JP2014198711A - Tetravalent peptides inhibiting ct, and cholera treatment agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel peptides inhibiting cholera toxin (CT) which target B-subunit (CTB) responsible for binding to the target cell of CT and to provide a cholera treatment agent.SOLUTION: In tetravalent peptides inhibiting CT, any one of peptide motifs in a specific sequence binds to each of four amino groups located at both ends of a molecule nuclear structure formed by binding three lysines (Lys), directly or through a spacer. In the tetravalent peptides inhibiting CT, an N terminal is acetylated.

Description

  The present invention relates to a CT-inhibiting tetravalent peptide and a cholera therapeutic agent.

  So far, the present inventors have developed a new multivalent peptide library method that inhibits strong interaction based on the cluster effect, and Shiga toxin produced by Enterohaemorrhagic E.coli (EHEC). A series of novel Stx-inhibiting peptides have been developed targeting (Stx) (Patent Documents 1 and 2). Furthermore, the present inventors have also developed a screening method using this multivalent peptide library. Specifically, a peptide having a known sequence is synthesized in a multivalent form so that a cluster effect can be exerted, and a spot-synthesized multivalent peptide is synthesized on Stx1a on a single cellulose sheet. 11 novel Stx1a-inhibiting peptides have been developed by screening using the high-affinity binding to the B subunit globo trisaccharide binding site, site 2, as an index (Patent Document 3).

  Cholera, on the other hand, is an infectious disease caused by Vibrio cholerae and is a disease that is required to be treated at the world level because it is widespread throughout the world, particularly in developing countries. This disease is mainly caused by severe water-soluble diarrhea, and if it is not treated appropriately, such as a replacement fluid, it will die with a high probability. Currently, vaccines against Vibrio cholerae are actively being developed, but on the other hand, many Vibrio cholerae are rapidly acquiring resistance to multiple types of antibiotics. Is an urgent need.

  The major virulence factor of Vibrio cholerae is cholera toxin (Cholera toxin; hereinafter referred to as CT) produced by this fungus. CT is a toxin of type AB5, which recognizes and binds to the ADP-ribosylating enzyme A-subunit of the protein and recognizes the receptor present on the target cell, and then binds the A-subunit into the cell. It is composed of B-subunit (CTB) pentamers that play a role in transport. The A-subunit that has entered the cell ADP-ribosylates Gα, a GTP-binding protein, and keeps it in an activated state, thereby causing a continuous outflow of ions and water through the channel. It is known that the CT receptor present on the target cell is ganglioside GM1, which is a kind of glycolipid. CTB1 molecule specifically recognizes the terminal sugar chain of GM1, and thus CTB pentamer can bind up to 5 molecules of GM1. It is known that the binding affinity is remarkably increased by this 5: 5 binding, and this phenomenon is called a cluster effect.

WO2006 / 001542 Publication JP 2011-079808 A JP 2011-158341 A

  As described above, in the research so far, the multivalent peptide library method has been mainly used for the development of Stx inhibitory peptides, but studies on inhibitory peptides against CT have not always been sufficiently conducted.

  An object of the present invention is to develop a CT inhibitory peptide and a cholera therapeutic agent by targeting CTB responsible for binding of CT to a target cell.

  In order to solve the above problems, the CT-inhibiting tetravalent peptide of the present invention is a CT-inhibiting tetravalent peptide that binds to cholera toxin and inhibits toxicity, and is formed by combining three lysines (Lys). In addition, any one of the peptide motifs of SEQ ID NOs: 1 to 6 is bound to each of the four amino groups located at the ends of the molecular nucleus structure directly or via a spacer. .

  In this CT-inhibiting tetravalent peptide, the N-terminus is preferably acetylated.

  The therapeutic agent for cholera of the present invention is characterized by containing the CT-inhibiting tetravalent peptide.

  The cholera toxin-inhibiting tetravalent peptide of the present invention and the cholera therapeutic agent containing the peptide can bind to CT with high binding affinity and effectively inhibit cytotoxicity.

