Novel peptide and use thereof

Abstract

The present invention relates to a novel peptide and a use thereof, and more particularly, to a novel peptide having a length of 4 to 10 a.a. And a structure of H-G-X1-X2-G-X3, which has an ability to inhibit abnormal protein aggregate formation and autophagy activity against protein aggregates.

Description

Technical Field

[0001] This invention relates to novel peptides and their uses, and more particularly to novel peptides capable of inhibiting the formation of abnormal protein aggregates and removing protein aggregates by autophagy, and to their novel uses in the treatment of neurodegenerative diseases. Background Technology

[0002] Zinc is a trace element 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. Furthermore, zinc homeostasis in neurons is known to play a crucial role in neuronal survival, and zinc deficiency in neurons is known to induce apoptosis and contribute to neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (Lien, H et al., BBRC. 268: 148-154, 2000). Conversely, excessive zinc in neurons is also known to induce cell damage, which can lead to acute brain injury, such as focal ischemia and seizures (Koh et al., Science. 272: 1013-1016, 1996).

[0003] Autophagy is an intracellular mechanism that breaks down organelles for energy or removes damaged organelles and abnormal or pathological protein aggregates during starvation. During autophagy, cytoplasmic components are surrounded by a double membrane and separated from other organelles to form autophagosomes. At this time, soluble light chain I (LC3I) in the cytoplasm is converted into light chain II (LC3II), which attaches to the autophagosome membrane. The autophagosome then fuses with lysosomes to form autolysosomes, which are degraded and recycled by several hydrolases present in the lysosome.

[0004] Recently, it has been shown that promoting autophagy can clear protein aggregates seen in neurodegenerative diseases, such as α-synuclein, β-amyloid, tau, superoxide dismutase-1 (SOD-1), huntingtin, and TARDNA-binding protein 43 (TDP-43). Indeed, there is evidence that defective autolysosomal function impairs the degradation of those protein aggregates, thereby blocking the flow of autophagy, and that this lack of 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 lysosomal function can be enhanced by zinc supply. On the other hand, it has also been reported that additional zinc supply can promote autophagy when the effective zinc chelator TPEN is applied to inhibit lysosomal membrane permeation (LMP) induced by H2O2, tamoxifen, and ethanol, thus reducing autophagy (Lee et al., Glia 57:1351-1361, 2009; Hwang et al., Biometals 23:997-1013, 2010; Liuzi et al., Biol. TraceElem. 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, no drugs have yet been developed to treat neurodegenerative diseases by maintaining zinc homeostasis within neurons. Summary of the Invention Technical issues

[0007] The object of this invention is to solve the above and other problems by providing novel peptides and their use in treating degenerative 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 neurodegenerative diseases) through autophagy or enhanced lysosomal function. Technical solution

[0008] In one aspect of the invention, a novel peptide having a length of 4 to 10 a.a. and the following structural formula 1 is provided, which has the ability to inhibit the formation of abnormal protein aggregates and autophagy activity against protein aggregates: HGX 1 -X 2 -GX 3 (Structure 1) (In the above structural formula, H represents L- or D-histidine, G represents glycine, and X represents...) 1 The absence of or presence of any D- or L-amino acids, X 2 It can be any D or L amino acid, and X 3 (It is absent or is any D- or L-amino acid other than valine).

[0009] In another aspect of the invention, a pharmaceutical composition for treating neurodegenerative diseases is provided, comprising the peptide as an active ingredient.

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

[0011] In another aspect of the invention, a method is provided for inhibiting the accumulation of pathogenic protein aggregates in the nervous system of a subject suffering from a neurodegenerative disease, the method comprising administering a therapeutically effective amount of the peptide to the subject. The 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 (ALS), Huntington's disease, etc., by preventing neuronal cell death and regulating intracellular zinc homeostasis. However, the scope of the present invention is not limited to these effects. Attached Figure Description

