Novel peptide with MARK4 inhibitory activity

Abstract

To provide a novel peptide with MARK4 inhibitory activity.SOLUTION: Provided is a peptide that has an amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 1: PALRLVDPQ, and an amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2; and does not have the function of the oligopeptides at positions of 44 to 70 in the SEQ ID NO: 2: MTKLATTEELPDEFVVVT Orf9b.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a peptide and the use (pharmaceutical use) of the peptide etc.

Background Art

[0002] Alzheimer's disease is a progressive brain disease. In patients with Alzheimer's disease, memory, thinking ability, etc. are gradually impaired, and eventually it becomes difficult to perform even simple tasks in daily life. The number of patients with Alzheimer's disease is increasing year by year, and the development of its treatment method is eagerly desired. From previous studies, it has been found that in the brains of patients with Alzheimer's disease, a protein called tau accumulates in aggregates, causing nerve cells to die. The present inventors have found and reported that Microtubule Affinity Regulating Kinase 4 (MARK4) makes tau more likely to accumulate in nerve cells by phosphorylating tau (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a novel peptide having MARK4 inhibitory activity. [Means for solving the problem]

[0005] Under these circumstances, the inventors conducted research on the function of the gene Orf9b (positions 28284 to 28577 of the nucleic acid sequence shown in accession No. NC_045512) present in the novel coronavirus (SARS-CoV-2). Specifically, the inventors investigated the effect of Orf9b on MARK4 and found that the Orf9b peptide inhibits the activity of MARK4. Furthermore, the inventors focused on the region of the Orf9b sequence in which the base sequence is relatively conserved across species, and after much trial and error, found that a peptide in which the portion with binding activity to TOM70 was modified while retaining some of the conserved region also exhibits inhibitory activity against MARK4. The present invention is based on these novel findings.

[0006] Accordingly, the present invention provides the following: Item 1. Having an amino acid sequence represented by Sequence ID No. 1, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by Sequence ID No. 1, Sequence ID 1: PALRLVDPQ It has an amino acid sequence represented by Sequence ID No. 2, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence represented by Sequence ID No. 2, and Sequence ID 2: MTKLATTEELPDEFVVVT A peptide that does not possess the function of the oligopeptide at positions 44-70 in Orf9b.

[0007] Item 2. The amino acid sequence represented by Sequence ID No. 1, or the amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by Sequence ID No. 1. The peptide according to claim 1, comprising an amino acid sequence represented by Sequence ID No. 2, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence represented by Sequence ID No. 2, linked via a spacer.

[0008] Item 3. The peptide according to item 1 or 2, having the amino acid sequence represented by SEQ ID NO: 3, or the amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 3: Sequence ID 3: PALRLVDPQGGMTKLATTEELPDEFVVVT

[0009] Item 4. A peptide according to any one of items 1 to 3, having MARK4 inhibitory activity.

[0010] Item 5. A MARK4 inhibitor comprising a peptide described in any one of items 1 to 4.

[0011] Item 6. A phosphorylation inhibitor of tau protein, comprising a peptide described in any one of items 1 to 4.

[0012] Article 7. A pharmaceutical product comprising any one of the peptides described in Articles 1 to 4.

[0013] Item 8. A pharmaceutical product as described in Item 7, for the prevention or treatment of a disease that can be treated by MARK4 inhibition. [Effects of the Invention]

[0014] This invention provides a novel peptide having MARK4 inhibitory activity. As mentioned above, MARK4 phosphorylates tau, making it easier for tau to accumulate in nerve cells. Therefore, the peptide of this invention is expected to suppress tau phosphorylation and tau accumulation in nerve cells by inhibiting MARK4, and is effective in preventing and / or treating Alzheimer's disease. [Brief explanation of the drawing]

[0015] [Figure 1] Orf9b and its subsequences are shown (sequences 17-24). [Figure 2] This shows a three-dimensional model of Orf9b from CoV-2. [Figure 3]Shows the three-dimensional models of the portions at positions 10 to 29 and 76 to 95 in Orf9b of CoV-2. [Figure 4] Shows the test results in Reference Example 1. [Figure 5] Shows the test results in Reference Example 2. [Figure 6] Shows the test results in Example 1 (MARK4). [Figure 7] Shows the test results in Example 1 (MARK2). [Figure 8] Shows the test results in Example 2.

