JP5897178B2 - Stx1 toxicity-inhibiting tetravalent peptide and disease therapeutic agent containing the same - Google Patents

Description

  The present invention relates to a Stx1 toxicity-inhibiting tetravalent peptide and a therapeutic drug containing the same.

  Shiga toxin (Stx) is a major virulence factor produced by enterohemorrhagic E. coli and is not only a gastrointestinal disorder but also a series of microvascular disorders, such as severe complications such as hemolytic uremic syndrome (HUS) )] Is known to cause.

  The inventor of this application has established a screening method using a multivalent peptide library based on the knowledge that a cluster effect exists in the binding between the B subunit of Stx1 and Stx2 and globotrisaccharide. (Patent Document 1).

  Here, the “cluster effect” is a phenomenon in which the binding affinity is remarkably increased by the interaction between multivalent and multivalent in the interaction between a certain functional molecule and its ligand as compared with the case of 1: 1. Say. In Patent Document 1, by making the peptide library multivalent, a cluster effect is exhibited with the B subunit of Stx2, and a Stx2 inhibitory peptide having a high binding affinity with Stx2 is specified.

  However, in the screening method of Patent Document 1, for example, a wild-type STX and a random multivalent library are brought into contact with this mutant, and for each amino acid obtained at each position, which amino acid is to what strength. A numerical result indicating whether or not is selected is obtained.

  For this reason, the selectivity of the amino acid selected by each position may be low depending on the kind of target protein or the target site. In addition, when there are no or few charged amino acids in the target site, hydrophobic amino acids such as Met, Val, IIe, Phe, and Trp are easily selected, which affects the selectivity of each position. For this reason, the binding motif selected as a result may not necessarily have sufficient binding property.

  Furthermore, since the screening method of Patent Document 1 determines the binding motif based on the amino acid selectivity at each position, a binding motif that combines highly selective amino acids does not necessarily have sufficient binding properties as a result. There was a case that did not.

  Thus, the inventor of this application, in the screening method of Patent Document 1, it is difficult to extract a binding motif having a high binding property, because it is possible to obtain a certain result in the identification of the binding motif, I came to realize that there are issues to be improved. Further, for example, in a position where it is difficult to judge selectivity, it is not realistic from the viewpoint of cost to incorporate a specific amino acid and evaluate the binding.

  In addition, the screening method of Patent Document 1 is limited to several binding motifs obtained at a time, and it was considered that there is a point to be improved in screening efficiency.

WO2006 / 001542 Publication

  The present invention has been made in view of the above circumstances, and in addition to the conventional screening method, a screening method capable of more strictly and efficiently identifying a peptide that binds to a target protein having a cluster effect. It is an issue to provide. Another object of the present invention is to provide a Stx1 toxicity-inhibiting tetravalent peptide obtained by this screening method and a therapeutic agent containing this peptide.

  The Stx1 toxicity-inhibiting tetravalent peptide of the present invention is a peptide that inhibits the cytotoxicity of Stx1, and each of the four amino groups located at the end of the molecular nucleus structure formed by combining three lysines (Lys). Further, any one of the peptides of SEQ ID NOs: 6 to 16 is characterized by being bound directly or via a spacer.

  The therapeutic agent of the present invention is a therapeutic agent for a disease caused by Stx1, and is characterized by containing the Stx1 toxicity-inhibiting tetravalent peptide.

  In the therapeutic agent of the present invention, the disease caused by Stx1 is preferably enterohemorrhagic E. coli infection or dysentery.

  According to the Stx1 toxicity-inhibiting tetravalent peptide of the present invention, the cytotoxicity of Stx1 can be effectively inhibited. Moreover, according to the therapeutic agent of this invention, the disease resulting from Stx1 can be treated.

