Peptides used to prevent or treat synucleinopathy

Abstract

To provide peptides used to prevent or treat synucleinopathy.SOLUTION: Provided is a peptide represented by the following (i) or (ii): (i) a peptide having an amino acid sequence represented by EEG(X1)QD(X2)EPEA (where, X1 and X2 are the same or different and are Y, F, or W); or (ii) a peptide having an amino acid sequence in which one or several are deleted, substituted, or added at amino acid sites other than X1 and X2 of the amino acid sequence of (i) above, and having an activity of binding to FABP3 protein.SELECTED DRAWING: Figure 3

Description

Technical Field

[0006]

[0001] The present invention relates to a peptide used for preventing or treating synucleinopathy.

Background Art

[0002] α-Synuclein is said to be the causative protein of synucleinopathy (Parkinson's disease, Lewy body dementia, multiple system atrophy), and synucleinopathy is characterized by the aggregation of α-synuclein in neurons and glial cells. In neuropathological tests of synucleinopathy, characteristic damage including abnormal aggregation of α-synuclein can be detected in neurons, nerve fibers or glial cells. It is known that when α-synuclein propagates between neurons or glial cells and aggregates and accumulates, it causes necrosis of neurons or glial cells throughout the brain, resulting in motor dysfunction, cognitive dysfunction, sleep disorder, and autonomic nerve disorder. Currently, the development of antibody therapy for α-synuclein is underway, but it has not yet reached a fundamental treatment due to the problem of side effects.

[0003] Patent Document 1 discloses an antibody that specifically binds to human α-synuclein, and a pharmaceutical preparation that inhibits cytotoxicity containing such an antibody.

[0004] Patent Document 2 discloses a pharmaceutical composition for preventing and / or treating synucleinopathy, which contains a peptide consisting of 9 amino acids including an amino acid sequence considered to be a mimotope of an epitope of α-synuclein.

[0005] Non-Patent Document 1 discloses that in the mouse brain, FABP3 enhances the diffusion of α-synuclein after injection of pre-formed fibrils of α-synuclein and mediates α-synuclein toxicity in the synucleopathy state, and that MF1, a ligand of FABP3, is an attractive therapeutic candidate for α-synucleinosis.

[0006] Non-patent document 2 is a review article concerning α-synuclein cleavage and disease, defining the N-terminal region (residues 1-60), NAC region (residues 61-95), and C-terminal region (residues 96-140) of α-synuclein. It describes that cleavage of the C-terminal side of α-synuclein increases the aggregation rate of the cleaved α-synuclein.

[0007] Non-patent document 3 discloses the development of amide-bridged nucleic acid-modified antisense oligonucleotides (AmNA-ASOs) that suppress the accumulation of α-synuclein protein, and the improvement of Parkinson's disease symptoms in animal models. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2020-73565 [Patent Document 2] Patent No. 5901714 [Non-patent literature]

[0009] [Non-Patent Document 1] Int J Mol Sci. 2020 Mar 23;21(6):2230 [Non-Patent Document 2] JBC Papers in Press, on May 18, 2020 as Manuscript REV120.011743 [Non-Patent Document 3] Scientific Reports (2019) 9:7567 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The problem that the present invention aims to solve is to provide peptides, antibodies, prophylactic or therapeutic agents for the prevention or treatment of synucleinopathy containing peptides or antibodies, and pharmaceutical compositions for the prevention or treatment of synucleinopathy containing peptides or antibodies, which are used for the prevention or treatment of synucleinopathy. [Means for solving the problem]

[0011] As a result of diligent research to solve the above problems, the present inventors focused on the complex formation between α-synuclein and FABP3, and found that by using a peptide derived from the C-terminal region of α-synuclein, the uptake of α-synuclein into nerve and glial cells and / or the propagation of α-synuclein between nerve and glial cells can be suppressed, thus completing the present invention.

[0012] The present invention encompasses the embodiments described below.

[0013] Item 1. (i) and (ii) below , or (iii) Peptides represented by: (i) Having an amino acid sequence represented by EEG(X1)QD(X2)EPEA Furthermore, the length of the amino acid is 13 or less. peptide. (In the formula, X1 and X2 are either the same or different, Y, F, or W.) (ii) A peptide having an amino acid sequence represented as EEG(X1)QD(X2)EPEA, where X1 is F or W and X2 is Y, F, or W, or where X1 is Y and X2 is F or W. (iii) Having an amino acid sequence represented by EEG(X1)QD(X2)EPEA, before Note A One or several amino acids are deleted in the amino acid sites other than X1 and X2 of the amino acid sequence. or Place exchange A peptide having a specific amino acid sequence and possessing the activity to bind to the FABP3 protein.

[0014] Item 2. A peptide consisting only of the amino acid sequence represented by EEG(X1)QD(X2)EPEA. (In the formula, X1 and X2 are either the same or different, Y, F, or W.) Item 3. The peptide according to item 1, wherein the length of the amino acids is 30 or less. term 4The peptide according to item 1, which binds to FABP3 through the interaction between the phenyl groups of the amino acids of X1 and X2 and the phenyl group of the 16th phenylalanine in the amino acid sequence of the native FABP3 protein One of the following three items described in

[0015] Item 5. A prophylactic or therapeutic agent for the prevention or treatment of synucleinopathy, comprising a peptide as described in any one of items 1 to 3 as an active ingredient.

