL1, DALV2) composed of amino acid sequences shown in SEQ ID NOs. 3 to 16 could resolve the resulting α-synuclein protein aggregates after transient transduction of α-synuclein protein into the HEK293T cell line. For this purpose, EGFP-α-synuclein A53T DNA was transiently transdone into the HEK293T cell line using Lipofectamine 2000. Analysis of the protein aggregates was performed using the same method as in Experimental Example 4.

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

[0071] Experimental Example 7: Analysis of the effect of mutant alloferon peptide on the formation of protein aggregates that appear after overexpression of mutant huntingtin protein. Subsequently, the inventors temporarily transduced mutant huntingtin protein into the HEK293T cell line and then investigated whether some mutant peptides (DALH3, DALH1, DAL1, DALV2) composed of amino acid sequences shown in SEQ ID NOs. 3 to 16 could resolve the resulting mutant huntingtin protein aggregates. For this purpose, GFP-mHttQ74 DNA was temporarily transduced into the HEK293T cell line using Lipofectamine 2000. The protein aggregates were analyzed using the same method as in Experimental Example 4.

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

[0073] Experiment Example 8: Animal experiments related to Alzheimer's disease Experiment 8-1: Changes in body weight due to administration of mutant alloferon peptide Based on the results of Experimental Examples 4 to 7, the inventors conducted animal experiments using disease model animals to confirm whether the mutant alloferon peptide according to one embodiment of the present invention can be used as a therapeutic agent for various degenerative neurological diseases.

[0074] First, to confirm whether or not toxicity would be observed during in vivo administration, a dementia model animal (5XFAD) was administered 20 mg / kg of a mutant alloferon peptide (DALV2) according to one embodiment of the present invention by intraperitoneal (IP) injection once daily, five days a week (Monday to Friday), for 25 weeks, while monitoring changes in body weight. As a comparison group, DAL, in which all amino acids are in the D form, was used.

[0075] As a result, as shown in Figure 7, a consistent weight gain was observed during the 25-week injection administration period, and no significant difference was observed between the experimental groups.

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

[0077] As a result, as can be seen in Figure 8, the peptide according to one embodiment of the present invention significantly restored memory and learning functions in Alzheimer's model animals.

[0078] 8-3: Analysis of whether or not the accumulation of β-amyloid aggregates in brain tissue is suppressed. To confirm whether the improvement in behavioral indices in Experimental Example 8-2 was due to the suppression of β-amyloid aggregate accumulation in the brain, the inventors sacrificed experimental animals that had completed the Morris water maze test shown in Figure 8, obtained brain tissue sections, and stained the β-amyloid aggregates with thioflavin S, which has a selective affinity for β-amyloid plaques.

[0079] Specifically, thioflavin S staining was performed by excising the brains of dementia model rats, freeze-drying them in OCT compound (Sakura finetec, USA), obtaining brain sections using coronal sectioning, and attaching them to glass slides coated with 0.1% poly-L-lysine. The obtained brain sections were washed with 70% ethanol for 1 minute, then again with 80% ethanol for 1 minute, and stained with 1% Thiflavin S solution (MilliporeSigma, USA) for 15 minutes. Subsequently, the sections were washed with 80% ethanol for 1 minute, then again with 70% ethanol for 1 minute, followed by two washes with distilled water, and β-amyloid senile plaques were observed using a fluorescence microscope.

[0080] Congo red staining to identify amyloid was performed as follows: Brain sections obtained from the coronal section were mounted on 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 with 0.5% Congo red solution (Thermo Fisher, USA) for 20 minutes. After washing the Congo red solution in PBS solution, the sections were rapidly immersed in an alkaline alcohol solution containing 0.01% sodium hydroxide and then removed 10 times. Subsequently, the sections were washed in PBS solution for 3 minutes, then in 95% ethanol for 3 minutes, and then in 100% ethanol for 3 minutes to dehydrate them, and amyloid was observed through a fluorescence microscope. The number of stained senile plaques and amyloid per brain section was counted and quantified using a statistical analysis program (Graph Prism).