[0013] Figure 1a and 1b These are a series of fluorescence micrographs showing that autophagic flux blocked by bafilomycin A-1 is restored by peptides according to one embodiment of the invention, wherein H4 cell lines (GL-H4) stably overexpressing GFP-LC3 protein are treated with various peptides according to one embodiment of the invention at a concentration of 20 μM in combination with bafilomycin, and the LC3-GFP spots of the H4 cell lines are photographed after 12 hours. Figure 1c and 1d This is a graph showing the fluorescence intensity of LC3-GFP spots 12 hours after treatment of the GL-H4 cell line with a peptide according to one embodiment of the invention in combination with bafloxacin at a concentration of 20 μM. It can be seen that the number of spots significantly increased after bafloxacin treatment due to the blockage of autophagy, while treatment with the peptide according to one embodiment of the invention reduced the number and size of spots, which is a result of autophagy elimination. In Figures 1C to 1D, * indicates a significant difference between treatment with the peptide according to one embodiment of the invention and treatment with bafloxacin alone. * p < 0.05 ** p < 0.01 *** p < 0.001).

[0014] Figure 2aThe results are Western blot analysis of the SOD1 protein expression level of protein samples obtained 18 hours after treatment with 100 nM bafloxacin alone or with 100 nM bafloxacin in combination with 30 μm of some peptides according to one embodiment of the invention, to study the degradation of SOD1 protein aggregates induced by treatment with some peptides (ZC303, ZC304, ZC309, ZC311) according to one embodiment of the invention. Figure 2b This is a graph showing the quantitative results of relative expression. In Figure 2b In the text, * indicates a significant difference compared to treatment with bafloxacin alone ( * p < 0.05).

[0015] Figure 3a The results are Western blot analysis of the expression level of mutant tau protein in protein samples obtained 18 hours after treatment with 100 nM bafloxacin alone or with 30 μM of bafloxacin in combination with some peptides according to one embodiment of the invention, on HEK293T cells transfected to express mutant tau protein, to study the degradation of mutant tau protein aggregates induced by treatment with some peptides (ZC303, ZC304, ZC309, ZC311) according to one embodiment of the invention. Figure 3b This is a graph showing the quantitative results of relative expression. In Figure 3b In the text, * indicates a significant difference compared to treatment with bafloxacin alone ( ** p < 0.01, *** p < 0.001).

[0016] Figure 4 This is a Western blot analysis result of the α-synuclein expression level of protein samples obtained 18 hours after treatment with 100 nM bafloxacin alone or with 30 μM of bafloxacin in combination with some peptides according to one embodiment of the invention, on HEK293T cells transfected to express α-synuclein, to study the degradation of α-synuclein aggregates induced by treatment with some peptides (ZC303, ZC304, ZC309, ZC311) according to one embodiment of the invention.

[0017] Figure 5This is a Western blot analysis result of the expression level of mutant huntingtin protein in protein samples obtained 18 hours after treatment with 100 nM bafloxacin alone or with 30 μM of bafloxacin in combination with some peptides according to one embodiment of the invention, on HEK293T cells transfected to express mutant huntingtin protein, in order to study the degradation of mutant huntingtin protein aggregates induced by treatment with some peptides (ZC303, ZC304, ZC309, ZC311) according to one embodiment of the invention.

[0018] Figure 6 It is a graph depicting the change in body weight over time after administration of a peptide according to one embodiment of the present invention to an animal model of Alzheimer's disease.

[0019] Figure 7 These are a series of fluorescence micrographs (left) and figures (right) showing the results of thioflavin S staining of β-amyloid aggregate plaques in cortical (top) and hippocampal (bottom) sections from Alzheimer's disease model animals administered a peptide according to an embodiment of the present invention.

[0020] Figure 8 These are a series of fluorescence micrographs (left) and figures (right) showing quantitative β-amyloid aggregate plaques stained with Congo red in cortical (top) and hippocampal (bottom) sections from Alzheimer's disease model animals administered with a peptide according to an embodiment of the present invention.

[0021] Figure 9a Western blot analysis results show the expression of various pathogenic aggregate-forming proteins in the cerebral cortex of Alzheimer's disease model animals administered a peptide according to an embodiment of the present invention, and Figure 9b These are a series of graphs showing the results of quantitative protein blot analysis.

[0022] Figure 10a Western blot analysis results show the expression of various pathogenic aggregate-forming proteins in the hippocampus of experimental animals administered a peptide according to an embodiment of the present invention, and Figure 10b These are a series of graphs showing the results of quantitative protein blot analysis.

[0023] Figure 11 This is a graph depicting the results of rotarod test analysis of motor function over time in ALS model animals treated with peptides according to one embodiment of the present invention.