It is a figure which shows the result of evaluation of GM1 binding activity of wild type CTB and CTB-E51A. It is a figure which shows the structure of the multivalent type peptide library used for the screening. It is a figure which shows the result of the screening by a multivalent type peptide library. The structure of each of the primary, secondary, and tertiary libraries and the results of screening using each library are shown in order from the top. The position number in the figure indicates the position from the N-terminal side of the degenerate position of each library. It is a figure which shows the binding activity with respect to CTB and CTB-E51A of GGR-tet and MA-tet. It is a figure which shows the effect of GGR-tet and MA-tet on the morphological change of the CHO cell by CT processing. It is the figure which illustrated the structure of the tetravalent peptide synthesize | combined on the cellulose sheet. (A) is a figure which showed the sheet | seat synthesis | combination of the peptide which substituted each position of GGRHRRR with Ala substitution or Glu substitution. Each tetravalent peptide synthesized on the sheet has a structure of MA-XXXXXXX-AU-. The sequence shown in the figure shows the sequence of the portion of -XXXXXXX-. (B) is a view showing a state in which the radioactivity of the binding between each sheet and labeled WT-CTB or labeled E51A-CTB is detected. (A) is a view showing sheet synthesis of peptides in which positions 1Gly, 2Gly, and 7Arg of GGRHRRR are substituted with amino acids. (B) is a view showing a state in which the radioactivity of the binding between each sheet and labeled WT-CTB or labeled E51A-CTB is detected. (A) is a view showing sheet synthesis of peptides in which positions 1Gly and 7Arg of GNRHRRR are substituted for positions 1Gly and 2Gly of GGRHRRL with various amino acids, respectively. (B) is a view showing a state in which the radioactivity of the binding between each sheet and labeled WT-CTB or labeled E51A-CTB is detected. It is a figure which shows the binding activity with respect to WT-CTB and E51A-CTB of YGR-tet. It is a figure which shows the binding activity with respect to WT-CTB and E51A-CTB of GNR-tet. It is a figure which shows the effect of YGR-tet and GNR-tet on the morphological change of the CHO cell by CT processing. It is a figure which shows the effect of YGR-tet and GNR-tet on cAMP production by CT. Intracellular cAMP production during CT treatment is shown as 100%.

  The CT inhibitory tetravalent peptide of the present invention inhibits CT cytotoxicity by binding to CT with high binding affinity.

The CT-inhibiting tetravalent peptide of the present invention has, on each of four amino groups located at the end of a molecular nucleus structure formed by combining three lysines (Lys),
The following peptide motifs;
Sequence number 1: Gly-Gly-Arg-His-Arg-Arg-Arg (GGRHRRR)
Sequence number 2: Tyr-Gly-Arg-His-Arg-Arg-Leu (YGRHRRL)
Sequence number 3: Gly-Asn-Arg-His-Arg-Arg-Gly (GNRHRRG)
Sequence number 4: Gly-Lys-Arg-His-Arg-Arg-Leu (GKRHRRL)
Sequence number 5: Arg-Gly-Arg-His-Arg-Arg-Leu (RGRHRRL)
Sequence number 6: Gly-Asn-Arg-His-Arg-Arg-Arg (GNRHRRR)
Any one of them is bound.

  That is, the CT-inhibiting tetravalent peptide of the present invention has, for example, the following chemical formula:

4 exemplifies a tetravalent peptide in which any one of the peptide motifs of SEQ ID NOs: 1 to 6 is incorporated in the XXXX portion located at the end of the molecular nucleus structure.

  Hereinafter, the CT inhibitory tetravalent peptide having the peptide motif of SEQ ID NO: 1 is “GGR-tet”, the CT inhibitory tetravalent peptide having the peptide motif of SEQ ID NO: 2 is “YGR-tet”, and has the peptide motif of SEQ ID NO: 3 A CT-inhibiting tetravalent peptide may be described as “GNR-tet”.

Further, in the above chemical formula 1, a form in which a spacer is bonded to each of the four amino groups located at both ends of the molecular nucleus structure is illustrated. It is also possible to directly bind the peptide motif of SEQ ID NO: 1. When the spacer is bound, any specific molecule and length may be used as long as the inhibitory activity against CT toxicity is not impaired. As the spacer, for example, a chain having an amino acid at the terminal and having a chain length of about 4 to 10 carbon atoms is preferable, and the CT toxicity inhibitory activity is particularly an amino hexanoic acid [NH] represented by “U” in the above chemical formula 1. _2- (CH ₂ ) ₅ —COOH] (aminocaproic acid) can be preferably exemplified. Moreover, as an amino acid contained in a spacer, an alanine (A) can be illustrated, for example.