It is the schematic which illustrated a mode that a bivalent and a tetravalent peptide were spot-synthesized on a cellulose sheet. FIG. 3 is a schematic view illustrating a method for optimizing the accumulation density of peptides on a sheet when Stx1 B-subunit (1BH) is a target protein. It is the figure which showed 1BH binding property for every accumulation density of the peptide on a sheet | seat. FIG. 3 is a schematic view illustrating a method for optimizing the spacer length of a peptide on a sheet when Stx1 B-subunit (1BH) is a target protein. It is the figure which showed 1BH binding property for every spacer length of the peptide on a sheet | seat. It is the figure which showed the optimal integration density and spacer length as a peptide on a sheet with respect to 1BH. It is a figure which shows the result of having identified the binding motif which targeted the site 2 of Stx1 using the random multivalent peptide library method according to the procedure of a prior application (WO2006 / 001542 gazette). It is the figure which illustrated the peptide sheet synthesis | combination of Xaa-Xaa-Xaa-Arg-Arg-Arg-Arg (sequence number 3). It is a figure which shows the 64 types of selected amino acid row | line | column. It is a figure which shows the density | concentration dependence of the binding activity with respect to 1BH and G62A of a peptide of 2 representative examples, respectively. According to the selection method, 11 peptides were used as candidates. It is the figure which showed 11 types of peptide candidates concretely. It is a figure which shows the inhibitory effect with respect to the cytotoxic activity of Stx1 with respect to Vero cell about 11 types of tetravalent peptide compounds.

  The peptide screening method of the present invention is a method for screening a peptide having a binding property to a target protein having a cluster effect.

  Examples of target proteins having a cluster effect include STX1, STX2, STX1, STX2 variants (STX1C, STX2c, 2e, 2d, 2f), cholera toxin produced by Vibrio cholerae, heat-labile enterotoxin produced by Escherichia coli E. coli, HA protein involved in influenza virus entry, gp120 involved in HIV entry, various selectins involved in cancer cell metastasis, and adiponectin related to diabetes.

The peptide screening method of the present invention comprises the following steps:
(A) determining a target protein binding motif in which one or more amino acid non-specific sites are present using a random multivalent library method;
(B) A synthetic nucleus structure of a bivalent peptide or a synthetic nucleus structure of a tetravalent peptide is formed directly or via a spacer from an amino group on the sheet, and the amino acid sequence of the target protein binding motif is formed from this peptide synthesis nucleus Elongating a divalent or tetravalent peptide comprising a multivalent peptide library in which amino acids at non-specific amino acid sites (excluding Cys) differ for each peptide;
(C) blotting the target protein to each bivalent or tetravalent peptide of the multivalent peptide library;
(D) determining that the peptide to which the target protein is bound is the target peptide;
including.

  Hereinafter, each step will be described.

  In step (A), first, the target protein binding motif is narrowed down by the “random multivalent library method” established by the inventors of this application.

  The details are disclosed in WO2006 / 00152, but as a general rule, based on the information of the receptor binding site of the target protein binding, a mutant in which the receptor binding site is functionally deleted is prepared, and a peptide library The method includes the identification of a binding site-specific peptide motif based on the amino selectivity by comparing the peptide motif that binds to the target protein and the peptide motif that binds to the mutant.

  The peptide library is a multivalent library in which the library itself is multivalent in order to exert a cluster effect. As the structure of the multivalent library, a structure in which a plurality of, for example, 2 to 5, lysines (Lys) are bound can be used from the viewpoint of design and synthesis ease and molecular size for cluster effect.

  Specific examples of the multivalent library having a molecular nucleus structure in which three lysines are bonded include the following.

  In this multivalent library, a peptide motif (XXXX portion) is bound to the molecular nucleus structure of lysine via a spacer molecule. Here, although AHA (amino-hexanoic acid) is employ | adopted as a spacer molecule | numerator, it is not limited to this, A spacer length can be designed according to a target protein. Moreover, terminal MA (Met-Ala) can be introduced at the time of screening for sequence check when performing amino acid sequencing. A (Ala) before AHA- is also for the same reason.

  And in the said multivalent library, 19 types of amino acids except Cys can be comprised in the XXXXXX part at random. The length of the XXX portion is appropriately determined according to the target protein. For example, when specifying a peptide having STX1 or 2 toxicity-inhibiting action, 7 amino acids can be assumed from the knowledge regarding molecular size.

  Then, for example, the target protein attached to the beads and each of the mutants are prepared and packed into a column to produce an affinity column. There, the multivalent library is added and allowed to bind sufficiently. Then, it is washed thoroughly with PBS or the like, and the library that has not been bound is washed away. Finally, the multivalent library remaining on the column is eluted with 30% acetic acid or the like. The collected fraction is dried up and amino acid sequence analysis is performed. As a result, for the XXXXXX part (degenerate position), a numerical result is obtained as to which amino acid is selected with what strength.

