JP5354206B2 - Antibacterial peptide - Google Patents
Antibacterial peptide Download PDFInfo
- Publication number
- JP5354206B2 JP5354206B2 JP2009505105A JP2009505105A JP5354206B2 JP 5354206 B2 JP5354206 B2 JP 5354206B2 JP 2009505105 A JP2009505105 A JP 2009505105A JP 2009505105 A JP2009505105 A JP 2009505105A JP 5354206 B2 JP5354206 B2 JP 5354206B2
- Authority
- JP
- Japan
- Prior art keywords
- peptide
- gly
- amino acid
- pro
- salt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
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Abstract
Description
本発明は抗菌活性を有する新規なペプチドならびにその生理学的に許容される塩に関する。さらに詳しくは、本発明は細菌類には毒性を示すが哺乳類細胞には毒性を示さず、細菌感染症の治療に有用なペプチドならびにその薬学的に許容される塩に関する。 The present invention relates to novel peptides having antibacterial activity and physiologically acceptable salts thereof. More particularly, the present invention relates to peptides and pharmaceutically acceptable salts thereof that are toxic to bacteria but not mammalian cells and are useful in the treatment of bacterial infections.
抗生物質は細菌感染症の治療に主要な役割を果たしているが、ほとんどの場合耐性菌の出現によりその効力が失われる。そこで、抗生物質とは作用機序の異なる抗菌剤が求められている。そのような抗菌剤として、抗菌性ペプチドが挙げられる。特に、膜作動性の抗菌性ペプチドは、微生物の細胞膜・外膜に作用し、そのバリアー能を破壊することで殺菌作用を示すが、原理的に耐性菌が出現し難いことが特徴である。 Antibiotics play a major role in the treatment of bacterial infections, but most often lose their potency due to the emergence of resistant bacteria. Therefore, an antibacterial agent having a different mechanism of action from antibiotics is required. Such antibacterial agents include antibacterial peptides. In particular, a membrane-acting antibacterial peptide acts on the cell membrane / outer membrane of microorganisms and exhibits a bactericidal action by destroying its barrier ability, but is characterized in that resistant bacteria are not likely to appear in principle.
この様な抗菌性ペプチドは天然にも数多く存在し、例えば、蜂の産生するMelittinが挙げられる。しかしながら、Meilittinは、細菌膜と同時に哺乳類細胞にも作用する。一方、アフリカツメガエルが分泌するMagainin 2は、細菌膜を選択的に破壊し、先天的な生体防御作用を担うと考えられている(例えば、非特許文献1)。MelittinやMagainin 2は、Lys残基やArg残基を含む塩基性ペプチドであり、α-ヘリックスを形成して、抗菌作用を示すことが報告されている。さらに、同様の作用を示す抗菌性ペプチドとして、カイコ由来のCecropin A、ヒト、昆虫、植物由来のDefensin等があり、静電相互作用と疎水性相互作用が細菌膜選択性に重要であることが報告されている(非特許文献2)。また、例えば、特許文献1には、抗菌剤、抗菌性医薬、点眼薬の用途に使用できるヒト角膜および結膜分泌アポ蛋白質由来の抗菌性ペプチド、これをコードするポリヌクレオチドが開示されている。 There are many such antibacterial peptides in nature, for example, Melittin produced by bees. However, Meilittin acts on mammalian cells as well as bacterial membranes. On the other hand, it is considered that Magainin 2 secreted by Xenopus laevis selectively destroys the bacterial membrane and bears an innate biological defense action (for example, Non-Patent Document 1). Melittin and Maginin 2 are basic peptides containing Lys residues and Arg residues, and have been reported to form an α-helix and exhibit antibacterial activity. In addition, as antibacterial peptides exhibiting the same action, there are Cecropin A derived from silkworm, Defensin derived from human, insect, plant, etc., and electrostatic interaction and hydrophobic interaction are important for bacterial membrane selectivity. It has been reported (Non-Patent Document 2). For example, Patent Document 1 discloses an antibacterial peptide derived from human cornea and conjunctival secretory apoprotein that can be used for antibacterial agents, antibacterial drugs, and eye drops, and a polynucleotide encoding the same.
人工的に製造された抗菌性ぺプチドとしては、例えば、特許文献2には、CAP11をベースにした新規なペプチド、これを有効成分とする抗菌剤、ならびに細胞に対するLPSの結合阻害剤、細菌感染治療剤やエンドトキシンショック抑制剤等の医薬が報告されている。特許文献3には、親水性アミノ酸残基の繰返し−疎水性アミノ酸部−塩基性アミノ酸部−ブリッジ部−塩基性アミノ酸部−疎水性アミノ酸部で表される抗菌性ペプチドが報告されている。特許文献4には、ペプチドの末端ではない2ヶ所に塩基性アミノ酸残基が2個以上連続している部位を含む、アミノ酸残基が7〜30個の抗菌性ペプチドが報告されている。特許文献5には、リゾチームをトリプシンで分解して得られる平均分子量が2〜6kDaの範囲にあるペプチド類を有効成分とする抗菌性ペプチド組成物が報告されている。特許文献6には、ウシラクトフェリンの加水分解物から分画された抗菌性ペプチドが報告されている。ヒト由来のものとしては、ヒト由来のタンパク質の中に存在する抗菌活性を有するタンパク質またはペプチドが知られている(例えば特許文献7を参照)。また、特許文献8には、ヒト由来で、LPS結合活性、抗菌活性およびLPS中和活性が非常に高いペプチドが報告されている。 As an artificially produced antibacterial peptide, for example, Patent Document 2 discloses a novel peptide based on CAP11, an antibacterial agent comprising this as an active ingredient, an inhibitor of LPS binding to cells, and bacterial infection Drugs such as therapeutic agents and endotoxin shock inhibitors have been reported. Patent Document 3 reports an antibacterial peptide represented by repeating hydrophilic amino acid residues-hydrophobic amino acid part-basic amino acid part-bridge part-basic amino acid part-hydrophobic amino acid part. Patent Document 4 reports an antibacterial peptide having 7 to 30 amino acid residues, including a portion where two or more basic amino acid residues are continuous at two positions that are not the ends of the peptide. Patent Document 5 reports an antibacterial peptide composition containing as an active ingredient peptides having an average molecular weight of 2 to 6 kDa obtained by decomposing lysozyme with trypsin. Patent Document 6 reports an antimicrobial peptide fractionated from a hydrolyzate of bovine lactoferrin. As a thing derived from a human, the protein or peptide which has the antibacterial activity which exists in the protein derived from a human is known (for example, refer patent document 7). Patent Document 8 reports a peptide derived from human and having very high LPS binding activity, antibacterial activity, and LPS neutralization activity.
これらの抗菌性ペプチドは、細菌膜と結合し、α-ヘリックスを形成したペプチドが細菌膜内でチャンネル様の構造をとって膜に細孔を形成することが提案されているが、α-ヘリックスを形成するペプチドは一般に熱や塩濃度などの外的環境に影響されやすく不安定であるという問題点があった。 These antibacterial peptides have been proposed that peptides that bind to bacterial membranes and form α-helices take a channel-like structure within the bacterial membrane and form pores in the membrane. In general, the peptides that form the are susceptible to the external environment such as heat and salt concentration and are unstable.
一方、本発明者らはコラーゲンのアミノ酸配列に基づいて、安定な三重らせん構造を形成するペプチドを報告している(例えば、非特許文献3、特許文献9)。本発明者らは、この様な知見をもとに、安定性の高い抗菌性ペプチドを作製できると考えた。
しかるに本発明は、安定性が高く、優れた抗菌活性を有する新規なペプチドならびにその生理学的に許容される塩を提供すること、さらには、細菌感染症などの治療に有用なペプチド並びにその薬学的に許容される塩を提供することを主な目的とする。さらに、本発明は、当該ペプチド又はその塩を利用した、抗菌剤、医薬製剤、医療用基材、化粧料、細菌感染粧又は敗血症の予防又は治療方法をも提供することを目的とするものである。 However, the present invention provides a novel peptide having high stability and excellent antibacterial activity, and a physiologically acceptable salt thereof, as well as a peptide useful for the treatment of bacterial infections and the like and its pharmaceutical The main object is to provide an acceptable salt. Furthermore, an object of the present invention is to provide a method for preventing or treating antibacterial agents, pharmaceutical preparations, medical base materials, cosmetics, bacterial infection makeup or sepsis using the peptide or a salt thereof. is there.
本発明者等は、鋭意検討を行ったところ、上記課題を解決し得る特定のアミノ酸配列を有するペプチドを見出した。すなわち、下記一般式(I)で表されるアミノ酸配列を有するペプチドであれば、安定なコラーゲン様の三重らせん構造を形成することができ、熱や塩濃度等の影響を受けにくく、優れた抗菌活性が発揮され得ることを見出した。本発明は、このような知見に基づいてさらに研究を重ねた結果得られたものである。 As a result of diligent studies, the present inventors have found a peptide having a specific amino acid sequence that can solve the above-mentioned problems. That is, a peptide having the amino acid sequence represented by the following general formula (I) can form a stable collagen-like triple helix structure, is hardly affected by heat, salt concentration, etc., and has excellent antibacterial properties. It has been found that the activity can be exerted. The present invention has been obtained as a result of further research based on such knowledge.
本発明は、以下の抗菌活性を有するペプチド又はその塩、これらを有効成分とする抗菌剤、医薬製剤、医療用基材、化粧料、細菌感染症又は敗血症の予防又は治療方法を提供するものである。
項1.以下の(1)又は(2)で表されるペプチド又はその塩;
(1)下記一般式(I)で表されるアミノ酸配列からなるペプチド
(Pro Hyp Gly)n-{(Lys X Gly)m-(Pro Hyp Gly)l-(Lys X Gly)p}α-(Pro Hyp Gly)r (I)
(ここで、式(I)中、nは1〜6の整数であり、mは1〜4の整数であり、lは0〜7の整数であり、pは1〜4の整数であり、rは1〜6の整数であり、αは1〜6の整数である。
Xは、Ile、Leu及びHypから選択されるいずれかのアミノ酸残基であり、
m+p個のXは、それぞれ同一又は異なっていてもよい。
n+m+l+p+r=4〜15である。
但し、l=0の時、m+p≧2を満たし、
α≧2のときm=1、l=0、p=1を満たす。)
(2)一般式(I)で表されるアミノ酸配列において、Lys、Gly及びX以外の1〜3個のアミノ酸残基が置換、欠失若しくは付加されたアミノ酸配列からなり、かつ抗菌活性を有するペプチド。
項2.前記(1)において、nは3〜6の整数であり、mは1〜4の整数であり、lは0〜7の整数であり、pは1〜4の整数であり、rは3〜6の整数であり、αは1であり、
XはIle又はLeuであり、
m+p個のXは、それぞれ同一又は異なっていてもよく、
n+m+l+p+r=8〜15であり、
l=0の時、m+p≧2を満たす、項1に記載のペプチド又はその塩。
項3.下記(1-1)又は(2-1)で表される項1に記載のペプチド又はその塩:
(1-1)以下の(i)〜(vi)のいずれかで表されるアミノ酸配列からなるペプチド。
(i)(Pro Hyp Gly)3 -(Lys Ile Gly)3 -(Pro Hyp Gly)4(配列番号:1)
(ii)(Pro Hyp Gly)3 -Lys Ile Gly Pro Hyp Gly Lys Ile Gly-(Pro Hyp Gly)4(配列番号:2)
(iii)(Pro Hyp Gly)4 -(Lys Ile Gly)2 -(Pro Hyp Gly)4(配列番号:3)
(iv)(Pro Hyp Gly)3-(Lys Hyp Gly Lys Leu Gly)2-(Pro Hyp Gly)3 (配列番号:4)、
(v)(Pro Hyp Gly)2-(Lys Leu Gly Lys Hyp Gly)3-(Pro Hyp Gly)2(配列番号:5)、
(vi)Pro Hyp Gly-(Lys Leu Gly Lys Hyp Gly)4-Pro Hyp Gly (配列番号:6)
(2-1)前記アミノ酸配列(i)〜(vi)において1〜3個のアミノ酸が置換、欠失、
若しくは付加されたアミノ酸配列からなるペプチドであって、抗菌活性を有するペプチドならびにその塩。
項4.前記アミノ酸配列のアミノ末端の水素がアセチル基、メトキシカルボニル基、ブトキシカルボニル基、1〜3残基のペプチドで置換された、項1〜4のいずれかに記載のペプチドならびにその薬学的に許容される塩。
項5.前記アミノ酸配列のカルボキシ末端のカルボキシル基がアミド基、メチルエステル基、ブチルエステル基、1〜3残基のペプチドで置換された、項1〜4のいずれかに記載のペプチドならびにその薬学的に許容される塩。
項6.コラーゲン様の三重らせん構造を含む項1〜5のいずれかに記載のペプチドならびにその薬学的に許容される塩。
項7.項1〜6のいずれかに記載のペプチド又はその塩を有効成分とする抗菌剤。
項8.項1〜7のいずれかに記載のペプチド又はその塩、及び薬学的に許容される担体又は基材を含む、医薬製剤。
項9.細菌感染症又は敗血症の予防又は治療用である項8に記載の医薬製剤。
項10.項1〜6のいずれかに記載のペプチド又はその塩が薬学的に許容される基材に担持されてなる医療用基材。
項11.前記薬学的に許容される基材が、アルギン酸ゲル、コラーゲンゲル、乳酸ポリマー及びセルロースからなる群より選択される少なくとも一種である、項10に記載の医療用基材。
項12.請求項1〜6のいずれかに記載のペプチド又はその塩を含有する化粧料。
項13.項1〜6のいずれかに記載のペプチド又はその塩の有効量を、細菌感染症又は敗血症に罹患した患者に投与することを特徴とする、細菌感染症又は敗血症の治療方法。
項14.項10又は11に記載の医療用基材を患部に適用することを特徴とする、細菌感染症の予防又は治療方法。
項15.項1〜6のいずれかに記載のペプチド又はその塩の、抗菌剤を製造するための使用。
項16.項1〜6のいずれかに記載のペプチド又はその塩の、細菌感染症又は敗血症の予防又は治療用の医薬製剤を製造するための使用。
項17.項1〜6のいずれかに記載のペプチド又はその塩の、細菌感染予防又は治療用医療基材の製造のための使用。
項18.