  Furthermore, the CT-inhibiting tetravalent peptide of the present invention preferably has a modified molecule at each end of the peptide motif of SEQ ID NO: 1.

Moreover, since NH ₂ is exposed to a positive charge when the terminal of the peptide motif is exposed, from the viewpoint of charge control, a molecule having no charge as a modified molecule at each terminal of the peptide motifs of SEQ ID NOs: 1 to 6, Is also considered to bind hydrophobic molecules. For example, when a CT-inhibiting tetravalent peptide or a therapeutic agent containing the same is orally administered, the terminal NH ₂ may be protected with an acetyl group for the purpose of stabilizing in order to suppress degradation by proteases in the digestive tract. it can. Thereby, the CT inhibitory tetravalent peptide of the present invention has an increased inhibitory activity on CT toxicity. Thus, the modified molecule at the end of the peptide motif can be appropriately selected.

  In addition, although the peptide illustrated by the said Chemical formula 1 has MA (Met-Ala) at the terminal, this has illustrated what was introduce | transduced in the screening in the below-mentioned Example, and the said Chemical formula One MA is not necessarily required in the CT-inhibiting tetravalent peptide of the present invention.

  And the preparation method of CT inhibition tetravalent peptide of this invention is not specifically limited, A well-known method can be employ | adopted suitably, For example, it can produce by methods, such as using a peptide synthesizer.

  As described above, the CT-inhibiting tetravalent peptide of the present invention is a tetravalent peptide having four of any one of the peptide motifs of SEQ ID NOs: 1 to 6, and strongly binds to CT due to the cluster effect. It exhibits affinity and can effectively inhibit CT cytotoxicity.

  And since the CT inhibitory tetravalent peptide of this invention has CT inhibitory activity, the chemical | medical agent containing this CT inhibitory tetravalent peptide becomes a cholera therapeutic agent.

  The cholera therapeutic agent can be prepared and stored by mixing with any pharmaceutically acceptable carrier, excipient or stabilizer in addition to the CT-inhibiting tetravalent peptide.

  Acceptable carriers, excipients, or stabilizers can be appropriately selected depending on the dosage form and administration route, provided that they are non-toxic to patients at the dosages and concentrations used.

  Specifically, for example, buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride) Benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides; serum albumin, gelatin Or proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Saccharides such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn -Protein complexes) and nonionic surfactants such as polyethylene glycol (PEG).

  In addition, it is desirable that the drug administered into the body is sterile. In addition, sustained-release preparations may be prepared. A suitable example of a sustained release formulation is a semi-permeable matrix (eg, in the form of a film or microcapsule) of a solid hydrophobic polymer. Examples of sustained release matrices are polyester hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polyactides (US Pat. No. 3,773,919), L-glutamic acid and γ-ethyl-L-glutamate. Non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer, poly- (D) -3-hydroxybutyric acid and the like can be exemplified.

  The cholera therapeutic agent formulated in this way can be administered systemically, for example, orally, locally, or intravenously, depending on the symptoms, but CT is produced in the intestinal tract, Oral administration is particularly preferred because the targeted details are mainly intestinal epithelial cells. In this case, it is preferable to appropriately modify the CT-inhibiting tetravalent peptide of the present invention so as not to be degraded by proteases in the digestive tract. Specifically, it is preferable to acetylate the N-terminus of the CT-inhibiting tetravalent peptide of the present invention, which can significantly enhance the therapeutic effect upon oral administration.

  In addition, the dose of the cholera therapeutic agent of the present invention can be determined according to the patient's weight, symptoms, etc., for example, about 5 to 100 mg / Kg per dose, and as a guideline, about 5 to 10 mg / Kg. The degree can be used as a guide.