[0016] Item 6. The prophylactic or therapeutic agent according to Item 7, wherein the synucleinopathy is selected from the group consisting of Parkinson's disease, Lewy body disease, and multiple system atrophy.

[0017] Item 7. A pharmaceutical composition comprising the peptide described in Item 1 or 2 or the antibody described in Item 3, for the prevention or treatment of synucleinopathy.

[0018] Section 8. Inhibitors of the interaction between α-synuclein and FABP3, comprising the peptide represented by (i) or (ii) below. . (i) A peptide having the amino acid sequence represented by EEG(X1)QD(X2)EPEA (In the formula, X1 and X2 are either the same or different, Y, F, or W.) (ii) A peptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sites other than X1 and X2 of the amino acid sequence of (i) above, and which has the activity to bind to the FABP3 protein. Item 9. An inhibitor of α-synuclein uptake into dopaminergic neurons, comprising the peptide represented by (i) or (ii) below. (i) A peptide having the amino acid sequence represented by EEG(X1)QD(X2)EPEA (In the formula, X1 and X2 are either the same or different, Y, F, or W.) (ii) A peptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sites other than X1 and X2 of the amino acid sequence of (i) above, and which has the activity to bind to the FABP3 protein.

Brief Description of Drawings

[0019] [Figure 1] Steric structures and binding sites of α-synuclein and FABP3. (A) Schematic diagram explaining the interaction between α-synuclein and FABP3. (B) Enlarged view of the C-terminus of α-synuclein in the part enclosed by the square in Fig. 1A. (C) Schematic diagram of the binding mode between α-synuclein in the native denatured structure and FABP3. The 133rd and 136th tyrosine residues of α-synuclein are involved, and in particular, the 133rd tyrosine residue strongly interacts with the phenyl group of the 16th phenylalanine located in the fatty acid binding pocket of FABP3 to form a complex of α-synuclein and FABP3. [Figure 2] Secondary structure of α-synuclein and characteristics of each domain. (A) Schematic diagram showing the structures of the N-terminus, NAC-terminus, and C-terminus of α-synuclein. (B) Amino acid sequences of the peptides in each region of Fig. 2(A). [Figure 3] Binding analysis of FABP3 and domain-specific α-synuclein. (A) Binding of FABP3 to full-length α-synuclein over time. (B) Binding of FABP3 to SynP2-10 over time. (C) Binding of FABP3 to SynP73-96 over time. (D) Binding of FABP3 to SynP130-140 over time. [Figure 4] Binding analysis of ABP3 and various mutant α-synuclein. (A) Peptide (Y133F) in which the 133rd tyrosine of SynP130-140 is substituted with phenylalanine. (B) Peptide (Y136F) in which the 136th tyrosine of SynP130-140 is substituted with phenylalanine. (C) Peptide (Y136W) in which the 136th tyrosine of SynP130-140 is substituted with tryptophan. (D) Peptide (Y133F / Y136F) in which the 133rd and 136th tyrosines of SynP130-140 are substituted with phenylalanine. (E) Peptide (Y133F / Y136W) in which the 133rd tyrosine of SynP130-140 is substituted with phenylalanine and the 136th tyrosine is substituted with tryptophan. (F) A peptide in which the tyrosine positions 133 and 136 of SynP130-140 are substituted with tryptophan (Y133W / Y136W). [Figure 5] Binding analysis and electron microscopy images of FABP3 and C-terminally deficient / mutant α-synuclein. (A) Formation of synuclein aggregation when an equal molar amount of FABP3 and SynP130-140 or its mutant peptide are simultaneously added to α-synuclein. ◆: Y133F / Y136F added, ■: Y133F added, ●: Full-length Syn, □: Y136W added, △: Y133F / Y136W added, ◇: Y133W / Y136W added, ○: Y136F added, ×: SynP130-140 added, (B) Micrographs showing synuclein aggregation when each SynP130-140 mutant peptide is added. [Figure 6]Synuclein-FABP3 binding antagonism by α-synuclein C-terminal peptide and its electron microscope images. (A) Formation of synuclein aggregation when α-synuclein is simultaneously added with an equimolar amount of FABP3 and 1 equivalent, 0.5 equivalent, or 0.1 equivalent of the SynP130-140 mutant peptide Y133F. (B) Micrographs showing synuclein aggregation at various Y133F concentrations (▲1 equivalent, ■0.5 equivalent, ◆0.1 equivalent, ●0 equivalent). [Figure 7] Time-dependent antagonistic effect of α-synuclein C-terminal peptide on synuclein-FABP3 and electron microscope images. (A) Formation of synuclein aggregation when an equimolar amount of FABP3 is added to α-synuclein to form a Syn-FABP3 complex, followed by the addition of SynP130-140 peptide. (B) Micrographs showing synuclein aggregation at 0 minutes (●), 180 minutes (■), 480 minutes (◆), and 1440 minutes (▲) after the addition of FABP3, indicated by triangles (▼) in Figure 7(A). [Figure 8] Inhibition of α-synuclein monomer uptake into FABP-expressing neurons by a peptide from the C-terminal region of α-synuclein. (A) Left column: control (addition of α-synuclein monomer), Middle column: addition of a peptide consisting of 20 amino acids from the C-terminal region of α-synuclein (20aa), Right column: addition of a peptide consisting of 10 amino acids from the C-terminal region of α-synuclein (10aa), Top row: tyrosine hydroxylase, the rate-limiting enzyme of dopamine (green), Middle row: fluorescent labeling of α-synuclein monomer with ATTO-550 (red), Bottom row: synthesis of the top and middle rows, (B) graph of ATTO fluorescence intensity for control, 20aa peptide addition, and 10aa peptide addition. **** indicates p<0.0001. (C) schematic diagram showing the position of the 20aa peptide and 10aa peptide in the C-terminal region of α-synuclein. [Figure 9]Suppression of α-synuclein monomer uptake into FABP-expressing neurons by a C-terminally deficient peptide of α-synuclein. (A) αS(WT): wild-type α-synuclein monomer, αS(Δ130-140): peptide deficient at amino acids 130-140 of the wild-type α-synuclein sequence. Top row: tyrosine hydroxylase (green), middle row: fluorescent labeling with ATTO-550, bottom row: synthesis of top and middle rows, (B) graph of ATTO fluorescence intensity for control (αS(WT)) and αS(Δ130-140). **** indicates p<0.0001. (C) schematic diagram showing the sequence of the αS(Δ130-140) peptide. [Figure 10] A list of FABP3 and various mutant / deficient α-synuclein or peptides, along with their dissociation constants. Kd, kon, and koff values ​​in binding experiments between FABP3 protein and various α-synuclein peptides. AA: Arachidonic acid. FABP3 F16S: A protein in which the 16th phenylalanine of wild-type FABP3 is replaced with serine. [Modes for carrying out the invention]