細菌感染症又は敗血症の治療用である、以下の(1)又は(2)で表されるペプチド又はその塩;
以下の(1)又は(2)で表されるペプチド又はその塩;
(1)下記一般式(I)で表されるアミノ酸配列からなるペプチド
(Pro Hyp Gly)n-{(Lys X Gly)m-(Pro Hyp Gly)l-(Lys X Gly)p}α-(Pro Hyp Gly)r (I)
(ここで、式(I)中、nは1〜6の整数であり、mは1〜4の整数であり、lは0〜7の整数であり、pは1〜4の整数であり、rは1〜6の整数であり、αは1〜6の整数である。
Xは、Ile、Leu及びHypから選択されるいずれかのアミノ酸残基であり、
m+p個のXは、それぞれ同一又は異なっていてもよい。
n+m+l+p+r=4〜15である。
但し、l=0の時、m+p≧2を満たし、
α≧2のときm=1、l=0、p=1を満たす。)
(2)一般式(I)で表されるアミノ酸配列において、Lys、Gly及びX以外の1〜3個のアミノ酸残基が置換、欠失若しくは付加されたアミノ酸配列からなり、かつ抗菌活性を有するペプチド。The present invention provides the following peptides or salts thereof having antibacterial activity, antibacterial agents containing these as active ingredients, pharmaceutical preparations, medical bases, cosmetics, methods for preventing or treating bacterial infections or sepsis. is there.
Item 1. A peptide represented by the following (1) or (2) or a salt thereof;
(1) Peptides having an amino acid sequence represented by the following general formula (I)
(Pro Hyp Gly) n -{(Lys X Gly) m- (Pro Hyp Gly) l- (Lys X Gly) p } α- (Pro Hyp Gly) r (I)
(Here, in formula (I), n is an integer of 1-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is an integer of 1 to 6, and α is an integer of 1 to 6.
X is any amino acid residue selected from Ile, Leu and Hyp,
The m + p Xs may be the same or different from each other.
n + m + l + p + r = 4-15.
However, when l = 0, m + p ≧ 2 is satisfied,
When α ≧ 2, m = 1, l = 0, and p = 1 are satisfied. )
(2) The amino acid sequence represented by the general formula (I) is composed of an amino acid sequence in which 1 to 3 amino acid residues other than Lys, Gly and X are substituted, deleted or added, and has antibacterial activity. peptide.
Item 2. In said (1), n is an integer of 3-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is 3- An integer of 6, α is 1,
X is Ile or Leu,
m + p Xs may be the same or different from each other,
n + m + l + p + r = 8-15,
Item 2. The peptide or salt thereof according to Item 1, which satisfies m + p ≧ 2 when l = 0.
Item 3. The peptide according to item 1 represented by the following (1-1) or (2-1) or a salt thereof:
(1-1) A peptide comprising an amino acid sequence represented by any one of (i) to (vi) below.
(I) (Pro Hyp Gly) 3- (Lys Ile Gly) 3- (Pro Hyp Gly) 4 (SEQ ID NO: 1)
(Ii) (Pro Hyp Gly) 3 -Lys Ile Gly Pro Hyp Gly Lys Ile Gly- (Pro Hyp Gly) 4 (SEQ ID NO: 2)
(Iii) (Pro Hyp Gly) 4 - (Lys Ile Gly) 2 - (Pro Hyp Gly) 4 ( SEQ ID NO: 3)
(iv) (Pro Hyp Gly) 3- (Lys Hyp Gly Lys Leu Gly) 2- (Pro Hyp Gly) 3 (SEQ ID NO: 4),
(v) (Pro Hyp Gly) 2- (Lys Leu Gly Lys Hyp Gly) 3- (Pro Hyp Gly) 2 (SEQ ID NO: 5),
(vi) Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) 4 -Pro Hyp Gly (SEQ ID NO: 6)
(2-1) In the amino acid sequences (i) to (vi), 1 to 3 amino acids are substituted, deleted,
Alternatively, a peptide comprising an added amino acid sequence and having antibacterial activity, and salts thereof.
Item 4. Item 5. The peptide according to any one of Items 1 to 4, wherein the amino terminal hydrogen of the amino acid sequence is substituted with an acetyl group, a methoxycarbonyl group, a butoxycarbonyl group, or a peptide having 1 to 3 residues, and a pharmaceutically acceptable peptide thereof. Salt.
Item 5. Item 5. The peptide according to any one of Items 1 to 4, wherein the carboxyl group at the carboxy terminus of the amino acid sequence is substituted with an amide group, a methyl ester group, a butyl ester group, or a peptide having 1-3 residues, and a pharmaceutically acceptable peptide thereof. Salt.
Item 6. Item 6. The peptide according to any one of Items 1 to 5, comprising a collagen-like triple helix structure, and a pharmaceutically acceptable salt thereof.
Item 7. Item 7. An antibacterial agent comprising the peptide or salt thereof according to any one of items 1 to 6 as an active ingredient.
Item 8. Item 8. A pharmaceutical preparation comprising the peptide according to any one of Items 1 to 7 or a salt thereof, and a pharmaceutically acceptable carrier or substrate.
Item 9. Item 9. The pharmaceutical preparation according to Item 8, which is used for prevention or treatment of bacterial infection or sepsis.
Item 11. Item 11. The medical substrate according to
Item 12. Cosmetics containing the peptide according to any one of claims 1 to 6 or a salt thereof.
Item 13. Item 7. A method for treating bacterial infection or sepsis, comprising administering an effective amount of the peptide or salt thereof according to any one of items 1 to 6 to a patient suffering from bacterial infection or sepsis.
Item 14. Item 12. A method for preventing or treating a bacterial infection, which comprises applying the medical substrate according to
Item 15. Item 7. Use of the peptide according to any one of Items 1 to 6 or a salt thereof for producing an antibacterial agent.
Item 16. Item 7. Use of the peptide according to any one of Items 1 to 6 or a salt thereof for producing a pharmaceutical preparation for preventing or treating bacterial infection or sepsis.
Item 17. Item 7. Use of the peptide according to any one of Items 1 to 6 or a salt thereof for producing a medical substrate for preventing or treating bacterial infection.
Item 18.
A peptide represented by the following (1) or (2) or a salt thereof for use in the treatment of bacterial infection or sepsis;
A peptide represented by the following (1) or (2) or a salt thereof;
(1) Peptides having an amino acid sequence represented by the following general formula (I)
(Pro Hyp Gly) n -{(Lys X Gly) m- (Pro Hyp Gly) l- (Lys X Gly) p } α- (Pro Hyp Gly) r (I)
(Here, in formula (I), n is an integer of 1-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is an integer of 1 to 6, and α is an integer of 1 to 6.
X is any amino acid residue selected from Ile, Leu and Hyp,
The m + p Xs may be the same or different from each other.
n + m + l + p + r = 4-15.
However, when l = 0, m + p ≧ 2 is satisfied,
When α ≧ 2, m = 1, l = 0, and p = 1 are satisfied. )
(2) The amino acid sequence represented by the general formula (I) is composed of an amino acid sequence in which 1 to 3 amino acid residues other than Lys, Gly and X are substituted, deleted or added, and has antibacterial activity. peptide.
本発明のペプチドによれば、安定な三重らせん構造を形成する抗菌性ペプチドを提供することができる。また、本発明のペプチドは、熱、塩濃度等の外的要因の影響を受けにくく、安定して優れた抗菌活性を発揮することができることから、医薬製剤として調製し、細菌感染症、敗血症等の予防又は治療に好適に使用することができる。 According to the peptide of the present invention, an antibacterial peptide that forms a stable triple helical structure can be provided. In addition, since the peptide of the present invention is not easily affected by external factors such as heat and salt concentration and can stably exhibit excellent antibacterial activity, it is prepared as a pharmaceutical preparation, such as bacterial infection, sepsis, etc. It can be suitably used for prevention or treatment.
さらに、本発明のポリペプチドを医療用基材に結合させて使用することもできる。例えば、人工コラーゲンゲル等に本発明のペプチドを結合させ、これを原料として製造された人工皮膚は、移植部分の細菌感染を予防することができる。 Furthermore, the polypeptide of the present invention can be used by binding to a medical substrate. For example, artificial skin produced by binding the peptide of the present invention to an artificial collagen gel or the like and using this as a raw material can prevent bacterial infection of the transplanted part.
加えて、本発明のペプチドは、ヒトに対して低刺激性であり、安定した抗菌性を保持し得ることから、化粧料に抗菌剤(又は防腐剤)として配合することもできる。化粧料に配合される従来の抗菌剤や防腐剤には、臭いの気になるものもあった。しかしながら、本発明のポリペプチドは臭い等の問題もなく、化粧料に配合される抗菌剤として好適に使用され得る。 In addition, since the peptide of the present invention is hypoallergenic to humans and can maintain stable antibacterial properties, it can also be incorporated into cosmetics as an antibacterial agent (or preservative). Some of the conventional antibacterial agents and preservatives blended in cosmetics are annoying. However, the polypeptide of the present invention can be suitably used as an antibacterial agent blended in cosmetics without problems such as odor.
本発明においては各種アミノ酸残基を次の略号で記述する。 In the present invention, various amino acid residues are described by the following abbreviations.
Ala :L−アラニン残基
Arg :L−アルギニン残基
Asn :L−アスパラギン残基
Asp :L−アスパラギン酸残基
Cys :L−システイン残基
Gln :L−グルタミン残基
Glu :L−グルタミン酸残基
Gly :グリシン残基
His :L−ヒスチジン残基
Hyp :L−ヒドロキシプロリン残基
Ile :L−イソロイシン残基
Leu :L−ロイシン残基
Lys :L−リジン残基
Met :L−メチオニン残基
Phe :L−フェニルアラニン残基
Pro :L−プロリン残基
Ser :L−セリン残基
Thr :L−トレオニン残基
Trp :L−トリプトファン残基
Tyr :L−チロシン残基
Val :L−バリン残基
また、本明細書においては、常法に従ってペプチドのアミノ酸配列を、そのN末端のアミノ酸残基が左側に位置し、C末端のアミノ酸残基が右側に位置するように記述する。Ala: L-alanine residue
Arg: L-arginine residue
Asn: L-asparagine residue
Asp: L-aspartic acid residue
Cys: L-cysteine residue
Gln: L-glutamine residue
Glu: L-glutamic acid residue
Gly: Glycine residue
His: L-histidine residue
Hyp: L-hydroxyproline residue
Ile: L-isoleucine residue
Leu: L-leucine residue
Lys: L-lysine residue
Met: L-methionine residue
Phe: L-phenylalanine residue
Pro: L-proline residue
Ser: L-serine residue
Thr: L-threonine residue
Trp: L-tryptophan residue
Tyr: L-tyrosine residue
Val: L-valine residue In the present specification, the amino acid sequence of a peptide is arranged according to a conventional method so that the N-terminal amino acid residue is located on the left side and the C-terminal amino acid residue is located on the right side. Describe.
[抗菌活性を有するペプチド及びその塩]
本発明の抗菌活性を有するペプチドとしては、以下の(1)又は(2)で表されるペプチドが挙げられる。
(1)下記一般式(I)で表されるアミノ酸配列からなるペプチド
(Pro Hyp Gly)n-{(Lys X Gly)m-(Pro Hyp Gly)l-(Lys X Gly)p}α-(Pro Hyp Gly)r (I)
式(I)中、例えば(Lys X Gly)mは、構成単位(Lys X Gly)がm個連結していることを
示すものである。[Peptides having antibacterial activity and salts thereof]
Examples of the peptide having antibacterial activity of the present invention include peptides represented by the following (1) or (2).
(1) Peptide having an amino acid sequence represented by the following general formula (I)
(Pro Hyp Gly) n -{(Lys X Gly) m- (Pro Hyp Gly) l- (Lys X Gly) p } α- (Pro Hyp Gly) r (I)
In the formula (I), for example, (Lys X Gly) m indicates that m structural units (Lys X Gly) are linked.
