JP6455983B2 - Artificial peptide having amyloid resolution and use thereof - Google Patents
Artificial peptide having amyloid resolution and use thereof Download PDFInfo
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- JP6455983B2 JP6455983B2 JP2015543878A JP2015543878A JP6455983B2 JP 6455983 B2 JP6455983 B2 JP 6455983B2 JP 2015543878 A JP2015543878 A JP 2015543878A JP 2015543878 A JP2015543878 A JP 2015543878A JP 6455983 B2 JP6455983 B2 JP 6455983B2
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
Description
本発明は、アミロイド分解能を有する人工ペプチド、当該人工ペプチドを含有するアミロイド分解剤、当該人工ペプチドを含有するアミロイド検出剤、当該人工ペプチドを用いるアミロイドの分解方法、当該人工ペプチドを用いるアミロイドの検出方法、当該人工ペプチドを含有する医薬組成物、当該人工ペプチドを含有する診断用組成物に関するものである。 The present invention relates to an artificial peptide having amyloid resolution, an amyloid degrading agent containing the artificial peptide, an amyloid detecting agent containing the artificial peptide, an amyloid decomposing method using the artificial peptide, and an amyloid detecting method using the artificial peptide The present invention relates to a pharmaceutical composition containing the artificial peptide and a diagnostic composition containing the artificial peptide.
アミロイドと呼ばれる線維状蛋白質が体内の種々の器官あるいは組織に沈着することにより発症する疾患は、アミロイドーシスと総称されている。アミロイドーシスに共通しているのはアミロイドと呼ばれるβシート構造に富んだ線維状蛋白質が全身の諸臓器あるいは局所に沈着し、その臓器や組織における機能異常を生じる点である。アミロイドとは、アミロイドβや変異トランスサイレチン、β２ミクログロブリンなど種々のアミロイド前駆体蛋白が生体内において凝集することにより形成される蛋白質凝集体の総称をいう。アミロイドは、いずれのアミロイド前駆体蛋白から形成されても、βシートに富んだ特徴的な構造を有している。 Diseases that develop when a fibrous protein called amyloid is deposited in various organs or tissues in the body are collectively called amyloidosis. A common feature of amyloidosis is that fibrillar protein called amyloid rich in β-sheet structure is deposited in various organs or regions throughout the body, resulting in functional abnormalities in the organs and tissues. Amyloid is a generic term for protein aggregates formed by aggregation of various amyloid precursor proteins such as amyloid β, mutant transthyretin, and β2 microglobulin in vivo. Amyloid has a characteristic structure rich in β-sheets, regardless of which amyloid precursor protein is formed.
厚生労働省特定疾患調査研究班新分類によれば、アミロイドーシスは全身諸臓器にアミロイドが沈着する全身性アミロイドーシスと、特定の臓器に限局して沈着を認める限局性アミロイドーシスとに分類される。全身性アミロイドーシスとしては、例えば、肝臓でアミロイドを作り出し、それが全身の器官に沈着して障害を起こす家族性アミロイドーシス、心臓および手関節等の大関節にアミロイドが沈着する老人性ＴＴＲアミロイドーシス、長期透析患者の骨、関節等に発症する透析アミロイドーシス、慢性関節リウマチ等の慢性の炎症性疾患に続発する急性期蛋白であるｓｅｒｕｍ ａｍｙｌｏｉｄ Ａ由来のアミロイドが沈着して発症する反応性ＡＡアミロイドーシス（続発性アミロイドーシス）、免疫グロブリン由来のアミロイドが全身諸臓器に沈着する免疫細胞性アミロイドーシスなどがある。限局性アミロイドーシスとしては、例えば、アミロイドが脳に蓄積するアルツハイマー病、脳血管アミロイドーシス、クロイツフェルト・ヤコブ病などの脳アミロイドーシスの他、ＩＩ型糖尿病に伴う膵島やインスリノーマにアミロイドが沈着したり、心房にアミロイドが沈着する内分泌アミロイドーシス、皮膚にアミロイドが沈着する皮膚アミロイドーシス、皮膚や肺に結節状のアミロイド沈着が生じる限局性結節性アミロイドーシスなどがある。 According to a new classification by the Ministry of Health, Labor and Welfare's Specified Disease Investigation Research Group, amyloidosis is classified into systemic amyloidosis in which amyloid is deposited in various organs and localized amyloidosis in which deposition is restricted to specific organs. Examples of systemic amyloidosis include familial amyloidosis in which amyloid is produced in the liver and deposited in systemic organs to cause damage, senile TTR amyloidosis in which amyloid is deposited in large joints such as the heart and wrist joints, and long-term dialysis Reactive AA amyloidosis (secondary amyloidosis) developed by deposition of amyloid derived from serum amyloid A, which is an acute phase protein secondary to chronic inflammatory diseases such as rheumatoid arthritis, and dialysis amyloidosis that develops in bones and joints of patients ), Immune cell amyloidosis in which immunoglobulin-derived amyloid is deposited in various organs. Examples of localized amyloidosis include, for example, amyloid deposits in pancreatic islets and insulinoma associated with type II diabetes, as well as brain amyloidosis such as Alzheimer's disease, cerebrovascular amyloidosis, and Creutzfeldt-Jakob disease, where amyloid accumulates in the brain. There are endocrine amyloidosis in which amyloid is deposited, cutaneous amyloidosis in which amyloid is deposited in the skin, and localized nodular amyloidosis in which nodular amyloid deposition occurs in the skin and lung.
限局性アミロイドーシスで高齢化に伴い、深刻な社会問題化しつつある疾患にアルツハイマー病がある。アルツハイマー病の病理的特徴として、脳の老人斑形成やタウ蛋白の異常リン酸化および神経原線維変化が挙げられる。このうち老人斑は、アミロイド蛋白（Ａβ蛋白）が凝集・蓄積することによって形成される。このアミロイド蛋白が蓄積することにより、活性酸素が生成されて神経細胞を破壊し、脳の萎縮が起こることが報告されている。しかも、アミロイド蛋白の凝集は、アルツハイマー病の症状が認められる数十年前から起こることが知られている。現在のアルツハイマー病治療薬は、神経賦活作用が主要であり、アミロイド蛋白の除去を目的としたものではないので対処療法に過ぎず、その効果はきわめて限定的である。アミロイド蛋白の凝集抑制、あるいは凝集したアミロイド蛋白の分解除去が可能な薬剤こそが、アルツハイマー病の予防および治療における原因治療薬となりうる。 Alzheimer's disease is a disease that is becoming a serious social problem with the aging of localized amyloidosis. Pathological features of Alzheimer's disease include senile plaque formation, abnormal phosphorylation of tau protein, and neurofibrillary tangles. Among these, senile plaques are formed by aggregation and accumulation of amyloid protein (Aβ protein). It has been reported that accumulation of this amyloid protein generates active oxygen, destroys nerve cells, and causes brain atrophy. Moreover, amyloid protein aggregation is known to occur several decades before the symptoms of Alzheimer's disease are observed. Current therapeutic agents for Alzheimer's disease mainly have neurostimulatory effects and are not intended for removal of amyloid protein, so they are only coping therapy, and their effects are extremely limited. A drug capable of inhibiting aggregation of amyloid protein or degrading and removing aggregated amyloid protein can be a causative therapeutic agent in the prevention and treatment of Alzheimer's disease.
多くの研究室でアミロイドおよびアミロイド―シスの研究が行なわれている。例えば特許文献１には、牡丹皮、桂皮、これらのエキスから選ばれる少なくとも１つを有効成分とする凝集アミロイドβ蛋白の分解剤が記載されている。しかし、未だアミロイド―シスを根治する薬が存在しないのが現状である。 Many laboratories are studying amyloid and amyloidosis. For example, Patent Document 1 describes a degrading agent for aggregated amyloid β protein containing at least one selected from peony skin, cinnamon bark, and extracts thereof as an active ingredient. However, there are no drugs that can cure amyloidosis.
本発明は、アミロイド分解能を有する人工ペプチドを提供し、当該人工ペプチドを用いるアミロイド分解剤、アミロイド検出剤、生理的条件下でアミロイドを分解可能なアミロイドの分解方法、簡便なアミロイド検出方法、アミロイド関連疾患の治療用医薬組成物、アミロイド関連疾患の診断用組成物を提供することを課題とする。 The present invention provides an artificial peptide having amyloid resolution, an amyloid degrading agent using the artificial peptide, an amyloid detecting agent, an amyloid degrading method capable of degrading amyloid under physiological conditions, a simple amyloid detecting method, and amyloid-related It is an object of the present invention to provide a pharmaceutical composition for treating a disease and a composition for diagnosing an amyloid-related disease.