  The therapeutic agent of the present invention contains a CT-inhibiting tetravalent peptide and can be effectively used for the treatment of cholera. Since cholera therapeutics targeting CT do not exhibit toxicity against Vibrio cholerae itself, there is no problem of resistance, and it is expected that the problem of drug resistance against antibiotics, which is currently spreading rapidly, can be overcome. Furthermore, the peptidic compound (peptide motif) incorporated into the CT-inhibiting tetravalent peptide of the present invention can be synthesized by sequentially adding amino acids to the tetravalent core structure, and can be conveniently prepared in the same manner as monovalent peptide synthesis. Bulk synthesis is possible.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.

<Example 1> Preparation of wild-type CTB and CTB mutant used for screening A large amount of CTB in a state of being fixed to beads is required for screening. Therefore, a large amount of recombinant CTB having a His-tag introduced at the C-terminal side of the CTB gene was prepared using an E. coli expression system and immobilized on the beads using Ni-beads.

  In creating a CTB mutant having a mutation at the receptor binding site, the terminal galactose binding site of GM1 was targeted. And from the knowledge about the crystal structure analysis of CTB, it was inferred that Glu51 was involved in the binding of GM1 to terminal galactose, and CTB-E51A in which Glu51 was replaced with Ala was similarly prepared in large quantities.

  The activity of the obtained wild-type CTB and CTB-E51A was evaluated by examining specific binding to GM1 using the ELISA method. Specifically, phosphatidylcholine: GM1 (or asialo-GM1) = 0.1: 0.025 (μg / plate, GM1 is 2.5%) is coated on an ELISA plate, and after combining recombinant CTB of each concentration eluted from the beads, it is washed. The remaining CTB was detected using a specific antibody.

  FIG. 1 shows the results of evaluation of GM1 binding activity of wild type CTB and CTB-E51A. In FIG. 1, “WT-GM1” indicates the binding activity between wild-type CTB and GM1, “WT-AGM1” indicates the binding activity between wild-type CTB and Asialo GM1, and “E51A-GM1” indicates CTB-E51A and GM1. "E51A-AGM1" indicates the binding activity between CTB-E51A and Asialo GM1. Asialo GM1 is a glycolipid obtained by removing terminal sialic acid from GM1.

  As shown in FIG. 1, it was confirmed that wild-type CTB strongly bound to GM1 in a concentration-dependent manner. On the other hand, it was confirmed that the maximum binding amount of wild-type CTB decreased to 1/4 for asialo GM1. From this result, it was shown that wild-type CTB specifically recognizes GM1.

  Furthermore, it was confirmed that CTB-E51A showed the maximum amount of binding to GM1 that was almost the same as that of wild type CTB, but the binding affinity was reduced to 1/2. Further, it was confirmed that the binding activity of CTB-E51A markedly decreased against asialo GM1, unlike the case of wild type CTB. This is because the terminal galactose plays an important role in binding to CTB because there is no terminal sialic acid in the case of Asialo GM1, but the site involved in the recognition of galactose is mutated in CTB-E51A. Therefore, it is considered that the binding activity was significantly reduced.

  From the above, it was confirmed that CTB-E51A had a reduced terminal galactose-dependent binding ability to GM1 without being greatly affected by the higher-order structure by mutagenesis.

<Example 2> Identification of an E51-specific binding motif using a multivalent peptide library method Based on the results of Example 1, screening with a multivalent peptide library was performed in comparison with CTB-E51A. Indices of high binding activity were used as indicators. It was speculated that this screening could obtain a motif that binds to CTB in an E51-dependent manner.

  The structure of the multivalent peptide library used for screening is shown in FIG. In the figure, M, A, and U represent Met, Ala, and aminocaproic acid, respectively. XXXX indicates a library portion, and X (degenerate position) indicates that synthesis was performed using a mixture of 19 amino acids other than Cys.

  First, as a primary library, screening was performed using a multivalent peptide library whose library part was XXXX (4 degenerate positions). First, each CTB-immobilized bead (CTB amount 250 μg) and a multivalent peptide library (300 μg) were incubated in 200 μl of PBS at 4 ° C. for 18 hours. After washing the beads, the peptide bound to CTB was eluted with 30% acetic acid, and after recovery, amino acid sequencing was performed sequentially from the N-terminal side.