ここで、式(I)中、n、m、l、p、r、α及びXは、以下の通りである。
nは1〜6の整数、好ましくは1〜5の整数、より好ましくは1〜4の整数である。
mは1〜4の整数、好ましくは1〜3の整数、より好ましくは1〜2の整数である。
lは0〜7の整数、好ましくは0〜5の整数、より好ましくは0〜3の整数である。
pは1〜4の整数、好ましくは1〜3の整数、より好ましくは1〜2の整数である。
rは1〜6の整数、好ましくは1〜5の整数、より好ましくは1〜4の整数である。
αは1〜6の整数、好ましくは1〜5の整数、より好ましくは1〜4の整数である。Here, in formula (I), n, m, l, p, r, α, and X are as follows.
n is an integer of 1-6, preferably an integer of 1-5, more preferably an integer of 1-4.
m is an integer of 1-4, preferably an integer of 1-3, more preferably an integer of 1-2.
l is an integer of 0 to 7, preferably an integer of 0 to 5, more preferably an integer of 0 to 3.
p is an integer of 1-4, preferably an integer of 1-3, more preferably an integer of 1-2.
r is an integer of 1-6, preferably an integer of 1-5, more preferably an integer of 1-4.
α is an integer of 1 to 6, preferably an integer of 1 to 5, more preferably an integer of 1 to 4.
Xは、同一又は異なって、Ile、Leu及びHypから選択されるいずれか少なくとも1種である。 X is the same or different and is at least one selected from Ile, Leu and Hyp.
また、一般式(I)に含まれるm+p個のXは、それぞれ同一又は異なっていてもよい。すなわち、m及びpがそれぞれ2以上である場合、(Lys X Gly)の繰り返し単位のなかでXが同一又は異なっていてもよい。具体的には、(Lys X Gly)mで示される構成単位が、繰り返し単位(Lys Ile Gly)のみによって構成されるアミノ酸配列、繰り返し単位(Lys Lue Gly)のみによって構成されるアミノ酸配列、繰り返し単位(Lys Hyp Gly)のみによって構成されるアミノ酸配列、又はこれらの繰り返し単位を任意の順で組み合わせたアミノ酸配列によって構成されるアミノ酸配列であることを示す。本発明の一般式(I)において、XはIle又はHypであることが好ましい。In addition, m + p Xs contained in the general formula (I) may be the same or different from each other. That is, when m and p are each 2 or more, X may be the same or different in the repeating unit of (Lys X Gly). Specifically, the structural unit represented by (Lys X Gly) m is an amino acid sequence composed only of a repeating unit (Lys Ile Gly), an amino acid sequence composed only of a repeating unit (Lys Lue Gly), and a repeating unit. It shows that the amino acid sequence is composed of only (Lys Hyp Gly), or is composed of amino acid sequences obtained by combining these repeating units in any order. In the general formula (I) of the present invention, X is preferably Ile or Hyp.
上記一般式(I)において、n+m+l+p+r=4〜15、好ましくは4〜13、より好ましくは4〜12である。但し、l=0の時、m+p≧2を満たし、α≧2のときは、m=1、l=0、且つn=1を満たす。 In the said general formula (I), it is n + m + l + p + r = 4-15, Preferably it is 4-13, More preferably, it is 4-12. However, when l = 0, m + p ≧ 2 is satisfied, and when α ≧ 2, m = 1, l = 0, and n = 1 are satisfied.
さらに、上記一般式(I)によって提供されるペプチドのアミノ酸配列は、抗菌活性の観点から、以下の態様が好ましい例として挙げられる。 Furthermore, as for the amino acid sequence of the peptide provided by the above general formula (I), the following embodiments are preferable examples from the viewpoint of antibacterial activity.
lが0のとき、nは1〜6;mは1〜4、好ましくは1〜3;pは1〜4、好ましくは1〜3;rは1〜6;α=1である。 When l is 0, n is 1-6; m is 1-4, preferably 1-3; p is 1-4, preferably 1-3; r is 1-6; α = 1.
lが1のとき、nは1〜6;mは1〜4、好ましくは1〜3;pは1〜4、好ましくは1〜3;rは1〜6;α=1である。 When l is 1, n is 1-6; m is 1-4, preferably 1-3; p is 1-4, preferably 1-3; r is 1-6; α = 1.
lが2のとき、nは1〜6;mは1〜4、好ましくは1〜3;pは1〜4、好ましくは1〜3;rは1〜6;α=1である。 When l is 2, n is 1-6; m is 1-4, preferably 1-3; p is 1-4, preferably 1-3; r is 1-6; α = 1.
lが3のとき、nは1〜6;mは1〜4、好ましくは1〜3;pは1〜4、好ましくは1〜3;rは1〜6;α=1である。 When l is 3, n is 1-6; m is 1-4, preferably 1-3; p is 1-4, preferably 1-3; r is 1-6; α = 1.
また、α≧2のとき、lは0;nは1〜6、好ましくは1〜4;mは1;pは1;rは1〜6、好ましくは1〜4である。 Further, when α ≧ 2, l is 0; n is 1 to 6, preferably 1 to 4; m is 1; p is 1; and r is 1 to 6, preferably 1 to 4.
また、lが0のとき、nは3〜6;mは1〜4;lは0〜7;pは1〜4;rは3〜6;α=1であり;XはIle又はLeuであり;m+p個のXは、それぞれ同一又は異なっていてもよく;n+m+l+p+r=8〜15であり;m+p≧2を満たす。 When l is 0, n is 3 to 6; m is 1 to 4; l is 0 to 7; p is 1 to 4; r is 3 to 6; α = 1; and X is Ile or Leu. Yes; m + p Xs may be the same or different; n + m + l + p + r = 8-15; m + p ≧ 2.
上記一般式(I)で表されるペプチドを構成するアミノ酸数は、本発明の効果を奏する限り特に限定されないが、通常12〜60、好ましくは15〜40、より好ましくは20〜35である。 The number of amino acids constituting the peptide represented by the general formula (I) is not particularly limited as long as the effect of the present invention is exhibited, but is usually 12 to 60, preferably 15 to 40, and more preferably 20 to 35.
さらに、上記一般式(I)で表されるペプチドとしては、具体的には、例えば下記(i)〜(vi)(配列番号1〜6)で表されるアミノ酸配列を有するものが挙げられる。
(i)(Pro Hyp Gly)3 -(Lys Ile Gly)3 -(Pro Hyp Gly)4(配列番号:1)
(ii)(Pro Hyp Gly)3 -Lys Ile Gly Pro Hyp Gly Lys Ile Gly-(Pro Hyp Gly)4(配列番号:2)
(iii)(Pro Hyp Gly)4 -(Lys Ile Gly)2 -(Pro Hyp Gly)4(配列番号:3)
(iv)(Pro Hyp Gly)3-(Lys Hyp Gly Lys Leu Gly)2-(Pro Hyp Gly)3(配列番号:4)
(v)(Pro Hyp Gly)2-(Lys Leu Gly Lys Hyp Gly)3-(Pro Hyp Gly)2(配列番号:5)
(vi)Pro Hyp Gly-(Lys Leu Gly Lys Hyp Gly)4-Pro Hyp Gly (配列番号:6)
(2)本発明には、さらに、一般式(I)で表されるアミノ酸配列において、1又は数個の任意のアミノ酸残基が置換、欠失若しくは付加されたアミノ酸配列からなり、かつ抗菌活性を有するペプチドも含まれる。Specific examples of the peptide represented by the general formula (I) include those having an amino acid sequence represented by the following (i) to (vi) (SEQ ID NOs: 1 to 6).
(i) (Pro Hyp Gly) 3- (Lys Ile Gly) 3- (Pro Hyp Gly) 4 (SEQ ID NO: 1)
(ii) (Pro Hyp Gly) 3 -Lys Ile Gly Pro Hyp Gly Lys Ile Gly- (Pro Hyp Gly) 4 (SEQ ID NO: 2)
(iii) (Pro Hyp Gly) 4 - (Lys Ile Gly) 2 - (Pro Hyp Gly) 4 ( SEQ ID NO: 3)
(iv) (Pro Hyp Gly) 3- (Lys Hyp Gly Lys Leu Gly) 2- (Pro Hyp Gly) 3 (SEQ ID NO: 4)
(v) (Pro Hyp Gly) 2- (Lys Leu Gly Lys Hyp Gly) 3- (Pro Hyp Gly) 2 (SEQ ID NO: 5)
(vi) Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) 4 -Pro Hyp Gly (SEQ ID NO: 6)
(2) The present invention further comprises an amino acid sequence in which one or several arbitrary amino acid residues are substituted, deleted or added in the amino acid sequence represented by the general formula (I), and has antibacterial activity. Also included are peptides having
上記(2)のペプチドにおいて、置換、欠失若しくは付加されるアミノ酸残基の数については、改変されたペプチドが抗菌活性を有している限り特に限定されない。 In the peptide of (2) above, the number of amino acid residues to be substituted, deleted or added is not particularly limited as long as the modified peptide has antibacterial activity.
例えば、アミノ酸残基の付加の場合であれば、当該付加されるアミノ酸残基の数としては、1〜3個、好ましくは1〜2個、さらに好ましくは1個である。 For example, in the case of addition of amino acid residues, the number of amino acid residues to be added is 1 to 3, preferably 1 to 2, and more preferably 1.
また、アミノ酸残基の欠失の場合であれば、当該欠失されるアミノ酸残基の数としては、1〜3個、好ましくは1〜2個、さらに好ましくは1個である。 In the case of deletion of amino acid residues, the number of amino acid residues to be deleted is 1 to 3, preferably 1 to 2, and more preferably 1.
さらに、アミノ酸残基の置換の場合であれば、当該置換されるアミノ酸残基の数としては、1〜3個、好ましくは1〜2個、さらに好ましくは1個である。 Further, in the case of substitution of amino acid residues, the number of amino acid residues to be substituted is 1 to 3, preferably 1 to 2, and more preferably 1.
また、上記(2)のペプチドにおいて、置換、欠失若しくは付加されるアミノ酸残基の位置は、改変されたペプチドが抗菌活性を有している限り、特に限定されない。ただし、アミノ酸の置換、欠失若しくは付加を行う場合は、前記一般式(I)においてLys、Gly及びX以外のアミノ酸残基について行うことが好ましい。 In the peptide (2), the position of the amino acid residue to be substituted, deleted or added is not particularly limited as long as the modified peptide has antibacterial activity. However, amino acid substitution, deletion or addition is preferably performed on amino acid residues other than Lys, Gly and X in the above general formula (I).
更に、上記(2)のペプチドにおいて、置換若しくは付加されるアミノ酸の種類については、改変されたペプチドが抗菌活性を有することを限度として、特に制限されない。付加されるアミノ酸の種類については、改変されたペプチドが抗菌活性を有している限りにおいて、天然アミノ酸、非天然アミノ酸の別を問わず、任意のアミノ酸を選択することができる。但し、置換については、各アミノ酸残基は側鎖官能基の性質に基づく相同性置換であることが望ましい。アミノ酸の置換の場合、例えば、ProはHypに、LeuはIle又はValにそれぞれ置換可能であり、その逆の置換も可能である。 Furthermore, in the peptide of (2), the type of amino acid to be substituted or added is not particularly limited as long as the modified peptide has antibacterial activity. As long as the modified peptide has antibacterial activity, any amino acid can be selected regardless of whether it is a natural amino acid or an unnatural amino acid. However, for substitution, each amino acid residue is preferably a homologous substitution based on the properties of the side chain functional group. In the case of amino acid substitution, for example, Pro can be substituted with Hyp, Leu can be substituted with Ile or Val, and vice versa.
本発明のペプチドは、アミノ末端の水素がアセチル基、メトキシカルボニル基、ブトキシカルボニル基、1〜3残基のペプチド等で置換されたものでも良い。また、C末端のカルボキシル基がアミド基、メチルエステル基、ブチルエステル基、1〜3残基のペプチド等で置換されたものでも良い。ここで、カルボキシル基が前記置換基で置換されている場合とは、それぞれ、カルボキシル基の-OHが、アミノ基、メトキシ基、ブトキシ基で置換されていることを指す。また、カルボキシル基がペプチドで置換されている場合とは、カルボキシル基と結合されるペプチド間で脱水縮合がおこり、ペプチド結合を形成することを指す。 The peptide of the present invention may be one in which the amino-terminal hydrogen is substituted with an acetyl group, a methoxycarbonyl group, a butoxycarbonyl group, a peptide having 1 to 3 residues, or the like. Further, the C-terminal carboxyl group may be substituted with an amide group, a methyl ester group, a butyl ester group, a peptide having 1 to 3 residues, or the like. Here, the case where the carboxyl group is substituted with the substituent means that —OH of the carboxyl group is substituted with an amino group, a methoxy group, or a butoxy group, respectively. Further, the case where the carboxyl group is substituted with a peptide means that dehydration condensation occurs between peptides bonded to the carboxyl group to form a peptide bond.
上記(1)又は(2)に示される本発明のペプチドは、薬学的に許容される塩を形成していても良く、その塩としては、例えば、塩酸、硫酸、燐酸、乳酸、酒石酸、マレイン酸、フマール酸、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 peptide of the present invention shown in the above (1) or (2) may form a pharmaceutically acceptable salt. Examples of the salt include hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid. Salts with acids such as acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, palmitic acid; alkali metals or alkaline earth metals such as sodium, potassium, calcium, or aluminum hydroxide or carbonic acid Salts with salts; salts with triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine, arginine and the like.
