Artificial peptides with peptide bond cleavage activity and their use

Abstract

To provide an artificial peptide that has a peptide bond cleaving ability and decomposes an amyloid β protein oligomer, an amyloid β protein oligomer decomposer including the artificial peptide, and a pharmaceutical composition for treating amyloid-related diseases.SOLUTION: The present invention discloses a peptide composed of an amino acid sequence represented by formula (I) X1AAX2AX3AHAASDAW (SEQ ID NO: 1) (X1 and X2 may be the same or different to represent V, I or L, and X3 is A or L) or a salt thereof; a peptide bond cleaving agent containing the peptide or a salt thereof; an amyloid β protein oligomer decomposer; and a composition for treating amyloid-related diseases.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an artificial peptide having peptide bond cleaving activity, a peptide bond cleaving agent containing the artificial peptide, an amyloid beta protein oligomer degrading agent containing the artificial peptide, and a pharmaceutical composition for treating amyloid-related diseases containing the artificial peptide. [Background technology]

[0002] It has recently become clear that Alzheimer's disease develops when amyloid beta protein oligomers, aggregates formed by the aggregation of several to several dozen molecules, disrupt synaptic function and cause neuronal death. The "oligomer hypothesis," which proposes that these oligomers disrupt synaptic function and cause hyperphosphorylation of tau protein, is currently the mainstream theory. It is now believed that oligomers, not only amyloid beta protein but also tau protein, are toxic. Therefore, drug targets should target amyloid beta protein oligomers and tau protein oligomers, rather than senile plaques or neurofibrils. Current Alzheimer's disease treatments, such as anticholinesterase inhibitors, are considered effective, but they are symptomatic treatments that do not target or inactivate oligomers and only moderately slow progression. Therefore, drugs capable of degrading and removing protein oligomers could potentially be causal treatments for the prevention and treatment of Alzheimer's disease.

[0003] Many laboratories are conducting research into Alzheimer's disease. For example, it has been reported that aducanumab reduces the deposition of amyloid plaques in the brain (Non-Patent Document 1). However, degradation of oligomers has not been confirmed, and the ENGAGE trial did not achieve its primary endpoint. Therefore, there is currently no drug that can cure this disease.

[0004] The present inventors have previously discovered and patented an artificial peptide that degrades amyloid fibrils (Patent Document 1). However, the present inventors have confirmed that this artificial peptide does not degrade amyloid β protein oligomers. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6455983 [Non-patent literature]

[0006] [Non-Patent Document 1] J. Sevigny, et al. The antibody aducanumab reduces Aβ plaques in Alzheimer's disease. Nature 537 50-56(2016) Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide an artificial peptide that has the ability to cleave peptide bonds and degrade amyloid β-protein oligomers, an agent for degrading amyloid β-protein oligomers that uses the artificial peptide, and a pharmaceutical composition for treating amyloid-related diseases. [Means for solving the problem]

[0008] In order to solve the above problems, the present invention includes the following inventions. [1] A peptide having an amino acid sequence represented by the following formula (I) or a salt thereof: (I) X1AAX2AX3AHAASDAW (SEQ ID NO: 1) (X1 and X2 are the same or different and each represent V, I or L, and X3 is A or L). [2] The peptide or salt thereof according to [1] above, wherein the formula (I) is IAALAAAHAASDAW (SEQ ID NO: 2). [3] The peptide or salt thereof according to [1] or [2] above, wherein the C-terminal amino acid is amidated. [4] A peptide bond cleaving agent comprising the peptide or salt thereof according to any one of [1] to [3] above. [5] An agent for degrading amyloid β-protein oligomers, comprising the peptide or salt thereof according to any one of [1] to [3] above. [6] A pharmaceutical composition for treating an amyloid-related disease, comprising the peptide or salt thereof according to any one of [1] to [3] above. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide an artificial peptide that has the ability to cleave peptide bonds and degrade amyloid β-protein oligomers, an agent for degrading amyloid β-protein oligomers that uses the artificial peptide, and a pharmaceutical composition for treating amyloid-related diseases. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the structures of the artificial peptides me5f and me5f5D confirmed by CD spectrum measurement. [Figure 2] FIG. 1 shows the results of evaluating the peptide bond cleavage ability using autolysis as an index by measuring CD spectra before and after incubation of the artificial peptide me5 at 37° C. for 3 days. [Figure 3] FIG. 1 shows the results of evaluating the peptide bond cleavage ability using autolysis as an index by measuring CD spectra before and after incubation of the artificial peptide me5f at 37° C. for 3 days. [Figure 4] FIG. 1 shows the results of evaluating the peptide bond cleavage ability using autolysis as an index by measuring CD spectra before and after incubation of the artificial peptide me5f5D at 37° C. for 3 days. [Figure 5]FIG. 1 shows the results of evaluating the ability of the artificial peptides me5f and me5f5D to decompose amyloid β protein oligomers by absorbance measurement. [Figure 6] FIG. 1 shows the results of dynamic light scattering measurement evaluating the ability of the artificial peptides me5f and me5f5D to decompose amyloid β protein oligomers. [Figure 7] FIG. 1 shows the results of evaluating the cytotoxicity of amyloid β protein oligomers treated with the artificial peptide me5f and untreated amyloid β protein oligomers using a WST-8 assay. [Figure 8] FIG. 1 shows the results of evaluating the albumin-decomposing ability of the artificial peptides me5f and me5f5D by measuring CD spectra before and after incubation with albumin at 37° C. for 7 days. [Figure 9] FIG. 1 shows the results of evaluating the ability of the artificial peptides me5f and me5f5D to decompose γ-globulin by measuring CD spectra before and after incubation with γ-globulin at 37° C. for 7 days. DETAILED DESCRIPTION OF THE INVENTION

