JPWO2010026735A1 - Amyloid β aggregation promoter peptide and use thereof - Google Patents

Abstract

It is an object of the present invention to provide a peptide that promotes aggregation of Aβ. Amino acid sequence represented by Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 1) (where Xaa1 is lysine or arginine, Xaa2 is leucine or isoleucine, Xaa3 is valine, isoleucine, leucine, lysine, phenylalanine or tyrosine, Xaa4 Is phenylalanine, isoleucine, leucine, lysine or valine, Xaa5 is phenylalanine, alanine, arginine, asparagine, cysteine, glycine, isoleucine, leucine, lysine, serine, tryptophan, tyrosine or valine, and Lys-Leu-Val-Phe- An amyloid β aggregation-promoting peptide is provided, including Phe (not SEQ ID NO: 7).

Description

  The present invention relates to a peptide that promotes aggregation of amyloid β and use thereof (such as a therapeutic agent and a therapeutic method for Alzheimer's disease).

  It has recently been reported that Alzheimer's disease is caused by an aggregate formed by oligomerizing a protein called amyloid β (hereinafter sometimes referred to as “Aβ”) in the brain. Aβ is an easily aggregated protein consisting of 39 to 43 amino acids. In the aggregation process of Aβ, oligomers are formed from monomers, and insoluble amyloid fibrils are formed as the aggregation proceeds further. Aβ is a peptide that is constantly produced in the normal brain. Aβ tends to aggregate and deposit, and previous reports indicate that Aβ aggregation precedes other symptoms of Alzheimer's disease.

  With the aim of establishing a therapeutic method for Alzheimer's disease, a search is being made for compounds that are effective in inhibiting or suppressing Aβ aggregation. For example, Patent Documents 1 and 2 disclose peptidic compounds effective for inhibiting Aβ aggregation. The peptidic compound is designed based on the region of Aβ17-21.

JP 2001-54343 A Special table 2001-500852 gazette

Arvi Rauk, Dalton Trans., 2008, 1273-1282 Dominic M. Walsh et al., Nature, Vol 416, 4 April 2002 R. Resende et al., Neuroscience, 2008 in press Jun Wang et al., J. Neurosci., June 18, 2008, 28 (25): 6388-6392

  Development of amyloid aggregation-inhibiting peptides (such as Patent Documents 1 and 2) and amyloid aggregation-inhibiting antibodies is underway based on the idea that inhibiting or suppressing Aβ aggregation is effective in the treatment of Alzheimer's disease. It has not yet been developed for the treatment of root treatment for diseases. It has been reported that the neurotoxicity of Aβ is higher in a soluble oligomer state than in an insoluble fibrotic state (Non-Patent Documents 1 to 4). According to this report, it is effective for the treatment of Alzheimer's disease to rapidly change the highly toxic oligomer state to the less toxic fibrosis state. Based on this idea, an object of the present invention is to provide a peptide that promotes Aβ aggregation (promotes fibrosis). It is another object of the present invention to provide a drug and a treatment method using the peptide.

In view of the above problems, the present inventors have repeatedly studied. First, attention was focused on the KLVFF (SEQ ID NO: 7) peptide (Aβ16-20), which is the nucleus of Aβ aggregation. And the binding property with respect to A (beta) of each peptide which consists of the said peptide as a seed was investigated. Next, the sequence information of peptides whose binding power was increased by substitution of one residue was synthesized, and amino acids effective for improving binding to Aβ were determined for each position. That is, the regularity characterizing the high binding peptide was found. Subsequently, a peptide array covering peptides composed of effective amino acid combinations was prepared, and high-binding peptides were searched using this. As a result, a peptide showing binding activity exceeding the seed peptide was found. Subsequently, Aβ aggregation (amyloid fibril formation) promoting activity was evaluated for three peptides having high binding activity by thioflavin T assay and electron microscope observation. As a result, all the peptides showed high activity. As described above, a peptide having a high Aβ aggregation promoting activity was successfully identified by a unique search method using the KLVFF peptide as a seed. In addition, the effectiveness of the search method was confirmed. Further investigations have confirmed the usefulness of successfully identified peptides in cell and animal experiments. The invention described below is mainly based on the above-mentioned results.
[1] Amino acid sequence represented by Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 1) (where Xaa1 is lysine or arginine, Xaa2 is leucine or isoleucine, Xaa3 is valine, isoleucine, leucine, lysine, phenylalanine or Tyrosine, Xaa4 is phenylalanine, isoleucine, leucine, lysine or valine, Xaa5 is phenylalanine, alanine, arginine, asparagine, cysteine, glycine, isoleucine, leucine, lysine, serine, tryptophan, tyrosine or valine, and Lys-Leu-Val An amyloid β aggregation-promoting peptide comprising -Phe-Phe (not SEQ ID NO: 7).
[2] The amino acid sequence is Lys-Ile-Ile-Phe-Val (SEQ ID NO: 2), Lys-Leu-Leu-Phe-Ile (SEQ ID NO: 3), Lys-Ile-Ile-Leu-Ile (SEQ ID NO: 2). 4) The peptide according to [1], which is Arg-Ile-Ile-Phe-Val (SEQ ID NO: 5) or Lys-Ile-Phe-Ile-Ile (SEQ ID NO: 6).
[3] An amyloid β aggregation promoter comprising the peptide of [1] or [2] or a salt thereof.
[4] A method for treating Alzheimer's disease, comprising administering the amyloid β aggregation promoter according to [3] to a subject.