このような本発明のペプチドは、コラーゲン様三重らせん構造を有することを1つの特徴とするものである。コラーゲン様三重らせん構造とは、コラーゲン分子に見られる特徴的な構造であり、ロープ状の超らせん構造を示す。コラーゲンには、プロリン、ヒドロキシプロリンとグリシンが特に多く含まれ、グリシンは3残基ごとに存在する。このような特徴が安定な三重らせん構造の形成に重要であるとされている。本発明においては、この様な構造が本発明のペプチドの安定性を高める役割があると考えられる。 Such a peptide of the present invention is characterized by having a collagen-like triple helix structure. The collagen-like triple helix structure is a characteristic structure found in collagen molecules, and shows a rope-like super helical structure. Collagen contains a particularly large amount of proline, hydroxyproline and glycine, and glycine is present every 3 residues. Such characteristics are considered to be important for the formation of a stable triple helical structure. In the present invention, such a structure is considered to have a role of enhancing the stability of the peptide of the present invention.
本発明のペプチドは、通常のペプチド合成方法によって得ることができ、例えば固相合成法または液相合成法によって調製されるが、固相合成法が操作上簡便である。固相合成法は、例えば、日本生化学会編「続生化学実験講座2 タンパク質の化学(下)」(昭和62年5月20日 株式会社東京化学同人発行)、第641−694頁に記載の方法を参考に実施することができる。 The peptide of the present invention can be obtained by an ordinary peptide synthesis method. For example, it is prepared by a solid phase synthesis method or a liquid phase synthesis method, but the solid phase synthesis method is simple in terms of operation. The solid-phase synthesis method is described in, for example, “Sequel Biochemistry Experiment Course 2 Protein Chemistry (2)” (published May 20, 1987, published by Tokyo Kagaku Dojin), pages 641-694. The method can be carried out with reference to the method.
本発明のペプチドの固相合成法による調製は、例えば、スチレン−ジビニルベンゼン共重合体などの反応溶媒に不溶性である重合体に目的とするペプチドのC末端に対応するアミノ酸をそれが有するα−COOH基を介して結合させ、次いで該アミノ酸に目的とするペプチドのN末端の方向に向かって、対応するアミノ酸またはペプチド断片を該アミノ酸またはペプチド断片が有するα−COOH基以外のα−アミノ基などの官能基を保護したうえで縮合させて結合させる操作と、該結合したアミノ酸またはペプチド断片におけるα−アミノ基などのペプチド結合を形成するアミノ基が有する保護基を除去する操作とを順次繰り返すことによってペプチド鎖を伸長させ、目的とするペプチドに対応するペプチド鎖を形成し、次いで該ペプチド鎖を重合体から脱離させ、かつ保護されている官能基から保護基を除去することにより目的とするペプチドを得、次いでこれを精製することによって実施される。 Preparation of the peptide of the present invention by a solid phase synthesis method includes, for example, an α-amino acid having an amino acid corresponding to the C-terminus of the target peptide in a polymer that is insoluble in a reaction solvent such as a styrene-divinylbenzene copolymer. An α-amino group other than the α-COOH group that the amino acid or peptide fragment has a corresponding amino acid or peptide fragment attached to the amino acid in the direction of the N-terminal of the target peptide, etc. The procedure of condensing and conjugating the functional group of the amino acid and the step of removing the protective group of the amino group that forms a peptide bond such as an α-amino group in the bound amino acid or peptide fragment is sequentially repeated. To elongate the peptide chain to form a peptide chain corresponding to the target peptide, and then the peptide chain Desorbed from the polymer, and obtain a peptide of interest by removing the protecting group from the functional group being protected, and then carried out by purification.
ここで、ペプチド鎖の重合体からの脱離および保護基の除去は、トリフルオロ酢酸を用いて同時に行うのが副反応を抑制する観点から好ましい。また、得られたペプチドの精製は逆相液体クロマトグラフィ−やゲルパーミエイションクロマトグラフィーで行うのが効果的である。 Here, the elimination of the peptide chain from the polymer and the removal of the protecting group are preferably performed simultaneously using trifluoroacetic acid from the viewpoint of suppressing side reactions. Moreover, it is effective to purify the obtained peptide by reverse phase liquid chromatography or gel permeation chromatography.
また、本発明のペプチドの塩は、通常の塩生成反応を利用することにより調製される。 Moreover, the salt of the peptide of this invention is prepared by utilizing normal salt formation reaction.
上記(1)又は(2)で表されるペプチドは、優れた抗菌活性を有する。本発明において抗菌活性を有するペプチドとは、細菌および/または真菌に対する制菌作用又は殺菌作用を有するペプチドであることを意味する。本発明のペプチド(又はその塩)の抗菌活性の測定は、通常以下の方法により行われる。DPPG(dipalmitoylphosphatidylglycerol)を主構成脂質とするリポソームからの内包する蛍光色素(carboxyfluorescein)の漏出量(Biochim Biophys Acta, 1070, 259-264, 1991)を測定して行われる。具体的な測定方法の例として、以下の方法が挙げられる。 The peptide represented by the above (1) or (2) has excellent antibacterial activity. In the present invention, the peptide having antibacterial activity means a peptide having antibacterial action or bactericidal action against bacteria and / or fungi. The antibacterial activity of the peptide of the present invention (or a salt thereof) is usually measured by the following method. It is carried out by measuring the leakage amount (Biochim Biophys Acta, 1070, 259-264, 1991) of a fluorescent dye (carboxyfluorescein) encapsulated from a liposome containing DPPG (dipalmitoylphosphatidylglycerol) as a main constituent lipid. Examples of specific measurement methods include the following methods.
<細菌膜モデルリポソームの作製>
ジパルミトイルホスファチジルグリセロール(DPPG:シグマP9789)0.7 mgを含む塩化
メチレン/メタノール混合溶液を褐色ガラスバイアルに入れ、窒素ガスを吹き込むことにより、バイアルの内壁に脂質膜を形成させる。カルボキシフルオレセイン(CF:シグマC7153)を20 mMの濃度で含む、1 mM EDTA/TBS溶液1 mLをガラスバイアルに加え、ボルテクスミキサーで脂質膜がバイアルの内壁から剥離するまで良く攪拌する。次に、60℃で20分間超音波処理を行った。さらに、PD10カラム(GEヘルスケアバイオサイエンス)で3-5 mLの分画を分取して、リポソーム溶液とする。<Preparation of bacterial membrane model liposome>
A methylene chloride / methanol mixed solution containing 0.7 mg of dipalmitoylphosphatidylglycerol (DPPG: Sigma P9789) is placed in a brown glass vial, and nitrogen gas is blown to form a lipid film on the inner wall of the vial. Add 1 mL of 1 mM EDTA / TBS solution containing carboxyfluorescein (CF: Sigma C7153) at a concentration of 20 mM to a glass vial, and stir well with a vortex mixer until the lipid membrane peels from the inner wall of the vial. Next, ultrasonic treatment was performed at 60 ° C. for 20 minutes. Furthermore, 3-5 mL fractions are collected with a PD10 column (GE Healthcare Bioscience) to obtain a liposome solution.
<哺乳類膜モデルリポソームの作製>
超音波処理を室温で行う以外は上記と同様の操作で、ホスファチジルコリン(PC:シグマP2772 Type XI-E)からなるリポソームを得る。<Production of mammalian membrane model liposome>
A liposome composed of phosphatidylcholine (PC: Sigma P2772 Type XI-E) is obtained in the same manner as described above except that sonication is performed at room temperature.
<抗菌性評価>
得られたDPPGリポソーム、PCリポソームのそれぞれをPBSで10倍希釈した溶液200 μLに対して、抗菌活性を評価する対象となるペプチドを最終濃度が0.25〜0.005 mMに成るように加え、15分間室温で静置する。その後、12,000 rpmで5分間遠心して得られた上清中のCFの量を、励起波長485 nm、蛍光波長535 nmで測定する。ペプチド溶液の代わりにPBSを加えた場合の蛍光を0%、最終濃度0.1%のTriton X-100を加えた場合の蛍光を100%として、各ペプチドによるCF漏出量を計算する。<Antimicrobial evaluation>
The peptide to be evaluated for antibacterial activity was added to 200 μL of the obtained DPPG liposome and PC liposome diluted 10-fold with PBS so that the final concentration would be 0.25 to 0.005 mM, and the mixture was allowed to stand at room temperature for 15 minutes. Leave at rest. Thereafter, the amount of CF in the supernatant obtained by centrifugation at 12,000 rpm for 5 minutes is measured at an excitation wavelength of 485 nm and a fluorescence wavelength of 535 nm. The amount of CF leakage due to each peptide is calculated assuming that the fluorescence when PBS is added instead of the peptide solution is 0% and the fluorescence when Triton X-100 at a final concentration of 0.1% is added is 100%.
上記試験においてPBSを加えた場合の蛍光を0%、最終濃度0.1%のTriton X-100を加えた場合の蛍光を100%とした場合に、CF漏出による蛍光が3%以上、好ましくは5%以上、より好ましくは10%以上のものを、本発明において抗菌活性を有するペプチドという。 In the above test, the fluorescence due to CF leakage is 3% or more, preferably 5% when the fluorescence when PBS is added is 0% and the fluorescence when Triton X-100 is added at a final concentration of 0.1% is 100%. Above, more preferably 10% or more is referred to as a peptide having antibacterial activity in the present invention.
また、黄色ブドウ球菌などに対する抗菌活性(参考文献:Nature, 443, 867-869, 2006)の測定方法に基づいて抗菌活性を評価することも可能である。上記リポソームからの蛍光色素(carboxyfluorescein)の漏出量による測定方法と、黄色ブドウ球菌などに対する抗菌活性の測定方法の少なくともいずれか一方、好ましくは両方によって抗菌活性が確認されたものを本発明のペプチドとする。 It is also possible to evaluate antibacterial activity based on a method for measuring antibacterial activity against Staphylococcus aureus (Reference: Nature, 443, 867-869, 2006). At least one of the method for measuring the leakage amount of fluorescent dye (carboxyfluorescein) from the liposome and the method for measuring antibacterial activity against Staphylococcus aureus, preferably the antibacterial activity confirmed by both and To do.
本発明の限定的な解釈を望むものではないが、本発明のペプチド(又はその塩)は、細菌の膜の構造に選択的に作用し、細菌の膜構造を破壊することによって抗菌活性を発揮するものであると考えられることから、幅広い種の細菌に対して有効に作用することができるものである。本発明のペプチドが優れた抗菌作用を示す細菌としては、グラム陰性細菌、グラム陽性細菌等、特に限定されず、例えば、黄色ブドウ球菌、レンサ球菌、緑膿菌、大腸菌等に対して優れた抗菌作用を発揮するものである。 Although not wishing to be limited to the interpretation of the present invention, the peptide (or salt thereof) of the present invention selectively acts on the structure of the bacterial membrane and exhibits antibacterial activity by destroying the bacterial membrane structure. Therefore, it can act effectively against a wide variety of bacteria. Bacteria exhibiting excellent antibacterial activity of the peptide of the present invention are not particularly limited, such as Gram-negative bacteria, Gram-positive bacteria, etc. It is effective.
さらに、本発明のペプチド又はその塩は、上記一般細菌のみならず、真菌に対しても有効に作用し得るものである。 Furthermore, the peptide of the present invention or a salt thereof can effectively act not only on the above general bacteria but also on fungi.
このように、本発明のポリペプチド又はその塩は、幅広い細菌及び真菌に対して優れた抗菌活性を有することから、当該ペプチド又は塩を1種単独又は2種以上を組み合わせて、抗菌剤又は抗真菌剤として使用することができる。また、本発明のポリペプチド又はその塩の抗菌作用(抗菌活性)に基づいて、医薬製剤として感染症の予防等を目的として使用することもできる。さらには、本発明のペプチド又はその塩を、防腐等を目的として医薬製剤、医療用基材、化粧料、食品、医薬部外品等の添加対象物に配合して使用することもできる。以下、それぞれの使用形態に応じて詳述する。 Thus, since the polypeptide of the present invention or a salt thereof has excellent antibacterial activity against a wide range of bacteria and fungi, the peptide or salt can be used alone or in combination of two or more to obtain an antibacterial agent or antibacterial agent. It can be used as a fungicide. Moreover, based on the antibacterial action (antibacterial activity) of the polypeptide of the present invention or a salt thereof, it can also be used as a pharmaceutical preparation for the purpose of preventing infectious diseases. Furthermore, the peptide of the present invention or a salt thereof can be used by adding it to an additive such as a pharmaceutical preparation, a medical base, a cosmetic, a food, or a quasi-drug for the purpose of preserving. Hereinafter, it explains in full detail according to each usage form.
