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Method of screening toxin-neutralizing peptide, stx2 inhibitory peptide and vero toxin-neutralizing agent

外国特許コード F110004050
整理番号 K05401WO
掲載日 2011年7月8日
出願国 欧州特許庁(EPO)
出願番号 05755811
公報番号 1782820
公報番号 1782820
出願日 平成17年6月28日(2005.6.28)
公報発行日 平成19年5月9日(2007.5.9)
公報発行日 平成26年7月30日(2014.7.30)
国際出願番号 JP2005012286
国際公開番号 WO2006001542
国際出願日 平成17年6月28日(2005.6.28)
国際公開日 平成18年1月5日(2006.1.5)
優先権データ
  • 2005JP012286 (2005.6.28) WO
  • 特願2004-189801 (2004.6.28) JP
  • 特願2004-295405 (2004.10.7) JP
発明の名称 (英語) Method of screening toxin-neutralizing peptide, stx2 inhibitory peptide and vero toxin-neutralizing agent
発明の概要(英語) A screening method comprises the following steps; (1) specifying a receptor binding site by introduction of mutation, and (2) specifying a binding site-specific peptide motif on the basis of an amino acid selection ratio by contrast between a peptide motif bound to a wild-type subunit and a peptide motif bound to a mutant functionally deficient in the binding site according to a peptide library method.
A peptide which inhibits a toxin whose receptor binding portion has a subunit structure is screened.
Accordingly, an STX2 inhibitor in which an STX2 inhibiting peptide is incorporated in a molecular nuclear structure portion having three molecules of lysine (Lys) peptide-linked thereto and which is easy to synthesize and can inhibit verotoxin is provided.
特許請求の範囲(英語) [claim1]
1. A method for screening for a polyvalent STX2 toxin-neutralizing peptide which can neutralize STX2, the method comprising the following steps: (a) providing STX2, (b) providing a mutant of STX2 which is functionally deficient in the site by which it binds to its receptor due to the presence of a mutation in the receptor-binding site of STX2, (c) providing a first polyvalent peptide library, wherein each entity in the first polyvalent peptide library comprises a core structure comprising a plurality of lysines and wherein degenerate peptides are bound to the terminal amino groups of the lysines, (d) contacting said first polyvalent peptide library with STX2 and the mutant, (e) contrasting a peptide motif bound to STX2 with a peptide motif bound to the mutant to obtain an amino acid selection ratio where the amino acid selection ratio is used to specify a receptor-binding site-specific binding peptide motif, and wherein the STX2-neutralizing peptide comprises the receptor-binding site-specific binding peptide motif.
[claim2]
2. The method of claim 1, wherein said core structure has three molecules of lysine (Lys) having the structure: (see diagramm)and said peptides are represented by MAXXXXA-AHA (SEQ ID NO: 5), to give a molecule of formula (MAXXXXA-AHA) 4-3Lys, wherein X is an amino acid which is not cysteine and AHA is aminohexanoic acid, and wherein said STX2 mutant comprises a tryptophan to alanine mutation at position 32 of the STX2B subunit and said contrasting step comprises: (a) determining a peptide motif within the peptides of the library that binds to STX2, (b) determining a peptide motif within the peptides of the library that binds to the mutant, (c) specifying a receptor-binding site-specific peptide motif by determining a first normalised value for each amino acid in the STX2 toxin-bound peptides based on the number of times each amino acid is present at each position in the STX2 toxin-bound peptides, and a second normalised value for each amino acid in the mutant-bound peptides based on the number of times each amino acid is present at each position in the mutant-bound peptides, and (d) dividing the first normalised value by the corresponding second normalised value, in order to obtain a selection ratio for each amino acid position.
[claim3]
3. A method as claimed in claim 2, wherein a second polyvalent peptide library is screened wherein the sequences of the peptides in the second peptide library are based on the sequences of the previously-obtained receptor-binding site-specific peptide motifs, and wherein the amino acids in the receptor-binding site-specific peptide motifs with high selection ratios are fixed in the peptides of the second library, in order to obtain a receptor-binding site-specific peptide motif with a higher selectivity for the receptor.
[claim4]
4. A method for screening for a toxin-neutralizing peptide according to claim 1, wherein in the first polyvalent peptide library, the peptides are bound to the terminal lysine amino groups via a spacer molecule.
[claim5]
5. An STX2 inhibiting peptide comprising a molecular core portion which has three molecules of lysine (Lys) having the structure: (see diagramm) wherein each terminal amino group in the Lys core structure is bound to a peptide motif, wherein each peptide motif is identical and is selected from the group consisting of: (1) FRRNRRN (SEQ ID NO: 1) (2) PPPRRRR (SEQ ID NO: 2) (3) PPRRNRR (SEQ ID NO: 3) (4) KRRNPRR (SEQ ID NO: 4).
[claim6]
6. An STX2 inhibiting peptide according to claim 5, wherein the peptide motif is incorporated via a spacer molecule.
[claim7]
7. An STX2 inhibiting peptide according to claim 6, wherein the spacer molecule is a molecule having a peptide or an amino group and a carboxyl group and having a hydrocarbon chain structure with from 4 to 10 carbon atoms.
[claim8]
8. An STX2 inhibiting peptide according to any of claims 5 to 7, wherein the peptide motif has a terminal modification molecule.
[claim9]
9. A peptide according to claim 8, wherein the terminal modification molecule is an uncharged molecule.
[claim10]
10. A peptide according to claim 9, wherein the terminal modification molecule has Met-Ala in the N-terminal.
[claim11]
11. An STX2 inhibiting peptide according to claim 5, wherein the molecular core structure has one of the following formulae attached to each terminal amino group:

Met-Ala-Xo-Ala-AHA- (a)

Acetyl-Xo-AHA- (b)

wherein AHA is an aminohexanecarboxylic acid group, and Xo represents any one of the peptides (1), (2), (3) or (4) as defined in claim 5.
[claim12]
12. A verotoxin neutralising agent comprising the peptide according to any one of claims 5 to 11 as an active ingredient.
  • 出願人(英語)
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • NISHIKAWA KIYOTAKA
国際特許分類(IPC)
欧州特許分類/主・副
  • G01N033/50D2D
  • K61K038/00
指定国 Contracting States: CH DE FR GB IT LI
参考情報 (研究プロジェクト等) PRESTO Host and Defense AREA
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