Binding activity to Aβ of each peptide obtained by single residue substitution of seed peptide (KLVFF: SEQ ID NO: 7). The activity of each peptide was shown as a relative value to the binding activity (1) of the seed peptide. Binding activity of each designed peptide to Aβ. The average value of the fluorescence intensity of the three spots was expressed as a relative value to the binding activity (1) of the seed peptide. Results of thioflavin T (ThT) assay. For each sample, the fluorescence intensity measured 6 hours after the start of incubation (left) and the fluorescence intensity measured 10 hours later (right) are shown. Fluorescence of ThT (wavelength 482 nM) was measured. Error bars are standard errors (n = 3). * P <0.05 Evaluation by TEM. A solution in which 100 μM Aβ and 100 μM sample peptide were mixed was incubated at 37 ° C., and a TEM image was taken after 6 hours. (A) TEM image for high-binding peptide, (B) TEM image for low-binding peptide, (C) TEM image (left) for seed peptide and TEM image without right peptide (right). The scale bars are 500 nm (upper TEM image) and 100 nm (lower TEM image). Comparison of amyloid fibril length calculated from TEM images. Results of MTT assay. Each peptide and β were mixed and incubated at 37 ° C. for 4 hours (ash) and 8 hours (white), and then incubated with PC-12 cells for 3 hours. As Aβ fibrosis progressed, cytotoxicity decreased, and it was suggested that cell viability was increased by mixing highly-binding peptides with Aβ. Binding activity to Aβ of D-substitution sequences of KLLFI, KIILI, and KIIFV. D-substitution sequences were prepared exhaustively and their activities were compared. Shows the binding activity of the top 10 sequences. KLVFF binding was evaluated as 1.0. L-form amino acids are shown in capital letters, and D-form amino acids are shown in lower case letters. Results of thioflavin T (ThT) assay for D-substituted peptides. For each sample, the fluorescence intensity measured 6 hours after the start of incubation (left) and the fluorescence intensity measured 10 hours later (right) are shown. Fluorescence of ThT (wavelength 482 nM) was measured. L-form amino acids are shown in capital letters, and D-form amino acids are shown in lower case letters. Error bars are standard errors (n = 3) Results of LDH assay. L-form amino acids are shown in capital letters, and D-form amino acids are shown in lower case letters. * P <0.05 (vs. Aβ group), ** p <0.01 (vs. Aβ group) Overview of animal testing. The animals used, the conditions of each test group (upper), and the experimental schedule (lower) are shown. Method of novel substance search test. Method of fear conditioning learning test. Results of novel substance search test. We searched freely for 5 minutes and asked for search preference. Mean ± standard error (n = 8) is shown. * P <0.05 (vs. control group, Fisher's LSD) # p <0.05 (vs. Aβ administration group or Aβ-low binding peptide administration group, Fisher's LSD) Results of fear conditioning learning test. (A) is the result of the situation dependence test, and (B) is the result of the sound stimulus dependence test. Mean ± standard error (n = 7-8) was shown. * p <0.05 (vs. control group, t-test) # p <0.05 (vs. Aβ-low binding peptide administration group, t-test)

  In this specification, according to a conventional notation method, peptides are represented such that the left end is the amino terminus (N terminus) and the right end is the carboxy terminus (C terminus). When the amino acid residue is L-form, the indication that it is L-form may be omitted.

