Aβ production inhibitory peptide and pharmaceutical composition, food and drink containing the same

Abstract

To provide an Aβ production inhibitory peptide capable of inhibiting Aβ production / release by targeting a new process related to Aβ aggregate formation without directly targeting β-secretase and γ-secretase, and also provide pharmaceutical compositions and foods and beverages, which contain the Aβ production inhibitory peptide.SOLUTION: Aβ production inhibitory peptides are multivalent peptides comprising two or more peptide motifs consisting of specific amino acid sequences. The Aβ production inhibitory peptide may be a tetravalent peptide containing four of the above peptide motifs.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an Aβ production inhibitory peptide and a pharmaceutical composition, food or drink containing the same. [Background technology]

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by a decline in memory and learning ability. Since amyloid beta (Aβ) aggregates are observed in the brain in the early stages of AD onset, it is believed that Aβ aggregation in the brain is the cause of AD onset. Therefore, inhibiting Aβ production and aggregation is thought to be an effective AD treatment strategy.

[0003] Aβ is produced by the sequential cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase. Specifically, APP is cleaved by β-secretase to produce the C99 peptide (C99), which is then cleaved by γ-secretase to produce Aβ (Aβ40 and Aβ42). The produced Aβ is then released outside the cell via vesicular transport and forms insoluble aggregates. Therefore, it is thought that Aβ aggregate formation can be suppressed by inhibiting any of these processes.

[0004] To date, for example, CNP520 (Non-Patent Document 1) has been developed as an inhibitor that directly targets β-secretase, and Semagacestat (Non-Patent Document 2) has been developed as an inhibitor that directly targets γ-secretase.

[0005] On the other hand, peptides have been developed that do not act directly on γ-secretase but bind to its substrate C99, thereby inhibiting Aβ production by γ-secretase (for example, Non-Patent Document 3). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5718574 [Patent Document 2] Patent No. 5897178 [Patent Document 3] Patent No. 6422046 [Patent Document 4] Patent No. 4744443 [Non-patent literature]

[0007] [Non-Patent Document 1] Alzheimers Dement, 2019, 5, 216-227. [Non-patent document 2] N. Engl. J. Med., 2013, 369, 341-350. [Non-patent document 3] Nat. Commun., 2013, 4:2529. doi: 10.1038 / ncomms3529 Summary of the Invention [Problem to be solved by the invention]

[0008] However, because β- and γ-secretases use multiple molecules other than APP and C99 as physiological substrates, conventional inhibitors such as those described in Non-Patent Documents 1 and 2 have been problematic in that they inhibit the cleavage of these molecules, resulting in side effects.

[0009] On the other hand, the peptide of Non-Patent Document 3 has not yet reached a level where its therapeutic effect on Alzheimer's disease (AD) has been fully established, and further investigation is required.

[0010] The present invention was made in consideration of the above circumstances, and aims to provide an Aβ production-inhibitory peptide that can inhibit the production and release of Aβ by targeting a new process related to Aβ aggregate formation, rather than directly targeting β- and γ-secretase, as well as pharmaceutical compositions, and foods and beverages containing the same. [Means for solving the problem]

[0011] In order to solve the above problems, the Aβ production inhibitory peptide of the present invention is characterized by being a multivalent peptide containing two or more peptide motifs consisting of the amino acid sequence of SEQ ID NO:1.

[0012] The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating a disease caused by Aβ aggregation, and is characterized by containing the Aβ production-inhibitory peptide.

[0013] The food and drink of the present invention is a food and drink for preventing or treating a disease caused by Aβ aggregation, and is characterized by containing the Aβ production inhibitory peptide. [Effects of the Invention]

[0014] The Aβ production-inhibitory peptide of the present invention can inhibit Aβ production and release by targeting a novel process related to Aβ aggregate formation, rather than directly targeting β- and γ-secretase. Therefore, the Aβ production-inhibitory peptide of the present invention can be used as a pharmaceutical composition for the prevention and treatment of diseases caused by Aβ aggregation (Alzheimer's disease).