[感染症の予防等を目的とする使用形態]
本発明のペプチド又はその塩は、上記抗菌活性に基づいて、医薬分野において有用な効果を奏し得る。本発明のペプチド又はその塩は、黄色ブドウ球菌、緑膿菌等の細菌及び真菌に対して優れた抗菌活性を有することから、本発明のペプチド又はその塩と薬学的に許容される基材又は担体と混合し、医薬製剤として調製し、使用することができる。医薬製剤として本発明のペプチド又はその塩を使用する場合、当該ペプチド又は塩を1種単独又は2種以上を組み合わせて使用することもできる。[Usage for the purpose of prevention of infectious diseases]
The peptide of the present invention or a salt thereof can exert a useful effect in the pharmaceutical field based on the antibacterial activity. Since the peptide of the present invention or a salt thereof has excellent antibacterial activity against bacteria and fungi such as Staphylococcus aureus and Pseudomonas aeruginosa, the peptide of the present invention or a salt thereof and a pharmaceutically acceptable substrate or It can be mixed with a carrier and prepared and used as a pharmaceutical preparation. When using the peptide of this invention or its salt as a pharmaceutical formulation, the said peptide or salt can also be used individually by 1 type or in combination of 2 or more types.
当該医薬製剤は、上記抗菌活性に基づく使用用途、即ち、感染症、敗血症等の予防又は治療に有用である。また、本発明のペプチド又はその塩は、真菌に対しても優れた抗菌活性を有することから、抗真菌剤として白癬(足白癬(いわゆる水虫)、爪白癬、手白癬等)、カンジダ症等の治療を目的する医薬製剤の形態に調製して使用することもできる。 The pharmaceutical preparation is useful for use based on the antibacterial activity, that is, for prevention or treatment of infectious diseases, sepsis and the like. In addition, since the peptide of the present invention or a salt thereof has excellent antibacterial activity against fungi, antifungal agents such as ringworm (foot ringworm (so-called athlete's foot), nail ringworm, hand ringworm, etc.), candidiasis, etc. It can also be prepared and used in the form of a pharmaceutical formulation for therapeutic purposes.
当該医薬製剤に使用される基材又は担体については、薬学的に許容されることを限度として特に制限されず、医薬の製剤化のために配合されている成分を広く使用することができる。また、当該医薬製剤には、上記成分の他に、添加剤や薬理活性成分を含んでいても良い。 The base material or carrier used in the pharmaceutical preparation is not particularly limited as long as it is pharmaceutically acceptable, and a wide variety of ingredients blended for pharmaceutical preparation can be used. The pharmaceutical preparation may contain additives and pharmacologically active ingredients in addition to the above ingredients.
当該医薬製剤の剤型としては、適用形態に応じて適宜設定されるが、一例として、錠剤、散剤、粉末剤、顆粒剤、カプセル剤等の固形製剤;及び液剤、乳剤、懸濁剤等の液状製剤;軟膏、クリーム等の半固形製剤が挙げられる。また、当該医薬製剤は、本発明のペプチド又はその塩をリポソ−ム化して製剤化したものであってもよい。 The dosage form of the pharmaceutical preparation is appropriately set according to the application form, but examples include solid preparations such as tablets, powders, powders, granules, capsules; and liquids, emulsions, suspensions, etc. Liquid preparations; semi-solid preparations such as ointments and creams. In addition, the pharmaceutical preparation may be prepared by liposomal formation of the peptide of the present invention or a salt thereof.
当該医薬製剤の投与形態については、例えば、静脈内投与、皮下投与、腹腔内投与、経口投与等の全身的投与;関節内投与等の局所投与等が挙げられ、用途や剤型等に応じて適宜設定することができる。また、疾患部位に塗布や散布により本発明のペプチドを、外用剤として直接投与する経皮投与の形態なども挙げられる。 Examples of the administration form of the pharmaceutical preparation include systemic administration such as intravenous administration, subcutaneous administration, intraperitoneal administration and oral administration; local administration such as intra-articular administration, etc. It can be set appropriately. Moreover, the form of transdermal administration which directly administers the peptide of this invention as an external preparation by application | coating or dispersion | spreading to a disease site etc. are mentioned.
また、全身に投与する場合にはペプチドを5%ブドウ糖液や生理食塩水などの薬学的に許容し得る溶液に溶解させて得られる溶液が好ましい。該溶液は薬理学的に許容される種々の添加剤を含んでいてもよい。さらにペプチド類をカプセル化またはリポソ−ム化することも可能である。局所投与する場合にも、5%ブドウ糖液や生理食塩水などの溶液や通常用いられる軟膏基剤やクリーム基材に混合することもできる。 When administered systemically, a solution obtained by dissolving the peptide in a pharmaceutically acceptable solution such as 5% glucose solution or physiological saline is preferable. The solution may contain various pharmacologically acceptable additives. It is also possible to encapsulate or liposome peptides. In the case of topical administration, it can be mixed with a solution such as 5% glucose solution or physiological saline, or a commonly used ointment base or cream base.
本発明の医薬製剤を、例えば静注する場合の投与量は、患者の性別や年齢、症状、投与形態、期待される効果等に基づいて、適宜設定することができる。当該医薬製剤の1日当たりの投与量の一例として、本発明のペプチド又はその塩の投与量に換算して、通常0.1μg/kg〜3g/kg(成人)であり、好ましくは1μg/kg〜500mg/kg(成人)、より好ましくは1μg/kg〜100mg/kg(成人)が挙げられる。 For example, the dosage when the pharmaceutical preparation of the present invention is intravenously administered can be appropriately set based on the sex, age, symptom, dosage form, expected effect and the like of the patient. As an example of the daily dose of the pharmaceutical preparation, it is usually 0.1 μg / kg to 3 g / kg (adult), preferably 1 μg / kg to the dose of the peptide of the present invention or a salt thereof. Examples include 500 mg / kg (adult), more preferably 1 μg / kg to 100 mg / kg (adult).
また、本発明の医薬製剤を外用剤の形態で用いる場合は、患部の大きさや症状に応じて適量を塗布すればよいが、例えば、1〜10cm2の患部にペプチドに換算して0.01μg〜100mg程度、好ましくは0.1μg〜10mgとなるように適用すればよい。In addition, when the pharmaceutical preparation of the present invention is used in the form of an external preparation, an appropriate amount may be applied according to the size and symptoms of the affected part. For example, 0.01 μg in terms of peptide in the affected part of 1 to 10 cm 2 What is necessary is just to apply so that it may become about -100 mg, Preferably it is 0.1 microgram-10 mg.
加えて、本発明は、上記ペプチド又はその塩の有効量を患者に投与することを特徴とする、細菌感染症、敗血症、白癬(足白癬(いわゆる水虫)、爪白癬、手白癬等)、カンジダ症等の予防又は治療方法をも提供し得るものである。当該治療方法における本発明のペプチド又はその塩の有効量及び投与方法は、上記投与量、投与方法に基づいて適宜設定することができる。 In addition, the present invention provides a bacterial infection, sepsis, ringworm (eg, tinea pedis (so called athlete's foot), nail ringworm, hand ringworm, etc.) It is also possible to provide a method for preventing or treating diseases. The effective amount and administration method of the peptide of the present invention or a salt thereof in the treatment method can be appropriately set based on the above-mentioned dose and administration method.
さらに、本発明は、本発明のペプチド又はその塩を、薬学的に許容される基材に担持させた医療用基材をも提供し得る。本発明に使用され得る基材としては、例えばアルギン酸ゲル、コラーゲンゲル、乳酸ポリマー、セルロースなどの生分解性基材が好ましい。このような基材に、本発明のペプチド又はその塩を物理的または化学的に結合させることによって担持させることができる。ここで、コラーゲンゲルとしては、人工的に製造されたものであってもよく、ウシ、ブタ等の動物から得られる天然由来のものであってもよいが、動物由来の病原体感染を防止する意味で、人工コラーゲンを用いることが好ましい。 Furthermore, this invention can also provide the medical base material which carry | supported the peptide of this invention, or its salt on the pharmaceutically acceptable base material. As the base material that can be used in the present invention, biodegradable base materials such as alginic acid gel, collagen gel, lactic acid polymer, and cellulose are preferable. Such a substrate can be supported by physically or chemically bonding the peptide of the present invention or a salt thereof. Here, the collagen gel may be artificially produced, or may be naturally derived from animals such as cows and pigs, but it means to prevent animal-derived pathogen infection. Therefore, it is preferable to use artificial collagen.
また、本発明のポリペプチド又はその塩を、生分解性基材と混合し、医療用基材を形成することもできる。例えば、人工コラーゲンゲルに本発明のペプチドを結合させ、人工皮膚の材料として使用することもできる。 In addition, the polypeptide of the present invention or a salt thereof can be mixed with a biodegradable substrate to form a medical substrate. For example, the peptide of the present invention can be bound to an artificial collagen gel and used as a material for artificial skin.
本発明は、このような医療用基材を、患部に適用することを特徴とする細菌感染症の予防又は治療方法をも提供するものである。例えば、本発明のペプチド又はその塩が担持された(又は混合された)人工皮膚を用いて創面や、分層皮膚移植、全層皮膚移植等による移植部分を覆い、細菌感染を予防することができる。当該医療用基材を用いた細菌感染症の予防又は治療方法においては、医療用基材の形態に応じて適宜適用方法を選択することができる。 The present invention also provides a method for preventing or treating bacterial infection, characterized by applying such a medical substrate to an affected area. For example, the artificial skin carrying (or mixed with) the peptide of the present invention or a salt thereof may be used to cover a wound surface, a grafted portion by split-layer skin grafting, full-thickness skin grafting, or the like to prevent bacterial infection. it can. In the prevention or treatment method of bacterial infection using the medical base material, an appropriate application method can be selected according to the form of the medical base material.
医療用基材に担持又は混合される本発明のペプチド又はその塩の使用量は、上記抗菌活性に基づいて適宜設定することができ、特に限定されないが、例えば医療用基材の乾燥重量あたり0.01〜10重量%、好ましくは0.05〜5重量%程度、より好ましくは0.1〜2重量%である。
[防腐を目的として添加対象物に配合する使用形態]
本発明のペプチド又はその塩は、幅広い抗菌活性を有することから、防腐を目的として医薬製剤、化粧料、医療用基材、食品、医薬部外品等の添加対象物に配合することができる。The amount of the peptide of the present invention or a salt thereof carried or mixed on a medical substrate can be appropriately set based on the antibacterial activity, and is not particularly limited. For example, it is 0 per dry weight of the medical substrate. 0.01 to 10% by weight, preferably about 0.05 to 5% by weight, more preferably 0.1 to 2% by weight.
[Usage form to be added to the additive for the purpose of antiseptic]
Since the peptide of the present invention or a salt thereof has a wide range of antibacterial activity, it can be blended with additives such as pharmaceutical preparations, cosmetics, medical bases, foods and quasi drugs for the purpose of preserving.
前記添加対象物が医薬製剤である場合、本発明のペプチド又はその塩が添加される製剤の形態は特に限定されないが、例えば、固形製剤、液状製剤、半固形製剤が挙げられる。 When the addition target is a pharmaceutical preparation, the form of the preparation to which the peptide of the present invention or a salt thereof is added is not particularly limited, and examples thereof include a solid preparation, a liquid preparation, and a semi-solid preparation.
また、前記添加対象物が化粧料である場合、その化粧料の形態は、特に制限されないが、例えば、クレンジング剤、皮膚洗浄料、マッサージ剤、軟膏、クリーム、ローション、オイル、パック、洗顔料、化粧水、乳液、ゼリー等が挙げられる。 When the additive is a cosmetic, the form of the cosmetic is not particularly limited. For example, a cleansing agent, a skin cleanser, a massage agent, an ointment, a cream, a lotion, an oil, a pack, a face wash, A lotion, a milky lotion, a jelly, etc. are mentioned.
従来、抗菌剤や防腐剤としてパラベン等が汎用されてきたが、使用の際に臭いが気になる等の問題があった。本発明のペプチドを抗菌剤(又は防腐剤)として配合した場合は、この様な臭いもない。また、本発明のペプチドは、熱、塩濃度等の影響を受けにくく、化粧料中においても長期間にわたって安定した抗菌活性を保持することができる。従って、本発明のペプチド又はその塩を従来の抗菌剤や防腐剤に代えて使用することができる。 Conventionally, parabens and the like have been widely used as antibacterial agents and preservatives, but there has been a problem such as annoying odors during use. When the peptide of the present invention is formulated as an antibacterial agent (or preservative), there is no such odor. In addition, the peptide of the present invention is hardly affected by heat, salt concentration, etc., and can maintain stable antibacterial activity for a long time even in cosmetics. Therefore, the peptide of the present invention or a salt thereof can be used in place of a conventional antibacterial agent or preservative.
さらに、前記添加対象物が医療用基材である場合、本発明のペプチド又はその塩を、アルギン酸ゲル、人工コラーゲンゲル、乳酸ポリマー、セルロースなどの生分解性基材に結合させるか、これらの生分解性基材に混合して用いることができる。 Further, when the object to be added is a medical base material, the peptide of the present invention or a salt thereof is bound to a biodegradable base material such as an alginate gel, an artificial collagen gel, a lactic acid polymer, cellulose, or the like. It can be used by mixing with a degradable substrate.