MODE FOR CARRYING OUT THE INVENTION

[0016] In the present invention, "protein" and "peptide" are used in a meaning including oligopeptides and polypeptides. Further, in the present specification, unless otherwise specified, "protein" and "peptide" include both peptides modified with sugar chains and the like and unmodified peptides.

[0017] In the present specification, "nucleic acid" is synonymous with nucleotide, oligonucleotide and polynucleotide, and may be any of DNA, RNA and DNA-RNA hybrid. Further, these may be double-stranded or single-stranded, and when referring to a nucleic acid molecule having a certain sequence, unless otherwise specified, it also comprehensively means a nucleic acid molecule (or nucleotide, oligonucleotide and polynucleotide) having a sequence complementary thereto. Further, these nucleic acid molecules may be cyclic or linear, and may be either synthetic or derived from organisms.

[0018] peptide The present invention has an amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 1: PALRLVDPQ It has an amino acid sequence represented by Sequence ID No. 2, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence represented by Sequence ID No. 2, and Sequence ID 2: MTKLATTEELPDEFVVVT This provides a peptide that does not possess the function of the oligopeptide at positions 44-70 in Orf9b.

[0019] In the present invention, Sequence IDs 1 and 2 are partial sequences of Orf9b (97 amino acids) derived from the novel coronavirus (severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)), respectively. More specifically, Sequence ID 1 is the amino acid sequence from positions 10 to 18 in Orf9b derived from SARS-CoV2, and Sequence ID 2 is the amino acid sequence from positions 78 to 95. The inventors anticipated that a region of Orf9b that is conserved among various viruses (e.g., SARS-CoV2, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), etc.) plays an important role, and focused on this region. As a result of diligent research, the inventors discovered that by modifying Orf9b so that the oligopeptide at positions 44 to 70 does not have function, while retaining the amino acid sequence portion represented by Sequence IDs 1 and 2, the activity against MARK4 is converted to inhibitory activity.

[0020] Figure 1 shows Orf9b and its partial sequence. As shown in Figure 1, the amino acid sequences at positions 10-29, 44-70, and 76-95 of Orf9b are conserved between CoV-1 and CoV-2. Figure 2 shows a three-dimensional model of Orf9b in CoV-2. Figure 3 shows a three-dimensional model of the 10-29 and 76-95 regions of Orf9b in CoV-2. As can be seen from Figures 2 and 3, the 10-29 and 76-95 regions of Orf9b in CoV-2 are located in close proximity. From the results of the examples described later and the three-dimensional models shown in Figures 2 and 3, it is considered that having the amino acid sequence portions represented by SEQ ID NOs: 1 and 2 in Orf9b, and not having the function of the oligopeptide at positions 44-70 in Orf9b, is important for MARK4 inhibitory activity. Furthermore, in the present invention, within the range in which MARK4 inhibitory activity is obtained, the amino acid sequence may be obtained by adding, deleting, and / or substituting one or more amino acids (for example, 1-2, 1, etc.) from the amino acid sequence shown in SEQ ID NO: 1, in place of the amino acid sequence portion shown in SEQ ID NO: 1. Examples of amino acid sequences obtained by adding, deleting, and / or substituting one or more amino acids (for example, 1-2, 1, etc.) from the amino acid sequence shown in SEQ ID NO: 1, in place of the amino acid sequence portion shown in SEQ ID NO: 1 include, PGLRLVDPQ (Sequence ID 4) PALRLVDPQG (Sequence ID 5) PLRLVDPQ (Sequence ID 6) These are some examples. Similarly, in the present invention, within the range in which MARK4 inhibitory activity is obtained, the amino acid sequence may be obtained by adding, deleting and / or substituting one or more amino acids (for example, 1-2, 1, etc.) from the amino acid sequence shown in SEQ ID NO: 2, in place of the amino acid sequence portion shown in SEQ ID NO: 2. Examples of amino acid sequences obtained by adding, deleting and / or substituting one or more amino acids from the amino acid sequence shown in SEQ ID NO: 2, MTKLAATTEELPDEFVVVT(Sequence ID 7) MTKLATTEELPDEFVVT (Sequence ID 8) MTKLGTTEELPDEFVVVT (Sequence ID 9) Examples include the following. Regarding the addition or deletion of amino acid sequences, the addition or deletion of neutral amino acids (amino acids other than acidic amino acids and basic amino acids) is preferred. Furthermore, regarding amino acid sequence substitutions, substitutions between neutral amino acids, substitutions between acidic amino acids, and substitutions between basic amino acids are preferred.