本発明は、上記課題を解決するために、以下の各発明を包含する。
［１］αへリックスを形成するペプチドのＣ末端側に、下記式（Ｉ）に示されるアミノ酸配列からなるペプチドが結合した構造を有することを特徴とするペプチドまたはその塩。
（Ｉ）ＨＸ１Ｘ２ＳＤＸ３Ｘ４（配列番号１）
（Ｘ１およびＸ２は同一または異なって任意のアミノ酸残基を表し、Ｘ３およびＸ４は同一または異なって疎水性アミノ酸を表す。）
［２］Ｘ１およびＸ２が同一または異なってＳ、Ｎ、Ａ、Ｖ、ＩまたはＬである前記［１］に記載のペプチドまたはその塩。
［３］Ｘ３およびＸ４が同一または異なってＡ、Ｖ、Ｉ、Ｌ、Ｗ、ＹまたはＦである前記［１］に記載のペプチドまたはその塩。
［４］Ｘ１およびＸ２が同一または異なってＡ、Ｓ、ＬまたはＮである前記［２］に記載のペプチドまたはその塩。
［５］Ｘ３がＡ、Ｖ、ＩまたはＬである前記［３］に記載のペプチドまたはその塩。
［６］Ｘ４がＷ、ＹまたはＦである前記［４］に記載のペプチドまたはその塩。
［７］式（Ｉ）に示されるアミノ酸配列からなるペプチドのＣ末端がアミドである請求項１に記載のペプチドまたはその塩。
［８］式（Ｉ）が、ＨＡＡＳＤＡＷ（配列番号２）、ＨＡＡＳＤＶＷ（配列番号３）、ＨＡＡＳＤＬＷ（配列番号４）またはＨＡＡＳＤＡＹ（配列番号５）である前記［１］〜［７］のいずれかに記載のペプチドまたはその塩。
［９］αへリックスを形成するペプチドのアミノ酸残基数が５０以下である前記［１］〜［８］請求項１〜５のいずれかに記載のペプチドまたはその塩。
［１０］αへリックスを形成するペプチドのアミノ酸残基数が１４以上である前記［９］に記載のペプチドまたはその塩。
［１１］αへリックスを形成するペプチドのアミノ酸配列が、ＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡ（配列番号６）である前記［１０］に記載のペプチドまたはその塩。
［１２］前記［１］〜［１１］のいずれかに記載のペプチドまたはその塩を含有するアミロイド分解剤。
［１３］前記［１］〜［１１］のいずれかに記載のペプチドまたはその塩を含有するアミロイド検出剤。
［１４］前記［１］〜［１１］のいずれかに記載のペプチドまたはその塩とアミロイドとを接触させる工程を含むことを特徴とするアミロイドの分解方法。
［１５］前記［１］〜［１１］のいずれかに記載のペプチドまたはその塩とアミロイドとを接触させる工程を含むことを特徴とするアミロイドの検出方法。
［１６］前記［１］〜［１１］のいずれかに記載のペプチドまたはその塩を有効成分として含有するアミロイド関連疾患の治療用医薬組成物。
［１７］前記［１］〜［１１］のいずれかに記載のペプチドまたはその塩を含有するアミロイド関連疾患の診断用組成物。The present invention includes the following inventions in order to solve the above problems.
[1] A peptide having a structure in which a peptide having an amino acid sequence represented by the following formula (I) is bound to the C-terminal side of a peptide that forms an α helix, or a salt thereof.
(I) HX 1 X 2 SDX 3 X 4 (SEQ ID NO: 1)
(X 1 and X 2 are the same or different and represent any amino acid residue, and X 3 and X 4 are the same or different and represent a hydrophobic amino acid.)
[2] The peptide or salt thereof according to [1] above, wherein X 1 and X 2 are the same or different and are S, N, A, V, I or L.
[3] The peptide or salt thereof according to [1] above, wherein X 3 and X 4 are the same or different and are A, V, I, L, W, Y, or F.
[4] The peptide or salt thereof according to [2] above, wherein X 1 and X 2 are the same or different and are A, S, L or N.
[5] The peptide or salt thereof according to [3], wherein X 3 is A, V, I or L.
[6] The peptide or salt thereof according to [4], wherein X 4 is W, Y or F.
[7] The peptide or salt thereof according to claim 1, wherein the C-terminal of the peptide consisting of the amino acid sequence represented by formula (I) is an amide.
[8] Any one of [1] to [7], wherein the formula (I) is HAASDAW (SEQ ID NO: 2), HAASV DW (SEQ ID NO: 3), HAASDLW (SEQ ID NO: 4), or HAASDAY (SEQ ID NO: 5). The described peptide or a salt thereof.
[9] The peptide or salt thereof according to any one of [1] to [8], wherein the peptide forming the α helix has 50 or less amino acid residues.
[10] The peptide or salt thereof according to [9], wherein the peptide forming the α helix has 14 or more amino acid residues.
[11] The peptide or salt thereof according to [10] above, wherein the amino acid sequence of the peptide forming the α helix is SAALEAKIAALERKIAALAA (SEQ ID NO: 6).
[12] An amyloid degrading agent containing the peptide or salt thereof according to any one of [1] to [11].
[13] An amyloid detection agent comprising the peptide according to any one of [1] to [11] or a salt thereof.
[14] A method for degrading amyloid, comprising the step of bringing the peptide or salt thereof according to any one of [1] to [11] into contact with amyloid.
[15] A method for detecting amyloid, comprising the step of bringing the peptide or salt thereof according to any one of [1] to [11] into contact with amyloid.
[16] A pharmaceutical composition for treating amyloid-related diseases, comprising the peptide or salt thereof according to any one of [1] to [11] as an active ingredient.
[17] A composition for diagnosing an amyloid-related disease, comprising the peptide according to any one of [1] to [11] or a salt thereof.
本発明によれば、アミロイド分解能を有する人工ペプチドを提供することができる。当該人工ペプチドは、アミロイド分解剤およびアミロイド検出剤として好適に使用することができる。当該ペプチドを用いることにより、アミロイドを生理的条件下で効率よく分解することができ、簡便にアミロイドを検出することができる。また、当該人工ペプチドは、アミロイド関連疾患の治療用医薬組成物およびアミロイド関連疾患の診断用組成物の有効成分として有用である。 According to the present invention, an artificial peptide having amyloid resolution can be provided. The artificial peptide can be suitably used as an amyloid degradation agent and an amyloid detection agent. By using the peptide, amyloid can be efficiently decomposed under physiological conditions, and amyloid can be easily detected. In addition, the artificial peptide is useful as an active ingredient of a pharmaceutical composition for treating amyloid-related diseases and a diagnostic composition for amyloid-related diseases.
〔ペプチド〕
本発明は、αへリックスを形成するペプチドのＣ末端側に、下記式（Ｉ）に示されるアミノ酸配列からなるペプチドが結合した構造を有する人工ペプチドを提供する。
（Ｉ）ＨＸ１Ｘ２ＳＤＸ３Ｘ４（配列番号１）
（Ｘ１およびＸ２は同一または異なって任意のアミノ酸残基を表し、Ｘ３およびＸ４は同一または異なって疎水性アミノ酸を表す。）
このような構造を有するペプチドは、アミロイド分解能を有することが本発明者らにより確認されている。〔peptide〕
The present invention provides an artificial peptide having a structure in which a peptide having an amino acid sequence represented by the following formula (I) is bound to the C-terminal side of a peptide that forms an α helix.
(I) HX 1 X 2 SDX 3 X 4 (SEQ ID NO: 1)
(X 1 and X 2 are the same or different and represent any amino acid residue, and X 3 and X 4 are the same or different and represent a hydrophobic amino acid.)
It has been confirmed by the present inventors that a peptide having such a structure has amyloid resolution.
式（Ｉ）のアミノ酸配列において、Ｘ１およびＸ２は特に限定されず、どのようなアミノ酸でもよいが、Ｓ、Ｎ、Ａ、Ｖ、ＩまたはＬであることが好ましく、Ｓ、Ｎ、ＡまたはＬであることがより好ましい。Ｘ１およびＸ２は同一であってもよく、異なっていてもよい。異なっている場合、いずれか一方がＡであることが好ましい。Ｘ１、Ｘ２の両方がＡであることがより好ましい。In the amino acid sequence of the formula (I), X 1 and X 2 are not particularly limited and may be any amino acid, but are preferably S, N, A, V, I or L, and S, N, A Or it is more preferable that it is L. X 1 and X 2 may be the same or different. When they are different, it is preferable that either one is A. More preferably, both X 1 and X 2 are A.
式（Ｉ）のアミノ酸配列において、Ｘ３およびＸ４は同一または異なって疎水性アミノ酸であることが好ましい。疎水性アミノ酸としては、Ａ、Ｖ、Ｉ、Ｌ、Ｗ、ＹまたはＦが好ましい。Ｘ３は、Ａ、Ｖ、ＩまたはＬであることがより好ましく、Ａ、ＶまたはＬであることがさらに好ましく、ＡまたはＬであることがさらに好ましく、Ａであることがさらに好ましい。Ｘ４は、Ｗ、ＹまたはＦであることがより好ましく、ＷまたはＹであることがさらに好ましく、Ｗであることがさらに好ましい。In the amino acid sequence of formula (I), X 3 and X 4 are preferably the same or different and are hydrophobic amino acids. As the hydrophobic amino acid, A, V, I, L, W, Y or F is preferable. X 3 is more preferably A, V, I or L, more preferably A, V or L, still more preferably A or L, and even more preferably A. X 4 is more preferably W, Y or F, further preferably W or Y, and further preferably W.
式（Ｉ）のアミノ酸配列として、ＨＡＡＳＤＡＷ（配列番号２）、ＨＡＡＳＤＶＷ（配列番号３）、ＨＡＡＳＤＬＷ（配列番号４）またはＨＡＡＳＤＡＹ（配列番号５）が好ましい。なかでもＨＡＡＳＤＡＷ（配列番号２）またはＨＡＡＳＤＡＹ（配列番号５）がより好ましく、ＨＡＡＳＤＡＷ（配列番号２）がさらに好ましい。 As the amino acid sequence of the formula (I), HAASDAW (SEQ ID NO: 2), HAASV DW (SEQ ID NO: 3), HAASDLW (SEQ ID NO: 4) or HAASDAY (SEQ ID NO: 5) is preferable. Of these, HAASDAW (SEQ ID NO: 2) or HAASDAY (SEQ ID NO: 5) is more preferable, and HAASDAW (SEQ ID NO: 2) is more preferable.