[0024] Figure 12 This is a graph showing the results of analysis on the change in body weight over time in ALS model animals treated with peptides according to one embodiment of the present invention.

[0025] Figure 13This is a graph depicting the results of analyzing survival over time in ALS model animals treated with peptides according to one embodiment of the present invention. Detailed Implementation Definition of terminology:

[0026] As used in this article, the term “zinc homeostasis” refers to the mechanism that maintains a constant level of intracellular zinc, which is known to involve zinc transporters, zinc-binding proteins (metallothionein, MT), and transcription factors (MTF1-2).

[0027] As used in this article, the term "neurodegenerative disease" refers to a disease characterized by the progressive loss of neural structure or function due to the 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 aggregate" refers to the abnormal aggregation of proteins such as amyloid or tau within cells to form insoluble fibrils. These abnormal or pathological protein aggregates are known to be neuropathological markers of various intermittent or hereditary neurodegenerative diseases. Detailed description of the invention

[0029] In one aspect of the invention, a novel peptide having a length of 4 to 10 a.a. and the following structural formula 1 is provided, which has the ability to inhibit the formation of abnormal protein aggregates and autophagy activity against protein aggregates: HGX 1 -X 2 -GX 3 (Structure 1) (In the above structural formula, H represents L- or D-histidine, G represents glycine, and X represents...) 1 The absence of or presence of any D- or L-amino acids, X 2 It can be any D or L amino acid, and X 3 (It is absent or is any D- or L-amino acid other than valine).

[0030] The peptide may contain at least one D amino acid.

[0031] In the peptide, X 1 It may be absent or may be a nonpolar or polar amino acid. The nonpolar amino acid may be alanine, valine, leucine, methionine, isoleucine, or proline, and the polar amino acid may be glutamine or asparagine. More preferably, X 1 It may be absent or may be D-type or L-type valine or glutamine.

[0032] The peptide comprises the structure of Formula 1 and may have a length of 4, 5, 6, 7, 8, 9 or 10 a.a.

[0033] In the peptide, X 2 These can be polar amino acids, nonpolar amino acids, or aromatic amino acids. Polar amino acids can be serine, cysteine, asparagine, glutamine, threonine, or tyrosine; nonpolar amino acids can be alanine, valine, leucine, isoleucine, methionine, or proline; and aromatic amino acids can be tyrosine, tryptophan, or phenylalanine. 2 More preferably, it is a D- or L-type amino acid selected from serine, histidine, valine, threonine, cysteine, and tryptophan.

[0034] In the peptide, X 3 It can be a polar or nonpolar amino acid, or an aromatic amino acid. Polar amino acids can be serine, cysteine, asparagine, glutamine, threonine, or tyrosine; nonpolar amino acids can be alanine, valine, leucine, isoleucine, methionine, or proline; and aromatic amino acids can be tyrosine, tryptophan, or phenylalanine. X3 may preferably be absent or may be a D or L amino acid selected from aspartic acid, serine, asparagine, tyrosine, histidine, and leucine.

[0035] Most preferably, the peptide may comprise an amino acid sequence selected from SEQ ID NO: 1 to 15 or consist of an amino acid sequence selected from SEQ ID NO: 1 to 15.

[0036] To investigate whether short peptides inhibit the formation of abnormal protein aggregates that induce autophagy, the inventors designed peptides in various combinations of D and L amino acids as shown in Table 1 below, and then performed L3 point assays. As a result, as... Figures 1a to 1d As shown, it was found that the peptide according to one embodiment of the invention promotes autophagy by eliminating the inhibition of autophagy induced by bafloxacin treatment. These results demonstrate that the peptide according to one embodiment of the invention can be used as a therapeutic agent for neurodegenerative diseases caused by abnormal protein aggregates by inhibiting the abnormal production of protein aggregates that accumulate in neurons and induce neuronal death, and by promoting autophagic and lysosomal activity against the produced abnormal protein aggregates.

[0037] In another aspect of the invention, a pharmaceutical composition for treating neurodegenerative diseases is provided, comprising the peptide as an active ingredient.