本発明のペプチド又はその塩を、防腐等を目的として上記添加対象物に配合する場合の配合量としては、ペプチドに換算して、添加対象物中0.01〜10重量%、好ましくは0.05〜5重量%、より好ましくは0.1〜2重量%が挙げられる。このとき本発明のペプチド又はその塩を、1種単独で又は2種以上を組み合わせて使用することができる。本発明のペプチド又はその塩を従来公知の方法に従って、上記添加対象物に配合することができる。 In the case where the peptide of the present invention or a salt thereof is blended in the above-mentioned addition target for the purpose of preserving or the like, the amount to be added is 0.01 to 10% by weight, preferably 0.05 to 5% in the addition target in terms of peptide. %, More preferably 0.1 to 2% by weight. At this time, the peptide of this invention or its salt can be used individually by 1 type or in combination of 2 or more types. The peptide of this invention or its salt can be mix | blended with the said addition target according to a conventionally well-known method.
以下、実施例により本発明を具体的に説明する。なお、本発明はこれらの実施例により限定されるものではない。
[実施例1]
(Pro Hyp Gly)3 -(Lys Ile Gly)3 -(Pro Hyp Gly)4(配列番号:1)で示されるアミノ酸配列からなるペプチドを、ペプチド自動合成装置を用いて固相合成法により合成した。すなわち、4−(Nα−9−(フルオレニルメトキシカルボニル)−グリシン)−オキシメチル−フェノキシ−メチル基を0.65 mmol/g(樹脂)の割合で含むスチレン−ジビニルベンゼン共重合体〔スチレンとジビニルベンゼンの構成モル比:99対1〕からなる粒状樹脂〔米国アプライド・バイオシステムズ社製、HMPグリシン〕0.1ミリモルを用い、目的とするペプチドのカルボキシル末端からアミノ末端に向かって順次対応するアミノ酸を結合させた。Hereinafter, the present invention will be described specifically by way of examples. In addition, this invention is not limited by these Examples.
[Example 1]
A peptide having the amino acid sequence represented by (Pro Hyp Gly) 3- (Lys Ile Gly) 3- (Pro Hyp Gly) 4 (SEQ ID NO: 1) was synthesized by a solid phase synthesis method using an automatic peptide synthesizer. . That is, a styrene-divinylbenzene copolymer containing 4- (Nα-9- (fluorenylmethoxycarbonyl) -glycine) -oxymethyl-phenoxy-methyl group at a rate of 0.65 mmol / g (resin) [styrene and divinyl Amino acid corresponding sequentially from the carboxyl terminus to the amino terminus of the target peptide using 0.1 millimole of granular resin (HMP glycine, manufactured by Applied Biosystems, USA) consisting of 99: 1 benzene molar ratio Were combined.
結合反応において、アミノ酸として、アプライド・バイオシステムズ社(米国)製のNα−9− (フルオレニルメトキシカルボニル)−L−プロリン〔Fmocプロリン〕、Nα−9−(フルオレニルメトキシカルボニル)−Nε−ブトキシカルボニル−L−リジン〔Fmocリジン〕、Nα−9−(フルオレニルメトキシカルボニル)−L−イソロイシン〔Fmocイソロイシン〕、Nα−9−(フルオレニルメトキシカルボニル)−グリシン〔Fmocグリシン〕、BACHEM社(ドイツ)製Nα−9−(フルオレニルメトキシカルボニル)−O−ブトキシカルボニル−L−ヒドロキシプロリン〔Fmocヒドロキシプロリン〕を、各結合ステップについてそれぞれ1ミリモルずつ用いた。In binding reaction, as the amino acid, Applied Biosystems (USA) for N alpha-9-(fluorenyl methoxy carbonyl) -L- proline [Fmoc-proline], N alpha-9-(fluorenyl methoxy carbonyl) -N epsilon - butoxycarbonyl -L- lysine [Fmoc lysine], N α -9- (fluorenyl methoxy carbonyl) -L- isoleucine [Fmoc isoleucine], N α -9- (fluorenyl methoxy carbonyl) - glycine [Fmoc-glycine], BACHEM Inc. of Germany made N alpha-9-(fluorenyl methoxy carbonyl) -O- butoxycarbonyl -L- hydroxyproline [Fmoc hydroxyproline], using one mmole respectively for each coupling step .
得られたペプチド樹脂を、5%の水を含むトリフルオロ酢酸10 mlで3時間処理した。得られた溶液をジエチルエーテルに加えて生じる沈殿をさらに数回ジエチルエーテルで洗浄して、ペプチドの脱保護と樹脂からの脱離を行った。粗生成物をPD10カラム(GEヘルスケアバイオサイエンス)で精製してペプチドを得た。得られた精製ペプチドをHPLC〔GEヘルスケアバイオサイエンス株式会社製AKTA explorer10XTシステム、カラム:ミリポアウオーターズ株式会社製ノバパックC18 3.9 mmφ×150 mm、移動相:トリフルオロ酢酸を0.05容量%含有するアセトニトリルと水の混合溶媒(アセトニトリル濃度を30分間で5容量%から50容量%に直線的に変化させた)、流速1.0 ml/min〕に付したところ、12.96 minに単一のピ−クが示された。 The obtained peptide resin was treated with 10 ml of trifluoroacetic acid containing 5% water for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was further washed several times with diethyl ether to deprotect the peptide and desorb from the resin. The crude product was purified with a PD10 column (GE Healthcare Bioscience) to obtain a peptide. The purified peptide thus obtained was subjected to HPLC [AKTA explorer10XT system manufactured by GE Healthcare Biosciences Co., Ltd., column: Novapack C18 manufactured by Millipore Waters Co., Ltd., 3.9 mmφ × 150 mm, mobile phase: acetonitrile containing 0.05% by volume of trifluoroacetic acid. And a mixed solvent of acetonitrile and water (acetonitrile concentration was linearly changed from 5 vol% to 50 vol% over 30 minutes), flow rate 1.0 ml / min], a single peak was shown at 12.96 min. It was done.
得られたペプチドをゲルパーミエーションクロマトグラフィー(GPC;GEヘルスケアバイオサイエンス(株)製、AKTApurifierシステム、カラム:Supedex peptide 10/30GL、流速:0.5 mL/min、溶離液:150 mMのNaClを含む10mM phosphate buffer(pH7.4))に供したところ、溶出体積10.18 mLに主ピークと9 mL付近に副ピークが認められた。また、得られたペプチドの円二色性スペクトル(CD)を測定したところ、223 nmに正のコットン効果、199 nmに負のコットン効果が観測され、3重らせん構造を含むことが確認された。
The obtained peptide is gel permeation chromatography (GPC; manufactured by GE Healthcare Biosciences, AKTApurifier system, column:
[実施例2及び3]
(Pro Hyp Gly)3 -Lys Ile Gly Pro Hyp Gly Lys Ile Gly-(Pro Hyp Gly)4(配列番号:2)で示されるアミノ酸配列をからなるペプチド(実施例2)、(Pro Hyp Gly)4 -(Lys Ile Gly)2-(Pro Hyp Gly)4(配列番号:3)で示されるアミノ酸配列からなるペプチド(実施例3)を、ペプチド自動合成装置を用いて固相合成法により合成した。[Examples 2 and 3]
(Pro Hyp Gly) 3 -Lys Ile Gly Pro Hyp Gly Lys Ile Gly- (Pro Hyp Gly) 4 (Example 2), (Pro Hyp Gly) 4 A peptide (Example 3) having an amino acid sequence represented by-(Lys Ile Gly) 2- (Pro Hyp Gly) 4 (SEQ ID NO: 3) was synthesized by a solid phase synthesis method using an automatic peptide synthesizer.
すなわち、実施例2では、4−(Nα−9−(フルオレニルメトキシカルボニル)−グリシン)−オキシメチル−フェノキシ−メチル基を0.65 mmol/g(樹脂)の割合で含むス
チレン−ジビニルベンゼン共重合体〔スチレンとジビニルベンゼンの構成モル比:99対1〕からなる粒状樹脂〔米国アプライド・バイオシステムズ社製、HMPグリシン〕の代わりに、4−(2’、4’−ジメトキシフェニル−フルオレニルメトキシカルボニル)−アミノメチル)−フェノキシアセトアミド−エチル基を0.62 mmol/g(樹脂)の割合で有するスチレン−ジビニルベンゼン共重合体〔スチレンとジビニルベンゼンの構成モル比:99対1〕からなる粒状樹脂〔米国アプライド・バイオシステムズ社製、Fmocアミドレジン〕を用いる以外は実施例1と同様にして合成、ペプチドの脱保護と樹脂からの脱離、精製を行った。実施例3についても実施例1と同様の方法に従ってペプチドを調製した。That is, in Example 2, styrene-divinylbenzene copolymer containing 4- (Nα-9- (fluorenylmethoxycarbonyl) -glycine) -oxymethyl-phenoxy-methyl group at a rate of 0.65 mmol / g (resin). 4- (2 ′, 4′-dimethoxyphenyl-fluorenyl) instead of a granular resin (HMP glycine, manufactured by Applied Biosystems, USA) composed of a polymer [constitutive molar ratio of styrene and divinylbenzene: 99: 1] Granular resin comprising a styrene-divinylbenzene copolymer having a methoxycarbonyl) -aminomethyl) -phenoxyacetamido-ethyl group at a ratio of 0.62 mmol / g (resin) [constitutive molar ratio of styrene and divinylbenzene: 99 to 1] Synthesis was performed in the same manner as in Example 1 except that [Fmoc amide resin, manufactured by Applied Biosystems, USA] was used. Desorption from deprotection and resin peptide was subjected to purification. Also in Example 3, the peptide was prepared according to the same method as in Example 1.
HPLCにおいて、実施例2では11.73 minに単一のピ−クが示された。また、実施例
3では12.19 minに単一のピ−クが示された。In HPLC, Example 2 showed a single peak at 11.73 min. In Example 3, a single peak was shown at 12.19 min.
GPCにおいて、実施例2では10.22 mLに主ピークと8.81 mLに副ピークが認められた。また、実施例3では10.16 mLに主ピークと8.74 mLに副ピークが認められた。 In GPC, in Example 2, a major peak was observed at 10.22 mL and a minor peak was observed at 8.81 mL. In Example 3, a main peak was observed at 10.16 mL and a minor peak was observed at 8.74 mL.
CDにおいて、実施例2では225 nmに正のコットン効果、199 nmに負のコットン効果が観測された。実施例3では225 nmに正のコットン効果、199 nmに負のコットン効果が観測された。この結果より、実施例2及び3のペプチドが、3重らせん構造を含むことが確認された。 In CD, in Example 2, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 199 nm. In Example 3, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 199 nm. From this result, it was confirmed that the peptides of Examples 2 and 3 contain a triple helical structure.
[比較例1及び2]
(Pro Hyp Gly)4 -Lys Ile Gly-(Pro Hyp Gly)5(配列番号:7)で示されるアミノ酸配列からなるペプチド(比較例1)を実施例1と同様の方法で合成し、ペプチドの脱保護と樹脂からの脱離、精製を行った。[Comparative Examples 1 and 2]
A peptide consisting of the amino acid sequence represented by (Pro Hyp Gly) 4 -Lys Ile Gly- (Pro Hyp Gly) 5 (SEQ ID NO: 7) (Comparative Example 1) was synthesized in the same manner as in Example 1, and Deprotection, desorption from the resin, and purification were performed.
得られた比較例1のペプチドは、HPLCにおいて11.03 minと11.44 minにピ−クが示された。また、GPCにおいて10.61 mLに主ピークと9.07 mLに副ピークが認められた。さらに、CDにおいて224 nmに正のコットン効果、199 nmに負のコットン効果が観測され、3重らせん構造を含むことが確認された。 The obtained peptide of Comparative Example 1 showed peaks at 11.03 min and 11.44 min on HPLC. In GPC, a major peak was observed at 10.61 mL and a minor peak was observed at 9.07 mL. Further, in CD, a positive cotton effect was observed at 224 nm and a negative cotton effect was observed at 199 nm, and it was confirmed that a triple helical structure was included.
(Pro Hyp Gly)10(配列番号:8)で示されるアミノ酸配列をからなるペプチド(比較例2)を、(株)ペプチド研究所から購入した。A peptide consisting of the amino acid sequence represented by (Pro Hyp Gly) 10 (SEQ ID NO: 8) (Comparative Example 2) was purchased from Peptide Institute.
GPCにおいて、8.81 mLに主ピークと10.14 mLに副ピークが認められた。CDにおいて、225 nmに正のコットン効果、197 nmに負のコットン効果が観測され、3重らせん構造を含むことが確認された。 In GPC, a major peak was observed at 8.81 mL and a minor peak at 10.14 mL. In CD, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 197 nm, confirming the inclusion of a triple helical structure.