[0020] In the brain, alpha-synuclein undergoes fibrillation and aggregation from monomers to oligomers and then to multimers such as protofibrils. Recent research suggests that the oligomerization of alpha-synuclein is involved in its propagation between nerve cells or glial cells, thereby exerting cytotoxicity. The inventors of this invention recently reported that when alpha-synuclein forms oligomers, fatty acid-binding protein 3 (FABP3) also generates oligomers with alpha-synuclein (Kawahata, I. et al., Int. J. Mol. Sci. 2019, 20, (21)). As the disease progresses, oligomerized alpha-synuclein propagates between nerve cells, is taken up by nerve cells at the destination, and causes cell death. FABP3 is involved in this uptake of alpha-synuclein into nerve and glial cells. In other words, oligomer formation by FABP3 and α-synuclein is thought to be involved in both the propagation and uptake of α-synuclein, and inhibiting or suppressing the interaction between FABP3 and α-synuclein would be useful for the prevention or treatment of synucleinopathy.

[0021] Therefore, the inventors of this application prepared peptides corresponding to the N-terminal region (region consisting of amino acids 2 to 10 from the N-terminus, SEQ ID NO: 2), the NAC region (region consisting of amino acids 73 to 96, SEQ ID NO: 3), and the C-terminal region (region consisting of amino acids 130 to 140, SEQ ID NO: 4) of human α-synuclein protein (140 amino acids, SEQ ID NO: 1, UniPlot: P37840), and investigated which region of α-synuclein is involved in interaction with FABP3.

[0022] Surprisingly, we found that the C-terminal region, rather than the N-terminal or NAC region, is significantly involved in the interaction with FABP3.

[0023] Figures 1A-C show schematic diagrams of the 1:1 complex formation between α-synuclein and FABP3. The C-terminal region of α-synuclein, shown by a single curve, binds to the fatty acid binding site of FABP3. The binding is largely influenced by the 16th Phe of FABP3 and the 133rd Tyr of the C-terminal region of α-synuclein. Furthermore, a stronger binding occurs when the 133rd position of the α-synuclein peptide is replaced with Phe. It is thought that the rest of α-synuclein, except for the C-terminal region, binds to the surface of FABP3 via charge or other means.

[0024] According to a first aspect of the present invention, a peptide represented by (i) or (ii) below is provided: (i) A peptide having the amino acid sequence represented by EEG(X1)QD(X2)EPEA (SEQ ID NO: 5). (In the formula, X1 and X2 are the same or different tyrosine (Y), phenylalanine (F), or tryptophan (W)) (ii) A peptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sites other than X1 and X2 of the amino acid sequence of (i) above, and which has the activity to bind to the FABP3 protein.