[0038] In the pharmaceutical composition, the neurodegenerative disease can be a neurodegenerative disease having abnormal protein aggregates formed as its etiology or pathology, wherein the abnormal protein aggregates are formed by abnormal aggregations of α-synuclein, β-amyloid, TDP-43, p62 protein, FUS protein, superoxide dismutase-1 (SOD-1), huntingtin protein, or tau protein. Neurodegenerative diseases can specifically include Alzheimer's disease (AD), which is associated with the accumulation of β-amyloid 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 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; or progressive supranuclear palsy, which is associated with the accumulation of tau protein aggregates. Other diseases associated with the accumulation of protein aggregates in neurons include Creuzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, meningeal hemangioma, and neuronal ceroid lipofuscin deposition disease. These neurodegenerative diseases and their association with the accumulation of protein aggregates in neurons are described in detail in the prior art. 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).

[0039] In the composition, the peptide can treat neurodegenerative diseases by preventing neuronal cell death, regulating intracellular zinc homeostasis, and promoting lysosomal function.

[0040] A pharmaceutical composition according to one embodiment of the present invention may contain a pharmaceutically acceptable carrier and may further contain other pharmaceutically acceptable excipients, excipients or diluents besides the carrier.

[0041] As used herein, the term "pharmaceutically acceptable" means a composition that is physiologically acceptable and, when administered to humans, generally does not cause allergic reactions such as gastrointestinal disturbances, dizziness, or similar reactions. Experimental 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, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. It may further include fillers, antiflocculators, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

[0042] Furthermore, pharmaceutical compositions according to one embodiment of the invention can be formulated using methods known in the art to allow for rapid, sustained, or delayed release of the active ingredient upon administration to mammals. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

[0043] The pharmaceutical composition according to one embodiment of the invention can be administered via various routes, such as oral, parenteral (e.g., via suppository), percutaneous, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, intrathecal, or intrathecal. It can also be administered as a sustained-release agent or by using an implantable device for continuous or repeated release. The dosage can be administered once daily or divided into multiple doses within any desired range, and there is no particular limitation on the duration of administration.

[0044] The pharmaceutical composition according to one embodiment of the present invention can be administered by conventional systemic or local administration, such as by intramuscular or intravenous injection. Furthermore, the peptide according to one embodiment of the present invention can also be administered orally.

[0045] Pharmaceutical compositions according to one embodiment of the invention can be formulated in any suitable form with any commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline solutions, and carriers for parenteral administration such as aqueous glucose solutions and glycols, 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, methylparaben, or propylparaben and chlorobutanol. Depending on the method of administration or formulation, compositions according to the invention may also contain suspending agents, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorbents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, and antioxidants. Pharmaceutically acceptable carriers and formulations suitable for use in the invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].

[0046] The dosage of the pharmaceutical composition according to one embodiment of the invention for a 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 condition, and other concurrently administered medications. The therapeutically active peptide can be administered in amounts from 100 ng to 1000 mg per kg of body weight, more preferably from 1 μg to 100 mg per kg of body weight, and most preferably from 5 to 40 mg per kg of body weight, while adjusting the dosage taking into account the above factors.

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

[0048] As used herein, the term "therapeutic effective dose" refers to an amount sufficient to treat a condition with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose can be determined based on factors including, and other factors known in the medical field, such as: individual type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and clearance rate, duration of treatment, and concomitant drugs. The pharmaceutical compositions of the present invention can be administered at doses from 0.1 mg / kg to 1 g / kg, more preferably from 1 mg / kg to 500 mg / kg. Simultaneously, the dose can be appropriately adjusted according to the patient's age, sex, and condition.

[0049] In another aspect of the invention, a method of treating a subject suffering from a neurodegenerative disease is provided, the method comprising administering to the subject the composition comprising a therapeutically effective amount of the peptide.

[0050] In another aspect of the invention, a method is provided for inhibiting the accumulation of pathogenic protein aggregates in the nervous system of a subject suffering from a neurodegenerative disease, the method comprising administering a therapeutically effective amount of the peptide to the subject.

[0051] In the above methods, the composition can be administered orally or parenterally as described above, and in the case of parenteral administration, it can be administered via any route of systemic or local administration. In the case of systemic administration, it can be administered by intravenous injection (IV), intraperitoneal injection (IP), or intramuscular injection (IM). In the case of local administration, it can be administered by intracranial administration, intrathecal administration, intraspinal administration, subcutaneous injection (SC), etc.