  That is, the value of each amino acid at the degenerate position when the target protein is used is divided by the value of the amino acid at the corresponding degenerate position when the mutant is used, and as a result, how many times that amino acid is compared with the mutant in the target protein. It is possible to obtain a numerical result indicating whether or not the selection has been made. At this time, for example, it can be standardized to be 19 when all 19 amino acid values are added. In this case, the value is 1 if there is no difference in selectivity between amino acids.

  From the results thus obtained, amino acids having a high selectivity at each position can be identified.

  However, in the present invention, due to problems such as amino acid selectivity as described above, for example, a position with relatively low selectivity (for example, selectivity of 1.2 or less) is a specific amino acid as an amino acid non-specific site. Not specified. That is, in step (A) of the present invention, a target protein binding motif in which one or more amino acid non-specific sites are present is determined in this way.

  Next, in step (B) of the present invention, a synthetic nucleus structure of a bivalent peptide or a synthetic nucleus structure of a tetravalent peptide consisting of Lys is spot-synthesized directly or via a spacer from an amino group on the sheet.

  Although a sheet | seat is not specifically limited, For example, a cellulose sheet, resin, etc. can be illustrated. And the amino group on a sheet | seat can be added by a well-known method. Moreover, for example, a commercial product in which a free amino group is added to a cellulose sheet can be used.

  Concerning peptide synthesis, condensation and removal of protecting groups can be performed according to conventionally known methods. Specifically, for example, an Fmoc peptide synthesis method or the like can be employed, and a commercially available peptide synthesizer can also be used.

  More specifically, in the condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used. Condensation of the protected amino acid may be performed, for example, by adding the protected amino acid together with an activating reagent and a racemization inhibitor (for example, HOBt, HOOBt), or after previously activating the protected amino acid as a symmetric anhydride, HOBt ester or HOBt ester This can be done by adding.

  The solvent used for the activation and condensation of the protected amino acid is appropriately selected from solvents that are known to be usable for protein condensation reactions. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; dimethyl sulfoxide and the like Sulfoxides; amines such as pyridine; ethers such as dioxane and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof can be used.

  The reaction temperature is appropriately selected from the range known to be used in protein condensation reactions, and is usually selected from the range of about -20 ° C to 50 ° C.

  Examples of the protective group for the amino group of the raw material amino acid include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Examples include trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, and Fmoc.

  The carboxyl group of the raw material amino acid is, for example, alkyl esterified (for example, straight chain such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, branched or Cyclic alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, It can be protected by t-butoxycarbonyl hydrazation, trityl hydrazation or the like.

The spacer is not specifically limited, but, for example, Ahx (amino hexanoic acid [NH ₂ — (CH ₂ ) ₅ —COOH]) can be employed. At this time, an optimal spacer length can be designed according to the target protein.

  For example, as illustrated in FIG. 1, a synthetic nucleus structure of a bivalent peptide or a synthetic nucleus structure of a tetravalent peptide is formed from an amino group on the sheet. Specifically, for example, a divalent or tetravalent synthetic nucleus structure can be formed using Fmoc-Lys or Fmoc-His. Fmoc-Lys is preferable in consideration of interaction with other amino acids. In addition, when a tetravalent synthetic nucleus structure is formed, it is considered to synthesize peptides with Fmoc-Lys twice in succession. The synthetic core structure is not particularly limited as long as it is a structure capable of extending a divalent or tetravalent peptide.

  As described above, a screening kit can be obtained by forming a divalent peptide synthetic nucleus structure or a tetravalent peptide synthetic nucleus structure directly or via a spacer from an amino group on a sheet.

  From this peptide synthesis core structure, a bivalent or tetravalent peptide consisting of the amino acid sequence of the target protein binding motif is extended, and the amino acid (except for Cys) of the amino acid non-specific site is different for each peptide on the sheet. Create a rally.

  That is, the binding motif having the amino acid non-specific site narrowed down in the step (A) is extended from the peptide synthesis nucleus structure, and at this time, the peptide in which the amino acid non-specific site is comprehensively substituted with 19 amino acids excluding Cys Can be synthesized to create a multivalent library with a known sequence.