[0015] The pharmaceutical composition and food and drink of the present invention can prevent and treat diseases caused by Aβ aggregation (Alzheimer's disease) by inhibiting the production and release of Aβ. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram showing the structure of a multivalent peptide and the structure of a multivalent peptide synthesized on a sheet. [Figure 2]This figure shows the results of the primary screening. A peptide library having the tetravalent sequence shown in Figure 2 was spot-synthesized on a sheet. The sheet was incubated overnight at 4°C in the presence of 10 micrograms / ml of biotinylated Aβ1-28. After washing, the sheet was colored with avidin-HRP to detect the bound Aβ1-28. [Figure 3] 1 shows the results of the secondary screening. A peptide library having the tetravalent sequence shown in FIG. 3 was spot synthesized on a sheet, and the same examination as in the primary screening was carried out. [Figure 4] 4 shows the results of the tertiary screening. A peptide library having the tetravalent sequence shown in FIG. 4 was spot synthesized on a sheet, and the same examination as in the primary and secondary screenings was carried out. [Figure 5] 5 shows the results of the fourth screening (I series). A peptide library having the tetravalent sequences shown in FIG. 5 was spot synthesized on a sheet, and the same investigation as in the first to third screenings was carried out. [Figure 6] 6 shows the results of the fourth screening (II series). A peptide library having the tetravalent sequences shown in FIG. 6 was spot synthesized on a sheet, and the same investigation as in the first to third screenings was carried out. [Figure 7] 7 shows the results of the fifth screening (I series). A peptide library having the tetravalent sequences shown in FIG. 7 was spot synthesized on a sheet, and the same investigation as in the first to fourth screenings was carried out. [Figure 8] 8 shows the results of the fifth screening (II series). A peptide library having the tetravalent sequences shown in FIG. 8 was spot synthesized on a sheet, and the same investigation as in the first to fourth screenings was carried out. [Figure 9] 9 shows the results of the sixth screening (Ia system). A peptide library having the tetravalent sequences shown in FIG. 9 was spot synthesized on a sheet, and the same investigation as in the first to fifth screenings was carried out. [Figure 10]10 is a diagram showing the results of the sixth screening (Ib system). A peptide library having the tetravalent sequences shown in FIG. 10 was spot synthesized on a sheet, and the same investigation as in the first to fifth screenings was carried out. [Figure 11] 11 shows the results of the sixth screening (II series). A peptide library having the tetravalent sequences shown in FIG. 11 was spot synthesized on a sheet, and the same investigation as in the first to fifth screenings was carried out. [Figure 12] 12 shows the results of the seventh screening (Ic, Id, Ie, If, IIa, and IIb systems). A peptide library having the tetravalent sequences shown in FIG. 12 was spot-synthesized on a sheet, and the same investigations as in the first to sixth screenings were carried out. [Figure 13] FIG. 1 shows the identification scheme (1st to 7th screenings) of Aβ1-28 high-affinity binding motifs using the tetravalent random peptide sheet screening method. The randomized 7 amino acids in the library part (XXXXXXX) are shown. [Figure 14] FIG. 1 shows the sequences of identified peptide motifs and the names of their tetravalent forms. [Figure 15] Figure showing the inhibitory effect of tetravalent peptides on the amount of Aβ released outside the cells. a) CHO cells stably expressing human APP were cultured with each tetravalent peptide (50 μM) for 48 hours, and then the amounts of C99 and Aβ in the cell lysate, as well as the amount of Aβ in the culture medium, were detected and quantified using the human Aβ N-terminal end-specific antibody 82E1. b) The effect of tetravalent peptides on the amount of C99 in cells is shown. c) The effect of tetravalent peptides on the amount of Aβ in cells is shown. d) The effect of tetravalent peptides on the amount of Aβ released outside the cells is shown. Mean ± SE (n=3), *, p<0.05 vs (-) by Dunnett test [Figure 16]This figure shows that LME-tet binds to Aβ40 and Aβ42 via the clustering effect. a) LME-tet (8.3 μM) or LME-mono (33 μM) was immobilized on an ELISA plate, and various concentrations of biotinylated Aβ1-28, Aβ40, or Aβ42 were bound to it. Biotinylated Aβ1-28 was detected using avidin-HRP, and Aβ40 and Aβ42 were detected by ELISA using specific antibodies. Mean ± SE (n=3). b) CHO cells stably expressing human APP were cultured with various concentrations of LME-mono for 48 hours, and the amounts of C99 and Aβ in the cell lysate, as well as the amount of Aβ in the culture medium, were detected and quantified using specific antibodies. When using LME-mono, a four-fold molar concentration compared to LME-tet can be used to achieve the same number of molar peptide chains. Mean ± SE (n=3), vs (-) by Dunnett test. [Figure 17] Figure 1 shows the inhibitory effect of tetravalent peptides on the amount of Aβ released from neurons. ad) Human neuroblastoma SH-SY5Y cells were cultured with LME-tet (25 μM) or LME-mono (100 μM) for 48 hours. C99 (b) and Aβ levels (c) in the cell lysate, and Aβ levels (d) in the culture medium were immunoprecipitated with the human Aβ N-terminus-specific antibody 6E10, and then detected and quantified with the specific antibody 82E1. Mean ± SE (n=3), vs (-) by Dunnett test. [Figure 18]Figure 1 shows that LME-tet binds to APP and inhibits β-secretase activity. a) Recombinant Flag-tagged APP633-685, which contains the β-secretase cleavage site, was used as a substrate to examine the cleavage activity of purified human β-secretase. Reactions were performed at 37°C for 4 hours in the presence of various concentrations of LME-mono or LME-tet. The cleavage product, Aβ33-Flag, in the reaction mixture was analyzed by Western blotting using the specific antibody 82E1. Mean ± SE (n=3), **, p<0.01, ***, p<0.001, vs (-) by Dunnett test. b) Recombinant Flag-tagged C99 was used as a substrate to examine the cleavage activity of HEK293 lysates expressing human γ-secretase. Reactions were performed at 37°C for 4 hours in the presence of various concentrations of LME-tet. The cleavage product, Aβ, in the reaction mixture was analyzed by Western blotting using the specific antibody 82E1. mean ± SE (n=3), vs (-) by Dunnett test [Figure 19] This figure shows that LME-tet reduces Aβ accumulation in the brain of AD model mice. 6-8 week-old APPNLGF mice were intraperitoneally administered PBS (n=6) or N-terminally acetylated LME-tet (71 mg / kg) (n=7) one week later, their brains were removed and the cerebral cortex and olfactory bulbs were collected. After homogenization, Aβ levels were quantified by Western blotting using the specific antibody 82E1. A standard curve was prepared using various amounts of purified Aβ, and quantification was performed. Each lane represents a sample from each individual. Mean ± SE, *, p<0.05 by Man-Witney U test DETAILED DESCRIPTION OF THE INVENTION