[0011] 〔peptide〕 The present invention provides an artificial peptide consisting of the amino acid sequence shown in the following formula (I) (hereinafter referred to as "the peptide of the present invention"). (I) X1AAX2AX3AHAASDAW (SEQ ID NO: 1) (X1 and X2 are the same or different and each represent V, I, or L, and X3 is A or L) The present inventors have confirmed that the peptide of the present invention has the ability to cleave peptide bonds and to degrade amyloid β protein oligomers (see Examples).

[0012] In the amino acid sequence shown in formula (I), X1 and X2 may be the same or different and may be V, I, or L. V, I, and L are all aliphatic amino acids and share chemical properties. In the amino acid sequence of formula (I), X3 may be A or L. A and L are aliphatic amino acids and share chemical properties. The amino acid sequence shown in formula (I) is preferably IAALAAAHAASDAW (SEQ ID NO: 2).

[0013] The peptides of the present invention can be produced by solid-phase synthesis (Fmoc method, Boc method) or liquid-phase synthesis according to known general peptide synthesis protocols. They can also be produced by a method using a transformant transfected with an expression vector containing DNA encoding the peptide of the present invention, or by a method using an in vitro transcription-translation system.

[0014] The peptide of the present invention has a C-terminus containing a carboxy group (-COOH) or a carboxylate group (-COO - R in the ester group may be, for example, a C group such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, or an n-butyl group. 1-6 Alkyl groups, such as cyclopentyl and cyclohexyl groups 3-8 Cycloalkyl groups, such as phenyl and α-naphthyl groups 6-12 Aryl groups, for example, phenyl-C such as benzyl and phenethyl groups 1-2 Alkyl group or α-naphthyl-C such as α-naphthylmethyl group 1-2 C such as alkyl group 7-14 In addition to aralkyl groups, examples include pivaloyloxymethyl groups, which are commonly used as oral esters. When the peptide of the present invention has a carboxy group or carboxylate group other than that at the C-terminus, the peptide of the present invention also includes those in which these groups are amidated or esterified.

[0015] The amino acids constituting the peptides of the present invention may have their side chains modified with any substituent, including, but not limited to, a fluorine atom, a chlorine atom, a cyano group, a hydroxyl group, a nitro group, an alkyl group, a cycloalkyl group, an alkoxy group, and an amino group. Furthermore, in the peptide of the present invention, the amino group of the N-terminal serine residue is protected by a protecting group (e.g., C group such as formyl group, acetyl group, etc.). 2-6 C such as alkanoyl group 1-6 The N-terminal side is cleaved in vivo and the glutamyl group generated is pyroglutamic acid-modified. The substituents on the amino acid side chains in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.). 2-6 C such as alkanoyl group 1-6 Also included are those protected with an acyl group or the like.

[0016] The peptides of the present invention may form salts or hydrates thereof. Pharmaceutically acceptable salts are preferred. Specific examples include salts with acids such as hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, and palmitic acid; salts with hydroxides or carbonates of alkali metals or alkaline earth metals such as sodium, potassium, and calcium, or aluminum; and salts with triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine, and arginine.