[0052] The method of any one of the preceding claims, wherein the neurodegenerative disease can be a neurodegenerative disease having abnormal protein aggregates formed as etiology or pathology, and the abnormal protein aggregates can be formed by abnormal aggregates of α-synuclein, β-amyloid, TDP-43, p62 protein, FUS protein, superoxide dismutase-1 (SOD-1), huntingtin protein or tau protein. Implementation of the invention

[0053] The invention will now be described in more detail with reference to the following embodiments. However, the 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 invention and to give full insight into the scope of the invention to those skilled in the art. Example: Design and synthesis of peptides

[0054] The inventors synthesized peptides having the amino acid sequences shown in Table 1 below, which were obtained from Anigen (Korea). Table 1. Peptides synthesized according to one embodiment of the present invention. Experimental Example 1: Cell Culture

[0055] This invention utilizes H4 cell lines (GL-H4) and HEK293T cell lines permanently transfected with the GFP-LC3 plasmid. The cell culture medium is minimum essential medium (MEM, WellGene) supplemented with 10% fetal bovine serum (Hyclone, USA) and a mixture of antibiotics and antifungal agents (WellGene, Korea), and the cells are cultured in a cell culture machine at 37°C and 5% CO2. Experimental Example 2: Analysis of Autophagic Flow Blocked by Bafloromycin A-1

[0056] To investigate the effect of a peptide comprising the amino acid sequence designated as SEQ ID NO: 1 to 15 on autophagic flux, the inventors treated an H4 cell line (GL-H4) expressing GFP-labeled LC3 with MEM, and then used the aforementioned peptide (20 μM) and the autophagy inhibitor bafloxacin A-1 (Baf A1, 100 nM) to inhibit autophagy. Subsequently, the effect of the peptide according to one embodiment of the present invention was investigated by fluorescence microscopy analysis.

[0057] exist Figure 1a and 1b The LC3 spots observed are due to the presence of the LC3-GFP protein within autophagosomes. If this protein is not degraded by lysosomes, the entire autophagy process is inhibited, leading to the accumulation of autophagosomes. Treatment of the GL-H4 cell line with 100 nM bafloxacin (Baf A1) to inhibit autophagic flux significantly increased the number and size of LC3 spots, while treatment with a peptide (20 μM) according to one embodiment of the invention reduced the number and size of LC3 spots (although to varying degrees). Figure 1a and 1b Quantification by measuring the fluorescence intensity at all points also demonstrated a similar decrease in fluorescence intensity after treatment with peptides (ZC303 to ZC317) according to one embodiment of the invention. Figure 1c and 1d This indicates that autophagosomes accumulated due to the blockage of autophagy are cleared by a peptide according to one embodiment of the invention. Experimental Example 3: Analysis of the effect of novel peptides on the formation of protein aggregates after SOD1 protein overexpression

[0058] The inventors performed Western blot analysis to investigate whether certain components of the peptides (ZC303, ZC304, ZC309, ZC311) containing the amino acid sequences designated as SEQ ID NO: 1 to 15 could eliminate SOD1 aggregates formed after transient transfection of SOD1 protein into the HEK293T cell line. Specifically, the inventors used Lipofectamine 2000 to transiently transfect EGFP-SOD1 G93A DNA into the HEK293T cell line. After transfection, the cell line was incubated at 37°C and 5% CO2 for 30 h, and the medium was replaced with minimum essential medium (MEM, Gibco, USA). After pretreatment with 100 nM bafloxacin for 30 min, posttreatment was performed by adding 30 μM of peptide and incubating for 18 hours. Following this, cell lysis was performed in 200 μl of Triton X-100 lysis buffer (30 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mN EDTA) supplemented with protease inhibitors and phosphatase inhibitors (2 μg / ml aprotinin, 2 μg / ml leucopeptide, 1 μg / ml pepsin inhibitor A, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 5 mM NaF, and 10 mM Na4P2O7) in 200 μl wells. The resulting cell lysates were incubated at 4 °C for 30 min. Protein quantification of the cell lysates was performed using a BCA protein assay kit (Pierce Biotechnology, USA). The lysates were then centrifuged at 17,000 × g for 20 min, and the supernatant was discarded; cell debris was collected to obtain protein extracts. The protein extracts were then resuspended in 5× sample buffer (300 mM Tris, pH 6.8, 10% SDS, 50% glycerol, 0.1% bromophenol blue, 2.5% mercaptoethanol, 100 mM DTT), mixed into aliquots, and denatured at 95 °C for 5 min to prepare the samples. Electrophoresis was then performed using an 8%–15% SDS-polyacrylamide gel, and the proteins separated by size were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membranes containing the transferred proteins were then blocked with 3% skim milk powder in TBST for 1 h. The blocked membranes were reacted with an anti-GFP antibody (Santa Cruz Biotechnology, USA), with an anti-actin antibody (Sigma, USA) used as a loading control. Antibodies recognizing specific proteins were added to a solution of 1% BSA dissolved in TBST. For all blots, a secondary antibody diluted 1:10,000 in 2% skim milk powder was used for reaction.Protein signals were identified using enhanced chemiluminescence (iNTRoNBiotechnology, Korea) and a bioimaging system (MF-Chemibis, Shimadzu Scientific Korea Corporation, Korea).