αへリックスを形成するペプチドのアミノ酸配列は限定されない。αへリックスを形成するペプチドのアミノ酸配列は、公知の二次構造予測ソフトウェア（例えば、Ｔａｎｇｏ（http://tango.crg.es/tango.jsp）など）を用いることにより容易にデザインすることができる。また、二次構造予測ソフトウェアによりデザインされたアミノ酸配列を有するペプチドがαへリックスを形成していることは、例えばＣＤ（円二色性）測定により確認することができる（実施例参照）。 The amino acid sequence of the peptide forming the α helix is not limited. The amino acid sequence of the peptide forming the α helix can be easily designed by using known secondary structure prediction software (eg, Tango (http://tango.crg.es/tango.jsp)). it can. Moreover, it can confirm that the peptide which has the amino acid sequence designed by the secondary structure prediction software forms alpha helix, for example by CD (circle dichroism) measurement (refer an Example).
αへリックスを形成するペプチドのアミノ酸残基数は特に限定されないが、５０以下であることが好ましく、４２以下であることがより好ましく、３５以下であることがさらに好ましく、２８以下であることがさらに好ましい。下限は特に限定されないが、１４以上であることが好ましく、２１以上であることがより好ましい。 The number of amino acid residues of the peptide forming the α helix is not particularly limited, but is preferably 50 or less, more preferably 42 or less, further preferably 35 or less, and preferably 28 or less. Further preferred. Although a minimum is not specifically limited, It is preferable that it is 14 or more, and it is more preferable that it is 21 or more.
αへリックスを形成するペプチドの好ましいアミノ酸配列として、例えばＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡ（配列番号６）が挙げられる。したがって、本発明のペプチドとしては、以下の（ａ）〜（ｄ）のアミノ酸配列からなるペプチドが好適である。
（ａ）ＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡＨＡＡＳＤＡＷ（配列番号７）
（ｂ）ＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡＨＡＡＳＤＶＷ（配列番号８）
（ｃ）ＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡＨＡＡＳＤＬＷ（配列番号９）
（ｄ）ＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡＨＡＡＳＤＡＹ（配列番号１０）A preferred amino acid sequence of the peptide forming the α helix includes, for example, SAALEAKIAALERKIAALAA (SEQ ID NO: 6). Accordingly, the peptide of the present invention is preferably a peptide consisting of the following amino acid sequences (a) to (d).
(A) SAALEKIAALERKIAALAAAHAASDAW (SEQ ID NO: 7)
(B) SAALEAKIAALELERKIAALAAAAHASDVW (SEQ ID NO: 8)
(C) SAALEAKIAALELERKIAALAAAHAASDLW (SEQ ID NO: 9)
(D) SAALEAKIAALERKIAAALAAAHAASDAY (SEQ ID NO: 10)
本発明のペプチドは、公知の一般的なペプチド合成のプロトコールに従って、固相合成法（Ｆｍｏｃ法、Ｂｏｃ法）または液相合成法により製造することができる。また、本発明のペプチドをコードするＤＮＡを含有する発現ベクターを導入した形質転換体を用いる方法や、ｉｎ ｖｉｔｒｏ転写・翻訳系を用いる方法により製造することができる。
得られたペプチドがアミロイド分解能を有することは、アミロイドとペプチドを接触させ、アミロイド分解物の生成、またはアミロイドの消失を測定することで確認することができる。例えば、質量分析やチオフラビンＴアッセイなどを用いることができる。The peptide of the present invention can be produced by a solid phase synthesis method (Fmoc method, Boc method) or a liquid phase synthesis method according to a known general peptide synthesis protocol. Further, it can be produced by a method using a transformant into which an expression vector containing a DNA encoding the peptide of the present invention has been introduced, or a method using an in vitro transcription / translation system.
It can be confirmed that the obtained peptide has amyloid resolution by contacting amyloid with the peptide and measuring the production of amyloid degradation products or the disappearance of amyloid. For example, mass spectrometry or thioflavin T assay can be used.
本発明のペプチドは、Ｃ末端がカルボキシル基（−ＣＯＯＨ）、カルボキシレート(−ＣＯＯ−)、アミド（−ＣＯＮＨ２）またはエステル（−ＣＯＯＲ）の何れであってもよい。好ましくはアミド（−ＣＯＮＨ２）である。エステルにおけるＲとしては、例えば、メチル、エチル、ｎ−プロピル、イソプロピルもしくはｎ−ブチルなどのＣ１−６アルキル基、例えば、シクロペンチル、シクロヘキシルなどのＣ３−８シクロアルキル基、例えば、フェニル、α−ナフチルなどのＣ６−１２アリール基、例えば、ベンジル、フェネチルなどのフェニル−Ｃ１−２アルキル基もしくはα−ナフチルメチルなどのα−ナフチル−Ｃ１−２アルキル基などのＣ７−１４アラルキル基のほか、経口用エステルとして汎用されるピバロイルオキシメチル基などが挙げられる。本発明のペプチドがＣ末端以外にカルボキシル基またはカルボキシレートを有している場合、それらの基がアミド化またはエステル化されているものも本発明のペプチドに含まれる。In the peptide of the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO − ), an amide (—CONH 2 ), or an ester (—COOR). Preferably an amide (-CONH 2). As R in the ester, for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl, α - C 6-12 aryl group such as naphthyl, for example, benzyl, C 7 - 14 aralkyl such as α- naphthyl -C 1-2 alkyl group such as a phenyl -C 1-2 alkyl or α- naphthylmethyl such phenethyl In addition to the group, a pivaloyloxymethyl group, which is widely used as an oral ester, can be mentioned. When the peptide of the present invention has a carboxyl group or a carboxylate other than the C-terminus, those in which these groups are amidated or esterified are also included in the peptide of the present invention.
本発明のペプチドを構成するアミノ酸は、側鎖が任意の置換基で修飾されたものでもよい。置換基は特に限定されないが、例えば、フッ素原子、塩素原子、シアノ基、水酸基、ニトロ基、アルキル基、シクロアルキル基、アルコキシ基、アミノ基などが挙げられる。 さらに、本発明のペプチドには、Ｎ末端のセリン残基のアミノ基が保護基（例えば、ホルミル基、アセチルなどのＣ２−６アルカノイル基などのＣ１−６アシル基など）で保護されているもの、分子内のアミノ酸の側鎖上の置換基（例えば、−ＯＨ、−ＳＨ、アミノ基、イミダゾール基、インドール基、グアニジノ基など）が適当な保護基（例えば、ホルミル基、アセチルなどのＣ２−６アルカノイル基などのＣ１−６アシル基など）で保護されているものも含まれる。The amino acid constituting the peptide of the present invention may be one in which the side chain is modified with an arbitrary substituent. Although a substituent is not specifically limited, For example, a fluorine atom, a chlorine atom, a cyano group, a hydroxyl group, a nitro group, an alkyl group, a cycloalkyl group, an alkoxy group, an amino group etc. are mentioned. Furthermore, in the peptide of the present invention, the amino group of the N-terminal serine residue is protected with a protecting group (for example, a C 1-6 acyl group such as a C 2-6 alkanoyl group such as formyl group or acetyl). A substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) is a suitable protecting group (eg, formyl group, acetyl, etc.) And those protected with a C 1-6 acyl group such as a C 2-6 alkanoyl group).
本発明のペプチドは塩を形成していてもよく、またそれらの水和物であってもよい。塩としては薬学的に許容される塩が好ましい。具体定期には、例えば、塩酸、硫酸、燐酸、乳酸、酒石酸、マレイン酸、フマル酸、シュウ酸、リンゴ酸、クエン酸、オレイン酸、パルミチン酸などの酸との塩；ナトリウム、カリウム、カルシウムなどのアルカリ金属もしくはアルカリ土類金属の、またはアルミニウムの水酸化物または炭酸塩との塩；トリエチルアミン、ベンジルアミン、ジエタノールアミン、ｔ−ブチルアミン、ジシクロヘキシルアミン、アルギニンなどとの塩などが挙げられる。 The peptides of the present invention may form a salt or a hydrate thereof. The salt is preferably a pharmaceutically acceptable salt. Specifically, for example, 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, palmitic acid; sodium, potassium, calcium, etc. And salts with alkali metal or alkaline earth metal or aluminum hydroxide or carbonate; and salts with triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine, arginine and the like.
本発明のペプチドは、元のペプチドの特性が保持される限り、Ｄ−アミノ酸を含んでもよく、非天然アミノ酸を含んでもよい。また、本発明のペプチドは、元のペプチドの特性が保持される限り、ペプチドに他の物質を連結してもよい。ペプチドに連結可能な他の物質としては、例えば、脂質、糖または糖鎖、アセチル基、天然または合成のポリマー等が挙げられる。また、本発明のペプチドは、元のペプチドの特性が保持される限り、ペプチドに、糖鎖付加、側鎖酸化、リン酸化等の修飾を行ってもよい。 The peptide of the present invention may contain D-amino acids or non-natural amino acids as long as the properties of the original peptide are retained. Moreover, as long as the peptide of this invention has the characteristic of the original peptide, you may connect another substance to a peptide. Examples of other substances that can be linked to peptides include lipids, sugars or sugar chains, acetyl groups, natural or synthetic polymers, and the like. In addition, the peptide of the present invention may be subjected to modifications such as glycosylation, side chain oxidation, and phosphorylation as long as the characteristics of the original peptide are maintained.