[0025] The amino acid symbols used herein follow the IUPAC amino acid abbreviations. The following represents single-letter codes, three-letter codes, and amino acids. A, Ala: Alanine M, Met: Methionine C, Cys: Cysteine; N, Asn: Asparagine D, Asp: Aspartic acid P, Pro: Proline E, Glu (Glutamic acid) Q, Gln (Glutamic acid) F, Phe Phenylalanine R, Arg Arginine G, Gly Glycine S, Serine Serine H, His: Histidine T, Thr: Threonine I, Ile isoleucine V, Val valine K, Lysine; W, Trp, Tryptophan L, Leu: Leucine; Y, Tyr: Tyrosine

[0026] The peptides of the first aspect of the present invention include not only peptides represented by (i) or (ii) in which each amino acid is unmodified, but also peptides represented by (i) or (ii) in which one or more amino acids are modified while maintaining the types of amino acids. Such modifications include oxidation by oxygen atom bonding, phosphorylation, N-acetylation, S-cysteine ​​formation, and the like. The peptide of the present invention binds to FABP3 through the interaction between the phenyl groups of amino acids X1 and X2 and the phenyl group of phenylalanine at position 16 in the amino acid sequence of the native FABP3 protein.

[0027] In one embodiment, the peptide of the present invention is the peptide represented by (i) above, where X1 is tyrosine and X2 is tyrosine, phenylalanine, or tryptophan. In another embodiment, the peptide of the present invention is the peptide represented in (i) above, where X1 is phenylalanine and X2 is tyrosine, phenylalanine, or tryptophan. When X1 is phenylalanine, the binding affinity to the FABP3 protein is increased compared to when X1 is tyrosine.

[0028] In another embodiment, the peptide of the present invention is the peptide represented by (i) above, where X1 is tryptophan and X2 is tyrosine, phenylalanine, or tryptophan.

[0029] In one embodiment, the peptide of the present invention is the peptide represented by (ii) above, having an amino acid sequence in which one or two amino acids are deleted, substituted, or added at the amino acid site, and having the activity to bind to the FABP3 protein. If one or two amino acids are substituted or added to an amino acid site, such substituted or added amino acids may be natural amino acids or non-natural amino acids (i.e., amino acids other than the 20 classical amino acids).

[0030] In another embodiment, the peptide of the present invention is the peptide represented in (ii) above, having an amino acid sequence in which one amino acid is deleted, substituted, or added at the amino acid site, and having the activity to bind to the FABP3 protein.

[0031] In another embodiment, the peptide of the present invention is the peptide represented in (ii) above, having an amino acid sequence in which one amino acid is deleted, substituted, or added at the amino acid site, and having activity to bind to the FABP3 protein, wherein X1 is tyrosine and X2 is tyrosine, phenylalanine, or tryptophan, or X1 is phenylalanine and X2 is tyrosine, phenylalanine, or tryptophan, or X1 is tryptophan and X2 is tyrosine, phenylalanine, or tryptophan.

[0032] In one embodiment, the peptide of the present invention is a peptide represented by (i) or (ii) above, wherein the length of the amino acids is 7 to 30, preferably 8 to 20, more preferably 9 to 20, and even more preferably 10 to 20, for example 9, 10, 11, 12, 13, 14, 15, or 16.

[0033] The peptide according to the first aspect of the present invention can bind to FABP3, and therefore competes with α-synuclein, thereby inhibiting or suppressing the interaction between FABP3 and α-synuclein. The inhibitory or suppressive effect of the peptide according to the first aspect of the present invention on the interaction between α-synuclein and FABP3 can be confirmed by an in vitro inhibition assay. When the interaction between α-synuclein and FABP3 is inhibited or suppressed, oligomer formation by α-synuclein and FABP3 is inhibited, thereby suppressing the propagation of α-synuclein oligomers between nerve cells and the uptake of α-synuclein into FABP3-expressing nerve cells. For this reason, the peptide according to the first aspect of the present invention is considered useful for the prevention or treatment of synucleinopathy.

[0034] The peptide according to the first aspect of the present invention can be produced as an isolated peptide by a chemical synthesis method well known to those skilled in the art, or it can be produced as part of another peptide.

[0035] According to a second aspect of the present invention, an antibody against the peptide of the first aspect of the present invention is provided. Such an antibody is specific to the peptide of the first aspect and recognizes part or all of the peptide of the first aspect of the present invention as an epitope, thereby inhibiting or suppressing the interaction between α-synuclein and FABP3. For this reason, the antibody of the second aspect of the present invention is also considered useful for the prevention or treatment of synucleinopathy.

[0036] The antibody according to the second aspect of the present invention can be produced by manufacturing the peptide according to the first aspect of the present invention by a chemical synthesis method well known to those skilled in the art, and then using the peptide as an antigen by a well known immunological method. The antibody according to the second aspect of the present invention may be either a polyclonal antibody or a monoclonal antibody. Furthermore, the antibody may include not only the complete antibody molecule but also its fragments, such as Fab, F(ab')2, ScFv, etc.

[0037] Since the peptide according to the first aspect of the present invention is derived from a partial sequence of α-synuclein, the peptide according to the first aspect of the present invention and the antibody according to the second aspect can inhibit the uptake of α-synuclein into FABP3-expressing nerve cells while avoiding side effects. Furthermore, it is possible to design peptides and antibodies that are stable in vivo and have good brain penetration.

[0038] A third aspect of the present invention provides a method for inhibiting the interaction between α-synuclein and FABP3, comprising exposing α-synuclein to the peptide of the first aspect of the present invention or the antibody of the second aspect of the present invention.