[0021] The peptide of the present invention may have a structure in which a peptide having the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence represented by SEQ ID NO: 1, and a peptide having the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence represented by SEQ ID NO: 2, are linked via a spacer. The spacer is not particularly limited as long as the effects of the present invention can be obtained, and a wide range of spacers used in the field of peptide design can be used. In such embodiments, the length of the spacer is not particularly limited, but examples include 1 to 20 amino acid residues, 1 to 10 amino acid residues, 1 to 5 amino acid residues, etc. Examples of amino acid sequences of spacers include the following. GG GGG GGGG (Sequence No. 13) GGGGG (Sequence No. 14) GGGGS (Sequence No. 15)

[0022] In the present invention, the oligopeptide at positions 44-70 in Orf9b is considered to be the binding site for TOM70. As described in the examples below, the peptide from which the binding site for TOM70 was removed showed inhibitory activity against MARK4. Therefore, it is considered that suppressing the binding activity to TOM70 affected the inhibitory activity against MARK4. Accordingly, in one embodiment, the present invention provides a peptide having the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and having the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 2, and having no binding activity to TOM70.

[0023] In the present invention, "lacking the function of the oligopeptide at positions 44-70 in Orf9b" includes not only the absence of the oligopeptide at positions 44-70 in Orf9b, but also modifications to the oligopeptide (for example, partial absence of the oligopeptide, modification of a portion of the oligopeptide, substitution, etc.) to the extent that the function of the oligopeptide at positions 44-70 in Orf9b is absent. Furthermore, in the present invention, "lacking the function of the oligopeptide at positions 44-70 in Orf9b" includes not only the complete loss of the function of the original oligopeptide at positions 44-70 in Orf9b (binding activity to TOM70), but also cases where the binding activity to TOM70 is reduced to 50% or less of the original Orf9b, typically 30% or less, and more typically 10% or less. The binding activity to TOM70 can be measured using the test method shown in Fig. 1b of Non-Patent Literature 2.

[0024] In a more specific embodiment, the present invention provides a peptide having an amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence represented by SEQ ID NO: 3: Sequence ID 3: PALRLVDPQGGMTKLATTEELPDEFVVVT.

[0025] The peptide of the present invention may have an amino acid sequence obtained by adding, deleting, and / or substituting one or more amino acids (for example, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1, etc.) from the amino acid sequence shown in SEQ ID NO: 3, within a range in which MARK4 inhibitory activity can be obtained.