〔アミロイド分解剤およびアミロイド分解方法〕
本発明のペプチドはアミロイド分解能を有するため、アミロイド分解剤として好適に用いることができる。本発明のペプチドをアミロイドと接触させることにより、アミロイド分解することができる。反応温度は、本発明のペプチドがαへリックス構造を保持できる温度であればよく、約１０℃〜約４５℃が好ましく、約２５℃〜約３７℃がより好ましい。反応時間は、用いるペプチドおよび分解対象アミロイドに応じて適宜最適な時間を選択すればよい。[Amyloid degradation agent and amyloid degradation method]
Since the peptide of the present invention has amyloid resolution, it can be suitably used as an amyloid degrading agent. By contacting the peptide of the present invention with amyloid, amyloid degradation can be achieved. The reaction temperature may be any temperature at which the peptide of the present invention can maintain the α-helix structure, preferably about 10 ° C. to about 45 ° C., more preferably about 25 ° C. to about 37 ° C. The reaction time may be appropriately selected depending on the peptide to be used and the amyloid to be decomposed.
〔アミロイド検出剤およびアミロイド検出方法〕
本発明のペプチドはアミロイド分解能を有するため、アミロイド検出剤として好適に用いることができる。アミロイドが存在するか否かを調べたい試料と本発明のペプチドを接触させることにより、アミロイドが存在する場合はアミロイド由来の分解物が生成するので、当該分解物の有無を確認することにより、試料中のアミロイドの存在を検出することができる。アミロイド由来の分解物の確認は、例えば、質量分析法、電気泳動法、クロマトグラフィー法などで行うことができる。好ましくは質量分析法である。質量分析法は、分解前および分解後のすべての分子の分子量を一度に精密に確定できるので、最も優れた手法である[Amyloid detection agent and amyloid detection method]
Since the peptide of the present invention has amyloid resolution, it can be suitably used as an amyloid detection agent. By contacting the sample of the present invention with the peptide of the present invention to determine whether amyloid is present or not, amyloid-derived degradation products are produced when amyloid is present. The presence of amyloid in it can be detected. Confirmation of the degradation product derived from amyloid can be performed by, for example, mass spectrometry, electrophoresis, chromatography, or the like. Mass spectrometry is preferred. Mass spectrometry is the best method because it can accurately determine the molecular weight of all molecules before and after degradation at once.
〔アミロイド関連疾患治療用医薬組成物〕
本発明のペプチドはアミロイド分解能を有するため、当該ペプチドまたはその塩はアミロイド関連疾患治療薬の有効成分として有用である。治療対象であるアミロイド関連疾患としては、免疫細胞性アミロイドーシス、反応性ＡＡアミロイドーシス（続発性アミロイドーシス）、家族性アミロイドーシス（遺伝性アミロイドーシス）、透析アミロイドーシス、老人性全身性アミロイドーシス等の全身性アミロイドーシス、アルツハイマー病、ダウン症候群、脳血管アミロイドーシス、遺伝性脳アミロイド・アンギオパチー、英国型家族性痴呆、クロイツフェルト・ヤコブ病、甲状腺髄様癌に伴うアミロイド、ＩＩ型糖尿病、インスリノーマ、限局性心房アミロイドーシス、皮膚アミロイドーシス、角膜アミロイド―シス、限局性結節性アミロイドーシス等の限局性アミロイドーシスが挙げられる。[Pharmaceutical composition for treatment of amyloid-related diseases]
Since the peptide of the present invention has amyloid resolution, the peptide or a salt thereof is useful as an active ingredient of a therapeutic drug for amyloid-related diseases. Examples of amyloid-related diseases to be treated include immune cell amyloidosis, reactive AA amyloidosis (secondary amyloidosis), familial amyloidosis (hereditary amyloidosis), dialysis amyloidosis, senile systemic amyloidosis, etc., Alzheimer's disease Down syndrome, cerebrovascular amyloidosis, hereditary cerebral amyloid angiopathy, British familial dementia, Creutzfeldt-Jakob disease, amyloid associated with medullary thyroid cancer, type II diabetes, insulinoma, focal atrial amyloidosis, cutaneous amyloidosis, cornea Examples include localized amyloidosis such as amyloidosis and localized nodular amyloidosis.
本発明の医薬組成物は、本発明のペプチド、その薬学的に許容される塩またはそれらの水和物を有効成分とし、薬学的に許容される担体または添加剤を適宜配合して製剤化することができる。具体的には錠剤、被覆錠剤、丸剤、散剤、顆粒剤、カプセル剤、液剤、懸濁剤、乳剤等の経口剤；注射剤、輸液、坐剤、軟膏、パッチ剤等の非経口剤とすることができる。担体または添加剤の配合割合については、医薬品分野において通常採用されている範囲に基づいて適宜設定すればよい。配合できる担体または添加剤は特に制限されないが、例えば、水、生理食塩水、その他の水性溶媒、水性または油性基剤等の各種担体；賦形剤、結合剤、ｐＨ調整剤、崩壊剤、吸収促進剤、滑沢剤、着色剤、矯味剤、香料等の各種添加剤が挙げられる。 The pharmaceutical composition of the present invention is formulated by using the peptide of the present invention, a pharmaceutically acceptable salt thereof or a hydrate thereof as an active ingredient, and appropriately blending a pharmaceutically acceptable carrier or additive. be able to. Specifically, oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions; parenterals such as injections, infusions, suppositories, ointments, patches, etc. can do. What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ | adopted in the pharmaceutical field | area. Carriers or additives that can be blended are not particularly limited. For example, various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorption Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.
錠剤、カプセル剤などに混和することができる添加剤としては、例えば、ゼラチン、コーンスターチ、トラガント、アラビアゴムのような結合剤、結晶性セルロースのような賦形剤、コーンスターチ、ゼラチン、アルギン酸などのような膨化剤、ステアリン酸マグネシウムのような潤滑剤、ショ糖、乳糖またはサッカリンのような甘味剤、ペパーミント、アカモノ油またはチェリーのような香味剤などが用いられる。調剤単位形態がカプセルである場合には、上記タイプの材料にさらに油脂のような液状担体を含有することができる。注射のための無菌組成物は通常の製剤業務（例えば有効成分を注射用水、天然植物油等の溶媒に溶解または懸濁させる等）に従って調製することができる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液（例えば、Ｄ−ソルビトール、Ｄ−マンニトール、塩化ナトリウムなど）などが用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン性界面活性剤（例、ポリソルベート８０ＴＭ、ＨＣＯ−５０）などと併用してもよい。油性液としては、例えば、ゴマ油、大豆油などが用いられ、溶解補助剤である安息香酸ベンジル、ベンジルアルコールなどと併用してもよい。また、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤（例えば、ベンジルアルコール、フェノールなど）、酸化防止剤などと配合してもよい。 Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be prepared according to normal pharmaceutical practice (for example, dissolving or suspending an active ingredient in a solvent such as water for injection or natural vegetable oil). As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80TM, HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage You may mix | blend with an agent (for example, benzyl alcohol, phenol, etc.), antioxidant, etc.
このようにして得られる製剤は安全で低毒性であるので、例えば、ヒトや他の哺乳動物（例えば、ラット、マウス、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど）に対して投与することができる。
投与量は、投与対象、対象臓器、症状、投与方法などにより差異はあるが、経口投与の場合、一般的に例えば、体重約６０ｋｇのヒトにおいては、１日当たり約０.１〜１００ｍｇ、好ましくは約１．０〜５０ｍｇ、より好ましくは約１．０〜２０ｍｇである。非経口的に投与する場合は、その１回投与量は投与対象、対象臓器、症状、投与方法などによっても異なるが、例えば注射剤では、通常例えば体重約６０ｋｇのヒトにおいては、１日当たり約０．０１〜３０ｍｇ程度、好ましくは約０．１〜２０ｍｇ程度、より好ましくは約０．１〜１０ｍｇ程度を静脈注射により投与するのが好都合である。１日当たりの総投与量は、単一投与量であっても分割投与量であってもよい。Since the preparation thus obtained is safe and has low toxicity, for example, it is administered to humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) can do.
The dose varies depending on the administration subject, target organ, symptom, administration method and the like, but in the case of oral administration, for example, generally about 0.1 to 100 mg per day in a human body weight of about 60 kg, preferably About 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, and the like. For example, in the case of an injection, it is usually about 0 per day for a human having a body weight of about 60 kg. It is convenient to administer about 0.1 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. The total daily dose may be a single dose or divided doses.
本発明には以下の各発明も含まれる。
哺乳動物に対し上記本発明のペプチド、その薬学的に許容される塩またはそれらの水和物の有効量を投与することを特徴とするアミロイド関連疾患の治療方法。
アミロイド関連疾患治療用医薬を製造するための上記本発明のペプチド、その薬学的に許容される塩またはそれらの水和物の使用。
アミロイド関連疾患の治療に使用するための上記本発明のペプチド、その薬学的に許容される塩またはそれらの水和物。The present invention includes the following inventions.
A method for treating an amyloid-related disease, which comprises administering an effective amount of the peptide of the present invention, a pharmaceutically acceptable salt thereof or a hydrate thereof to a mammal.