[0017] The peptides of the present invention may contain D-amino acids or unnatural amino acids, as long as the properties of the original peptide are maintained. Furthermore, the peptides of the present invention may be linked to other substances, as long as the properties of the original peptide are maintained. Examples of other substances that can be linked to peptides include other peptides, lipids, sugars or sugar chains, acetyl groups, natural or synthetic polymers, and the like. Furthermore, the peptides of the present invention may be modified, such as by glycosylation, side chain oxidation, or phosphorylation, as long as the properties of the original peptide are maintained.

[0018] [Peptide bond cleaving agent] The peptides of the present invention have the ability to cleave peptide bonds, and therefore can be suitably used as peptide bond cleaving agents. It has been confirmed that the peptides of the present invention can selectively cleave peptide bonds in flexible regions of proteins, rather than cleaving all peptide bonds in proteins. Therefore, it has been confirmed that the peptides of the present invention do not degrade albumin or gamma globulin (see Reference Examples).

[0019] By contacting the peptide of the present invention with a protein having a flexible region, the peptide bond in the flexible region of the protein can be cleaved. The reaction temperature is not particularly limited, but is preferably about 10°C to about 45°C, and more preferably about 25°C to about 37°C. The optimal reaction time can be selected appropriately depending on the protein to be decomposed.

[0020] [Amyloid β protein oligomer degrading agent] The peptides of the present invention have the ability to degrade amyloid β-protein oligomers, and therefore can be suitably used as agents for degrading amyloid β-protein oligomers. Amyloid β-protein oligomers can be degraded by contacting the peptides of the present invention with the amyloid β-protein oligomers. The reaction temperature is not particularly limited, but is preferably about 10°C to about 45°C, and more preferably about 25°C to about 37°C. The optimal reaction time can be selected appropriately depending on the amyloid β-protein oligomers to be degraded.

[0021] [Pharmaceutical composition for treating amyloid-related diseases] Since the peptide of the present invention has the ability to degrade amyloid β protein oligomers, the peptide or a salt thereof is useful as an active ingredient of a therapeutic agent for amyloid-related diseases. Examples of amyloid-related diseases to be treated include systemic amyloidoses such as immune cell-mediated amyloidosis, reactive AA amyloidosis (secondary amyloidosis), familial amyloidosis (hereditary amyloidosis), dialysis-related amyloidosis, and senile systemic amyloidosis, as well as localized amyloidoses such as Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, hereditary cerebral amyloid-angiopathy, British familial dementia, Creutzfeldt-Jakob disease, amyloid associated with medullary thyroid carcinoma, type II diabetes, insulinoma, localized atrial amyloidosis, cutaneous amyloidosis, corneal amyloidosis, and localized nodular amyloidosis.

[0022] The pharmaceutical compositions of the present invention can be formulated by appropriately blending pharmaceutically acceptable carriers or additives with the peptide of the present invention, its pharmaceutically acceptable salt, or a hydrate thereof as an active ingredient. Specifically, they can be oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions; or parenteral preparations such as injections, infusions, suppositories, ointments, and patches. The proportion of the carrier or additive may be appropriately determined based on the range commonly used in the pharmaceutical field. The carrier or additive that can be blended is not particularly limited, and examples include various carriers such as water, physiological saline, other aqueous solvents, and aqueous or oily bases; and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0023] Examples of additives that can be incorporated into tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth, and gum arabic, excipients such as crystalline cellulose, bulking agents such as corn starch, gelatin, and alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose, or saccharin, and flavoring agents such as peppermint, saffron oil, and cherry. When the dosage unit form is a capsule, a liquid carrier such as oil or fat may be further contained in addition to the above-mentioned materials. Sterile compositions for injection can be prepared according to conventional pharmaceutical practices (for example, by dissolving or suspending the active ingredient in a solvent such as water for injection or natural vegetable oil). Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), and the like, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80™, HCO-50). Examples of oily solutions include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Furthermore, the injection may also contain buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives (e.g., benzyl alcohol, phenol, etc.), antioxidants, etc.

[0024] The preparations thus obtained are safe and of low toxicity and can be administered to, for example, humans and other mammals (for example, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). The dosage varies depending on the subject, target organ, symptoms, administration method, etc., but in the case of oral administration, for example, for a human weighing approximately 60 kg, it is generally about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the subject, target organ, symptoms, administration method, etc., but for example, in the case of injections, for example, for a human weighing approximately 60 kg, it is convenient to administer about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg by intravenous injection. The total daily dosage may be a single dose or divided doses.