[0059] As a result, it can be seen that, compared with the control, bafloxacin increased SOD1 protein aggregates, such as... Figure 2a and 2b As shown in the figure. Quantitative analysis also demonstrated that some peptides according to one embodiment of the invention reduced protein aggregates, and that some peptides according to one embodiment of the invention eliminated aggregate accumulation. Experimental Example 4: Analysis of the effect of novel peptides on protein aggregate formation after overexpression of mutant tau protein

[0060] The inventors then investigated whether certain components of the peptides (ZC305, ZC306, ZC307, ZC311) containing the amino acid sequences designated as SEQ ID NO: 1 to 15 could eliminate mutant tau protein aggregates formed after transient transfection of mutant tau protein into the HEK293T cell line. For this purpose, EGFP-TauP301L DNA was transiently transfected into the HEK293T cell line using Lipofectamine 2000. Protein aggregate analysis was performed in the same manner as in Experimental Example 3 above.

[0061] As a result, in Figure 3a As can be seen, compared with the control, bafloxacin increased mutant tau protein aggregates, and quantification also showed that some peptides according to one embodiment of the present invention reduced protein aggregates. Experimental Example 5: Analysis of the effect of novel peptides on protein aggregate formation after α-synuclein overexpression

[0062] The inventors then investigated whether certain components of peptides (ZC308, ZC310, ZC311, ZC312) containing the amino acid sequences designated as SEQ ID NO: 1 to 15 could eliminate α-synuclein aggregates formed after transient transfection of α-synuclein into the HEK29T cell line. For this purpose, EGFP-α-synuclein A53T DNA was transiently transfected into the HEK293T cell line using Lipofectamine 2000. Protein aggregate assays were performed in the same manner as in Experimental Example 3 above.

[0063] As a result, in Figure 4 As can be seen, compared with the control, bafloxacin increases α-synuclein aggregates, while some of the peptides according to one embodiment of the invention reduce protein aggregates. Experimental Example 6: Analysis of the effect of novel peptides on protein aggregate formation after overexpression of mutant huntingtin protein

[0064] The inventors then investigated whether certain components of the peptides (ZC311, ZC314, ZC315, and ZC316) containing the amino acid sequences designated as SEQ ID NO: 1 to 15 could eliminate mutant huntingtin aggregates formed after transient transfection of mutant huntingtin protein into the HEK293T cell line. For this purpose, GFP-mHttQ74 DNA was transiently transfected into the HEK293T cell line using Lipofectamine 2000. Protein aggregate analysis was performed in the same manner as in Experimental Example 3 above.

[0065] As a result, in Figure 5 As can be seen, compared with the control, bafloxacin increased mutant huntingtin protein aggregates, and some peptides according to one embodiment of the present invention reduced protein aggregates. Experimental Example 7: Animal Study of Alzheimer's Disease 7-1: Analyze changes in body weight

[0066] Based on the results of experiments 4 to 6 above, the inventors conducted animal studies using disease model animals to determine whether the peptide according to one embodiment of the present invention can be used as a therapeutic agent for various neurodegenerative diseases.

[0067] First, to determine whether the peptide administered in vivo was toxic, dementia model animals (5XFAD) were administered 20 mg / kg of the peptide (ZC311) prepared in Example 9 of this invention via intraperitoneal (IP) injection once daily, five days a week (Monday to Friday), for 25 weeks to determine changes in body weight. As controls, wild-type mice and Alzheimer's model mice were administered saline alone.