[0017] The present inventors have focused on the fact that the Aβ sequence is shared by both APP and C99, and have conceived the idea that if a series of high-affinity peptides can be obtained using a partial region of Aβ, Aβ1-28 (the region from the amino terminus of Aβ that is not embedded in the membrane to Lys at position 28), as a probe, and that if peptides that inhibit Aβ production and release at the cellular level can be identified from the obtained peptides, they could be established as AD therapeutic drugs with a new mechanism of action. Furthermore, focusing on the tendency of APP and Aβ to form multimers, the inventors have considered that the use of a multivalent peptide sheet screening method originally developed by the present inventors (Patent Documents 1-3) would be effective in obtaining such high-affinity peptides.

[0018] Hereinafter, one embodiment of the Aβ production inhibitory peptide, pharmaceutical composition, and food and drink of the present invention will be described.

[0019] (Aβ production inhibitor peptide) The Aβ production inhibitory peptide of the present invention comprises: SEQ ID NO: 1: Pro-Lys-Leu-Arg-Met-Lys-Glu (PKLRMKE) The Aβ production-inhibitory peptide of the present invention is a multivalent peptide containing two or more peptide motifs consisting of the amino acid sequence shown in SEQ ID NO: 1. That is, the Aβ production-inhibitory peptide of the present invention includes, for example, a bivalent peptide containing two peptide motifs shown in SEQ ID NO: 1, a tetravalent peptide containing four peptide motifs, and the like. Of these, the Aβ production-inhibitory peptide of the present invention is preferably a tetravalent peptide containing four peptide motifs shown in SEQ ID NO: 1.

[0020] In the Aβ production inhibitory peptide of the present invention, the form and synthesis method of the multivalent peptide are not particularly limited. Specifically, for example, in peptide synthesis, condensation and removal of protecting groups can be performed according to conventionally known methods. For example, solid-phase peptide synthesis, Fmoc peptide synthesis, etc. can be used, and the synthesis can also be performed using a commercially available peptide synthesizer.