  For each degenerate position, the amount ratio of 19 detected amino acids was calculated and corrected so that the sum was 19. Furthermore, for each amino acid, the value obtained when wild-type CTB was used was divided by the value obtained when CTB-E51A was used to calculate the ratio, and the sum of the values for all amino acids was again calculated. Was corrected to be 19. Therefore, if there is no difference in the selectivity of each amino acid between wild type CTB and CTB-E51A, the value is all 1. As an index of selectivity, when this value exceeded 1.2, it was determined that strong selectivity was observed for wild-type CTB.

  As a result of the screening using the primary library, it was shown that basic amino acids were selected for all four degenerate positions, in particular, Arg was frequently selected (the upper part of FIG. 3).

  Therefore, as a secondary library, Arg was mainly introduced and three degenerate positions were introduced on both sides (middle of Fig. 3). When screening with the secondary library was performed, all positions were It was shown that a basic amino acid was selected, and in particular, Arg selectivity at the +2 position of immobilized Arg was the highest (middle in FIG. 3).

  Therefore, a tertiary library centered on the RXR motif was created (bottom of Fig. 3), and screening with the tertiary library was performed. Finally, the motif that arranged the most strongly selected amino acids at each position, GGRHRRR ( SEQ ID NO: 1) was identified (bottom of FIG. 3).

<Example 3> Evaluation of CTB binding ability of GGR-tet The motif of Gly-Gly-Arg-His-Arg-Arg-Arg (GGRHRRR) identified in Example 2 is shown in FIG. A tetravalent peptide (peptidic compound) incorporated in the XXXX part of the type peptide library was synthesized. Hereinafter, this tetravalent peptide is referred to as “GGR-tet” (tet means a tetramer). As a negative control, MA-tet, a peptidic compound in which only the XXXX part of the multivalent peptide library shown in FIG. 2 was deleted and all other structures were retained, was also synthesized.

  The binding activity of GGR-tet and MA-tet to wild type CTB and CTB-E51A was examined by ELISA. Specifically, ELISA plates are coated with various concentrations of GGR-tet and MA-tet, and after binding CTB (1.0 μg / ml) or CTB-E51A (1.0 μg / ml), they are washed and remain. CTB or CTB-E51A was detected using anti-CTB antibody.

  The results are shown in FIG. “GGR-tet WT-CTB” in FIG. 4 indicates the binding activity between GGR-tet and wild-type CTB, and “GGR-tet E51A-CTB” indicates the binding activity between GGR-tet and CTB-E51A. “MA-tet WT-CTB” indicates the binding activity between MA-tet and wild-type CTB, and “MA-tet E51A-CTB” indicates the binding activity with CTB-E51A. .

  As a result, it was confirmed that MA-tet did not show any binding activity to either wild type CTB or CTB-E51A.

  It was confirmed that GGR-tet exhibits strong binding activity to wild-type CTB in a concentration-dependent manner. On the other hand, although GGR-tet showed a binding activity to CTB-E51A in a concentration-dependent manner, it was confirmed that its maximum binding activity was reduced to about 1/2 compared to CTB. This indicates that GGR-tet is bound to CTB in an E51-dependent manner.

<Example 4> Evaluation of CT inhibition ability of GGR-tet
It is known that the morphology of CHO cells changes markedly by CT treatment and becomes spindle-shaped. This system is a simple and widely used CT activity evaluation system. Therefore, the effect of GGR-tet on the morphological change of CHO induced by CT treatment was examined.

  CHO cells were treated with 0.5 μg / ml CT in the presence or absence of GGR-tet and MA-tet (30,100 μg / ml), and changes in cell morphology were observed under a microscope for 3 hours at 37 ° C. did. About 300 cells were observed per treatment, and the proportion of cells that were recognized as having a markedly expanded morphology and spindle shape was calculated. In untreated cells, the proportion of spindle-shaped cells is only about 10%, whereas when CT treatment is performed, the proportion increases to 60% or more.

  At this time, coexistence of MA-tet without any motif in the structure has almost no effect on the ratio, but when GGR-tet coexists, cells that show spindle shape in a volume-dependent manner It was confirmed that the ratio decreased to 38% or less at 100 μg / ml.