Use of the peptide of the present invention, a pharmaceutically acceptable salt thereof or a hydrate thereof for producing a medicament for treating amyloid-related diseases.
The peptide of the present invention, a pharmaceutically acceptable salt thereof or a hydrate thereof for use in the treatment of amyloid-related diseases.
〔アミロイド関連疾患診断用組成物〕
本発明のペプチドはアミロイド分解能を有するため、当該ペプチドまたはその塩はアミロイド関連疾患診断用組成物の成分として有用である。本発明のアミロイド関連疾患診断用組成物は、生体から摘出した臓器、組織、血液中のアミロイドの有無を検出することによりアミロイド関連疾患の診断を行うことができる。具体的には、アミロイドの存在が疑われる臓器、組織、血液等を採取し、これに本発明のペプチドを接触させ、アミロイドが存在する場合はアミロイド由来の分解物が生成するので、当該分解物の有無を確認することにより、アミロイド関連疾患であるか否かを診断することができる。アミロイド由来の分解物の確認は、例えば、質量分析法、電気泳動法、クロマトグラフィー法などで行うことができる。[Amyloid-related disease diagnostic composition]
Since the peptide of the present invention has amyloid resolution, the peptide or a salt thereof is useful as a component of a composition for diagnosing amyloid-related diseases. The composition for diagnosing amyloid-related diseases of the present invention can diagnose amyloid-related diseases by detecting the presence or absence of amyloid in organs, tissues, and blood extracted from a living body. Specifically, organs, tissues, blood, etc. suspected of the presence of amyloid are collected and brought into contact with the peptide of the present invention. When amyloid is present, a degradation product derived from amyloid is generated. By confirming the presence or absence, it is possible to diagnose whether or not the disease is an amyloid-related disease. Confirmation of the degradation product derived from amyloid can be performed by, for example, mass spectrometry, electrophoresis, chromatography, or the like.
本発明には以下の各発明も含まれる。
本発明のペプチドまたはその塩と生体由来試料とを接触させる工程を含むことを特徴とするアミロイド関連疾患の診断方法。
アミロイド関連疾患診断用組成物を製造するための上記本発明のペプチドまたはその塩の使用。
アミロイド関連疾患の診断に使用するための上記本発明のペプチドまたはその塩。The present invention includes the following inventions.
A method for diagnosing an amyloid-related disease, comprising the step of bringing the peptide of the present invention or a salt thereof into contact with a biological sample.
Use of the peptide of the present invention or a salt thereof for producing a composition for diagnosing amyloid-related diseases.
The peptide of the present invention or a salt thereof for use in diagnosis of amyloid-related diseases.
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.
〔実施例１：人工ペプチドの合成および構造確認〕
（１）ペプチドの合成
５種類のペプチド（ｍｅ５〜ｍｅ９）を、ペプチド合成機（Pioneer, Peptide synthesis System; Applied Biosystems社製）を用いて、Ｆ−ｍｏｃ固相法により合成した。ｍｅ５〜ｍｅ９のアミノ酸配列は以下のとおりであり、いずれもＣ末端はアミドである。
ｍｅ５：ＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡＨＡＡＳＤＡＷ（配列番号７）
ｍｅ６：ＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡＨＡＡＳＤＶＷ（配列番号８）
ｍｅ７：ＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡＨＡＡＳＤＬＷ（配列番号９）
ｍｅ８：ＳＡＡＬＥＡＫＩＡＡＬＥＲＫＩＡＡＬＡＡＡＨＡＡＳＤＡＹ（配列番号１０）
ｍｅ９：ＳＡＡＬＥＡＲＧＡＡＬＥＲＲＩＡＡＬＡＡＡＨＡＡＳＤＡＷ（配列番号１１）[Example 1: Synthesis and structure confirmation of artificial peptide]
(1) Peptide synthesis Five types of peptides (me5 to me9) were synthesized by a F-moc solid phase method using a peptide synthesizer (Pioneer, Peptide synthesis System; Applied Biosystems). The amino acid sequences of me5 to me9 are as follows, and in any case, the C-terminus is an amide.
me5: SAALEAKIAALERKIAAALAAAHAASDAW (SEQ ID NO: 7)
me6: SAALEAKIAALERKIAALAAAAHASDVW (SEQ ID NO: 8)
me7: SAALEAKIAALERKIAAALAAAHAASDLW (SEQ ID NO: 9)
me8: SAALEAKIAALERKIAAALAAAHAASDAY (SEQ ID NO: 10)
me9: SAALEARGAALERRIAAALAAAHAASDAW (SEQ ID NO: 11)
（２）ＣＤ（円二色性）測定
合成した各ペプチドをＴｒｉｓバッファー（ｐＨ７．３）にそれぞれ溶解して、１００μＭのペプチド溶液を調製し、ＣＤを測定した。測定には、Ｊａｓｃｏ Ｊ−７２０ ｓｐｅｃｔｒｏｐｏｌａｒｉｍｅｔｅｒ（日本分光社製）を使用し、光路長０．１ｍｍのタイコセルを用いて２００μＬのペプチド溶液について測定した。測定条件は、温度２５℃、走査波長２５０ｎｍ〜１９０ｎｍ、データ間隔０．２ｎｍ、走査速度１００ｎｍ／ｍｉｎ、レスポンス２ｓｅｃ、バンド幅１ｎｍ、感度１０ｍｄｅｇ、積算回数８回とした。(2) CD (Circular Dichroism) Measurement Each synthesized peptide was dissolved in Tris buffer (pH 7.3) to prepare a 100 μM peptide solution, and CD was measured. For the measurement, Jasco J-720 spectropolarimeter (manufactured by JASCO Corporation) was used, and a 200 μL peptide solution was measured using a Tyco cell having an optical path length of 0.1 mm. The measurement conditions were a temperature of 25 ° C., a scanning wavelength of 250 nm to 190 nm, a data interval of 0.2 nm, a scanning speed of 100 nm / min, a response of 2 sec, a bandwidth of 1 nm, a sensitivity of 10 mdeg, and an integration count of 8 times.
（３）結果
結果を図１に示した。図１から、ｍｅ５〜ｍｅ８はαへリックス構造、ｍｅ９はランダムコイル構造であることが明らかとなった。(3) Results The results are shown in FIG. From FIG. 1, it became clear that me5 to me8 have an α helix structure and me9 has a random coil structure.
〔実施例２：人工ペプチドによるアミロイド分解能の検討（その１）〕
（１）βラクトグロブリンのアミロイド線維化
βラクトグロブリンを２４ｍｇ／１．２ｍＬの濃度で純水に溶解させ、塩酸によってｐＨ２．０に調整し、８０℃で２４時間インキュベートして、アミロイド線維を形成させた。線維化の確認はＡＦＭ（原子間力顕微鏡）観察およびチオフラビンＴによる蛍光測定によって行った。
（２）使用ペプチド
実施例１で合成したｍｅ５を使用した。対照として、以下に示すアミノ酸配列からなるｍｅ４を実施例１と同じペプチド合成機を用いてＦ−ｍｏｃ固相法により合成し（Ｃ末端はアミド）、使用した。データを示していないが、ＣＤ測定によりｍｅ４がαへリックス構造であることを確認した。ｍｅ４はｍｅ５と異なり、プロテアーゼの活性中心（ＳＨＤ）がαへリックスを形成するペプチドのＮ末端側に設計されている。
ｍｅ４：ＨＮＬＳＤＡＷＩＡＡＬＥＡＫＩＡＡＬＥＡＫＩＡＡＬＡＲＲ（配列番号１２）
（３）チオフラビンＴアッセイ
２０ｍＭのＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．３）にペプチドを溶解し、１００μＭのペプチド溶液を調製した。このペプチド溶液にチオフラビンＴを０．０４７ｍｇ／ｍＬとなるように加え、さらにアミロイド線維化βラクトグロブリンを約２００μＭとなるように添加した。光路長１ｃｍの蛍光測定用セルに、ペプチド混合溶液を１ｍＬ添加し、４４２ｎｍの励起光で４５０ｎｍ−６００ｎｍまで経時変化を追って測定した。反応温度は２５℃または３７℃で行った。蛍光測定にはＦＰ−６５００（JASCO社製）を用いた。[Example 2: Examination of amyloid resolution by artificial peptide (part 1)]
(1) β-lactoglobulin amyloid fibrils β-lactoglobulin is dissolved in pure water at a concentration of 24 mg / 1.2 mL, adjusted to pH 2.0 with hydrochloric acid, and incubated at 80 ° C. for 24 hours to form amyloid fibrils I let you. Fibrosis was confirmed by AFM (atomic force microscope) observation and fluorescence measurement with thioflavin T.
(2) Peptide used The me5 synthesized in Example 1 was used. As a control, me4 having the amino acid sequence shown below was synthesized by the F-moc solid phase method using the same peptide synthesizer as in Example 1 (C-terminal is amide) and used. Although data is not shown, it was confirmed by CD measurement that me4 has an α helix structure. Unlike me5, me4 is designed on the N-terminal side of the peptide in which the active center (SHD) of the protease forms an α helix.
me4: HNLSDAWIAALEAKIAALEAKIAALAR (SEQ ID NO: 12)
(3) Thioflavin T assay The peptide was dissolved in 20 mM Tris-HCl buffer (pH 7.3) to prepare a 100 µM peptide solution. To this peptide solution, thioflavin T was added to a concentration of 0.047 mg / mL, and amyloid fibrillated β-lactoglobulin was further added to a concentration of about 200 μM. 1 mL of the peptide mixed solution was added to a fluorescence measurement cell having an optical path length of 1 cm, and the time-dependent change was measured from 450 nm to 600 nm with 442 nm excitation light. The reaction temperature was 25 ° C. or 37 ° C. FP-6500 (manufactured by JASCO) was used for fluorescence measurement.