[0021] Preferably, the peptide synthesis method on a sheet (Patent Document 1) already proposed by the present inventors can be considered. A divalent or tetravalent peptide synthesis core structure can be formed from amino groups on a sheet of cellulose, resin, or the like. Specifically, for example, Fmoc-Lys or Fmoc-His can be used to form divalent or tetravalent synthetic core structures. Considering interactions with other amino acids, Fmoc-Lys is preferred. Branching points can be established by introducing Fmoc-Lys, and amino acid synthesis from the branching points can be designed to be divalent.

[0022] Furthermore, when forming a tetravalent synthetic core structure, peptide synthesis using Fmoc-Lys twice in succession is considered. The synthetic core structure is not specifically limited as long as it can elongate a divalent or tetravalent peptide.

[0023] Furthermore, a spacer can be appropriately introduced into a divalent or tetravalent peptide. The spacer is not particularly limited, but an example is Ahx (amino hexanoic acid [NH-(CH-)-COOH]). Furthermore, trifluoroacetic acid, for example, can be appropriately used to cleave a synthetic peptide.

[0024] Furthermore, when the Aβ production inhibitory peptide of the present invention is in the form of a tetravalent peptide, it has the following molecular core structure (chemical formula 1) formed by the bonding of three lysines (Lys):

[0025] [ka]

[0026] An example of such a tetravalent peptide is one in which the peptide motif of SEQ ID NO: 1 is bound directly or via a spacer to each of the four amino groups located at the ends of the peptide.

[0027] A more preferred embodiment is, for example, a tetravalent peptide in which either the peptide (A) or (B) above is incorporated into each of the four XXXX moieties located at the ends of a molecular core structure consisting of three lysines (Lys) in the following chemical formula 2.

[0028] [ka]

[0029] In the above chemical formula 2, the position where the peptide motif of SEQ ID NO: 1 is incorporated is conveniently indicated as "XXXX." Furthermore, while the above chemical formula 2 illustrates a configuration in which a spacer is attached to each of the four amino groups located at the ends of the molecular core structure, the peptide motif of SEQ ID NO: 1 can also be attached directly to each of the four amino groups without the intermediary of a spacer. When a spacer is attached, it is sufficient as long as it does not impair the binding ability to APP or the inhibitory effect on β-secretase activity. The specific molecule and length of the spacer are not limited, and it can be designed appropriately. A preferred spacer is, for example, one with a chain length of approximately 4 to 10 carbon atoms and an amino acid at its terminal, and a particularly preferred example is amino hexanoic acid [NH2-(CH2)5-COOH] (aminocaproic acid) shown in the above chemical formula 2. An example of an amino acid contained in the spacer is alanine (A).

[0030] This form of Aβ production inhibitory peptide (tetravalent peptide) can also be prepared by known methods, for example, using a peptide synthesizer, etc. For example, the peptide motif of SEQ ID NO: 1 incorporated into the "XXXX" portion of Chemical Formula 2 can be synthesized by sequentially adding amino acids to a tetravalent core structure.

[0031] Furthermore, the Aβ production inhibitory peptide may have a modifying molecule at each end of the peptide motif incorporated into the XXXX portion of Chemical Formula 2. Since an exposed NH2 at the peptide terminus results in a positive charge, from the perspective of charge regulation, it is also possible to attach an uncharged molecule or even a hydrophobic molecule as a modifying molecule to each terminus. Furthermore, for example, when a pharmaceutical composition containing the Aβ production inhibitory peptide of the present invention is orally administered, the terminal NH2 can be protected with an acetyl group for the purpose of stabilization to prevent degradation by proteases in the gastrointestinal tract. Thus, the modifying molecule at the terminus of the peptide motif can be appropriately selected depending on the desired effect, and modifications such as phosphorylation, methylation, adenylylation, and glycosylation may be added. Furthermore, the Aβ production inhibitory peptide may also contain one or more amino acids, for example, at the C-terminus or N-terminus of the peptide motif.