  Specifically, when there are three non-amino acid specific sites, 19 × 19 × 19 = 6859 kinds of peptides can be spot-synthesized. At this time, the type of peptide (the number of spots) can be limited based on the prediction of non-amino acid specific sites, the type and number of sheets, and the performance of the peptide synthesizer.

  The accumulation density of peptides extending from amino groups on the sheet can also be appropriately designed according to the target protein.

  Next, as step (C) of the present invention, the target protein is blotted onto each bivalent peptide or tetravalent peptide of the multivalent peptide library. Then, as step (D) of the present invention, the peptide to which the target protein is bound is determined to be the target peptide.

  In the multivalent peptide library prepared in step (B), since each peptide on the sheet is bivalent or tetravalent, a labeled target protein is blotted to produce a cluster effect with the target protein. be able to. For this reason, the high coupling | bonding between a peptide and a target protein is securable. A known method can be appropriately employed for labeling the target protein. For example, fluorescein, tetramethylrhodamine and the like can be exemplified as the fluorescent substance in the case of fluorescent labeling.

  In order to wash away the unbound target protein, the sheet is appropriately washed, and the binding properties of the target protein and the peptide on the sheet can be evaluated by various known analysis methods. For example, the target protein can be fluorescently labeled and its photointensity can be quantitatively evaluated.

  In the screening method of the present invention, since a divalent or tetravalent peptide having a known sequence is spot-synthesized on a sheet, if a spot that has been confirmed to be highly binding to the target protein is extracted, it is set in step (A). It is possible to reliably and easily identify amino acid candidates at non-specific amino acid sites.

  In addition, for example, in order to identify a motif that specifically binds to a specific binding site in the target protein, separately, a mutant in which the binding site is functionally deleted is prepared, and this mutant is stored in a multivalent peptide library. Each peptide can be blotted and compared with the result of the binding evaluation of the target protein (wild type).

  As described above, in the screening method of the present invention, in addition to the conventional random multivalent library method, the peptides that bind to the target protein having the cluster effect can be more strictly and efficiently identified through the above steps. can do. Therefore, peptides that specifically bind to STX1 or STX2, STX1c, STX2c, STX2e, STX2d, STX2f, cholera toxin, heat-labile enterotoxin, HA protein, gp120, selectin, adiponectin and the like that are known to have a cluster effect By confirming the action of these peptides, diseases caused by the target protein such as enterohemorrhagic E. coli infection, dysentery, cholera, protozoan E. coli infection, influenza, HIV, It can be used as a cancer cell metastasis, diabetes treatment, or a prophylactic agent. The screening method of the present invention is extremely useful as such drug discovery screening, for example.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.
<1> Establishment of Sheet Synthesis Technology for Multivalent Peptide Library (1) Synthesis of Divalent Peptide As shown in the schematic diagram of FIG. 1 in order to improve the disadvantages of the multivalent random peptide library of Patent Document 1. Then, it was studied to spot-synthesize divalent and tetravalent peptides of known sequence on a cellulose sheet. The synthetic core structure in FIG. 1 shows lysine from which Fmoc has been removed.

  For spot synthesis, a spot peptide synthesizer from intavis AG was used. This instrument is capable of spot synthesizing 384 peptides at a time on a cellulose sheet.

  Here, a bivalent peptide is synthesized on a sheet. For this purpose, Fmoc Lys of [Chemical Formula 2] was introduced into the peptide extending from the amino group on the sheet to provide a branch point, and the amino acid synthesis from the branch point was designed to be divalent.

(2) Optimization of peptide accumulation density
In order to optimize the accumulation density of peptides on the sheet when Stx1 B-subunit (1BH) was used as a target protein, divalent peptides were synthesized at three densities. This is in order to evaluate the influence of steric hindrance when the peptide synthesis is carried out bivalently. At this time, SEQ ID NO: 1: Met-Met-Ala-Arg-Arg-Arg-Arg (MMARRRR) was employed as a binding motif for the target protein. First, as shown in FIG. 2, Fmoc-βAla and Boc-βAla are used in each ratio (100%, 30%, 10%) during the synthesis of the first amino acid. In order to remove only Fmoc, piperidine treatment was performed, and when Boc-βAla entered, subsequent amino acid elongation was prevented. For this reason, the density of the extended amino acid decreases as the ratio of Boc-βAla used increases.