[0026] Examples of amino acid sequences obtained by adding, deleting, and / or substituting one or more amino acids from the amino acid sequence shown in Sequence ID No. 3 include the following: PALRLVDPQGGGMTKLATTEELPDEFVVVT (Sequence ID 10) PALRLVDPQGMTKLATTEELPDEFVVVT (Sequence ID 11) PALRLVDPQGAMTKLATTEELPDEFVVVT (Sequence ID 12)

[0027] In this embodiment, the amino acid sequence shown in SEQ ID NO: 3 may have additions, deletions, and / or substitutions to the amino acid sequence portion shown in SEQ ID NOs: 1 and 2, but it is preferable that the amino acid sequence portion other than the portion shown in SEQ ID NOs: 1 and 2 has additions, deletions, and / or substitutions to the amino acid sequence portion.

[0028] The peptide of the present invention has MARK4 inhibitory activity. In a typical embodiment, the peptide of the present invention is useful because it selectively inhibits the activity of MARK4. Furthermore, the peptide of the present invention is characterized by inhibiting the activity of MARK4 through an allosteric reaction, rather than being a competitive inhibitor that attacks the catalytic active site of MARK4.

[0029] Pharmaceuticals In another embodiment, the present invention provides a pharmaceutical product comprising the peptide of the present invention or a nucleic acid expressing the peptide. As described above, the peptide of the present invention has MARK4 inhibition. Therefore, in a preferred embodiment, the present invention provides a pharmaceutical product comprising the peptide of the present invention for the prevention or treatment of diseases that can be treated by MARK4 inhibition. Examples of diseases that can be prevented and / or treated by MARK4 inhibition include Alzheimer's disease, tauopathy, cancer (malignant neoplasm), and diabetes. The peptide of the present invention can inhibit the phosphorylation of tau protein by inhibiting the activity of MARK4. Therefore, the pharmaceutical product of the present invention is preferably used for the prevention and / or treatment of Alzheimer's disease. Since conventional Alzheimer's treatments target amyloid, the pharmaceutical product of the present invention, which inhibits the phosphorylation of tau protein, can be an important option in the treatment of Alzheimer's disease.

[0030] The pharmaceutical product of the present invention may contain the peptide of the present invention itself, or it may contain a nucleic acid that expresses the peptide. In this specification, the peptide of the present invention or the nucleic acid that expresses the peptide may be referred to as the peptide of the present invention or the nucleic acid. Examples of nucleic acids that express the peptide of the present invention include nucleic acids (DNA, RNA, etc.) that encode the peptide of the present invention.

[0031] In the present invention, the active ingredient of the present invention, the peptide or nucleic acid itself, may be used as a pharmaceutical, or it may be used as a pharmaceutical composition in combination with various pharmaceutically acceptable carriers (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, viscosity modifiers, etc.).

[0032] Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol; polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride.

[0033] Examples of chelating agents include edetates such as disodium edetate, disodium calcium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, as well as ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate, and citric acid.

[0034] Examples of stabilizers include sodium bisulfite.

[0035] Examples of pH adjusting agents include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or bicarbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol.

[0036] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, and other quaternary ammonium salts, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine.

[0037] Examples of antioxidants include sodium bisulfite, anhydrous sodium sulfite, sodium pyrosulfite, and concentrated mixed tocopherols.

[0038] Examples of solubilizers include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol, and D-mannitol. Examples of viscosity-concentrating agents include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, chondroitin sulfate sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol.

[0039] In embodiments of the pharmaceutical composition, the content of peptides or nucleic acids in the composition is not particularly limited and can be appropriately set from conditions such as 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more, etc.

[0040] The formulation form is not particularly limited and can include various forms such as oral preparations like tablets, pills, capsules, powders, granules, and syrups; parenteral preparations like injections (intravenous, intramuscular, local injections, etc.), mouthwashes, drips, topical preparations (ointments, creams, patches, inhalants), and suppositories. Among the above formulation forms, injections are preferred, for example.

[0041] Furthermore, the pharmaceutical product of the present invention may contain the peptide of the present invention encapsulated in a vector such as a liposome. Alternatively, the pharmaceutical product of the present invention may contain a nucleic acid expressing the peptide of the present invention encapsulated in a vector such as a plasmid vector or a viral vector. In these embodiments, liposomes, plasmid vectors, viral vectors, etc., that are widely used in the pharmaceutical field can be used.