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

[0082] 8-4: Analysis of the effects on the expression of various aggregated proteins in the brain Based on the results of Experimental Examples 8-2 and 8-3, the inventors investigated the expression levels of pathogenic aggregate-forming proteins other than β-amyloid, such as tau protein, p62, and LC3, in brain tissue through Western blot analysis.

[0083] Specifically, after sacrificing experimental animals that had completed behavioral experiments, cerebral cortex and hippocampal tissue were obtained. After lysing the tissue, Western blot analysis was performed 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 (APP), respectively.

[0084] As a result, as can be seen from Figures 11A to 12B, all six proteins were increased in the Alzheimer's disease model mice compared to the control group (normal mice) not only in the cerebral cortex but also in the hippocampus. In contrast, experimental animals administered with DAL1 (the comparison group) or the peptide according to one embodiment of the present invention (DALV2) showed significantly lower expression of the aggregate-forming proteins in both the cerebral cortex and hippocampus. Western blot analysis of the cerebral cortex showed that DALV2 exhibited even superior protein aggregate dissolution ability compared to the D-type allopherone peptide (DAL1), in which all amino acids are substituted with D-type amino acids. Western blot analysis of the hippocampal region showed that DAL1 did not show a significant reduction in β-amyloid levels, while DALV2 showed a significant reduction. This result indicates that the peptide according to one embodiment of the present invention can suppress the formation of pathogenic protein aggregates in the brain even after in vivo administration, demonstrating the superior inhibitory effect of DALV2.

[0085] Experimental Example 9: Effect Analysis on an Animal Model of Amyotrophic Lateral Sclerosis (ALS) To confirm whether the mutant alloferon peptide according to one embodiment of the present invention has a therapeutic effect against amyotrophic lateral sclerosis (ALS) in actual vivo conditions, the inventors have used an animal model of amyotrophic lateral sclerosis (SOD1) G93AThe peptide (DALV2) according to one embodiment of the present invention was repeatedly administered subcutaneously (SC) at a dose of 40 mg / kg five days a week (Monday to Friday) from 112 days to 180 days of age. After the experiment was completed and the experimental animals were sacrificed, spinal cord sections were obtained from the lumbar region and Western blot analysis was performed using an anti-SOD-1 antibody.

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

[0087] Although the present invention has been described with reference to the embodiments described above, these are merely illustrative, and those skilled in the art will understand that a wider variety of modifications and equivalent other embodiments are possible. Therefore, the true scope of technical protection of the present invention must be determined by the technical idea of ​​the claims. [Industrial applicability]