The first aspect of the present invention relates to a peptide that promotes aggregation of Aβ (hereinafter referred to as “the peptide of the present invention” for convenience of explanation). As described above, amyloid fibrils are formed when Aβ aggregation proceeds. Therefore, when the peptide of the present invention is applied, Aβ fibrosis can be promoted. The peptides of the present invention comprise the following sequences:
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 1)

In the above sequence, Xaa1 to Xaa5 each represent the following amino acid.
Xaa1: Lysine (Lys) or Arginine (Arg)
Xaa2: Leucine (Leu) or isoleucine (Ile)
Xaa3: Valine (Val), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Phenylalanine (Phe) or Tyrosine (Tyr)
Xaa4: phenylalanine (Phe), isoleucine (Ile), leucine (Leu), lysine (Lys) or valine (Val)
Xaa5: Phenylalanine (Phe), Alanine (Ala), Arginine (Arg), Asparagine (Asn), Cysteine (Cys), Glycine (Gly), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Serine (Ser ), Tryptophan (Trp), tyrosine (Tyr) or valine (Val)

The above regularity regarding the amino acid at each position was derived as a result of the assay shown in Examples described later. The combination of amino acids at each position Xaa1 to Xaa5 is arbitrary unless it becomes Lys-Leu-Val-Phe-Phe (SEQ ID NO: 7). Examples of preferred combinations are shown below.
Lys-Ile-Ile-Phe-Val (SEQ ID NO: 2)
Lys-Leu-Leu-Phe-Ile (SEQ ID NO: 3)
Lys-Ile-Ile-Leu-Ile (SEQ ID NO: 4)
Arg-Ile-Ile-Phe-Val (SEQ ID NO: 5)
Lys-Ile-Phe-Ile-Ile (SEQ ID NO: 6)
All of these peptides showed strong binding activity to Aβ (see Examples described later). Moreover, as a result of selecting the upper 3 sequences and examining their Aβ aggregation promoting activity, all showed high activity.

  In the above sequence (any of SEQ ID NOs: 1 to 6) contained in the peptide of the present invention, the amino acid at each position may be L-form or D-form. L-form amino acids and D-form amino acids may be mixed. On the other hand, considering the solubility and the ability to pass through the blood-brain barrier, the number of constituent amino acids is generally better. Therefore, the number of amino acids in the peptide of the present invention is preferably 4-6. It has been confirmed that peptides composed of D-form amino acids show the desired activity (columns of Examples described later).

  The peptide of the present invention can be prepared by a known peptide synthesis method (for example, solid phase synthesis method, liquid phase synthesis method). The peptides of the present invention may be prepared using genetic engineering techniques. That is, the peptide of the present invention can be prepared by introducing a nucleic acid encoding the peptide of the present invention into a suitable host cell and recovering the peptide expressed in the transformant. The recovered peptide is purified as necessary. The recovered peptide can be subjected to an appropriate substitution reaction and converted into a desired modified peptide. In addition, when the peptide of the present invention exists in nature, it can also be prepared by operations such as extraction and purification.

  In the peptide of the present invention, some or all of the amino acids may be modified. The modified peptide is referred to as a modified peptide in this specification. “Modified peptide” as used herein refers to substituting a part of the basic structure composed of the above-mentioned sequences (SEQ ID NOs: 1 to 6) (may be plural places) with other atomic groups or the like. Or a compound having a structure different from the basic structure at least partially by modification such as addition of other molecules. A person skilled in the art can design peptide modifications such as substitutions based on the above sequences using known or conventional means. Moreover, it can be said that it is easy for those skilled in the art to prepare a target modified substance using well-known or usual means based on this design, and to investigate the property and action.

  As a typical example of the modified peptide in the present invention, a peptide derivative in which a part of an amino acid residue (atom or atomic group) is substituted with another atom or atomic group can be exemplified. Such peptide derivatives can be prepared by any manufacturing process designed to obtain the peptide derivative as a final product. Therefore, when a target peptide derivative is a peptide in which a part (for example, an atomic group that is a part of a side chain) is apparently substituted by a specific atomic group, the target peptide derivative is Even if it is produced by a substitution reaction using the specific peptide as a starting material, which is an apparently basic peptide, or an appropriate substitution reaction using a peptide having another structure as a starting material, It may be manufactured by a process).