The synthesized bivalent peptides of each density were blotted with STX1B-subunit (1BH) to examine the optimum density. Here, Fuji-film BAS-2500 was used and exposed to an imaging plate for analysis. It was shown that the binding of 1BH was the highest under the conditions using 100% Fmoc-βAla (synthetic conditions with the highest density) (FIG. 3). This indicates that even when a divalent peptide is extended with 100% Fmoc-βAla in order to increase the binding efficiency, it does not cause steric hindrance.
(3) Optimization of spacer length In order to optimize the distance (degree of freedom) of the peptide synthesized on the sheet from the sheet, a bivalent peptide having three types of spacer lengths was synthesized. As shown in FIG. 4, the number of Ahx (amino hexanoic acid [NH _2- (CH ₂ ) ₅ -COOH]) as a spacer existing between βAla and Lys (Fmoc) as a branch point is 1, 2, The binding strength when synthesized with 3 and blotted with 1BH was compared.

As a result, it was shown that the binding of 1BH was the highest under the condition of one spacer (Ahx) (FIG. 5).
(4) Based on the results of (2) and (3) above, the bivalent peptide on the sheet had a density of 100% and the number of spacers (Ahx) was 1, as shown in FIG.

<2> Identification of STX1 Site 2 Specific Binding Motif Using Random Multivalent Peptide Library Method According to the procedure of the prior application (WO2006 / 001542), STX1 site 2 was determined using the random multivalent peptide library method. The target binding motif was identified. For screening, as a multivalent peptide library, the following chemical formula,

A tetravalent peptide consisting of

  At this time, G62A was used for subtraction as a site 2 mutant. G62A is a mutant in which the 62nd Gly of Stx1 B-subunit is replaced with Ala. This amino acid is an amino acid present at an important position constituting the site 2, and by this mutation introduction, binding activity to the receptor (Gb3), binding to the target cell, and toxicity are greatly reduced. Site 2 has a flat structure compared to other sites, and there are sufficient amino acids around the 62nd Gly constituting site 2 to achieve electrostatic or hydrophobic interaction. It has also been found not to have.

  And this mutation reduces the ability to bind to the receptor Gb3, but this mutation only slightly changes -H to -CH3. In other words, since the difference between the wild type and the mutant is slight, it is considered that the difference in amino acid selectivity is difficult to obtain, and it was expected that the binding motif by the screening would be difficult to obtain. As shown in FIG. 7, one motif of KRRRRRR (Lys-Arg-Arg-Arg-Arg-Arg-Arg-Arg: SEQ ID NO: 2) could be identified.

  The obtained binding motif was incorporated into each of the four XXX parts of [Chemical Formula 3] to synthesize a tetravalent peptide compound (hereinafter referred to as KRR-tet).

  From the examination using the ELISA method, it was revealed that KRR-tet has about twice the binding activity to wild-type 1BH compared to G62A, that is, shows the binding specificity of Stx1 to site 2.

  In addition, when the toxicity of KRR-tet to Vero cells was examined, it was revealed that this peptide exhibits a strong toxicity to cells since it is very basic. Therefore, it was judged that it was difficult to use KRR-tet as an inhibitor.

<3> Identification of STX1 Site 2 Specific Binding Inhibitor Using Sheet Synthesis Technique of Bivalent Peptide Library From the above screening results of <2>, in order to bind to site 2 of STX1, it is shown in FIG. Thus, we focused on the point that Arg (R) is selected for positions 4-7.

  Therefore, an attempt was made to apply the sheet synthesis technique of the bivalent peptide library established in <1> using Xaa-Xaa-Xaa-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 3) as a target protein binding motif. Xaa represents an amino acid non-specific site.

  Specifically, in Xaa-Xaa-Ala-Arg-Arg-Arg-Arg (SEQ ID NO: 4), 361 (19 × 19) species in which two Xaas are each one of 19 amino acids other than Cys 19 divalent peptides in which Xaa is any one of 19 amino acids other than Cys in Ala-Ala-Xaa-Arg-Arg-Arg-Arg (SEQ ID NO: 5) < By extending the synthetic core structure of the bivalent peptide on the single sheet studied in 1> and spot-synthesizing peptides (380 species) with different amino acids (except for Cys) at non-specific amino acid sites for each peptide, A known multivalent peptide library was created.