[0088] A peptide according to one embodiment of the present invention can be developed as a therapeutic agent for various degenerative neurological diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis, which are caused by pathogenic protein aggregates, by suppressing abnormal hyperphosphorylation of pathogenic proteins and the resulting aggregation formation in the central nervous system, and by removing pre-existing protein aggregates. Furthermore, the present invention includes the following embodiments. [Aspect 1] A novel peptide having the ability to suppress the formation of abnormal protein aggregates, comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, having a length of 12 to 20a.a., wherein, excluding glycine, 1 to 4 amino acids are L-type amino acids and the remaining amino acids are D-type amino acids. [Aspect 2] If the peptide contains the amino acid sequence shown in SEQ ID NO: 1, the peptide according to embodiment 1 contains at least one of the following: i) At least one of the four histidines is a D-type amino acid; ii) At least one of the valine amino acids at the 3rd and 11th positions is an L-type amino acid, or both are D-type amino acids; iii) The fourth amino acid, serine, is a D-type amino acid; iv) The eighth amino acid, glutamine, is a D-type amino acid; and v) Two or more of the above i through iv are D-type amino acids. [Aspect 3] If the peptide contains the amino acid sequence shown in Sequence ID No. 2, the peptide according to Embodiment 1 contains at least one of the following: i) At least one of the four histidines is a D-type amino acid: ii) At least one of the valine amino acids at the 3rd and 10th positions is an L-type amino acid, or both are D-type amino acids; iii) The fourth amino acid, serine, is a D-type amino acid; and iv) At least two of the amino acids from i to iii are D-type amino acids. [Aspect 4] 1) In the L-type allopherone peptide of SEQ ID NO: 1, at least 3 of the 4 histidines are substituted with D-type histidines, and at least 2 of the 2 valine, the 3rd amino acid serine, and the 8th amino acid glutamine are D-type amino acids, or 2) The peptide according to embodiment 1, wherein the L-type allopherone peptide of Sequence ID No. 1 has four histidine amino acids that are all D-type amino acids, and the eighth amino acid, glutamine, is either an L-type or D-type amino acid or deleted. [Aspect 5] The peptide according to embodiment 1, comprising any one amino acid sequence from among the amino acid sequences shown in SEQ ID NOs. 3 to SEQ ID NOs. 14. [Aspect 6] The peptide according to embodiment 1, comprising any one amino acid sequence from SEQ ID NOs. 3 to 5, SEQ ID NOs. 7, 8, 10, and SEQ ID NOs. 12 to 14. [Aspect 7] A pharmaceutical composition for the treatment of degenerative neurological diseases, comprising a peptide described in any one of embodiments 1 to 6 as an active ingredient. [Aspect 8] The pharmaceutical composition according to embodiment 7, wherein the degenerative neurological disease is a degenerative neurological disease having the formation of abnormal protein aggregates as its etiology or pathological phenomenon. [Aspect 9] The pharmaceutical composition according to embodiment 8, wherein the abnormal protein aggregates are formed by abnormal aggregation of α-synuclein, β-amyloid, TDP-34, p62, FUS protein, superperoxide disequilibrium-1 (SOD-1), huntingtin protein, or tau protein. [Aspect 10] The pharmaceutical composition according to embodiment 8, wherein the degenerative brain disease having the formation of the aforementioned abnormal protein aggregates 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, Ritico-Bodig disease, frontotemporal lobar degeneration, corticobasal degeneration, or progressive supranuclear palsy. [Aspect 11] A method for treating an individual suffering from a degenerative neurological disease, comprising the step of administering a therapeutically effective amount of a peptide described in any one of embodiments 1 to 6 to the individual. [Aspect 12] A method for suppressing the accumulation of pathogenic protein aggregates in the nervous system of an individual suffering from a degenerative neurological disease, comprising the step of administering a therapeutically effective amount of a peptide described in any one of embodiments 1 to 6 to the individual.

Claims

1. A peptide having a length of 12 to 20 a.a., comprising any one amino acid sequence from the amino acid sequences shown in SEQ ID NOs: 3 to 14, which has the function of removing abnormal protein aggregates by promoting self-predation.

2. The peptide according to claim 1, comprising any one amino acid sequence from SEQ ID NOs. 3 to 5, SEQ ID NOs. 7, SEQ ID NOs. 8, SEQ ID NOs. 10, and SEQ ID NOs. 12 to 14.

3. A pharmaceutical composition for the treatment of degenerative neurological diseases, comprising the peptide described in claim 1 or claim 2 as an active ingredient.

4. The pharmaceutical composition according to claim 3, wherein the degenerative neurological disease is a degenerative neurological disease having the formation of abnormal protein aggregates as its etiology or pathological phenomenon.

5. The pharmaceutical composition according to claim 4, wherein the abnormal protein aggregates are formed by abnormal aggregation of α-synuclein, β-amyloid, TDP-34, p62, FUS protein, superperoxide disequilibrium-1 (SOD-1), huntingtin protein, or tau protein.

6. The pharmaceutical composition according to claim 4, wherein the degenerative neurological disease having the formation of the abnormal protein aggregates as an etiology or pathological phenomenon is Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), chronic traumatic encephalopathy, Ritico Bodig disease, frontotemporal lobar degeneration, corticobasal degeneration, or progressive supranuclear palsy.

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