  Other atoms or atomic groups herein include hydroxyl groups, halogens (fluorine, chlorine, bromine, iodine, etc.), alkyl groups (methyl groups, ethyl groups, n-propyl groups, isopropyl groups, etc.), hydroxyalkyl groups ( Examples thereof include hydroxymethyl group, hydroxyethyl group, etc., alkoxy groups (methoxy group, ethoxy group, etc.), acyl groups (formyl group, acetyl group, malonyl group, benzoyl group, etc.) and the like.

  The modified peptide of the present invention includes those in which the functional group in the constituent amino acid residue is protected by an appropriate protecting group. Acyl groups, alkyl groups, monosaccharides, oligosaccharides, polysaccharides and the like can be used as protecting groups used for such purposes. Such a protecting group is linked by an amide bond, an ester bond, a urethane bond, a urea bond, or the like according to the peptide site to which the protecting group is bound, the kind of the protecting group to be used, or the like.

  As a further example of the modified peptide of the present invention, one modified by addition of a sugar chain can be mentioned. In addition, various peptide derivatives classified into alkylamine, alkylamide, sulfinyl, sulfonylamide, halide, amide, aminoalcohol, ester, aminoaldehyde, etc. by replacing the N-terminal or C-terminal with other atoms etc. It is an example of the modified peptide of the invention.

  In addition, the peptide derivative comprised by combining the various modification methods demonstrated above is also an example of the peptide modification body of this invention.

  The second aspect of the present invention relates to the use of the peptide of the present invention. One embodiment of this aspect is an Aβ aggregation promoter. The Aβ aggregation promoter of the present invention contains the peptide of the present invention or a salt thereof as an active ingredient. The above-mentioned modified peptide or a salt thereof can also be used as an active ingredient. The salt here is a pharmaceutically acceptable salt, and the kind thereof is not particularly limited. The “pharmaceutically acceptable salt” is, for example, an acid addition salt, a metal salt, an ammonium salt, or an organic amine addition salt. Examples of acid addition salts include inorganic acid salts such as trifluoroacetate, hydrochloride, sulfate, nitrate, phosphate, hydrobromide, acetate, maleate, fumarate, citrate, Organic acid salts such as benzene sulfonate, benzoate, malate, oxalate, methanesulfonate, and tartrate are listed. Examples of the metal salt include alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt. Examples of ammonium salts include salts such as ammonium and tetramethylammonium. Examples of organic amine addition salts include morpholine addition salts and piperidine addition salts. These salts can be prepared by conventional means.

  Formulation of the Aβ aggregation promoter of the present invention can be performed according to a conventional method. When formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, etc.) Can be contained. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories.

  The Aβ aggregation promoter of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (or preventive effect) (that is, a therapeutically effective amount). The amount of the active ingredient in the Aβ aggregation promoter of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set within a range of, for example, about 0.1 wt% to about 95 wt% so as to achieve a desired dose. To do.

  The Aβ aggregation promoter of the present invention is administered orally or parenterally (intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, brain, depending on the dosage form. Applied to the subject by internal injection, etc.). These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. , Sheep, dogs, cats, chickens, quails, etc.). In a preferred embodiment, the Aβ aggregation promoter of the present invention is applied to humans.

  The dose of the Aβ aggregation promoter of the present invention is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dose, the patient's symptoms, age, sex, weight, etc. are generally considered. A person skilled in the art can set an appropriate dose in consideration of these matters. For example, the dose can be set so that the amount of the active ingredient per day is about 0.1 mg to about 100 mg, preferably about 1 mg to about 50 mg for an adult (body weight about 60 kg). As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's symptoms and the duration of effect of the active ingredient can be taken into consideration.

  The disease (application disease) to be treated by the Aβ aggregation promoter of the present invention is typically Alzheimer's disease. That is, the present invention also provides a method for treating Alzheimer's disease, which comprises administering the Aβ aggregation promoter of the present invention to a subject. The subject, administration route, administration schedule, etc. are as described above.