[0032] The Aβ production-inhibitory peptide of the present invention exerts a cluster effect (a phenomenon in which binding affinity is significantly enhanced by the formation of multivalent-multivalent interactions) to efficiently bind to intracellular APP and inhibit β-secretase activity, thereby inhibiting the production of C99 and ultimately suppressing Aβ production and release. Furthermore, the Aβ production-inhibitory peptide of the present invention targets a novel process related to Aβ aggregate formation and does not directly target β-secretase or γ-secretase, thereby eliminating the risk of side effects caused by inhibiting the cleavage of molecules that serve as substrates for these enzymes. Therefore, the Aβ production-inhibitory peptide of the present invention can be used in pharmaceutical compositions for the prevention or treatment of diseases caused by Aβ aggregation.

[0033] (Pharmaceutical composition) The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating diseases caused by Aβ aggregation, and contains the Aβ production-inhibitory peptide of the present invention described above. Therefore, the pharmaceutical composition of the present invention can inhibit the production and release of Aβ as described above, and can prevent or treat Alzheimer's disease.

[0034] The pharmaceutical composition of the present invention may use the Aβ production inhibitory peptide of the present invention as an active ingredient as is, or may be formulated by adding pharmaceutically acceptable ingredients.

[0035] The pharmaceutical composition may be in a dosage form for oral use or a dosage form for parenteral use. Examples of dosage forms for oral use include powders, granules, tablets, capsules, elixirs, microcapsules, etc. Examples of dosage forms for parenteral use include injections, drip infusions, ointments, inhalants, suppositories, etc.

[0036] The pharmaceutical composition may contain a pharmaceutically acceptable carrier, such as a known excipient, disintegrant, binder, lubricant, surfactant, buffer, solubilizer, stabilizer, isotonicity agent, suspending agent, emulsifier, solvent, thickener, mucolytic agent, wetting agent, preservative, etc.

[0037] The pharmaceutical composition may further contain additives, such as lubricants such as magnesium stearate, sweeteners such as sucrose, lactose, and saccharin, flavorings such as peppermint and rhododendron oil, stabilizers for benzyl alcohol and phenol, buffers such as phosphates and sodium acetate, solubilizers such as benzyl benzoate and benzyl alcohol, antioxidants, and preservatives.

[0038] The pharmaceutical composition can be formulated by appropriately combining the above-mentioned carriers, additives, etc. and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.

[0039] The dosage of the pharmaceutical composition varies depending on the patient's weight, age, symptoms, administration method, etc., but a person skilled in the art can appropriately select an appropriate dosage. The dosage when the Aβ production-inhibitory peptide of the present invention is administered to a human varies depending on symptoms, the patient's age, sex, weight, sensitivity difference, administration method, administration interval, type of active ingredient, and type of formulation, and is not particularly limited, but can be, for example, 100 μg to 1000 mg administered once or in divided doses.

[0040] (Food and beverages) The food and drink products (foods and beverages) of the present invention are for preventing or treating diseases caused by Aβ aggregation and contain the Aβ production-inhibitory peptide of the present invention described above. Therefore, the food and drink products of the present invention can inhibit the production and release of Aβ as described above, and can prevent or treat Alzheimer's disease.

[0041] Examples of foods and beverages include health foods and beverages (supplements, dietary supplements, health supplements, nutritionally balanced foods, etc.) in the form of tablets, tablets, chewable tablets, powders, powders, capsules, granules, drinks, etc., soft drinks, tea drinks, jelly drinks, sports drinks, coffee drinks, carbonated drinks, vegetable drinks, fruit juice drinks, fermented vegetable drinks, fermented fruit juice drinks, fermented milk drinks (yogurt, etc.), lactic acid bacteria drinks, milk drinks, powdered drinks, cocoa drinks, alcoholic drinks, confectionery, jellies, etc. Furthermore, the "food and drink" of the present invention may contain ingredients commonly used in the field of food engineering. Specific examples include various oils and fats (e.g., vegetable oils such as soybean oil, corn oil, safflower oil, olive oil, etc., and animal oils and fats such as beef tallow and sardine oil), herbal medicines (e.g., royal jelly, ginseng, etc.), amino acids (e.g., glutamine, cysteine, leucine, arginine, etc.), polyhydric alcohols (e.g., ethylene glycol, polyethylene glycol, propylene glycol, glycerin, sugar alcohols such as sorbitol, erythritol, xylitol, maltitol, mannitol, etc.), natural polymers (e.g., gum arabic, agar, water-soluble corn fiber, gelatin, xanthan gum, etc.), and the like. Examples of suitable surfactants include gluten, gluten hydrolysates, lecithin, starch, dextrin, etc.), vitamins (e.g., vitamin C, B vitamins, etc.), minerals (e.g., calcium, magnesium, zinc, iron, etc.), dietary fiber (e.g., mannan, pectin, hemicellulose, etc.), surfactants (e.g., glycerin fatty acid esters, sorbitan fatty acid esters, etc.), purified water, diluents, stabilizers, isotonicity agents, pH adjusters, buffers, humectants, solubilizers, suspending agents, colorants, flavoring agents, odorants, fragrances, antioxidants, sweeteners, taste-providing components, and acidulants.