[0068] As a result, such Figure 6 As shown, weight gain was consistent throughout the 25-week injection period, with no difference between the experimental groups. 7-2: Analysis of the effect on the accumulation of β-amyloid protein aggregates in the brain

[0069] To determine whether the peptide according to one embodiment of the invention can inhibit the accumulation of β-amyloid aggregates in the brain, a pathological phenomenon of Alzheimer's disease, even under actual in vivo conditions, as shown in the results of experiments 4 and 5 above, brain tissue sections obtained from experimental animals that were sacrificed 25 weeks after peptide administration were stained with thioflavone S and Congo red, which have selective affinity for β-amyloid plaques.

[0070] Specifically, for thioflavone S staining, brains from dementia model mice were harvested, freeze-dried in an OCT compound (Sakurafintec, USA), and brain sections were obtained via coronal sectioning and fixed onto slides coated with 0.1% poly-L-lysine. The obtained brain sections were washed in 70% ethanol for 1 min, then again in 80% ethanol for 1 min, and stained in 1% thioflavone S solution (MilliporeSigma, USA) for 15 min. After this, they were sequentially washed in 80% ethanol for 1 min, then again in 70% ethanol for 1 min, and then twice in distilled water. Finally, β-amyloid senile plaques were observed using a fluorescence microscope.

[0071] Congo red staining was performed to identify amyloid protein as follows. Brain sections obtained from coronal sections were fixed onto slides coated with 0.1% poly-L-lysine. The obtained brain sections were washed in PBS (phosphate-buffered saline) for 2 min and then stained in 0.5% Congo red solution (Thermo Fisher, USA) for 20 min. After washing with Congo red solution in PBS, the sections were rapidly immersed and removed 10 times in an alkaline alcohol solution containing 0.01% sodium hydroxide. The sections were then washed in PBS for 3 min, then in 95% ethanol for 3 min, and finally in 100% ethanol for 3 min to dehydrate. Amyloid senile plaques were observed under a fluorescence microscope, and the number of stained senile plaques and amyloid protein in each brain section was counted and quantified using a statistical program (Graph Prism).

[0072] As a result, such Figure 7 and 8 As shown, a significant number of β-amyloid plaques were identified in Alzheimer's model animals treated with PBS alone, but the accumulation of β-amyloid aggregates was significantly inhibited in Alzheimer's model animals treated with a peptide according to an embodiment of the present invention. 7-3: Analysis of the effects on the expression of different aggregate-forming proteins in the brain

[0073] Based on the results of the above experimental examples 7-2, the inventors used Western blot analysis to study the expression of pathogenic aggregate-forming proteins (e.g., tau protein, p62, and LC3) in brain tissues other than β-amyloid protein.

[0074] Specifically, cortical and hippocampal tissues were obtained from experimental animals euthanized after the experiment. The tissues were lysed and subjected to Western blot analysis in the same manner as in Experiment Example 3, 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.

[0075] As a result, such Figures 9a to 10b As shown, compared with control mice, all six proteins were increased in the cerebral cortex and hippocampus of Alzheimer's disease model mice, while all animals treated with the peptide (ZC311) according to an embodiment of the present invention showed significantly lower expression of the aforementioned aggregate-forming proteins in the cerebral cortex and hippocampus. These results indicate that the peptide according to an embodiment of the present invention can inhibit the formation of pathogenic protein aggregates in the brain even after in vivo administration. Experimental Example 8: Confirming the effectiveness of this treatment for amyotrophic lateral sclerosis (ALS) 8-1: Rotating bar test

[0076] To determine whether the peptide according to one embodiment of the present invention has a therapeutic effect on amyotrophic lateral sclerosis (ALS) in vivo, the inventors administered the peptide (ZC311) according to one embodiment of the present invention to amyotrophic lateral sclerosis model animals (G93A-SOD1) aged 112 to 200 days old at a dose of 40 mg / kg by repeated subcutaneous (SC) injections five days a week (Monday to Friday).

