JP7774972B2 - Artificial peptides with peptide bond cleavage activity and their use - Google Patents

Description

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

It has recently become clear that Alzheimer's disease develops when oligomers, aggregates formed by the aggregation of several to several dozen amyloid beta proteins, inhibit synaptic function and lead to neuronal death. The "oligomer hypothesis," which proposes that these oligomers inhibit synaptic function and cause hyperphosphorylation of tau protein, is currently the mainstream theory. It is now believed that oligomers, not only amyloid beta proteins but also tau proteins, are toxic. Therefore, drug targets should be amyloid beta protein oligomers and tau protein oligomers, rather than senile plaques or neurofibrils. Current Alzheimer's disease treatments, including anticholinesterase inhibitors, are considered effective, but they are symptomatic treatments that do not aim to remove or inactivate oligomers and only slow progression to a certain extent. Therefore, drugs that can degrade and remove protein oligomers could potentially be causal treatments for the prevention and treatment of Alzheimer's disease.

Research into Alzheimer's disease is being conducted in many laboratories. For example, it has been reported that the drug aducanumab reduces the deposition of amyloid plaques of amyloid beta protein 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, meaning that there is currently no drug that can cure this disease.

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

Patent No. 6455983

J. Sevigny, et al. The antibody aducanumab reduces Aβ plaques in Alzheimer’s disease. Nature 537 50-56(2016)

The 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.

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) X ₁ AAX ₂ AX ₃ AHAASDAW (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], 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.

The present invention provides 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.

FIG. 1 shows the structures of artificial peptides me5f and me5f5D confirmed by CD spectrum measurement. 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. 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. 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. 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. FIG. 1 shows the results of dynamic light scattering measurement evaluation of the ability of the artificial peptides me5f and me5f5D to decompose amyloid β protein oligomers. 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. FIG. 1 shows the results of evaluating the albumin decomposition ability of the artificial peptides me5f and me5f5D by measuring CD spectra before and after incubation with albumin at 37° C. for 7 days. FIG. 10 shows the results of evaluating the ability of the artificial peptides me5f and me5f5D to decompose gamma globulin by measuring CD spectra before and after incubation with gamma globulin at 37° C. for 7 days.

〔peptide〕
The present invention provides an artificial peptide (hereinafter referred to as "the peptide of the present invention") consisting of the amino acid sequence represented by the following formula (I):
(I) X ₁ AAX ₂ AX ₃ AHAASDAW (SEQ ID NO: 1)
( _X1 and _X2 are the same or different and each represent V, I, or L, and _X3 represents 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).

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).

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 into which an expression vector containing DNA encoding the peptide of the present invention has been introduced, or by a method using an in vitro transcription/translation system.

In the peptides of the present invention, the C-terminus may be any of a carboxy group (—COOH), a carboxylate group (—COO ⁻ ), an amide group (—CONH ₂ ), or an ester group (—COOR). An amide group (—CONH ₂ ) is preferred. Examples of R in the ester group include C _1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, or n-butyl; C _3-8 cycloalkyl groups such as cyclopentyl or cyclohexyl; C _6-12 aryl groups such as phenyl or α-naphthyl; C _7-14 aralkyl groups such as phenyl-C _1-2 alkyl groups such as benzyl or phenethyl, or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl; and pivaloyloxymethyl groups, which are commonly used as oral esters. When the peptides of the present invention have a carboxy group or carboxylate group other than that at the C-terminus, peptides in which these groups are amidated or esterified are also included in the peptides of the present invention.

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, the peptides of the present invention also include those in which the amino group of the N-terminal serine residue is protected with a protecting group (e.g., a _C1-6 acyl group such as a formyl group or a _C2-6 alkanoyl group such as acetyl), those in which the glutamyl group generated by in vivo cleavage of the N-terminus is pyroglutamated, and those in which substituents on the side chains of amino acids in the molecule (e.g., —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected with appropriate protecting groups (e.g., a _C1-6 acyl group such as a formyl group or a _C2-6 alkanoyl group such as acetyl).

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.

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, etc. 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.

[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).

By contacting the peptides of the present invention with a protein having a flexible region, the peptide bonds 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 degraded.

[Amyloid β protein oligomer degrading agent]
Since the peptide of the present invention has the ability to degrade amyloid β-protein oligomers, it can be suitably used as an agent for degrading amyloid β-protein oligomers. By contacting the peptide of the present invention with amyloid β-protein oligomers, the amyloid β-protein oligomers can be degraded. 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.

[Pharmaceutical composition for treating amyloid-related diseases]
Since the peptide of the present invention has the ability to decompose 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 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.

The pharmaceutical composition of the present invention can be formulated by appropriately blending a pharmaceutically acceptable carrier or additive with the peptide of the present invention, its pharmaceutically acceptable salt, or a hydrate thereof as the active ingredient. Specifically, it can be formulated as 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 blending ratio of the carrier or additive may be appropriately set based on the range commonly used in the pharmaceutical field. There are no particular limitations on the carrier or additive that can be blended; examples include various carriers such as water, 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.

Additives that can be incorporated into tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth, and gum arabic; fillers such as crystalline cellulose; leavening agents such as corn starch, gelatin, and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; and flavorings such as peppermint, saffron oil, or cherry. When the dosage unit is a capsule, the above-mentioned materials may further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be prepared according to conventional pharmaceutical practice (for example, by dissolving or suspending the active ingredient in a solvent such as water for injection or natural vegetable oil). Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), and may be used in combination with appropriate solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80™, HCO-50). Oily solutions include, for example, sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Additionally, the solution may be formulated with 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.