[0042] The amount of food and drink to be ingested will vary depending on the patient's weight and age, symptoms of the patient, administration method, etc., but a person skilled in the art can appropriately select an appropriate dosage.

[0043] The Aβ production inhibitory peptide of the present invention and the pharmaceutical composition, food and drink containing the same are not limited to the above embodiments. [Example]

[0044] The present invention will be described below with reference to examples, but the Aβ production inhibitory peptide of the present invention and the pharmaceutical composition, food and drink containing the same are not limited to the following examples in any way.

[0045] (Principle of high-affinity motif identification technology using tetravalent random peptide sheet screening method) Using a technology for sheet synthesis of bivalent or tetravalent peptide libraries on a nitrocellulose sheet (Patent Documents 1-3), the following tetravalent peptide library is synthesized on the sheet (Figure 1). (ZXXXXXX-Ahx)4-3Lys Z represents 19 fixed amino acids excluding Cys, X represents a mixture of 19 amino acids excluding Cys, and Ahx represents caproic acid as an example of a spacer (the basic structure is the same as in Figure 1). If Z is shifted from position 1 to 7 (Figure 1), a total of 133 (=19*7) types of libraries will be spot-synthesized (primary library). Biotin-labeled Aβ1-28 (with biotinylated Lys introduced at the carboxy terminus) is bound to this sheet, and the optimal amino acid and its fixed position (here, tentatively designated XXXB4XXX) are determined using its binding activity as an indicator. Binding is detected using a colorimetric method using avidin-conjugated peroxidase.

[0046] Next, based on the obtained information, 114 (=19*6) types consisting of (ZXXB4XXX-Ahx)4-3Lys are spot-synthesized (secondary library). Similar procedures are performed to determine the optimal amino acid and its fixed position. Similar screening is repeated (up to the seventh library, since there are seven Xs) to determine the optimal amino acid for each X, and the optimal motif, B1B2B3B4B5B6B7, is determined. This screening method determines the optimal amino acid for each position, eliminating the redundancy problem that occurs with motif determination methods using amino acid sequencing (Patent Document 4), and significantly increasing the number of inhibitory motifs that can ultimately be obtained. Tetravalent peptide compounds in which the identified motifs are incorporated into the same core structure as the multivalent peptide library can be used as final inhibitor candidates.

[0047] Example 1: Identification of Aβ1-28 high-affinity motifs using tetravalent random peptide sheet screening method (First screening) The binding activity of each peptide was evaluated by blotting a sheet on which a tetravalent primary random peptide library (library part: XXXXXXX-Ahx) was spot-synthesized with biotin-labeled Aβ1-28 (Figure 2, Table 1). As a result, it was found that the amount of binding to Aβ1-28 was greatest when the sixth position was Lys.

[0048] [Table 1]

[0049] (Second screening) Therefore, a secondary library with XXXXXKX-Ahx in the library section was created and similar studies were performed (Figure 3, Table 2). Similar analysis revealed that the amount of binding to Aβ1-28 was greatest when the first position was Pro.

[0050] [Table 2]

[0051] (Third screening) Therefore, we created a tertiary library with PXXXXKX-Ahx in the library section and performed the same study (Figure 4, Table 3). Similar analysis revealed that the binding to Aβ1-28 was greatest when the seventh position was Glu. Furthermore, to ensure diversity, we also adopted a separate lineage with Thr at the third position.

[0052] [Table 3]

[0053] (4th screening) Therefore, we created a fourth library with PXXXXKE-Ahx (series I) and PXTXXKX-Ahx (series II) in the library section and performed a similar study. As a result, the most strongly selected clones had Lys at the second position in series I (Fig. 5, Table 4), and the most strongly selected clones had Lys at the second position in series II (Fig. 6, Table 5).