[試験例1]
<細菌膜モデルリポソームの作製>
ジパルミトイルホスファチジルグリセロール(DPPG:シグマP9789)0.7 mgを含む塩化
メチレン/メタノール混合溶液を褐色ガラスバイアルに入れ、窒素ガスを吹き込むことにより、バイアルの内壁に脂質膜を形成させた。カルボキシフルオレセイン(CF:シグマC7153)を20 mMの濃度で含む、1 mM EDTA/TBS溶液1 mLをガラスバイアルに加え、ボルテクスミキサーで脂質膜がバイアルの内壁から剥離するまで良く攪拌した。次に、60℃で20分間超音波処理を行った。さらに、PD10カラム(GEヘルスケアバイオサイエンス)で3〜5 mL(好ましくは3〜4 mL)の分画を分取して、リポソーム溶液とした。[Test Example 1]
<Preparation of bacterial membrane model liposome>
A methylene chloride / methanol mixed solution containing 0.7 mg of dipalmitoyl phosphatidylglycerol (DPPG: Sigma P9789) was placed in a brown glass vial, and nitrogen gas was blown to form a lipid film on the inner wall of the vial. 1 mL of a 1 mM EDTA / TBS solution containing carboxyfluorescein (CF: Sigma C7153) at a concentration of 20 mM was added to a glass vial, and stirred well with a vortex mixer until the lipid membrane was peeled off from the inner wall of the vial. Next, ultrasonic treatment was performed at 60 ° C. for 20 minutes. Further, a 3 to 5 mL (preferably 3 to 4 mL) fraction was fractionated with a PD10 column (GE Healthcare Bioscience) to obtain a liposome solution.
<哺乳類膜モデルリポソームの作製>
超音波処理を室温で行う以外は上記と同様の操作で、ホスファチジルコリン(PC:シグマP2772 Type XI-E)からなるリポソームを得た。<Production of mammalian membrane model liposome>
A liposome composed of phosphatidylcholine (PC: Sigma P2772 Type XI-E) was obtained in the same manner as above except that the ultrasonic treatment was performed at room temperature.
<抗菌性評価>
得られたDPPGリポソーム、PCリポソームのそれぞれをPBSで10倍希釈した溶液200 μLに対して、実施例1〜3、比較例1及び2のペプチドを最終濃度が0.25〜0.005 mMに成るように加え、15分間室温で静置した。その後、12,000 rpmで5分間遠心して得られた上清中のCFの量を、励起波長485 nm、蛍光波長535 nmで測定した。ペプチド溶液の代わりにPBSを加えた場合の蛍光を0%、最終濃度0.1%のTriton X-100を加えた場合の蛍光を100%として、各ペプチドによるCF漏出量を計算した。<Antimicrobial evaluation>
The peptides of Examples 1-3 and Comparative Examples 1 and 2 were added to 200 μL of the obtained DPPG liposome and PC liposome diluted 10-fold with PBS so that the final concentration would be 0.25 to 0.005 mM. And left at room temperature for 15 minutes. Thereafter, the amount of CF in the supernatant obtained by centrifugation at 12,000 rpm for 5 minutes was measured at an excitation wavelength of 485 nm and a fluorescence wavelength of 535 nm. The amount of CF leakage due to each peptide was calculated assuming that the fluorescence when PBS was added instead of the peptide solution was 0% and the fluorescence when Triton X-100 having a final concentration of 0.1% was added was 100%.
細菌膜モデルのDPPGリポソームに対して、実施例1のペプチドは、50 μMで20 %、実施例2のペプチドは、125 μMで11 %、実施例3では25 μMで3 %のCF漏出を示した。哺乳類細胞膜モデルのPCリポソームに対してはいずれの濃度でもCF漏出は認められず、細菌選択的な抗菌活性を発現することが示された(図1を参照)。 For the bacterial membrane model DPPG liposome, the peptide of Example 1 shows 20% CF leakage at 50 μM, the peptide of Example 2 shows 11% at 125 μM, and Example 3 shows 3% CF leakage at 25 μM. It was. No leakage of CF was observed at any concentration against PC liposomes of mammalian cell membrane model, indicating that bacteria-selective antibacterial activity was expressed (see FIG. 1).
これに対して、比較例1、2のペプチドでは、いずれのリポソームに対しても全ての濃度範囲で全くCF漏出は認められなかった。 In contrast, in the peptides of Comparative Examples 1 and 2, no CF leakage was observed in any concentration range for any liposome.
[実施例4]
(Pro Hyp Gly)3-(Lys Hyp Gly Lys Leu Gly)2-(Pro Hyp Gly)3(配列番号:4)で示されるアミノ酸配列からなり、カルボキシ末端のカルボキシル基がアミド基で置換されたペプチド、すなわち(Pro Hyp Gly)3-(Lys Hyp Gly Lys Leu Gly)2-(Pro Hyp Gly)3-NH2をペプチド自動合成装置を用いて固相合成法により合成した。[Example 4]
A peptide having the amino acid sequence represented by (Pro Hyp Gly) 3- (Lys Hyp Gly Lys Leu Gly) 2- (Pro Hyp Gly) 3 (SEQ ID NO: 4), wherein the carboxyl group at the carboxy terminus is substituted with an amide group That is, (Pro Hyp Gly) 3- (Lys Hyp Gly Lys Leu Gly) 2- (Pro Hyp Gly) 3 -NH 2 was synthesized by a solid phase synthesis method using an automatic peptide synthesizer.
すなわち、4−(2’、4’−ジメトキシフェニル−フルオレニルメトキシカルボニル)−アミノメチル)−フェノキシアセトアミド−エチル基を0.62ミリモル/g(樹脂)の割合で含むスチレン−ジビニルベンゼン共重合体〔スチレンとジビニルベンゼンの構成モル比:99対1〕からなる粒状樹脂〔米国アプライド・バイオシステムズ社製、Fmocアミドレジン〕0.1ミリモルを用い、目的とするペプチドのカルボキシル末端からアミノ末端に向かって順次対応するアミノ酸を結合させた。結合反応において、アミノ酸として、米国アプライド・バイオシステムズ社製のNα−9− (フルオレニルメトキシカルボニル)−L−プロリン〔Fmocプロリン〕、Nα−9−(フルオレニルメトキシカルボニル)−Nε−ブトキシカルボニル−L−リジン〔Fmocリジン〕、Nα−9− (フルオレニルメトキシカルボニル)−L−ロイシン〔Fmocロイシン〕、Nα−9− (フルオレニルメトキシカルボニル)−グリシン〔Fmocグリシン〕、独国BACHEM社製Nα−9−(フルオレニルメトキシカルボニル)−O−ブトキシカルボニル−L−ヒドロキシプロリン〔Fmocヒドロキシプロリン〕を、各結合ステップについてそれぞれ1ミリモルずつ用いた。That is, styrene-divinylbenzene copolymer containing 4- (2 ′, 4′-dimethoxyphenyl-fluorenylmethoxycarbonyl) -aminomethyl) -phenoxyacetamido-ethyl group at a rate of 0.62 mmol / g (resin) Using 0.1 mmol of a granular resin (Fmoc amide resin, manufactured by Applied Biosystems, USA) composed of a polymer (constituent molar ratio of styrene and divinylbenzene: 99 to 1) from the carboxyl terminal to the amino terminal of the target peptide. The corresponding amino acids were combined sequentially. In the binding reaction, N α -9- (fluorenylmethoxycarbonyl) -L-proline [Fmoc proline], N α -9- (fluorenylmethoxycarbonyl) -N manufactured by Applied Biosystems, Inc. epsilon - butoxycarbonyl -L- lysine [Fmoc lysine], N α -9- (fluorenyl methoxy carbonyl) -L- leucine [Fmoc-leucine], N α -9- (fluorenyl methoxy carbonyl) - glycine [Fmoc glycine], a Germany BACHEM Inc. N alpha-9-(fluorenyl methoxy carbonyl) -O- butoxycarbonyl -L- hydroxyproline [Fmoc hydroxyproline], using one mmole respectively for each coupling step.
得られたペプチド樹脂を、5%の水を含むトリフルオロ酢酸10mlで3時間処理した。得られた溶液をジエチルエーテルに加えて生じる沈殿をさらに数回ジエチルエーテルで洗浄して、ペプチドの脱保護と樹脂からの脱離を行った。粗生成物をPD10カラム(GEヘルスケアバイオサイエンス)で精製してペプチドを得た。得られた精製ペプチドをHPLC〔GEヘルスケアバイオサイエンス株式会社製AKTA explorer10XTシステム、カラム:ミリポアウオーターズ株式会社製ノバパックC18 3.9mmφ×150mm、移動相:トリフルオロ酢酸を0.05容量%含有するアセトニトリルと水の混合溶媒(アセトニトリル濃度を30分間で5容量%から50容量%に直線的に変化させた)、流速1.0ml/min〕に付したところ、10.7 minに単一のピ−クが示された。 The obtained peptide resin was treated with 10 ml of trifluoroacetic acid containing 5% water for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was further washed several times with diethyl ether to deprotect the peptide and desorb from the resin. The crude product was purified with a PD10 column (GE Healthcare Bioscience) to obtain a peptide. The purified peptide thus obtained was HPLC [AKTA explorer 10XT system manufactured by GE Healthcare Biosciences Co., Ltd., column: Novopak C18 3.9 mmφ × 150 mm manufactured by Millipore Waters Co., Ltd., mobile phase: acetonitrile containing 0.05% by volume of trifluoroacetic acid. And a mixed solvent of acetonitrile and water (acetonitrile concentration was linearly changed from 5 vol% to 50 vol% over 30 minutes), flow rate of 1.0 ml / min], a single peak was observed at 10.7 min. Indicated.
得られたペプチドをゲルパーミエーションクロマトグラフィー(GPC; GEヘルスケアバイオサイエンス(株)製、AKTApurifierシステム、カラム:Supedex peptide 10/30 GL、流速:0.5mL/min、溶離液:150mMのNaClを含む10mM phosphate buffer(pH 7.4))に供したところ、溶出体積10.1mLに主ピークと7.5mL付近に副ピークが認められた。また、得られたペプチドの円二色性スペクトル(CD)を測定したところ、223nmに正のコットン効果、200nmに負のコットン効果が観測され、3重らせん構造を含むことが確認された。
The obtained peptide is gel permeation chromatography (GPC; manufactured by GE Healthcare Biosciences, AKTApurifier system, column:
[実施例5及び6]
(Pro Hyp Gly)2-(Lys Leu Gly Lys Hyp Gly)3-(Pro Hyp Gly)2(配列番号:5) で示されるアミノ酸配列からなり、カルボキシ末端のカルボキシル基がアミド基で置換されたペプチド、すなわち(Pro Hyp Gly)2-(Lys Leu Gly Lys Hyp Gly)3-(Pro Hyp Gly)2-NH2 (実施例5)、
Pro Hyp Gly-(Lys Leu Gly Lys Hyp Gly)4-Pro Hyp Gly (配列番号:6)で示されるアミノ酸配列からなり、カルボキシ末端のカルボキシル基がアミド基で置換されたペプチド、すなわちPro Hyp Gly-(Lys Leu Gly Lys Hyp Gly)4-Pro Hyp Gly-NH2(実施例6)をペプチド自動合成装置を用いて固相合成法により実施例4と同様にして合成し、ペプチドの脱保護と樹脂からの脱離、精製を行った。[Examples 5 and 6]
(Pro Hyp Gly) 2- (Lys Leu Gly Lys Hyp Gly) 3- (Pro Hyp Gly) 2 (SEQ ID NO: 5) A peptide in which the carboxyl group at the carboxy terminus is substituted with an amide group (Pro Hyp Gly) 2- (Lys Leu Gly Lys Hyp Gly) 3- (Pro Hyp Gly) 2 -NH 2 (Example 5),
Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) 4 -Pro Hyp Gly (SEQ ID NO: 6), a peptide in which the carboxyl group at the carboxy terminus is substituted with an amide group, ie, Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) 4- Pro Hyp Gly-NH 2 (Example 6) was synthesized in the same manner as in Example 4 by solid phase synthesis using an automatic peptide synthesizer, and peptide deprotection and resin were synthesized. From and purified.
HPLCにおいて、実施例5では10.2 minに単一のピ−クが、実施例6では10.1 minに単一のピ−クが示された。GPCにおいて、実施例5では10.1 mLに主ピークと7.5 mLに副ピークが、実施例6では10.1 mLに主ピークと7.5 mLに副ピークが認められた。また、CDにおいて、実施例5では222 nmに正のコットン効果、198 nmに負のコットン効果が、実施例6では220 nmに正のコットン効果、197 nmに負のコットン効果が観測され、3重らせん構造を含むことが確認された。 In HPLC, Example 5 showed a single peak at 10.2 min and Example 6 showed a single peak at 10.1 min. In GPC, in Example 5, a main peak was observed at 10.1 mL and a minor peak was observed at 7.5 mL, and in Example 6, a major peak was observed at 10.1 mL and a minor peak was observed at 7.5 mL. In CD, a positive cotton effect was observed at 222 nm in Example 5, a negative cotton effect at 198 nm, a positive cotton effect at 220 nm, and a negative cotton effect at 197 nm in Example 6. It was confirmed to contain a heavy helical structure.