1. Search for high-binding peptides by single residue substitution of seed peptide
A single-residue substitution peptide (a peptide with a single residue different from the seed peptide sequence) using KLVFF (SEQ ID NO: 7), which is the core of Aβ aggregation / fibrosis, as a seed peptide was spot-synthesized on a cellulose membrane. . Using the thus obtained peptide array, the binding ability of each peptide to Aβ was measured by the following method. First, the peptide array was washed with phosphate buffered saline (PBS) and then blocked with bovine serum albumin (BSA). Next, Aβ (1-42) (4349-v, Peptide Institute) fluorescently labeled with FAM was reacted with the peptides on the array at 37 ° C. for 1 hour in the dark. Subsequently, after washing with PBS to remove unbound Aβ, the amount of Aβ binding for each peptide spot was calculated by fluorescence intensity measurement, and the values obtained by subtracting the fluorescence intensity of the back (blank spot) were compared for each sequence. The binding property to Aβ was measured.

The measurement results are shown in the table of FIG. The numerical values in the table are relative activities when the binding activity of the seed peptide is 1. When the first amino acid from the N-terminal of the seed peptide is substituted from lysine (K) to arginine (R) (1.01: first row, second column mass), high activity is observed. Similarly, high activity is observed when the second amino acid from the N-terminal of the seed peptide is replaced with isoleucine (I) (1.07), and the third amino acid from the N-terminal of the seed peptide is isoleucine (I). Substitution (0.97), leucine (L) substitution (1.20), lysine (K) substitution (0.95), phenylalanine (F) substitution (1.22) and tyrosine (Y) substitution (1.02) Yes, the fourth amino acid from the N-terminal of the seed peptide is substituted with isoleucine (I) (1.56), substituted with leucine (L) (1.01), substituted with lysine (K) (0.91) and valine (V ) For the fifth amino acid from the N-terminal of the seed peptide (0.98), arginine (R) (0.98), asparagine (N) Substitution (0.90), Cysteine (C) Substitution (0.86), glycine (G) substitution (0.97), isoleucine (I) substitution (1.41), leucine (L) substitution (1.63), lysine (K) substitution (1.34) Substitution with serine (S) (0.96), substitution with tryptophan (W) (1.03), substitution with tyrosine (Y) (1.23) and substitution with valine (V) (1.12). From these results, the following (a) to (e) are derived as conditions for being a high-binding peptide.
(a) The first amino acid from the N-terminus is lysine or arginine.
(b) The second amino acid from the N-terminus is leucine or isoleucine.
(c) The third amino acid from the N-terminus is valine, isoleucine, leucine, lysine, phenylalanine or tyrosine.
(d) The fourth amino acid from the N-terminus is phenylalanine, isoleucine, leucine, lysine or valine.
(e) The fifth amino acid from the N-terminal is phenylalanine, alanine, arginine, asparagine, cysteine, glycine, isoleucine, leucine, lysine, serine, tryptophan, tyrosine or valine.

  Subsequently, all the peptide sequences (2 × 2 × 6 × 5 × 13 = 1,560 sequences) that meet the conditions (a), (b), (c), (d), and (e) are covered. Peptide arrays (6) were prepared by spot synthesis. Using this peptide array, the binding of each peptide to Aβ was examined. The results are shown in FIG. Five high binding peptides (KIIFV (SEQ ID NO: 2), KLLFI (SEQ ID NO: 3), KIILI (SEQ ID NO: 4), RIIFV (SEQ ID NO: 5), KIFII (SEQ ID NO: 6)) were found (left table). ). The table on the right shows peptides with low binding activity.

2. Aβ aggregation-promoting activity of high-binding peptides (1) Thioflavin T (ThT) assay Next, three high-binding peptides (KIIFV, KLLFI, KIILI) were subjected to ThT assay to promote Aβ aggregation-promoting activity (amyloid fibril formation-promoting activity ) Was evaluated. A seed peptide (KLVFF) and a low binding peptide (KLIIN (SEQ ID NO: 8), ELIFV (SEQ ID NO: 9)) were used as controls. The method of ThT assay was as follows. A solution (sample solution) in which 100 μM Aβ (1-42) (4349-v, Peptide Institute) and 100 μM sample peptide were mixed was incubated at 37 ° C. 6 μl was sampled 6 hours and 10 hours after each sample solution, and 54 μl of phosphate buffered saline (PBS) containing 100 μM ThT was mixed, and then optimal fluorescence of amyloid fibrils (excitation wavelength 450 nm, fluorescence wavelength 482 nm). ) Was measured.

  The results of the ThT assay (average of three independent measurements) are shown in FIG. Compared with the control, the fluorescence intensity at the time point after 6 hours is higher in the high-binding peptide. That is, the fluorescence intensity increased in a shorter time than the control, indicating that the high-binding peptide promotes amyloid fibril formation.