[0054] [Table 4]

[0055] [Table 5]

[0056] (5th screening) Therefore, a fifth library was created with PKXXXKE-Ahx (series I) and PKTXXKX-Ahx (series II) in the library section, and similar studies were performed. As a result, the most strongly selected clones were those with Met at the fifth position in series I (Figure 7, Table 6) and those with Glu at the fourth position in series II (Figure 8, Table 7). Furthermore, to ensure diversity, a clone with Phe at the third position was also selected as a separate clone for series I.

[0057] [Table 6]

[0058] [Table 7]

[0059] (6th screening) Therefore, we created a sixth library with PKXXMKE-Ahx (Ia lineage), PKFXXKE-Ahx (Ib lineage), and PKTEXKX-Ahx (II lineage) in the library section and performed similar experiments. As a result, in lineage Ia, Lys at the third position was most strongly selected. Furthermore, to ensure diversity, a separate lineage with Arg at the fourth position was also adopted for lineage Ia (Figure 9, Table 8). In lineage Ib, Lys at the fourth position was most strongly selected. Furthermore, to ensure diversity, a separate lineage with Arg at the fourth position was also adopted for lineage Ib (Figure 10, Table 9). In lineage II, Lys at the fifth position was most strongly selected. Furthermore, to ensure diversity, a separate lineage with Arg at the fifth position was also adopted for lineage II (Figure 11, Table 10).

[0060] [Table 8]

[0061] [Table 9]

[0062] [Table 10]

[0063] (7th screening) Therefore, we created a seventh library containing PKKXMKE-Ahx (Ic lineage), PKXRMKE-Ahx (Id lineage), PKFKXKE-Ahx (Ie lineage), PKFRXKE-Ahx (If lineage), PKTEKKX-Ahx (IIa lineage), and PKTERKX-Ahx (IIb lineage) in the library section, and performed similar studies. As a result, 15 species with strong binding activity were selected (Figure 12, Table 11, Table 12).

[0064] [Table 11]

[0065] [Table 12]

[0066] Furthermore, because the Ib series was not included in the top 15, the top two were selected separately from the Ie and If series. The outline of the first to seventh screenings (Figure 13) and the final selected motifs (Figure 14) are shown below.

[0067] Example 2: Effects of each tetravalent peptide on intracellular C99 and Aβ levels, and extracellular Aβ levels Each peptide motif obtained in Example 1 was incorporated into the library portion (XXXXXXX) of the molecular core structure shown in Figure 1 and Chemical Formula 2 to create a tetravalent peptide (Figure 14).

[0068] CHO cells stably expressing human APP were cultured with each tetravalent peptide (50 μM) for 48 hours, and the amounts of C99 and Aβ in the cell lysate, as well as the amount of Aβ in the culture medium, were detected and quantified using specific antibodies.

[0069] As a result, it was confirmed that none of the tetravalent peptides had a significant effect on the intracellular C99 and Aβ levels, but only LME-tet significantly reduced the amount of Aβ released into the extracellular space (Figure 15a-d).

[0070] As shown in Figure 14, LME-tet has a molecular core structure (Figure 1, chemical formula 2) formed by the binding of three lysines (Lys) in the library part (XXXXXXXXX). SEQ ID NO: 1: Pro-Lys-Leu-Arg-Met-Lys-Glu (PKLRMKE) It is a tetravalent peptide into which four peptide motifs consisting of the amino acid sequence of

[0071] Example 3: Binding of LME-tet to Aβ1-28, Aβ40, and Aβ42 The binding activity of LME-tet or a monomeric form (LME-mono) with the same peptide motif (PKLRMKE) as LME-tet to Aβ1-28, Aβ40, and Aβ42 was measured by ELISA.

[0072] The results confirmed that LME-tet bound to both molecules with high affinity, whereas LME-mono showed almost no binding activity (Fig. 16a). These results indicate that LME-tet binds to these molecules with high affinity through a clustering effect. Furthermore, LME-mono had almost no effect on either the amount of intracellular C99 and Aβ or the amount of Aβ released into the cells (Fig. 16b). These results indicate that LME-tet efficiently inhibits Aβ release into the cells through a clustering effect achieved by adopting a multivalent structure.