[0025] The present invention also includes the following inventions. A method for treating an amyloid-related disease, which comprises administering to a mammal an effective amount of the peptide of the present invention or a salt thereof. Use of the peptide of the present invention or a salt thereof for producing a pharmaceutical composition for treating an amyloid-related disease. The peptide of the present invention or a salt thereof for use in treating an amyloid-related disease. [Example]

[0026] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0027] Example 1: Synthesis of artificial peptides and confirmation of their structures (1) Synthesis of artificial peptides Two artificial peptides (me5f and me5f5D) were synthesized by the F-moc solid-phase method using a peptide synthesizer (Pioneer Peptide synthesis System; Applied Biosystems). The amino acid sequences of me5f and me5f5D are as follows, and both have an amide group at the C-terminus. me5f:IAALAAAHAASDAW (SEQ ID NO: 2) me5f5D:IAALDAAHAASAAW (SEQ ID NO: 3)

[0028] (2) CD (Circular Dichroism) Measurement Each synthesized peptide was dissolved in Tris-HCl buffer (pH 7.0) to prepare a 100 μM peptide solution, and CD spectra were measured. A Jasco J-720 spectropolarimeter (JASCO Corporation) was used to measure 200 μL of peptide solution in a quartz cell with a 1 mm path length. Measurement conditions were: temperature 25°C, scanning wavelength 250 nm to 190 nm, data interval 0.2 nm, scanning speed 100 nm / min, response time 2 sec, bandwidth 1 nm, sensitivity 10 mdeg, and number of integrations 8.

[0029] (3) Results The results are shown in Figure 1. Figure 1 reveals that both me5f and me5f5D have a random coil structure.

[0030] Example 2: Examination of peptide bond cleavage ability of artificial peptides (1) Peptide used The me5f and me5f5D synthesized in Example 1 were used. As a comparative example, me5 consisting of the amino acid sequence shown below was synthesized by the F-moc solid-phase method using the same peptide synthesizer as in Example 1 (the C-terminus is an amide group) and used. Although data is not shown, me5 was confirmed to have an α-helical structure by CD measurement, unlike me5f and me5f5D. me5:SAALEAKIAALERKIAALAAAHAASDAW (SEQ ID NO: 4)

[0031] (2) CD (Circular Dichroism) Measurement Each peptide was dissolved in 20 mM Tris-HCl buffer (pH 7.3) to prepare a 100 μM peptide solution. CD spectra were measured for samples immediately after preparation and for samples incubated at 37°C for 3 days. A Jasco J-720 spectropolarimeter (JASCO Corporation) was used to measure 300 μL of peptide solution in a quartz cell with a 1 mm path length. The measurement conditions were: temperature 25°C, scanning wavelength 250 nm to 190 nm, data interval 0.2 nm, scanning speed 100 nm / min, response 2 sec, bandwidth 1 nm, sensitivity 10 mdeg, and accumulation 8 times.

[0032] (3) Results The results for me5 are shown in Figure 2, those for me5f in Figure 3, and those for me5f5D in Figure 4. As is clear from Figures 2 to 4, the average molar ellipticity hardly decreased with me5, whereas the average molar ellipticity decreased significantly with me5f and me5f5D. When peptides are decomposed, they become amino acids, and because the average molar ellipticity of amino acids is very small, the average molar ellipticity decreases when peptides are decomposed. These results indicate that me5f and me5f5D undergo autolysis by cleaving peptide bonds, whereas me5 does not cleave peptide bonds. These results reveal that the peptide structure (random coil structure) is important for peptide bond cleavage.

[0033] Example 3: Investigation of the ability of artificial peptides to decompose amyloid β protein oligomers (1) Preparation of oligomers Amyloid β protein (Peptide Institute) was dissolved in HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) at a concentration of 0.5 mg / mL, distilled under low pressure, and then dissolved in ultrapure water. The solution was sonicated in an ice bath for 5 minutes and centrifuged at 14,000 rpm for 10 minutes. The supernatant was incubated for 20 hours at 25°C to produce amyloid β protein oligomers.

[0034] (2) Absorbance measurement The artificial peptides used were me5f and me5f5D, synthesized in Example 1. Ultrapure water was used as a control instead of peptide. A 20 mM Tris-HCl buffer solution (pH 7.3) was prepared so that the amyloid β protein oligomer concentration was 3 μM and the peptide concentration was 50 nM. 200 μL of the peptide mixture was added to a Y-shaped cell with a 1 cm path length, and the absorbance at 210 nm and 224 nm was measured over time. A V-650 spectrophotometer (Jasco) was used to measure the absorbance.