（４）結果
ｍｅ５の反応温度２５℃の結果を図２に示した。図２から明らかなように、チオフラビンＴの蛍光は４９０ｎｍにピークが見られるが、反応開始後５分の時点で、ほぼピークが消失した。
ｍｅ５の反応温度３７℃の結果を図３に示した。図３から、反応温度が２５℃の場合（図２）より分解速度は遅いが、ヒトの体温に相当する３７℃でもアミロイドを分解可能であることが明らかになった。
ｍｅ４の反応温度２５℃の結果を図４に示した。図４から、４９０ｎｍのピークは時間の経過に伴ってむしろ増大しており、アミロイドは全く分解されなかった。この結果から、プロテアーゼの活性中心（ＳＨＤ）の位置が重要であることが明らかになった。(4) Results The results for the reaction temperature of me5 at 25 ° C. are shown in FIG. As is clear from FIG. 2, the fluorescence of thioflavin T has a peak at 490 nm, but almost disappeared at 5 minutes after the start of the reaction.
The result of the reaction temperature of me5 at 37 ° C. is shown in FIG. From FIG. 3, it was clarified that amyloid can be decomposed even at 37 ° C. corresponding to human body temperature, although the decomposition rate is slower than when the reaction temperature is 25 ° C. (FIG. 2).
The result of the reaction temperature of me4 at 25 ° C. is shown in FIG. From FIG. 4, the peak at 490 nm increased rather with time, and no amyloid was degraded. This result revealed that the position of the active center (SHD) of the protease is important.
〔実施例３：人工ペプチドによるアミロイド分解能の検討（その２）〕
（１）ＡＦＭ（原子間力顕微鏡）による観察
ペプチドとして、実施例１で合成したｍｅ５〜９の５種類を使用した。実施例２で調製した１００μＭのペプチド溶液にアミロイド線維化βラクトグロブリンを２００μＭとなるように添加し、２５℃に保温して経時的にサンプリングした。ｍｅ５のみ３７℃でも行った。対照としてペプチドが存在しない２０ｍＭのＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．３）にアミロイド線維化βラクトグロブリンを２００μＭとなるように添加し、２５℃に保温して経時的にサンプリングした。サンプルをマイカ上に載せ１分後にミリＱ水で３回洗浄後乾燥させたものを観察に供した。ＡＦＭにはＳＰＭ−９５００Ｊ２（島津製作所）を使用し、カンチレバーはＮＣＨＲ−２０（NANOWORLD INNOVATION TECHNOLOGY）を使用した。[Example 3: Examination of amyloid resolution by artificial peptide (part 2)]
(1) Observation with AFM (Atomic Force Microscope) Five types of me5-9 synthesized in Example 1 were used as peptides. Amyloid fibrillated β-lactoglobulin was added to the 100 μM peptide solution prepared in Example 2 to a concentration of 200 μM, and the mixture was kept at 25 ° C. and sampled over time. Only me5 was performed at 37 ° C. As a control, amyloid fibrillated β-lactoglobulin was added to a 20 mM Tris-HCl buffer (pH 7.3) containing no peptide so as to have a concentration of 200 μM, and kept at 25 ° C. and sampled over time. The sample was placed on mica, washed one minute later with milli-Q water and dried for observation. SPM-9500J2 (Shimadzu Corporation) was used for the AFM, and NCHR-20 (NANOWORLD INNOVATION TECHNOLOGY) was used for the cantilever.
（２）結果
ｍｅ５の２５℃の結果を図５に示した。アミロイドは６０分以内で相当程度分解しており、１日後にはほとんど観察されなくなった。
ｍｅ５の３７℃の結果を図６に示した。アミロイドは１８０分後には相当程度分解していた。
ｍｅ６（２５℃）の結果を図７に、ｍｅ８（２５℃）の結果を図８にそれぞれ示した。いずれの人工ペプチドを用いた場合も、アミロイドは１日後には相当程度分解していた。
ｍｅ８（２５℃）の結果を図９に示した。アミロイドは６時間後には相当程度分解していた。
ｍｅ９（２５℃）の結果を図１０に示した。アミロイドは６時間後でも分解されなかった。したがって、αへリックス構造を有しない人工ペプチドはアミロイド分解能がないことが示唆された。
人工ペプチド非存在下（２５℃）の結果を図１１に示した。１８０分後でもアミロイドは分解しておらず、本実験条件ではアミロイドが自己分解しないことが示された。(2) Results The results of me5 at 25 ° C. are shown in FIG. Amyloid was considerably degraded within 60 minutes, and was hardly observed after 1 day.
The result of me5 at 37 ° C. is shown in FIG. The amyloid was considerably degraded after 180 minutes.
The result of me6 (25 ° C.) is shown in FIG. 7, and the result of me8 (25 ° C.) is shown in FIG. When any artificial peptide was used, amyloid was considerably degraded after 1 day.
The result of me8 (25 ° C.) is shown in FIG. The amyloid was degraded to a considerable extent after 6 hours.
The result of me9 (25 ° C.) is shown in FIG. Amyloid was not degraded even after 6 hours. Therefore, it was suggested that the artificial peptide having no α-helix structure has no amyloid resolution.
The results in the absence of artificial peptide (25 ° C.) are shown in FIG. Even after 180 minutes, amyloid was not decomposed, and it was shown that amyloid does not self-decompose under these experimental conditions.
〔実施例４：人工ペプチドによるアミロイド分解能の検討（その３）〕
（１）インスリンのアミロイド線維化
インスリンを１．０ｍｇ／ｍＬの濃度で２５ｍＭ ＨＣｌ（ｐＨ１．６）１００ｍＭ ＮａＣｌに溶解し、６５℃で２４時間インキュベートして、アミロイド線維を形成させた。線維化の確認はＡＦＭ観察およびチオフラビンＴによる蛍光測定によって行った。[Example 4: Examination of amyloid resolution by artificial peptide (part 3)]
(1) Amyloid fibrillation of insulin Insulin was dissolved in 25 mM HCl (pH 1.6) 100 mM NaCl at a concentration of 1.0 mg / mL and incubated at 65 ° C. for 24 hours to form amyloid fibrils. Fibrosis was confirmed by AFM observation and fluorescence measurement with thioflavin T.
（２）チオフラビンＴアッセイ
ペプチドには実施例１で合成したｍｅ５を使用した。２０ｍＭのＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．３）にペプチドを溶解し、１００μＭのペプチド溶液を調製した。このペプチド溶液にチオフラビンＴを０．０４７ｍｇ／ｍＬとなるように加え、さらにアミロイド線維化インスリンを１００μＭとなるように添加した。光路長１ｃｍの蛍光測定用セルに、ペプチド混合溶液を１ｍＬ添加し、４４２ｎｍの励起光で４５０ｎｍ−６００ｎｍまで経時変化を追って測定した。蛍光測定にはＦＰ−６５００（JASCO社製）を用いた。(2) Thioflavin T assay As the peptide, me5 synthesized in Example 1 was used. The peptide was dissolved in 20 mM Tris-HCl buffer (pH 7.3) to prepare a 100 μM peptide solution. To this peptide solution, thioflavin T was added to a concentration of 0.047 mg / mL, and amyloid fibrillated insulin was further added to a concentration of 100 μM. 1 mL of the peptide mixed solution was added to a fluorescence measurement cell having an optical path length of 1 cm, and the time-dependent change was measured from 450 nm to 600 nm with 442 nm excitation light. FP-6500 (manufactured by JASCO) was used for fluorescence measurement.
（３）ＡＦＭによる観察
２０μＭのペプチド溶液にアミロイド線維化インスリンを２０μＭとなるように添加し、２５℃に保温して経時的にサンプリングした。サンプルをマイカ上に載せ１分後にミリＱ水で３回洗浄後乾燥させたものを観察に供した。ＡＦＭにはＳＰＭ−９５００Ｊ２（島津製作所）を使用し、カンチレバーはＮＣＨＲ−２０（NANOWORLD INNOVATION TECHNOLOGY）を使用した。(3) Observation by AFM Amyloid fibrillated insulin was added to a 20 μM peptide solution so as to be 20 μM, and the mixture was kept at 25 ° C. and sampled over time. The sample was placed on mica, washed one minute later with milli-Q water and dried for observation. SPM-9500J2 (Shimadzu Corporation) was used for the AFM, and NCHR-20 (NANOWORLD INNOVATION TECHNOLOGY) was used for the cantilever.
（４）結果
チオフラビンＴアッセイの結果を図１２に示した。反応開始後約９０分でチオフラビンＴの蛍光ピークは半減した。
ＡＦＭによる観察結果を図１３に示した。アミロイドは１２０分以内で相当程度分解しており、３日後にはほとんど観察されなくなった。(4) Results The results of the thioflavin T assay are shown in FIG. About 90 minutes after the start of the reaction, the fluorescence peak of thioflavin T was halved.
The observation result by AFM is shown in FIG. Amyloid was degraded to a considerable extent within 120 minutes and was hardly observed after 3 days.