[0073] A similar study was performed using human neuroblastoma SH-SY5Y cells, which are used as a model of neurons, and it was confirmed that, similar to the results above, the amount of Aβ released outside the cells tended to decrease only in the case of LME-tet treatment (Figure 17a-d).

[0074] Example 4: Inhibitory effect of LME-tet on APP cleavage by β-secretase APP is cleaved by β-secretase to produce C99, which is then cleaved by γ-secretase to produce Aβ (Aβ40 and Aβ42). That is, APP and C99 contain the sequences Aβ1-28, Aβ40, and Aβ42.

[0075] From this, 1) LME-tet may bind to APP and inhibit its cleavage by β-secretase, thereby suppressing C99 production; or 2) LME-tet may inhibit Aβ production by binding to C99 and inhibiting its cleavage by γ-secretase. was considered.

[0076] Therefore, the inhibitory effect of LME-mono or LME-tet on the cleavage activity of purified human β-secretase was evaluated in vitro using recombinant Flag-tagged APP633-685, which contains a β-secretase cleavage site, as a substrate.

[0077] The results confirmed that LME-tet efficiently inhibited β-secretase activity, whereas LME-mono showed almost no inhibitory activity (Fig. 18a). We then examined the effect of LME-tet on the cleavage activity of human γ-secretase expressed HEK293 lysate using recombinant Flag-tagged C99 as a substrate, but no inhibitory activity was observed (Fig. 18b). These results suggest that in APP-expressing cells, LME-tet efficiently binds to APP via the clustering effect, inhibiting the subsequent β-secretase activity and thereby inhibiting the production of C99, ultimately suppressing the production and release of Aβ.

[0078] Example 5: LME-tet reduces Aβ accumulation in the brain of AD model mice The AD model mouse (APPNLGF mouse) contains a humanized APP Aβ sequence and a genetic mutation found in familial AD patients. Aβ accumulation is observed in the brain from 6 weeks of age onward, and by 2 months of age, it forms the same Aβ aggregates as AD patients. 6-8 week-old mice were intraperitoneally injected with PBS or LME-tet (71 mg / kg) 1 week later, followed by brain excision and collection of the cerebral cortex and olfactory bulb. After homogenization, Aβ levels were quantified by Western blotting using specific antibodies. LME-tet was acetylated at its N-terminus to prevent protease degradation and enhance in vivo stability. The acetylated LME-tet-treated group demonstrated significantly reduced Aβ accumulation in the brain compared to the control group (Figure 19).

[0079] (summary) Using a tetravalent random peptide sheet screening method, we identified 19 high-affinity Aβ1-28 binding motifs. Furthermore, among the 19 peptide compounds with tetravalent binding motifs, we found that LME-tet potently inhibits extracellular release of Aβ. LME-tet efficiently binds to intracellular APP via the clustering effect, inhibiting β-secretase activity and thereby inhibiting C99 production, ultimately suppressing Aβ production and release. Furthermore, in APPNLGF mice, LME-tet significantly reduced intracerebral Aβ accumulation. Previously, no molecule had been identified that binds to APP and inhibits β-secretase activity. LME-tet therefore has unique potential as an Aβ production inhibitor and a potential preventive and therapeutic agent for AD.

Claims

1. An Aβ production inhibitory peptide characterized by being a multivalent peptide containing two or more peptide motifs consisting of the amino acid sequence of SEQ ID NO:

1.

2. The Aβ production inhibitory peptide of claim 1, which is a tetravalent peptide containing four of the peptide motifs.

3. A molecular core structure formed by the combination of three lysines (Lys) is shown in the following chemical formula 1: 【Chemistry 1】 The Aβ production inhibitory peptide of claim 2, characterized in that it is a tetravalent peptide in which the peptide motif is bound directly or via a spacer to each of the four amino groups located at the ends of the peptide motif.

4. 4. An Aβ production inhibitory peptide according to any one of claims 1 to 3, characterized in that the peptide motif has a modified molecule at the N-terminus thereof.

5. A pharmaceutical composition for preventing or treating a disease caused by Aβ aggregation, comprising: A pharmaceutical composition comprising an Aβ production inhibitory peptide according to any one of claims 1 to 4.

6. 6. The pharmaceutical composition of claim 5, wherein the disease is Alzheimer's disease.

7. A food or drink for the prevention or treatment of Alzheimer's disease, A food or drink comprising the Aβ production inhibitor peptide of any one of claims 1 to 4.

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