  Here, Ala has no charge, no large hydrophobicity, small volume, and most other amino acids (except Gly) have Ala structure in it. ing. The multivalent peptide library follows the optimum conditions (spacer length, optimum density) determined in <1> above.

  FIG. 8 shows the results of blotting these sheets with the labels 1BH and G62A. Labels 1BH and G62A were prepared by incubating radiolabeled NaI, 1BH and G62A on a glass tube coated with IODO-Gen (Pierce) as an oxidizing agent, and removing unreacted substances by gel filtration.

  Then, the density of each spot was quantified, the sensitivity was quantified, and the difference in radioactivity between 1BH and G62A was corrected. The ratio, the value of 1BH / G62A, was calculated for the corresponding spots in the sheet blotted with 1BH and the sheet blotted with G62A. If the value of 1BH / G62A is large, it can be determined that the specificity for 1BH site 2 is high.

  The 64 peptide motifs were finally extracted using two points, ie, strong blotting with 1BH and high ratio of 1BH / G62. FIG. 8 shows eight typical spots.

  Furthermore, FIG. 9 shows the extracted 64 types of amino acid sequence information. These 64 species were re-synthesized as divalent peptides and blotted with different concentrations of labeled 1BH and G62A, followed by the same analysis, and the concentration dependence of the binding activity of each peptide to 1BH and G62A, respectively. It was investigated.

  Two representative examples are shown in FIG. As a result, 11 types of peptides were identified anew with selection criteria based on binding motifs that strongly bind to 1BH in a concentration-dependent manner and binding motifs with a high ratio of 1BH / G62. The identified binding motif is shown in FIG. 11 as SEQ ID NOs: 6-16.

Furthermore, these binding motifs were respectively incorporated into the four XXX parts of the above [Chemical Formula 3], and 11 types of tetravalent peptide compounds were synthesized as final STX1 site 2-specific inhibitor candidates.

  The 11 tetravalent peptide compounds synthesized were examined for the inhibitory effect of the cytotoxic activity of STX1 on Vero cells.

  The results are shown in FIG. Among them, IIA-RRRR (SEQ ID NO: 7), KGA-RRRR (SEQ ID NO: 8), KMA-RRRR (SEQ ID NO: 9), FRA-RRRR (SEQ ID NO: 11), PQA-RRRR (SEQ ID NO: 12), YTA- It has been clarified that a compound having the RRRR (SEQ ID NO: 13) motif in tetravalent has a very strong toxicity inhibitory activity.

  In addition, a compound (MMA-tet, MMA-RRRR (SEQ ID NO: 1) motif, AAA-RRRR (Ala-Ala-Ala Arg-Arg-Arg-Arg) (SEQ ID NO: 17) motif separately identified by the present inventor AAA-tet) is also presented for comparison. AAA-tet is included in the study at the same time as the basic compound of the compounds identified in this sheet synthesis screening because the first three amino acids are all Ala. Of the 11 species identified this time, it was revealed that more than half showed activity superior to AAA-tet.

  As described above, the random multivalent library method of the prior application (WO2006 / 001542) was able to identify KRRRRRR (SEQ ID NO: 2). For example, the above 11 types that strongly bind to 1BH in a concentration-dependent manner. The binding motif has not been extracted. That is, in the method of the prior application (WO2006 / 001542), for example, it is difficult to judge the selectivity for those having a low amino acid selection ratio (for example, amino acids having a selection ratio of 1.2 or less), and in some cases, they are not extracted. However, in the present invention, such extraction omission can be covered by using the multivalent peptide library of <2> above. Therefore, a peptide that binds to a target protein having a cluster effect can be identified more strictly and efficiently.

Claims (3)

  A peptide that inhibits the cytotoxicity of Stx1, and each of the four amino groups located at the ends of the molecular nucleus structure formed by binding three lysines (Lys), A Stx1 toxicity-inhibiting tetravalent peptide characterized in that any one of the above is bound directly or via a spacer.   A therapeutic agent for a disease caused by Stx1, comprising the Stx1 toxicity-inhibiting tetravalent peptide of claim 1.   The therapeutic agent according to claim 2, wherein the disease caused by Stx1 is enterohemorrhagic Escherichia coli infection or dysentery.