The preparations thus obtained are safe and of low toxicity and can be administered to, for example, humans and other mammals (e.g., 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, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day. 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, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day by intravenous injection. The total daily dosage may be a single dose or divided doses.

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.

The present invention will be explained in detail below using examples, but the present invention is not limited to these examples.

Example 1: Synthesis of artificial peptides and confirmation of their structures
(1) Synthesis of Artificial Peptides Two types of 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)

(2) CD (Circular Dichroism) Measurements. 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 optical 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 number of integrations 8.

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

Example 2: Examination of peptide bond cleavage ability of artificial peptides
(1) Peptides 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)

(2) CD (Circular Dichroism) Measurements: 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 a sample immediately after preparation and a sample incubated at 37°C for 3 days. A Jasco J-720 spectropolarimeter (manufactured by JASCO Corporation) was used to measure 300 μL of peptide solution in a quartz cell with a 1 mm optical 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 number of accumulations 8.

(3) Results The results for me5 are shown in Figure 2, the results for me5f in Figure 3, and the results 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 since 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 cleave peptide bonds and undergo autolysis, but me5 does not cleave peptide bonds. These results reveal that the structure of the peptide (random coil structure) is important for peptide bond cleavage.

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 at 25°C for 20 hours to prepare amyloid β protein oligomers.

(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 solution was added to a Y-shaped cell with a 1 cm optical path length, and the absorbance at 210 nm and 224 nm was measured over time. A V-650 spectrophotometer (Jasco) was used for absorbance measurements.

(3) Dynamic Light Scattering Measurements The artificial peptides used were me5f and me5f5D synthesized in Example 1. As controls, ultrapure water was added instead of the peptide, and untreated amyloid β-protein oligomers were used. A solution was prepared in 20 mM Tris-HCl buffer (pH 7.3) so that the amyloid β-protein 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.

(4) Results The results of evaluating peptide bond cleavage activity based on absorbance measurement are shown in Figure 5. Figure 5 shows the results of normalization based on the results of absorbance measurement, with the peak absorbance (Abs _{210 nm} - Abs _{224 nm} ) of the control sample to which ultrapure water was added being set to 1 and the peak absorbance of the sample to which me5f was added 7 days later being set to 0. The absorbance of the sample to which me5f was added was shown to decrease with each passing day. These results demonstrate that me5f degrades most of the peptide bonds in amyloid β protein oligomers.

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

Example 4: Examination of the Reduction of Toxicity of Amyloid β Protein Oligomers by Artificial Peptides (1) WST-8 Assay PC12 cells were cultured in a 96-well plate, and 100 μM amyloid β protein oligomers treated with 1 μM me5f, 100 μM amyloid β protein oligomers not treated with me5f, or untreated amyloid fibrils were added to the wells. After incubation at 37°C for 1 day, the absorbance at 450 nm was measured to verify cytotoxicity. This experiment was commissioned to the National Institute of Advanced Industrial Science and Technology (AIST). The amyloid fibrils used were prepared by dissolving amyloid β42 (Peptide Institute) in dimethyl sulfoxide (DMSO) to a concentration of 5 mM, sonicating at 37°C for 10 minutes, diluting with 10 mM HCl to a concentration of 100 μM, and incubating at 37°C for 48 hours.

(2) Results The results of the WST-8 assay are shown in Figure 7. The cell viability of amyloid β oligomers without me5f treatment was 78%, whereas the cell viability of amyloid β oligomers with me5f treatment was 90%. Therefore, it was demonstrated that me5f reduces the toxicity of amyloid β oligomers.

[Reference example: Examination of protein degradation ability other than amyloid β protein oligomers]
(1) Investigation of albumin and gamma globulin degradation ability In order to confirm that the peptides of the present invention do not cause side effects such as degradation of representative proteins that may be present in the blood, the albumin and gamma globulin degradation abilities of me5f and me5f5D were investigated.
Each protein was obtained as a reagent and prepared at 0.1 mg/mL using 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. A Jasco J-720 spectropolarimeter (manufactured by JASCO Corporation) was used for the measurements, using a quartz cell with an optical path length of 1 mm.

(2) Results The results for albumin are shown in Figure 8. As is clear from Figure 8, the CD spectrum hardly changed even after 7 days of incubation with me5f or me5f5D, indicating that no structural changes occurred. In other words, it was demonstrated that me5f and me5f5D do not degrade albumin.
The results for γ-globulin are shown in Figure 9. As is clear from Figure 9, even after adding me5f or me5f5D and incubating for 7 days, the CD spectrum showed almost no change, indicating that no structural change occurred. In other words, it was demonstrated that me5f and me5f5D do not degrade γ-globulin.

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 (5)

A peptide consisting of the amino acid sequence IAALAAAHAASDAW (SEQ ID NO: 2) or a salt thereof. 2. The peptide or salt thereof according to claim 1, wherein the C-terminal amino acid is amidated. A peptide bond cleaving agent comprising the peptide or salt thereof according to claim 1 or 2 . An agent for degrading amyloid β-protein oligomers, comprising the peptide or salt thereof according to claim 1 or 2 . A pharmaceutical composition for treating an amyloid-related disease, comprising the peptide or salt thereof according to claim 1 or 2 .