[0039] The above inhibition method may be an in vivo method or an in vitro method.

[0040] A fourth aspect of the present invention provides a method for inhibiting the uptake of α-synuclein into FABP3-expressing cells, comprising exposing α-synuclein to the peptide of the first aspect of the present invention or the antibody of the second aspect of the present invention.

[0041] The above inhibition method may be an in vivo or in vitro method. Examples of FABP3-expressing cells include, but are not limited to, dopaminergic neurons and glial cells.

[0042] According to a fifth aspect of the present invention, a kit for inhibiting the interaction between α-synuclein and FABP3 is provided, comprising the peptide of the first aspect of the present invention or the antibody of the second aspect.

[0043] The above kit may include other reagents and / or equipment for measuring free α-synuclein or the aggregation of α-synuclein with FABP, negative controls and positive controls for expression level measurement, etc.

[0044] According to a sixth aspect of the present invention, a prophylactic or therapeutic agent for the prevention or treatment of synucleinopathy is provided, comprising the peptide of the first aspect or the antibody of the second aspect as an active ingredient.

[0045] According to a seventh aspect of the present invention, a pharmaceutical composition comprising a peptide of the first aspect or an antibody of the second aspect is provided for the prevention or treatment of synucleinopathy.

[0046] Synucleinopathies may be selected from the group consisting of Parkinson's disease (PD), Lewy body disease (LBD), and multiple system atrophy (MSA). Lewy body disease (LBD) includes Parkinson's disease with dementia (PDD), and Lewy body disease includes dementia with Lewy body disease (DLB).

[0047] The preventive or therapeutic agent of the sixth embodiment and the pharmaceutical composition of the seventh embodiment can be administered in various forms, such as injections, oral preparations, or nasal preparations, and preferably by injection (intravenous, subcutaneous, intradermal, intrathecal, etc.). Each of these administration forms can be manufactured by a formulation method known and commonly used by those skilled in the art.

[0048] The target population for the preventive or therapeutic agent of the sixth embodiment and the pharmaceutical composition of the seventh embodiment described above is mammals, preferably mice, rats, hamsters, ferrets, dogs, cats, monkeys, or humans, and more preferably humans.

[0049] The preventive or therapeutic agent of the sixth embodiment and the pharmaceutical composition of the seventh embodiment may contain a pharmaceutical carrier as needed. Examples of pharmaceutical carriers include various organic or inorganic carrier substances commonly used as formulation materials, such as excipients, binders, disintegrants, lubricants, coatings, solvents, solubilizers, suspending agents, isotonic agents, pH adjusters, buffers, and micellars. Furthermore, the preventive or therapeutic agent of the sixth embodiment and the pharmaceutical composition of the seventh embodiment may also contain formulation additives such as preservatives, antioxidants, colorants, flavoring / odorizing agents, and stabilizers as needed.

[0050] The amount of peptide of the first embodiment to be incorporated into each of the above-mentioned dosage units is not constant, depending on the symptoms of the patient to whom it is to be administered, or the dosage form, etc., but generally it is 0.1 ng to 1000 mg, preferably 10 ng to 10 mg per dosage unit.

[0051] The daily dose of the peptide according to the first embodiment having the above-described dosage form varies depending on the patient's symptoms, weight, age, sex, etc., and cannot be determined in general terms. However, it is generally preferable for an adult (weighing 50 kg) to administer 0.1 ng to 1000 mg per day, preferably 10 ng to 10 mg, once or twice to three times a day, over a period of one week, every other week, every four weeks (one month), or every other month.

[0052] The amount of antibody of the second embodiment to be incorporated into each of the above-mentioned dosage units is not constant, depending on the symptoms of the patient to whom it is to be administered, or the dosage form, etc., but generally it is 0.1 ng to 1000 mg, preferably 10 ng to 10 mg per dosage unit.

[0053] The daily dose of the antibody in the second embodiment having the above-described administration form varies depending on the patient's symptoms, weight, age, sex, etc., and cannot be determined in general terms. However, it is generally preferable for an adult (weighing 50 kg) to administer 0.1 ng to 1000 mg per day, preferably 10 ng to 10 mg, once or twice to three times a day, over a period of one week, every other week, every four weeks (one month), or every other month.

[0054] All patent applications and disclosures cited herein are incorporated herein by reference in their entirety.

[0055] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0056] Example 1: Interaction between FABP3 and full-length or partial α-synuclein peptides (method) First, Figures 2A and 2B describe the regions corresponding to the structure of the natural protein α-synuclein. Figure 2A shows the regions of the peptides used in this example (SynP2-10, SynP73-96, and SynP130-140), and Figure 2B shows the amino acid sequences of these peptides (SEQ ID NOs. 2, 3, and 4).