[0035] (3) Dynamic light scattering measurement The artificial peptides used were me5f and me5f5D, synthesized in Example 1. Ultrapure water was added instead of the peptides, and untreated amyloid β oligomers were used as controls. A solution was prepared in 20 mM Tris-HCl buffer (pH 7.3) so that the amyloid β oligomer concentration was 10 μM and the peptide concentration was 500 nM. The solution was incubated at 37°C for 7 days, then filtered using a 0.45 μm filter, and 16 μL was added to a quartz cell for measurement. A Zetasizer Nano-S (Malvern Instruments) was used for dynamic light scattering measurements.

[0036] (4) Results The results of evaluating peptide bond cleavage activity based on absorbance measurement are shown in Figure 5. Figure 5 shows the absorbance peak (Abs) of the control sample containing ultrapure water. 210nm -Abs 224nm The results were normalized by setting the absorbance at 1 and the peak absorbance of the sample with me5f added 7 days later as 0. The absorbance of the sample with me5f added decreased with each passing day. These results demonstrate that me5f degrades most of the peptide bonds in amyloid beta protein oligomers.

[0037] The results of dynamic light scattering measurements are shown in Figure 6. Only when amyloid β oligomers were treated with me5f were peaks of amyloid β protein degradation products of 1–2 nm in size detected. This demonstrates that me5f can cleave the peptide bonds of amyloid β oligomers.

[0038] Example 4: Examination of reducing the toxicity of amyloid β protein oligomers by artificial peptides (1) WST-8 assay PC12 cells were cultured in 96-well plates, and 100 μM amyloid β oligomers treated with 1 μM me5f, 100 μM amyloid β oligomers not treated with me5f, or untreated amyloid fibrils were added to the wells. After incubation at 37°C for 1 day, cytotoxicity was assessed by measuring absorbance at 450 nm. This experiment was conducted at the request of the National Institute of Advanced Industrial Science and Technology (AIST). Amyloid fibrils were prepared by dissolving amyloid β42 (Peptide Institute) in dimethyl sulfoxide (DMSO) to 5 mM, sonicating at 37°C for 10 minutes, diluting with 10 mM HCl to 100 μM amyloid β42, and incubating at 37°C for 48 hours.

[0039] (2) Results The results of the WST-8 assay are shown in Figure 7. Cell viability was 78% in the presence of untreated amyloid β oligomers, whereas cell viability was 90% in the presence of me5f-treated amyloid β oligomers. Therefore, me5f was found to reduce the toxicity of amyloid β oligomers.

[0040] [Reference example: Examination of protein degradation ability other than amyloid β protein oligomers] (1) Albumin and gamma globulin decomposition ability To confirm that the peptides of the present invention do not cause side effects such as degradation of typical proteins that may be present in the blood, the albumin and γ-globulin decomposition abilities of me5f and me5f5D were examined. Each protein was obtained as a reagent and prepared at 0.1 mg / mL in 20 mM Tris-HCl buffer (pH 7.3), and me5f and me5f5D synthesized in Example 1 were prepared at 500 nM. Each protein solution alone was used as a control. CD spectra were measured immediately after sample preparation and after incubation at 37°C for 7 days. Measurements were performed using a Jasco J-720 spectropolarimeter (manufactured by JASCO Corporation) in a quartz cell with a 1 mm optical path length.

[0041] (2) Results The results for albumin are shown in Figure 8. As is clear from Figure 8, the CD spectrum remained almost unchanged even after 7 days of incubation with me5f or me5f5D, indicating that no structural changes occurred. This indicates that me5f and me5f5D do not degrade albumin. The results for γ-globulin are shown in Figure 9. As is clear from Figure 9, the CD spectrum remained almost unchanged even after 7 days of incubation with me5f or me5f5D, indicating that no structural changes occurred. This indicates that me5f and me5f5D do not degrade γ-globulin.

[0042] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A peptide or salt thereof consisting of the amino acid sequence IAALAAAHAASDAW (sequence number 2).

2. 2. The peptide or salt thereof according to claim 1, wherein the C-terminal amino acid is amidated.

3. A peptide bond cleaving agent comprising the peptide or salt thereof according to claim 1 or 2.

4. An agent for degrading amyloid β-protein oligomers, comprising the peptide or salt thereof according to claim 1 or 2.

5. A pharmaceutical composition for treating an amyloid-related disease, comprising the peptide or salt thereof according to claim 1 or 2.

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