[比較例3]
(Pro Hyp Gly)3-Arg Leu Gly Pro Hyp Gly Arg Leu Gly-(Pro Hyp Gly)4(配列番号:9)で示されるアミノ酸配列からなり、カルボキシ末端のカルボキシル基がアミド基で置換されたペプチド、すなわち(Pro Hyp Gly)3-Arg Leu Gly Pro Hyp Gly Arg Leu Gly-(Pro Hyp Gly)4-NH2(比較例3)を実施例1と同様の方法で合成、ペプチドの脱保護と樹脂からの脱離、精製を行った。ただし、合成ステップにおいて、米国アプライド・バイオシステムズ社製のNα−9−(フルオレニルメトキシカルボニル)−NG−(2,2,5,7,8−ペンタメチルクロマン−6−スルフォニル)−L−アルギニン〔Fmocアルギニン〕を、各結合ステップについてそれぞれ1ミリモルずつ用いた。[Comparative Example 3]
(Pro Hyp Gly) 3 -Arg Leu Gly Pro Hyp Gly Arg Leu Gly- (Pro Hyp Gly) 4 (SEQ ID NO: 9) peptide having a carboxy-terminal carboxyl group substituted with an amide group That is, (Pro Hyp Gly) 3 -Arg Leu Gly Pro Hyp Gly Arg Leu Gly- (Pro Hyp Gly) 4 -NH 2 (Comparative Example 3) was synthesized in the same manner as in Example 1, peptide deprotection and resin From and purified. However, in the synthesis step, made in the US Applied Biosystems N alpha-9-(fluorenyl methoxy carbonyl) -N G - (2,2,5,7,8-pentamethyl chroman-6-sulfonyl) - 1 mmol of L-arginine [Fmoc arginine] was used for each binding step.
HPLCにおいて12.2 minにピ−クが示された。GPCにおいて10.6 mLに主ピークと7.5 mLに副ピークが認められた。また、CDにおいて222 nmに正のコットン効果、199 nmに負のコットン効果が観測され、3重らせん構造を含むことが確認された。 A peak was shown at 12.2 min on HPLC. GPC showed a main peak at 10.6 mL and a minor peak at 7.5 mL. In CD, a positive cotton effect was observed at 222 nm and a negative cotton effect was observed at 199 nm, confirming that a triple helical structure was included.
実施例4、5、6及び比較例3のペプチドについて、上記試験例1に示される方法に従って抗菌活性を評価した。 The antimicrobial activity of the peptides of Examples 4, 5, 6 and Comparative Example 3 was evaluated according to the method shown in Test Example 1 above.
細菌膜モデルのDPPGリポソームに対して、実施例4のペプチドは、18 μMで10%、実施例5のペプチドは、3.5 μMで17%、実施例6では7 μMで18%のCF漏出を示した。哺乳類細胞膜モデルのPCリポソームに対してはいずれの濃度でもCF漏出は認められず、細菌選択的な抗菌活性を発現することが示された(図1を参照)。 For the bacterial membrane model DPPG liposome, the peptide of Example 4 shows 10% CF leakage at 18 μM, the peptide of Example 5 shows 17% CF at 3.5 μM, and Example 6 shows 18% CF leakage at 7 μM. It was. No leakage of CF was observed at any concentration against PC liposomes of mammalian cell membrane model, indicating that bacteria-selective antibacterial activity was expressed (see FIG. 1).
これに対して、比較例3のペプチドでは、いずれのリポソームに対しても全ての濃度範囲で全くCF漏出は認められなかった。 In contrast, with the peptide of Comparative Example 3, no CF leakage was observed in any concentration range for any liposome.
以上の結果から明らかなように、本発明により提供される新規なペプチドは、細菌の細胞膜に対する選択的な抗菌活性を示し、細菌感染症などの治療や雑菌の繁殖抑制等に有用である。 As is clear from the above results, the novel peptide provided by the present invention exhibits selective antibacterial activity against bacterial cell membranes, and is useful for the treatment of bacterial infections, the suppression of the propagation of germs, and the like.
[処方例]
本発明のペプチド又はその塩を用いた化粧料の処方例を以下に示すが、本発明はこれに限定されない。
処方例1.
下記成分を混合し、保湿液を得た。
人工コラーゲン水溶液(0.3%) 99.5部
配列番号1のペプチド1%水溶液 0.5部
[Prescription example]
Although the formulation example of the cosmetics using the peptide of this invention or its salt is shown below, this invention is not limited to this.
Formulation Example 1
The following components were mixed to obtain a moisturizing liquid.
Artificial collagen aqueous solution (0.3%) 99.5 parts 0.5% peptide 1% aqueous solution of SEQ ID NO: 1
本発明のペプチドは、熱、塩濃度等の外的要因の影響を受けにくく、安定して優れた抗菌活性を発揮することができることから、医薬製剤として調製し、細菌感染症、敗血症等の予防又は治療に使用することができる。 The peptide of the present invention is not easily affected by external factors such as heat and salt concentration, and can stably exhibit excellent antibacterial activity. Therefore, it is prepared as a pharmaceutical preparation to prevent bacterial infection, sepsis, etc. Or it can be used for treatment.
また、本発明のポリペプチドを医療用基材に結合させて使用することもできる。例えば、人工コラーゲンゲル等に本発明のペプチドを結合させ、これを原料として製造された人工皮膚は、移植部分の細菌感染を予防することができる。 The polypeptide of the present invention can also be used by binding to a medical substrate. For example, artificial skin produced by binding the peptide of the present invention to an artificial collagen gel or the like and using this as a raw material can prevent bacterial infection of the transplanted part.
さらに、本発明のペプチドは、ヒトに対して低刺激性であり、安定した抗菌性を保持し得ることから、化粧料に抗菌剤として配合することもできる。化粧料に配合される従来の抗菌剤には、臭いの気になるものもあった。しかしながら、本発明のポリペプチドは臭い等の問題もなく、化粧料に配合される抗菌剤として好適に使用され得る。 Furthermore, since the peptide of the present invention is hypoallergenic to humans and can maintain stable antibacterial properties, it can also be formulated as an antibacterial agent in cosmetics. Some of the conventional antibacterial agents blended in cosmetics are annoying. However, the polypeptide of the present invention can be suitably used as an antibacterial agent blended in cosmetics without problems such as odor.
配列番号1は、実施例1において得られる人工ペプチドである。
配列番号2は、実施例2において得られる人工ペプチドである。
配列番号3は、実施例3において得られる人工ペプチドである。
配列番号4は、実施例4において得られる人工ペプチドである。
配列番号5は、実施例5において得られる人工ペプチドである。
配列番号6は、実施例6において得られる人工ペプチドである。
配列番号7は、比較例1において得られる人工ペプチドである。
配列番号8は、比較例2の人工ペプチドである。
配列番号9は、比較例3において得られる人工ペプチドである。SEQ ID NO: 1 is an artificial peptide obtained in Example 1.
SEQ ID NO: 2 is an artificial peptide obtained in Example 2.
SEQ ID NO: 3 is an artificial peptide obtained in Example 3.
SEQ ID NO: 4 is an artificial peptide obtained in Example 4.
SEQ ID NO: 5 is an artificial peptide obtained in Example 5.
SEQ ID NO: 6 is an artificial peptide obtained in Example 6.
SEQ ID NO: 7 is an artificial peptide obtained in Comparative Example 1.
SEQ ID NO: 8 is the artificial peptide of Comparative Example 2.
SEQ ID NO: 9 is an artificial peptide obtained in Comparative Example 3.
Claims (16)
(1)下記一般式(I)で表されるアミノ酸配列からなるペプチド:
(Pro Hyp Gly)n-{(Lys X Gly)m-(Pro Hyp Gly)l-(Lys X Gly)p}α-(Pro Hyp Gly)r (I)
(ここで、式(I)中、nは1〜6の整数であり、mは1〜4の整数であり、lは0〜7の整数であり、pは1〜4の整数であり、rは1〜6の整数であり、αは1〜6の整数である。
Xは、Ile、Leu及びHypから選択されるいずれかのアミノ酸残基であり、
m+p個のXは、それぞれ同一又は異なっていてもよい。
n+m+l+p+r=4〜15である。
但し、l=0の時、m+p≧2を満たし、
α≧2のときm=1、l=0、p=1を満たす。)、
(2)一般式(I)で表されるアミノ酸配列において、Lys、Gly及びX以外の1〜3個のアミノ酸残基が置換されたアミノ酸配列からなり、
該置換が、
ProからHypへの置換、または
HypからPro への置換、
から選択される少なくとも1つであるペプチド。 A peptide having antibacterial activity represented by the following (1) or (2) or a salt thereof;
(1) A peptide consisting of an amino acid sequence represented by the following general formula (I):
(Pro Hyp Gly) n -{(Lys X Gly) m- (Pro Hyp Gly) l- (Lys X Gly) p } α- (Pro Hyp Gly) r (I)
(Here, in Formula (I), n is an integer of 1-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is an integer of 1 to 6, and α is an integer of 1 to 6.
X is any amino acid residue selected from Ile, Leu and Hyp,
The m + p Xs may be the same or different from each other.
n + m + l + p + r = 4-15.
However, when l = 0, m + p ≧ 2 is satisfied,
When α ≧ 2, m = 1, l = 0, and p = 1 are satisfied. ),
(2) The amino acid sequence represented by the general formula (I) consists of an amino acid sequence in which 1 to 3 amino acid residues other than Lys, Gly and X are substituted,
The substitution is
Pro to Hyp replacement, or
Hyp to Pro replacement,
A peptide which is at least one selected from.
XはIle又はLeuであり、
m+p個のXは、それぞれ同一又は異なっていてもよく、
n+m+l+p+r=8〜15であり、
l=0の時、m+p≧2を満たす、
請求項1に記載のペプチド又はその塩。 In said (1), n is an integer of 3-6, m is an integer of 1-4, l is an integer of 0-7, p is an integer of 1-4, r is 3- An integer of 6, α is 1,
X is Ile or Leu,
m + p Xs may be the same or different from each other,
n + m + l + p + r = 8-15,
When l = 0, m + p ≧ 2 is satisfied.
The peptide according to claim 1 or a salt thereof.
(1−1)以下の(i)〜(vi)のいずれかで表されるアミノ酸配列からなるペプチド:
(i)(Pro Hyp Gly)3 -(Lys Ile Gly)3 -(Pro Hyp Gly)4(配列番号:1)
(ii)(Pro Hyp Gly)3 -Lys Ile Gly Pro Hyp Gly Lys Ile Gly-(Pro Hyp Gly)4(配列番号:2)
(iii)(Pro Hyp Gly)4 -(Lys Ile Gly)2 -(Pro Hyp Gly)4(配列番号:3)
(iv)(Pro Hyp Gly)3-(Lys Hyp Gly Lys Leu Gly)2-(Pro Hyp Gly)3(配列番号:4)、
(v)(Pro Hyp Gly)2-(Lys Leu Gly Lys Hyp Gly)3-(Pro Hyp Gly)2(配列番号:5)、
(vi)Pro Hyp Gly-(Lys Leu Gly Lys Hyp Gly)4-Pro Hyp Gly (配列番号:6)、(2−1)前記アミノ酸配列(i)〜(vi)において1〜3個のアミノ酸が置換されたアミノ酸配列からなるペプチドであって、
該置換が、
ProからHypへの置換、
HypからPro への置換、
LeuからIle又はValへの置換、及び、
Ile又はValからLeuへの置換
からなる群から選択される少なくとも1つの置換である、
抗菌活性を有するペプチドならびにその塩。 The peptide or salt thereof according to claim 1, which is represented by the following (1-1) or (2-1);
(1-1) A peptide comprising an amino acid sequence represented by any one of (i) to (vi) below:
(i) (Pro Hyp Gly) 3- (Lys Ile Gly) 3- (Pro Hyp Gly) 4 (SEQ ID NO: 1)
(ii) (Pro Hyp Gly) 3 -Lys Ile Gly Pro Hyp Gly Lys Ile Gly- (Pro Hyp Gly) 4 (SEQ ID NO: 2)
(iii) (Pro Hyp Gly) 4 - (Lys Ile Gly) 2 - (Pro Hyp Gly) 4 ( SEQ ID NO: 3)
(iv) (Pro Hyp Gly) 3- (Lys Hyp Gly Lys Leu Gly) 2- (Pro Hyp Gly) 3 (SEQ ID NO: 4),
(v) (Pro Hyp Gly) 2- (Lys Leu Gly Lys Hyp Gly) 3- (Pro Hyp Gly) 2 (SEQ ID NO: 5),
(vi) Pro Hyp Gly- (Lys Leu Gly Lys Hyp Gly) 4 -Pro Hyp Gly (SEQ ID NO: 6), (2-1) 1-3 amino acids in the amino acid sequences (i) to (vi) A peptide comprising a substituted amino acid sequence,
The substitution is
Replacement of Pro to Hyp,
Hyp to Pro replacement,
Substitution of Leu to Ile or Val, and
At least one substitution selected from the group consisting of substitution of Ile or Val to Leu,
Peptides having antibacterial activity and salts thereof.
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JPH11215983A (en) * | 1998-01-30 | 1999-08-10 | Agriculture Forestry And Fisheries Technical Information Society | Antifungal agent with peptide as active component |
JPH11335396A (en) * | 1998-03-25 | 1999-12-07 | Seikagaku Kogyo Co Ltd | New antimicrobial peptide |
JP2000319300A (en) * | 1999-05-14 | 2000-11-21 | Meiji Milk Prod Co Ltd | Antibicrobial peptide |
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JP2006045214A (en) * | 2004-07-06 | 2006-02-16 | Juntendo | Antibacterial peptide |
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