[0057] The interaction between FABP3 and wild-type α-synuclein (Syn) or peptides containing a portion of the amino acid sequence of wild-type α-synuclein (SynP2-10, SynP73-96, and SynP130-140) was measured using a quartz crystal microbalance (QCM measurement device (AffinixQNμ, Initium)). The Ni-NTA method was used to immobilize FABP3 onto the quartz substrate. Specifically, after washing with 1% SDS and Piranha solution (H2SO4:H2O2=3:1), 100 μl of 0.5 mM 3,3'-dithiobis[N-(5-amino-5-carboxypentyl)propionamide-N',N'-diacetic acid]dihydrochloride (C2-NTA, Dojindo) solution was added, and the mixture was allowed to stand at room temperature for 10 minutes. After washing with purified water, 500 μl of Ni solution (20 mM HEPES-NaOH) was added. Buffer (pH 7.5, 150 mM NaCl, 50 mM EDTA, 10 mM NiSO4) was added and allowed to stand at room temperature for 10 minutes. After washing with purified water, 100 nM of FABP3 with a His-Tag fused to the N-terminus, diluted in 50 mM Tris-HCl pH 7.4, was added to a quartz sensor and immobilized for 1 minute. Wild-type α-synuclein and three peptides (SynP2-10, SynP73-96, and SynP130-140), diluted in 50 mM Tris-HCl pH 7.4, were added at concentrations of 2.5 nM, 5 nM, 10 nM, 15 nM, and 20 nM, respectively, and measured using a QCM instrument. Measurements were performed in 500 μl of solution at 25 °C and 1000 rpm for 3 minutes. After measurement, Kd, ​​kon, koff were identified using AQUA2.0 software (Initium). The following were calculated for each.

[0058] (result) Figures 3A-D are graphs showing the interactions between FABP3 and the full-length α-synuclein (Syn) (A), SynP2-10 peptide (B), SynP73-96 peptide (C), and SynP130-140 peptide (D), respectively. As shown in Figure 3A, the full-length α-synuclein (Syn) bound to FABP3 in a dose-dependent manner. As shown in Figures 3B and C, the SynP2-10 peptide in the N-terminal region and the SynP73-96 peptide in the NAC region of α-synuclein hardly bound to FABP3, and surprisingly, as shown in Figure 3D, the SynP130-140 peptide in the C-terminal region bound to FABP3 in a dose-dependent manner. The SynP130-140 peptide is the peptide corresponding to the C-terminal portion (100-140) of the C-terminal region of α-synuclein.

[0059] Example 2: Interaction between FABP3 and various mutants of SynP130-140 (method) We created several mutants of the SynP130-140 peptide, including: one in which the tyrosine at position 133 was replaced with phenylalanine (Y133F); one in which the tyrosine at position 136 was replaced with phenylalanine (Y136F); one in which the tyrosine at position 133 was replaced with tryptophan (Y133W); one in which both the tyrosine at position 133 and position 136 were replaced with phenylalanine (Y133F / Y136F); one in which the tyrosine at position 133 was replaced with phenylalanine and the tyrosine at position 136 was replaced with tryptophan (Y133F / Y136W); and one in which both the tyrosine at position 133 and position 136 were replaced with tryptophan (Y136F / Y136W). The interaction between FABP3 and various mutants of the peptide SynP130-140 was investigated using the same method as in Example 1. Measurements were performed by adding each peptide to immobilized FABP3 at an arbitrary concentration.

[0060] (result) As shown in Figure 4A, changing the tyrosine at position 133 to phenylalanine doubled the binding affinity. As shown in Figures 4B and C, changing the tyrosine at position 136 to phenylalanine or tryptophan did not increase the affinity, maintaining an affinity for FABP3 at the same level as tyrosine (Y). As shown in Figure 4D, changing two tyrosine positions to phenylalanine resulted in an affinity for FABP3 at the same level as a single mutation (Y133F), i.e., twice the binding affinity of the natural sequence. These results suggest that the presence of phenylalanine at position 133 is important. As shown in Figure 4E, changing position 136 to tryptophan in addition to the mutation from tyrosine at position 133 to phenylalanine did not increase the affinity by the amount of the mutation at position 136. As shown in Figure 4F, changing two tyrosine positions (Y) to tryptophan (W) did not change the affinity compared to the natural sequence. This shows that the affinity is maintained by the mutation to tryptophan, but it does not necessarily increase.

[0061] Example 3: Inhibition of amyloid fibril formation by adding FABP3 to α-synuclein, and promotion of aggregation by adding peptides. (method) FABP3:α-synuclein (Syn):SynP130-140 mutant peptides were coexisted in a 1:1:1 molar ratio. Amyloid fibril formation of α-synuclein was observed using thioflavin T (Thio-T) fluorescence, or the morphology of the amyloid fibrils at the final stage was measured using an electron microscope (TEM). An ARVO X4 (Perkin Elmer) was used to measure the shaking and time-dependent fluorescence values ​​during the Syn fibril formation process. For the measurements, 150 μl / well of the sample was added to a 96-well plate (8×12 well plate; Greiner, Kremsmuenster, Austria), and fluorescence (Ex: 440 nm, Em: 486 nm) was measured every 15 minutes while shaking was continued at 37°C. The measurement samples were prepared by adding α-synuclein, FABP3, and peptides (SynP130-140, SynP130-140 Y133F, Y136F, Y136W, Y133F / Y136F, Y133F / Y136W, Y133W / Y136W) to 500 μl of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, and 20 μM Thio-T, respectively, to a concentration of 69 μM each. The samples obtained 1800 minutes after measurement were measured using a transmission electron microscope (TEM) (JEOL JEM-1400Plus). 10 μl of the sample was added to a collodion membrane (400 mesh; Nisshin EM, Tokyo, Japan), allowed to stand at room temperature for 2 minutes, washed with 5 μl of purified water, stained with 5 μl of 10% EM stain solution for 1 minute, and then washed with 5 μl of purified water. The measurements were taken after it had been air-dried overnight.