[0042] The amount of the peptide or nucleic acid of the present invention in the formulation cannot be specified in general terms as it varies depending on the route of administration, the patient's age, weight, symptoms, etc. However, it should be an amount that corresponds to a daily dose of approximately 0.1 to 5000 mg, more preferably 1 to 1000 mg, of the peptide or nucleic acid for an adult (weighing 50 kg). If administered once a day, this amount should be contained in one formulation; if administered three times a day, one-third of this amount should be contained in one formulation.

[0043] The pharmaceutical product of the present invention may further include, in addition to the peptide or nucleic acid of the present invention, a therapeutic agent for a disease that can be prevented and / or treated by MARK4 inhibition. Examples of therapeutic agents for diseases that can be prevented and / or treated by MARK4 inhibition include Alzheimer's disease treatments such as donepezil, galantamine, rivastigmine, and memantine hydrochloride; cancer treatments such as trastuzumab and paclitaxel; and diabetes treatments such as thiazolidinediones and metformin.

[0044] In such embodiments, the pharmaceutical product of the present invention may be a combination formulation containing not only the peptide or nucleic acid of the present invention and another therapeutic agent for a disease that can be prevented and / or treated by MARK4 inhibition in a single formulation, but also a combination formulation containing the peptide or nucleic acid of the present invention and another therapeutic agent for a disease that can be prevented and / or treated by MARK4 inhibition in a single formulation.

[0045] The pharmaceutical product of the present invention is administered to mammals, etc. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cattle, horses, sheep, and the like.

[0046] MARK4 Inhibitor As described above, the peptide of the present invention has MARK4 inhibitory activity. Accordingly, in one embodiment, the present invention provides a MARK4 inhibitor comprising the above-mentioned peptide or a nucleic acid expressing the peptide. In this embodiment, the details of the peptide of the present invention and the nucleic acid expressing the peptide, as well as the method of use thereof, are the same as in the "Peptide" and "Pharmaceuticals" sections above. MARK4 inhibitory activity can be measured, for example, by the method described in the examples below.

[0047] Tau protein phosphorylation inhibitors As mentioned above, MARK4 makes tau more likely to accumulate in nerve cells by phosphorylating it. Therefore, in one embodiment, the present invention provides a tau protein phosphorylation inhibitor comprising the above-mentioned peptide or a nucleic acid expressing the peptide. In this embodiment as well, the details of the peptide and the nucleic acid expressing the peptide, and the method of use thereof are the same as in the "Peptide" and "Pharmaceuticals" sections above.

[0048] The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0049] Reference example 1 We expressed the full-length Orf9b gene derived from SARS-CoV-2 in primary mouse neuronal cells and examined tau phosphorylation using a phosphorylation antibody. Specifically, the full-length Orf9b gene (291 bp) was artificially synthesized using a contract service (Eurofins), and the inserted gene was incorporated into a plasmid vector expressing it as a fusion protein with a C-terminal 3xFLAG tag under a CAG promoter to construct the expression vector. Primary mouse neuronal cells were prepared by dispersing the cerebral cortex of mouse embryos (16-day embryos) with trypsin. After gene transfer of the plasmid vector to the dispersed cells using electroporation (Lonza Nucleofector), the cells were seeded in culture dishes coated with polylysine. As a control, an empty plasmid vector (pCAG vector) was similarly gene-transfected. After culturing for 6 days, whole cell lysates were prepared, and tau phosphorylation was examined by Western blotting using phosphorylation antibodies (p-S356, Abcam ab92682), and whole tau was examined by Western blotting using an anti-tau monoclonal antibody (Tau-5). The results are shown in Figure 4. As shown in Figure 4, expression of full-length Orf9b reduced tau phosphorylation. Actin was detected as a loading control using an anti-actin antibody (SIGMA), and Orf9b was detected using an anti-FLAG monoclonal antibody (M2).