(2) Observation by transmission electron microscope (TEM) The Aβ aggregation promoting activity (amyloid fibril formation promoting activity) of the high binding peptide was evaluated by TEM. A solution (sample solution) in which 100 μM Aβ (1-42) (4349-v, Peptide Institute) and 100 μM sample peptide were mixed was incubated at 37 ° C., and TEM images were taken after 6 hours (FIGS. 4A to 4C). ). Moreover, the average length of the formed amyloid fibril was computed from the TEM image (FIG. 5). In the case of a high-binding peptide, it can be seen that aggregation / fibrosis is proceeding (FIGS. 4A and 5). In particular, clear fiber formation is observed in KIIFV and KIILI (left and right in FIG. 4A). On the other hand, in the case of a low-binding peptide, the degree of aggregation / fibrosis is lower than that of the control (KLVFF) (FIGS. 4B and C, FIG. 5). As described above, observation by TEM also suggested that the high-binding peptide has an activity of promoting amyloid fibril formation.

(3) MTT assay Rat adrenal medulla-derived pheochromocytoma PC-12 was seeded in a 96-well plate at a cell concentration of 2.5 × 10 ⁴ cells / well and cultured (overnight). Next, after removing the medium in each well, 100 μl of a solution in which the same amount of medium was mixed with the Aβ solution (4,8 hour incubation) was added to each well. After culturing for 3 hours, the solution was removed, washed twice with medium, 100 μl of medium was added, and the percentage of viable cells in each sample was determined as MTT (3- (4, 5-Dimethyl thial-2-yl) -2, 5- Diphenyltetrazalium Bromide) was added in an amount of 20 μl, and assayed. Absorbance measurement (450 nm) was performed every hour after the addition, and the measurement was performed until 4 hours passed. The measurement results are shown in FIG. It can be seen that the high-binding peptide reduces the cytotoxicity caused by Aβ. In contrast, low binding peptides do not show similar activity.

3. Screening of D-form high-binding peptides The binding activity to Aβ when each candidate sequence was comprehensively replaced with D-form was tested according to the case of L-form peptides. The results are shown in FIGS. 7 and 8 (lower case letters represent D-form amino acids). It can be seen that even when D-substitution is carried out, strong binding is exhibited (FIG. 7). In addition, the D-substituted peptide (kiili, Kiili) also has an amyloid fibril formation promoting action similar to the L-peptide (KIILI) (FIG. 8).

4). Peptide evaluation using N18 cells
Aβ (1-42) (4349-v, Peptide Institute) and synthetic peptides (KIILI, kiili, KLVFF, ELIFV. Lower case letters represent D-form amino acids) were dissolved in DMSO, and MEM medium was added to prepare a sample solution. On the day before the assay, 100 μl of N18 cell suspension was seeded in a 96-well plate (5000 cells / well) and cultured overnight. 50 μl of the sample solution was added so that the final concentrations of Aβ and peptide were 10 μM, and cultured for 3 days. 150 μl of PBS was added to each well, and LDH assay (LDH test Wako, in the case of positive control, cells were lysed and total LDH amount was measured) was performed. As shown in FIG. 9, the cytotoxicity when the high binding peptide was used was low, indicating that the high binding peptide inhibited the cytotoxicity caused by Aβ. Since the cytotoxicity of the D-substituted high-binding peptide (kiili) was particularly low, the peptide was used in the subsequent experiments.

5). Animal test (1) Experimental animals used (Fig. 10)
Six week old male C57BL / 6J mice were used. An Aβ1-42 solution (100 μM) was prepared aseptically, incubated for 4 hours, and then microinjected into the ventricle of the mouse at a flow rate of 3 μl for 3 minutes (Aβ administration group: n = 8). In the Aβ-high binding peptide administration group, the high binding peptide (kiili) was dissolved in the Aβ1-42 solution and administered in the same manner. In the Aβ-low binding peptide administration group, the low binding peptide (ELIFV) was dissolved in the Aβ1-42 solution and administered in the same manner. A control group (administered with physiological saline) and a peptide administration group (administered after dissolving a high-binding peptide in physiological saline) were also prepared. Breeding conditions were a temperature of 21 ± 1 ° C, humidity of 55 ± 5%, food and water ad libitum, and a 12 hour light / dark cycle (light period 9: 00-21: 00). This experiment was approved by the Animal Experiment Committee of Nagoya University and conducted with ethical considerations.