〔実施例５：人工ペプチドによるアミロイド分解能の検討（その４）〕
（１）アミロイドβ４０の線維化
アミロイドβ４０（ペプチド研究所より購入または神戸大学理学研究科茶谷研究室より提供）を２０ｍＭとなるように、２００ｍＭ ＮａＣｌ含有５０ｍＭリン酸緩衝液（ｐＨ７．５）中に溶解し、３７℃の条件下で４８時間インキュベートして、アミロイド線維を形成させた。線維化の確認はＡＦＭ観察およびチオフラビンＴによる蛍光測定によって行った。[Example 5: Examination of amyloid resolution by artificial peptide (part 4)]
(1) Fibrosis of amyloid β40 Amyloid β40 (purchased from Peptide Laboratories or provided from Kobe University's Graduate School of Science, Chatani Laboratory) in 50 mM phosphate buffer (pH 7.5) containing 200 mM NaCl so as to be 20 mM. It was dissolved and incubated at 37 ° C. for 48 hours to form amyloid fibrils. Fibrosis was confirmed by AFM observation and fluorescence measurement with thioflavin T.
（２）チオフラビンＴアッセイ
ペプチドには実施例１で合成したｍｅ５を使用した。２０ｍＭのＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．３）にペプチドを溶解し、１００μＭのペプチド溶液を調製した。このペプチド溶液にチオフラビンＴを０．０４７ｍｇ／ｍＬとなるように加え、さらに線維化アミロイドβ４０を１００μＭとなるように添加した。光路長１ｃｍの蛍光測定用セルに、ペプチド混合溶液を１ｍＬ添加し、４４２ｎｍの励起光で４５０ｎｍ−６００ｎｍまで経時変化を追って測定した。蛍光測定にはＦＰ−６５００（JASCO社製）を用いた。(2) Thioflavin T assay As the peptide, me5 synthesized in Example 1 was used. The peptide was dissolved in 20 mM Tris-HCl buffer (pH 7.3) to prepare a 100 μM peptide solution. To this peptide solution, thioflavin T was added to a concentration of 0.047 mg / mL, and fibrotic amyloid β40 was further added to a concentration of 100 μM. 1 mL of the peptide mixed solution was added to a fluorescence measurement cell having an optical path length of 1 cm, and the time-dependent change was measured from 450 nm to 600 nm with 442 nm excitation light. FP-6500 (manufactured by JASCO) was used for fluorescence measurement.
（３）ＡＦＭによる観察
２０μＭのペプチド溶液に線維化アミロイドβ４０を２０μＭとなるように添加し、２５℃に保温して経時的にサンプリングした。サンプルをマイカ上に載せ１分後にミリＱ水で３回洗浄後乾燥させたものを観察に供した。ＡＦＭにはＳＰＭ−９５００Ｊ２（島津製作所）を使用し、カンチレバーはＮＣＨＲ−２０（NANOWORLD INNOVATION TECHNOLOGY）を使用した。(3) Observation by AFM Fibrous amyloid β40 was added to a 20 μM peptide solution so as to be 20 μM, kept at 25 ° C., and sampled over time. The sample was placed on mica, washed one minute later with milli-Q water and dried for observation. SPM-9500J2 (Shimadzu Corporation) was used for the AFM, and NCHR-20 (NANOWORLD INNOVATION TECHNOLOGY) was used for the cantilever.
（４）結果
チオフラビンＴアッセイの結果を図１４に示した。反応開始後約６０分でチオフラビンＴの蛍光ピークは半減した。
ＡＦＭによる観察結果を図１５に示した。アミロイドは６０分以内で相当程度分解しており、１日後にはほとんど観察されなくなった。(4) Results The results of the thioflavin T assay are shown in FIG. About 60 minutes after the start of the reaction, the fluorescence peak of thioflavin T was halved.
The observation result by AFM is shown in FIG. Amyloid was considerably degraded within 60 minutes, and was hardly observed after 1 day.
〔実施例６：人工ペプチドによるアミロイド分解能の検討（その５）〕
（１）アミロイドβ４２の線維化
アミロイドβ４２（ペプチド研究所）の０．５ｍｇをＤＭＳＯ２０μＬに溶かし１０分間超音波処理をし、１０ｍＭのＨＣｌで１００μＭのアミロイドβ４２溶液を作製し、３０分間超音波処理し、０．４５μｍのフィルターを通し、３７℃で２日間インキュベートして、アミロイド線維を形成させた。線維化の確認はＡＦＭ観察およびチオフラビンＴによる蛍光測定によって行った。[Example 6: Examination of amyloid resolution by artificial peptide (5)]
(1) Fibrosis of amyloid β42 0.5 mg of amyloid β42 (Peptide Laboratories) was dissolved in 20 μL of DMSO and sonicated for 10 minutes. A 100 μM amyloid β42 solution was prepared with 10 mM HCl, and sonicated for 30 minutes. And passed through a 0.45 μm filter and incubated at 37 ° C. for 2 days to form amyloid fibrils. Fibrosis was confirmed by AFM observation and fluorescence measurement with thioflavin T.
（２）チオフラビンＴアッセイ
ペプチドには実施例１で合成したｍｅ５を使用した。３０ｍＭ ＮａＣｌ含有２．５ｍＭリン酸緩衝液（ｐＨ７．４）にペプチドを溶解し、１００μＭのペプチド溶液を調製した。このペプチド溶液にチオフラビンＴを０．０４７ｍｇ／ｍＬとなるように加え、さらに線維化アミロイドβ４２を１００μＭとなるように添加した。光路長１ｃｍの蛍光測定用セルに、ペプチド混合溶液を１ｍＬ添加し、４４２ｎｍの励起光で４５０ｎｍ−６００ｎｍまで経時変化を追って測定した。蛍光測定にはＦＰ−６５００（JASCO社製）を用いた。(2) Thioflavin T assay As the peptide, me5 synthesized in Example 1 was used. The peptide was dissolved in 2.5 mM phosphate buffer (pH 7.4) containing 30 mM NaCl to prepare a 100 μM peptide solution. To this peptide solution, thioflavin T was added to a concentration of 0.047 mg / mL, and fibrotic amyloid β42 was further added to a concentration of 100 μM. 1 mL of the peptide mixed solution was added to a fluorescence measurement cell having an optical path length of 1 cm, and the time-dependent change was measured from 450 nm to 600 nm with 442 nm excitation light. FP-6500 (manufactured by JASCO) was used for fluorescence measurement.
（３）ＡＦＭによる観察
２０μＭのペプチド溶液に線維化アミロイドβ４２を２０μＭとなるように添加し、２５℃に保温して経時的にサンプリングした。サンプルをマイカ上に載せ１分後にミリＱ水で３回洗浄後乾燥させたものを観察に供した。ＡＦＭにはＳＰＭ−９５００Ｊ２（島津製作所）を使用し、カンチレバーはＮＣＨＲ−２０（NANOWORLD INNOVATION TECHNOLOGY）を使用した。(3) Observation by AFM Fibrous amyloid β42 was added to a 20 μM peptide solution so as to be 20 μM, kept at 25 ° C., and sampled over time. The sample was placed on mica, washed one minute later with milli-Q water and dried for observation. SPM-9500J2 (Shimadzu Corporation) was used for the AFM, and NCHR-20 (NANOWORLD INNOVATION TECHNOLOGY) was used for the cantilever.
（４）結果
チオフラビンＴアッセイの結果を図１６に示した。反応開始後約１０分でチオフラビンＴの蛍光ピークは半減した。
ＡＦＭによる観察結果を図１７に示した。アミロイドは１８０分以内で相当程度分解しており、１日後にはほとんど観察されなくなった。(4) Results The results of the thioflavin T assay are shown in FIG. About 10 minutes after the start of the reaction, the fluorescence peak of thioflavin T was halved.
The observation result by AFM is shown in FIG. Amyloid was considerably degraded within 180 minutes, and was hardly observed after 1 day.
〔実施例７：人工ペプチドによるアミロイド分解能の検討（その６）〕
線維化アミロイドβ４２を、アミロイド量に対して１００分の１量のペプチドを加えた場合でも分解可能かどうかを検討した。[Example 7: Examination of amyloid resolution by artificial peptide (6)]
It was examined whether fibrotic amyloid β42 can be degraded even when a peptide in an amount of 1/100 of the amount of amyloid is added.
（１）チオフラビンＴアッセイ
ペプチドには実施例１で合成したｍｅ５を使用した。線維化アミロイドβ４２は実施例６と同じものを使用した。ＰＢＳにペプチドを溶解し、０．１μＭのペプチド溶液を調製した。このペプチド溶液にチオフラビンＴを０．０４７ｍｇ／ｍＬとなるように加え、さらに線維化アミロイドβ４２を１０μＭとなるように添加した。光路長１ｃｍの蛍光測定用セルに、ペプチド混合溶液を１ｍＬ添加し、４４２ｎｍの励起光で４５０ｎｍ−６００ｎｍまで経時変化を追って測定した。蛍光測定にはＦＰ−６５００（JASCO社製）を用いた。(1) Thioflavin T assay As the peptide, me5 synthesized in Example 1 was used. The same fibrotic amyloid β42 as in Example 6 was used. The peptide was dissolved in PBS to prepare a 0.1 μM peptide solution. To this peptide solution, thioflavin T was added to a concentration of 0.047 mg / mL, and fibrotic amyloid β42 was further added to a concentration of 10 μM. 1 mL of the peptide mixed solution was added to a fluorescence measurement cell having an optical path length of 1 cm, and the time-dependent change was measured from 450 nm to 600 nm with 442 nm excitation light. FP-6500 (manufactured by JASCO) was used for fluorescence measurement.