[0062] (result) Figure 5A shows fluorescence observations of amyloid fibril formation over time, and Figure 5B is a micrograph showing synuclein aggregation when the same mutant peptide used in Example 2 was used. When an equimolar amount of FABP3 was added to α-synuclein, synuclein aggregation was completely suppressed (● in the graph). However, when an equimolar amount of FABP3 and a peptide with P130-140 (× in the graph) or a peptide with a mutation at position 133 to F (◆, ■ in the graph) were added to α-synuclein simultaneously, the peptides bound to FABP3 more strongly than α-synuclein (full length, Kd=0.4nM) (Kd=0.2-0.1nM), so the peptides bound to FABP3 first, and the α-synuclein that could not bind formed amyloid fibril aggregation.

[0063] Example 4: Concentration dependence of SynP130-140 Y133F peptide on amyloid fiber aggregation formation FABP3, α-synuclein (Syn), and a mutant peptide (SynP130-140 Y133F) in which the tyrosine at position 133 of SynP130-140 was mutated to phenylalanine were co-existed in a 1:1:1 molar ratio. Amyloid fibril formation of α-synuclein was observed by fluorescence observation with thioflavin T (Thio-T), or the morphology of the amyloid fibrils at the final stage was measured by electron microscopy (TEM). Thio-T and TEM measurements were performed in the same manner as in Example 3. In the Thio-T measurements, SynP130-140 Y133F was added in amounts of 0.1 equivalent (6.9 μM), 0.5 equivalent (34.5 μM), and 1 equivalent (69 μM) relative to 69 μM of α-synuclein and 69 μM of FABP3.

[0064] (result) Figure 6A shows fluorescence observations of amyloid fibril formation over time, and Figure 6B shows micrographs of synuclein aggregation using SynP130-140 Y133F peptide at various concentrations. When an equivalent molar amount of FABP3 was added to α-synuclein, amyloid fibril aggregation of α-synuclein was completely suppressed. When Y133F peptide (Kd=0.1nM) was simultaneously added at 0.1 equivalent, 0.5 equivalent, and 1 equivalent, the Y133F peptide preferentially bound to FABP3 in a volume-dependent manner, and the α-synuclein that could not bind formed amyloid aggregation. The graph with black circles representing 0 equivalent shows amyloid fibril aggregation formation when only FABP3 and α-synuclein are present.

[0065] Example 5: Effect of SynP130-140 peptide addition time on amyloid fiber aggregation formation. The formation of amyloid fibrils from freed α-synuclein after the addition of SynP130-140 peptide to a complex of α-synuclein and FABP3 was observed by fluorescence microscopy or TEM at the final time. Thio-T and TEM measurements were performed in the same manner as in Example 3. In Thio-T measurements, SynP130-140 was added from the beginning (0 hours), and measurements were taken after addition at 3 hours, 8 hours, and 24 hours.

[0066] (result) Figure 7A shows the time-dependent fluorescence observation of amyloid fibril formation at each addition time, and Figure 6B shows micrographs of synuclein aggregation when the SynP130-140 Y133F peptide is added at each addition time. When an equivalent mole of FABP3 is added to α-synuclein (Syn), a 1:1 Syn-FABP3 complex is formed. When the C-terminal peptide (P130-140) is then added at the time indicated by the arrow, an equivalent mole is added. Since P130-140 has a stronger affinity for FABP3, it dissociates Syn from FABP3, and SynP130-140 binds to FABP3. It was confirmed that the Syn dissociated from FABP3 forms amyloid fibril aggregation.

[0067] Example 6: Uptake of α-synuclein in dopaminergic neurons and antagonism of uptake by C-terminal peptide. (method) Cultured dopamine neurons were prepared according to the previously described method (Kawahata et al. 2019). Specifically, the uterus was aseptically removed by midline abdominal incision under isoflurane deep anesthesia from C57BL6N mice at 15.5 days of gestation. The fetal midbrain was extracted in ice-cold Hanks equilibrium salt solution (HBSS) under a stereomicroscope, and the tissue was dispersed by treating it with 37°C neuronal dispersion (Sumitomo Bakelite) for 30 minutes. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded. After dispersing the cell pellet, removal solution (Sumitomo Bakelite) was added, and the mixture was centrifuged at 900 rpm for 5 minutes. The supernatant was removed, and the pellet was suspended in Eagle's minimal essential medium (EMEM) and seeded on a poly-L-lysine coated chamber plate (Iwaki). On day 10 of culture, 1 μM ATTO-550-labeled α-synuclein monomer was added to the culture medium for visualization analysis of α-synuclein. The culture was then treated for 48 hours in the presence or absence of either the synthesized α-synuclein C-terminal 20-residue peptide DNEAYEMPSEEGYQDYEPEA (1 μM) (SEQ ID NO: 6) or the 10-residue peptide EGYQDYEPEA (1 μM) (SEQ ID NO: 6). The antagonistic effect of the peptides on the uptake of α-synuclein into dopaminergic neurons was evaluated by quantitative analysis of the intracellular fluorescence intensity of ATTO-550-labeled α-synuclein (NIH Image J software). The values ​​are average SEM (n>20), and one-way ANOVA with Tukey test was performed for all groups. A conceptual diagram of the synthesized α-synuclein peptide is shown in C.