[0050] Reference example 2 The kinase activity of MARK4 in cell extracts was measured after expressing full-length Orf9b derived from SARS-CoV-2 in primary cultured mouse nerve cells. Specifically, nerve cells transfected with Orf9b were cultured for 6 days, as in Reference Example 1, and then the cells were harvested. After disrupting the cells with a cell extraction buffer containing 0.5% Nonidet P-40, the supernatant was collected as the cell extract after centrifugation at 17,000 xg for 15 minutes. MARK4 in the cell extract was immunoprecipitated using an anti-MARK4 antibody (Cell Signaling #4834) and magnetic beads (Thermo Fisher Dynabeads Protein G). The kinase activity was then measured in vitro using radiolabeled ATP[γ-32P] with human tau (2N4R type) expressed and purified from E. coli as a substrate. The results are shown in Figure 5. As shown in Figure 5, MARK4 activity decreased upon Orf9b expression.

[0051] Example 1 The effects of this peptide on MARK4 (Figure 6) and MARK2 (Figure 7) were investigated by in vitro kinase assay. Specifically, MARK4 and MARK2, with a MYC tag added to the C-terminus, were expressed on HEK293, and MARK4 and MARK2 were immunoprecipitated from cell extracts using an anti-Myc monoclonal antibody (4A6), similar to Reference Example 2. The peptide was added to the reaction mixture at final concentrations of 1, 10, 20, and 100 μM, and kinase activity was measured in vitro using radiolabeled ATP[γ-32P] with the peptide (CHKtide) as the substrate. The results are shown in Figures 6 and 7. As shown in Figure 6, this peptide partially inhibited MARK4 activity. On the other hand, as shown in Figure 7, no inhibitory effect was observed on MARK2.

[0052] Example 2 Primary mouse neuronal cells were treated with a peptide to which the cell membrane-permeable peptide sequence TAT(YGRKKRRQRRR) (SEQ ID NO: 16), consisting of 11 amino acids, was added to the N-terminus of the original peptide. Tau phosphorylation was then examined using a phosphorylation antibody. Specifically, neuronal cells were prepared in the same manner as in Reference Examples 1 and 2 and cultured for 4 days. After adding the TAT-added peptide at a final concentration of 10 μM to the culture medium, cells were harvested at each time interval. Whole cell lysates were prepared, and tau phosphorylation was examined by Western blotting using phosphorylation antibodies (p-S356, Abcam ab92682), and total tau was examined by Western blotting using an anti-tau monoclonal antibody (Tau-5). The results are shown in Figure 8. As shown in Figure 8, tau phosphorylation and tau expression decreased in a time-dependent manner. Actin and GAPDH were detected as loading controls using anti-actin antibody (SIGMA) and anti-GAPDH antibody (Fujifilm Wako).

Claims

1. A peptide having the amino acid sequence PALRLVDPQG n MTKLATTEELPDEFVVVT (where n is an integer from 2 to 5).

2. The peptide according to claim 1, having the amino acid sequence represented by Sequence ID No. 3: Sequence ID 3: PALRLVDPQGGMTKLATTEELPDEFVVVT

3. The peptide according to claim 1 or 2, having MARK4 inhibitory activity.

4. A MARK4 inhibitor comprising the peptide described in any one of claims 1 to 3 or a nucleic acid expressing the peptide.

5. A phosphorylation inhibitor for tau protein, comprising the peptide described in any one of claims 1 to 3 or a nucleic acid expressing the peptide.

6. A pharmaceutical product comprising a peptide according to any one of claims 1 to 3 or a nucleic acid expressing the peptide.

7. The pharmaceutical agent according to claim 6 for preventing or treating a disease that can be treated by MARK4 inhibition.

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