（２）ＡＦＭによる観察
２００ｎＭのペプチド溶液に線維化アミロイドβ４２を２０μＭとなるように添加し、２５℃に保温して経時的にサンプリングした。サンプルをマイカ上に載せ１分後にミリＱ水で３回洗浄後乾燥させたものを観察に供した。ＡＦＭにはＳＰＭ−９５００Ｊ２（島津製作所）を使用し、カンチレバーはＮＣＨＲ−２０（NANOWORLD INNOVATION TECHNOLOGY）を使用した。(2) Observation by AFM Fibrous amyloid β42 was added to a 200 nM peptide solution so as to be 20 μM, kept at 25 ° C., and sampled over time. The sample was placed on mica, washed one minute later with milli-Q water and dried for observation. SPM-9500J2 (Shimadzu Corporation) was used for the AFM, and NCHR-20 (NANOWORLD INNOVATION TECHNOLOGY) was used for the cantilever.
（３）結果
チオフラビンＴアッセイの結果を図１８に示した。反応開始後約９０分でチオフラビンＴの蛍光ピークは半減した。
ＡＦＭによる観察結果を図１９に示した。アミロイドは１８０分以内で相当程度分解しており、２日後にはほとんど観察されなくなった。
これらの結果から、本発明のペプチドは、アミロイド線維に対して化学量論的（例えば１：１）に反応して分解するのではなく、１／１００の量比においても触媒（酵素）的に作用して分解することが示された。(3) Results The results of the thioflavin T assay are shown in FIG. About 90 minutes after the start of the reaction, the fluorescence peak of thioflavin T was halved.
The observation result by AFM is shown in FIG. Amyloid was considerably degraded within 180 minutes and was hardly observed after 2 days.
From these results, the peptide of the present invention is not decomposed in a stoichiometric (for example, 1: 1) reaction with respect to amyloid fibrils, but catalytically (enzymatically) even at a quantitative ratio of 1/100. It was shown to act and decompose.
〔参考例：アミロイド以外の蛋白質分解能の検討〕
（１）ＧＦＰおよびミオグロビン分解能の検討
２０μＭのＧＦＰ溶液を調製し、実施例１で合成したｍｅ５を１００μＭになるように加え、経時的に蛍光を測定した。また、１．２ｍｇ／ｍｌのミオグロビン溶液を調製し、ｍｅ５を１００μＭになるように加え、経時的に可視紫外分光を測定した。溶媒はどちらも２０ｍＭのＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．３）を用いた。
結果を表１に示した。表１から明らかなように、ＧＦＰの蛍光強度およびミオグロビンの吸光強度は６０分まで変化がなかった。この結果から、ｍｅ５はＧＦＰおよびミオグロビンを分解しないことが示された。[Reference example: Examination of protein resolution other than amyloid]
(1) Examination of GFP and myoglobin resolution A 20 μM GFP solution was prepared, me5 synthesized in Example 1 was added to 100 μM, and fluorescence was measured over time. A 1.2 mg / ml myoglobin solution was prepared, me5 was added to 100 μM, and visible ultraviolet spectroscopy was measured over time. As the solvent, 20 mM Tris-HCl buffer (pH 7.3) was used.
The results are shown in Table 1. As is clear from Table 1, the fluorescence intensity of GFP and the absorbance intensity of myoglobin did not change until 60 minutes. This result showed that me5 does not degrade GFP and myoglobin.
（２）ヘモグロビン、γグロブリン、アルブミンおよびネイティブβラクトグロブリン分解能の検討
本発明のペプチドにより、血中あるいは組織中に存在し得る代表的な蛋白質が分解される副作用が生じないことを確認するために、本発明のペプチドのヘモグロビン、γグロブリン、アルブミンおよびネイティブβラクトグロブリン分解能について検討した。
各蛋白質を試薬として入手し、２０ｍＭのＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．３）を用いて各蛋白質の１０ｍｇ／ｍＬ溶液をそれぞれ調製した。実施例１で合成したｍｅ８の５ｍｇ／ｍＬ溶液を調製し、蛋白質溶液と等量混合して試料とした。対照として、各蛋白質溶液のみを試料とした。２５℃に保温して１時間後にサンプリングし、質量分析に供した。ＡＸＩＭＡ−ＣＦＲ（島津製作所）を用い、ＭＡＬＤＩ−ＴＯＦ−ＭＳ法で質量分析を行った。(2) Examination of hemoglobin, gamma globulin, albumin and native β-lactoglobulin resolution To confirm that the peptide of the present invention does not cause a side effect of degrading a typical protein that may be present in blood or tissue. The peptides of the present invention were examined for hemoglobin, γ globulin, albumin and native β lactoglobulin resolution.
Each protein was obtained as a reagent, and a 10 mg / mL solution of each protein was prepared using 20 mM Tris-HCl buffer (pH 7.3). A 5 mg / mL solution of me8 synthesized in Example 1 was prepared and mixed with an equal amount of protein solution to prepare a sample. As a control, only each protein solution was used as a sample. The sample was kept at 25 ° C. for 1 hour and sampled for mass spectrometry. Mass spectrometry was performed by MALDI-TOF-MS method using AXIMA-CFR (Shimadzu Corporation).
ヘモグロビンの結果を図２０に示した。（ａ）がヘモグロビン単独の結果、（ｂ）がヘモグロビンにｍｅ８を添加した結果である。図２０（ａ）、（ｂ）から明らかなように、ｍｅ８を添加しても分解された断片は検出されず、ｍｅ８はヘモグロビンを分解しないことが示された。
γグロブリンの結果を図２１に示した。（ａ）がγグロブリン単独の結果、（ｂ）がγグロブリンにｍｅ８を添加した結果である。図２１（ａ）、（ｂ）から明らかなように、ｍｅ８を添加しても分解された断片は検出されず、ｍｅ８はγグロブリンを分解しないことが示された。
アルブミンの結果を図２２に示した。（ａ）がアルブミン単独の結果、（ｂ）がアルブミンにｍｅ８を添加した結果である。図２２（ａ）、（ｂ）から明らかなように、ｍｅ８を添加しても分解された断片は検出されず、ｍｅ８はアルブミンを分解しないことが示された。
ネイティブβラクトグロブリンの結果を図２３に示した。（ａ）がネイティブβラクトグロブリン単独の結果、（ｂ）がネイティブβラクトグロブリンにｍｅ８を添加した結果である。図２３（ａ）、（ｂ）から明らかなように、ｍｅ８を添加しても分解された断片は検出されず、ｍｅ８はネイティブβラクトグロブリンを分解しないことが示された。つまり、ｍｅ８はアミロイド線維化したβラクトグロブリンを分解できるが、アミロイド線維化していないネイティブβラクトグロブリンは分解できないことが明らかになった。The result of hemoglobin is shown in FIG. (A) is the result of hemoglobin alone, and (b) is the result of adding me8 to hemoglobin. As is apparent from FIGS. 20A and 20B, no fragment was detected even when me8 was added, indicating that me8 does not degrade hemoglobin.
The results for γ globulin are shown in FIG. (A) is the result of γ globulin alone, and (b) is the result of adding me8 to γ globulin. As is clear from FIGS. 21 (a) and 21 (b), no fragment was detected even when me8 was added, indicating that me8 does not degrade γ globulin.
The results for albumin are shown in FIG. (A) is the result of albumin alone, and (b) is the result of adding me8 to albumin. As is clear from FIGS. 22 (a) and 22 (b), no fragment was detected even when me8 was added, indicating that me8 did not degrade albumin.
The results for native β-lactoglobulin are shown in FIG. (A) is a result of native β-lactoglobulin alone, and (b) is a result of adding me8 to native β-lactoglobulin. As is apparent from FIGS. 23 (a) and (b), no fragment was detected even when me8 was added, indicating that me8 did not degrade native β-lactoglobulin. That is, it was revealed that me8 can degrade amyloid fibrillated β-lactoglobulin, but not native amyloid fibrillated native β-lactoglobulin.
今回実験に供した人工ペプチドｍｅ４、ｍｅ５、ｍｅ６、ｍｅ７、ｍｅ８およびｍｅ９の構造およびアミロイド分解能を表２に示した。 Table 2 shows the structures and amyloid resolution of the artificial peptides me4, me5, me6, me7, me8, and me9 used in this experiment.
なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。 The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.
Claims (7)
（Ｉ）ＨＸ(I) HX １1 ＸX ２2 ＳＤＸSDX ３3 ＸX ４4 （配列番号１）(SEQ ID NO: 1)
（Ｘ(X １1 およびＸAnd X ２2 はＡであり、ＸIs A and X ３3 はＡ、ＶまたはＬであり、ＸIs A, V or L and X ４4 はＷ、ＹまたはＦである。）Is W, Y or F. )
（Ｉ）ＨＸ(I) HX １1 ＸX ２2 ＳＤＸSDX ３3 ＸX ４4 （配列番号１）(SEQ ID NO: 1)
（Ｘ(X １1 およびＸAnd X ２2 はＡであり、ＸIs A and X ３3 はＡ、ＶまたはＬであり、ＸIs A, V or L and X ４4 はＷ、ＹまたはＦである。）Is W, Y or F. )
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