(2) New substance search test method (Figs. 10 and 11)
After the mice were acclimatized to an acrylic experimental apparatus for 3 days (10 minutes / day), a training trial was performed. In the training trial, two different objects were placed in the device, and the device was freely searched for 10 minutes. Retention was performed 24 hours after the training trial. In the holding trial, one of the two objects placed in the device during the training trial was replaced with a novel object, and the mouse was allowed to search freely for 5 minutes. In training trials and holding trials, we measured the time of each search (contact, smelling behavior, etc.) for two objects. In the retention trial, the ratio of the search time of the novel object to the total search time of both objects was used as the exploratory preference, and used as an index of cognitive memory (Mouri et al., FASEB J. 21, 2135-2148, 2007 Mizoguchi et al., Psychopharmacology (Berl). 196, 233-241, 2008; Mizoguchi et al., J Pharmacol Exp Ther.).

(3) Fear conditioning learning test method (Figs. 10 and 12)
Associative learning was evaluated using the fear-conditioned learning test (Mouri et al., FASEB J. 21, 2135-2148, 2007; Nagai et al., FASEB J. 17, 50-52, 2003). Mice were placed in a clear acrylic cage with a stainless steel grid and given a 20 second sound stimulus (80 dB), followed by an electrical stimulus (0.6 mA) during the last 5 seconds. This combination stimulus was set as one set and repeated four times at intervals of 15 seconds to perform fear conditioning. Situation-dependent and sound stimulus-dependent tests were performed 24 hours after fear conditioning. In the former, mice were placed in a grid-conditioned acrylic white cage with fear conditioning, and freezing behavior was measured for 2 minutes in the absence of sound and electrical stimulation. In the latter case, mice were placed in an acrylic black cage with wood chips on the floor, and the freezing behavior was measured for 1 minute when a continuous sound stimulus was applied. Each result was expressed as a percentage (%) of freezing action time relative to the total measurement time.

(4) Statistical processing All experimental results are shown as mean ± standard error. The statistical test used statistical analysis software Excel statistics. A t-test was performed on data consisting of two independent groups. Data consisting of independent multigroups were analyzed by Fisher's LSD after one-way analysis of variance. A significance level of p <0.05 was evaluated as a difference between groups.
(5) Results The results of the novel substance search test are shown in FIG. It can be seen that the search preference of the Aβ-high binding peptide administration group is significantly high. That is, it was suggested that the high binding peptide has an inhibitory activity against the impairment of recognition memory by Aβ. On the other hand, as a result of the fear conditioned learning test, it was suggested that the high-binding peptide also exhibits inhibitory activity against the disturbance of associative learning by Aβ (FIG. 14).

  The present invention provides a peptide that promotes aggregation and fibrosis of Aβ. According to the peptide of the present invention, it is possible to promote the transition of a highly toxic Aβ oligomer to a less toxic fibrous state. The peptide of the present invention having this action is highly expected to be applied to the treatment of Alzheimer's disease.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (4)

Amino acid sequence represented by Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO: 1) (where Xaa1 is lysine or arginine, Xaa2 is leucine or isoleucine, Xaa3 is valine, isoleucine, leucine, lysine, phenylalanine or tyrosine, Xaa4 Is phenylalanine, isoleucine, leucine, lysine or valine, Xaa5 is phenylalanine, alanine, arginine, asparagine, cysteine, glycine, isoleucine, leucine, lysine, serine, tryptophan, tyrosine or valine, and Lys-Leu-Val-Phe- An amyloid β aggregation-promoting peptide comprising Phe (not SEQ ID NO: 7).   The amino acid sequence is Lys-Ile-Ile-Phe-Val (SEQ ID NO: 2), Lys-Leu-Leu-Phe-Ile (SEQ ID NO: 3), Lys-Ile-Ile-Leu-Ile (SEQ ID NO: 4), The peptide according to claim 1, which is Arg-Ile-Ile-Phe-Val (SEQ ID NO: 5) or Lys-Ile-Phe-Ile-Ile (SEQ ID NO: 6).   An amyloid β aggregation promoter comprising the peptide according to claim 1 or 2 or a salt thereof.   A method for treating Alzheimer's disease, comprising administering the amyloid β aggregation promoter according to claim 3 to a subject.

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