[0068] (result) Cultured dopamine neurons took up ATTO-550-labeled α-synuclein (Figure 8A, control, red), but simultaneous treatment with 20 or 10 residues of synuclein C-terminal peptide inhibited the intracellular uptake of fluorescently labeled α-synuclein (Figure 8A, 20aa, 10aa, Figure 8B). **** indicates p < 0.0001 compared to the control group (Figure 8B). No significant difference in the inhibitory effect on synuclein uptake was observed between the 20-residue and 10-residue synuclein C-terminal peptide groups (Figure 8B). These peptides belong to the acidic region of synuclein C-terminal (Figure 8C).

[0069] Example 7: Intracellular uptake characteristics of wild-type α-synuclein and C-terminal deficiency mutants in dopamine neurons (method) Cultured dopamine neurons were prepared in the same manner as described above (Example 6). On day 10 of culture, 1 μM ATTO-550 labeled α-synuclein monomer, or, conversely to Example 6, ATTO-550 labeled α-synuclein monomer lacking the 130-140 C-terminal residues, was added to the culture medium for visualization analysis of α-synuclein, and their uptake into neuronal cells was examined. The amount of wild-type α-synuclein or α-synuclein lacking the 130-140 C-terminal residues was evaluated by quantitative analysis of the intracellular fluorescence intensity of ATTO-550 labeled α-synuclein (NIH Image J software). Numerical values ​​were obtained using average SEM (n > 20), and a comparison between the two groups was performed using the Student's T-test. A conceptual diagram of the synthesized α-synuclein lacking the 130-140 C-terminal residues is shown in Figure 9C.

[0070] (result) Cultured dopaminergic neurons took up ATTO-550-labeled α-synuclein (αS, WT), but α-synuclein lacking the 130-140 residues at the C-terminus (αS, Δ130-140) was not taken up into neurons (Figure 9A, red, B). **** indicates p < 0.0001 compared to the control group (Figure 9B).

[0071] Example 8: Binding experiment between FABP and α-synuclein peptide (method) The affinity of FABP3 for interaction with various peptides or α-synuclein was quantitatively determined using a QCM measuring device. The detailed experimental procedure is as described in Example 1.

[0072] (result) The binding results of FABP3 to various peptides are shown in Figure 10. The binding of FABP3 to α-synuclein involves the C-terminal 130-140 residues of α-synuclein. Furthermore, the results of mutations in FABP3 and the α-synuclein C-terminal peptide indicate that the phenyl group of phenylalanine at position 16 in the amino acid sequence of the FABP3 protein interacts with the phenyl group, tyrosine, or tryptophan at position 133 and / or the phenyl group, tyrosine, or tryptophan at position 136 of α-synuclein.

Claims

1. A peptide having an amino acid sequence represented as EEG(X1)QD(X2)EPEA, with an amino acid length of 30 or less, where X1 is F or W and X2 is Y, F, or W, or where X1 is Y and X2 is F or W.

2. A peptide consisting solely of the amino acid sequence represented by EEG(X1)QD(X2)EPEA. (In the formula, X1 is F or W and X2 is Y, F, or W, or X1 is Y and X2 is F or W)

3. The peptide according to claim 1 or 2, which binds to FABP3 via the interaction between the phenyl groups of the amino acids X1 and X2 and the phenyl group of the 16th phenylalanine in the amino acid sequence of the native FABP3 protein.

4. A preventive or therapeutic agent for the prevention or treatment of synucleinopathy, comprising the peptide described in either claim 1 or 2 as an active ingredient.

5. The preventive or therapeutic agent according to claim 4, wherein the synucleinopathy is selected from the group consisting of Parkinson's disease, Lewy body dementia, and multiple system atrophy.

6. A pharmaceutical composition comprising the peptide according to claim 1 or 2 for the prevention or treatment of synucleinopathy.

7. An inhibitor of the interaction between α-synuclein and FABP3, comprising a peptide having the amino acid sequence represented by EEG(X1)QD(X2)EPEA and having an amino acid length of 20 or less (wherein X1 and X2 are the same or different, and are Y, F, or W).

8. An inhibitor of α-synuclein uptake into dopaminergic neurons, comprising a peptide having an amino acid sequence represented as EEG(X1)QD(X2)EPEA and having an amino acid length of 20 or less (wherein X1 and X2 are the same or different, Y, F, or W).

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