[0077] Then, the animals were subjected to rotarod tests twice a week at intervals of up to 3-4 days to assess their motor function. The rotarod test was performed using a rotarod fatigue meter (catalog number DJ-345, Dae Jong, Seoul, South Korea) to assess impaired coordination, a major symptom of basal ganglia lesions, by measuring the time it took for the test animals (n=11, 8, and 10) to fall from a bar at a constant speed of 15 rpm during three repetitions over 300 seconds. 300 seconds was set as the perfect score, and the highest score was used.

[0078] As a result, such Figure 11 As shown, ALS model mice began to exhibit ataxia after 116 days and became completely ataxia by 150 days, while the development of ataxia was delayed in animals treated with a peptide (ZC311) according to one embodiment of the invention. 8-2: Weight Changes

[0079] The inventors measured the change in body weight over time during the behavioral analysis of the above experimental example 8-1.

[0080] As a result, such Figure 12 As shown, ALS model animals gradually lose weight over time, a phenomenon not altered by the administration of the peptide according to an embodiment of the present invention. 8-3: Survival Rate

[0081] Amyotrophic lateral sclerosis (ALS) is a devastating disease that progresses after onset and ultimately leads to death. Animal models of ALS also eventually become 100% fatal some time after onset. The inventors sought to investigate how a peptide according to one embodiment of the present invention affects the survival of ALS model animals.

[0082] like Figure 13 As shown, the survival rate of animals undergoing the experiments in Experimental Example 8-1 was measured over time, and it was found that ALS model mice began to die at approximately 140 days, and all animals died by 178 days (50% survival at 162 days). In contrast, ALS model mice treated with the peptide according to one embodiment of the invention began to die at a similar time to the control group, but the time of death was delayed (50% survival at 183 days). These results indicate that the peptide according to one embodiment of the invention can delay ataxia (a key symptom of ALS) and subsequent death.

[0083] The present invention has been described with reference to the above-described embodiments and experimental examples, but these are merely illustrative, and those skilled in the art will understand that various modifications and equivalent embodiments and experimental examples can be derived from them. Therefore, the true scope of protection of the present invention should be determined by the technical concept of the appended claims. Industrial applicability

[0084] By inhibiting the abnormal hyperphosphorylation of pathogenic proteins and the resulting aggregate formation in the central nervous system, and by removing parasitic protein aggregates, the peptide according to one embodiment of the 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 4 to 10 a.a. and the following structural formula 1, which has the ability to inhibit the formation of abnormal protein aggregates and the autophagic activity against the protein aggregates: HGX 1 -X 2 -GX 3 (Structural Formula 1) (In the above structural formula, H is L- or D-histidine, G is glycine, X is 1 No D or L amino acids are present, X 2 is any D- or L-amino acid, and X 3 There is no D- or L-amino acid other than valine.

2. The peptide according to claim 1, wherein the peptide comprises at least one D-amino acid.

3. The peptide according to claim 1, wherein said X 1 Absent or nonpolar or polar amino acids.

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

6. The peptide according to claim 3, wherein said X 1 There is no valine or glutamine present or it is in the D or L form.

7. The peptide according to claim 1, wherein X2 is a polar amino acid, a nonpolar amino acid or an aromatic amino acid.

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

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

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

11. The peptide according to claim 1, wherein said X 2 It is a D-type or L-type amino acid selected from serine, histidine, valine, threonine, cysteine ​​and tryptophan.

12. The peptide according to claim 1, wherein said X 3 There is no polar or non-polar amino acid, or an amphipathic amino acid.

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

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

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

16. The peptide of claim 12, wherein the X3 is absent or is a D or L amino acid selected from aspartic acid, serine, asparagine, tyrosine, histidine and leucine.

17. The peptide according to claim 1, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 15.

18. A pharmaceutical composition for treating a neurodegenerative disease, comprising the peptide according to any one of claims 1 to 17 as an active ingredient.

19. The pharmaceutical composition according to claim 18, wherein the neurodegenerative disease has the formation of abnormal protein aggregates as its etiology or pathology.

20. The pharmaceutical composition according to claim 18, wherein the abnormal protein aggregates are formed by abnormal aggregation of α-synuclein, β-amyloid protein, TDP-43, p62 protein, FUS protein, superoxide dismutase-1 (SOD-1), huntingtin protein or tau protein.

21. The pharmaceutical composition of claim 19, 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.

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

23. A method of 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 17.

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