  This indicates that GGR-tet inhibited the action of CT on CHO by binding to the receptor binding site of CT targeting E51.

  As shown in the above examples, a novel CTB binding motif (SEQ ID NO: 1) was identified by screening a multivalent peptide library targeting the receptor binding site of CTB, and this motif was incorporated into the molecule. Four tetravalent peptides (peptidic compounds) having four: GGR-tet was confirmed to bind to CTB with high affinity and efficiently inhibit the action of CT on target cells. Therefore, it became clear that it can be used as a therapeutic agent for cholera (CT inhibitor) by containing GGR-tet as an active ingredient.

  <Example 5> Identification of CTB binding motif using multivalent peptide sheet synthesis technology Since the CT inhibitory effect by GGR-tet was confirmed, more binding force based on GGR-tet peptide motif (GGRHRRR) I tried to obtain a motif with excellent inhibitory effect.

  First, in order to identify which amino acids of the GGRHRRR motif are essential for binding to CTB, amino acids at each position were replaced with Ala, and basic amino acids were subjected to Glu substitution with different charges. The peptide was synthesized as a tetravalent peptide on the sheet so that the cluster effect could be exhibited, and the influence on the binding to WT-CTB and E51A-CTB was examined.

  The method for synthesizing the tetravalent peptide on the sheet was performed according to the procedure described in Japanese Patent Application Laid-Open No. 2011-158341 proposed by the present inventor. Specifically, a spot peptide synthesizer from intavis AG was used for the synthesis. First, the amino group present on the cellulose sheet (Intavis AG) was reacted with Fmoc-βAla and then with Fmoc-aminocaproic acid to ensure a sufficient spacer length. Next, in order to branch the peptide chain into four valences, FmocLys (Fmoc) was reacted twice in succession, and an elongation reaction was performed so as to give the subsequent sequences evenly to the four amino groups formed. . By this operation, the tetravalent peptide synthesized on the cellulose sheet has the same nuclear structure as that of the multivalent peptide library shown in FIG. 2 (FIG. 6). FIG. 7A shows the motif sequence of each tetravalent peptide spot-synthesized on the sheet by the above operation and positional information on the sheet.

Furthermore, the sheet on which the tetravalent peptide compound was spot-synthesized was blocked with 5% Skim Milk Powder, and then ¹²⁵ I-His-labeled WT-CTB (1 μg / ml) or ¹²⁵ I-His-labeled E51A-CTB ( 1 μg / ml), each at 4 ° C. for 1 hour, washed, washed with BAS-2500 to detect the radioactivity bound on the sheet (FIG. 7B), and quantitative analysis was performed (Table 1) . Specifically, when blotting with labeled WT-CTB and blotting with labeled E51A-CTB, the density (PSL) of each spot was quantified, and the total PSL value of each spot was the number of spots. The correction was displayed so that there was 19. That is, if there is no difference in binding between the compounds, all values are 1.0. In addition, as an indicator of binding specificity, the value obtained by dividing the corrected binding amount to WT-CTB (WT-CTB binding force) by the corrected binding amount to E51A-CTB (E51A-CTB binding force) WT-CTB / E51A-CTB (binding ratio) was calculated, and this value was corrected and displayed so that the total was 19. Furthermore, as an indicator that the binding strength to WT-CTB and the binding ratio are both excellent, the value obtained by multiplying the corrected binding amount to WT-CTB and the corrected binding ratio (WT-CTB binding) Force x binding ratio).

  The results are shown in Table 1.

  As shown in Table 1, especially when the 3rd to 6th amino acids from the N-terminus are each replaced with Ala, the amount of binding to WT-CTB decreases compared to GGRHRRR, and any base present in GGRHRRR It was clarified that the amount of binding to WT-CTB was reduced even when the sex amino acid was replaced with Glu.

  Furthermore, it became clear that the amount of binding to WT-CTB decreased even if any two of the four Args present in GGRHRRR were replaced with Ala. On the other hand, even if the 1st, 2nd, and 7th amino acids from the N-terminus are replaced with Ala, no significant reduction in binding force is observed. Furthermore, all of these three amino acid substitution products showed high WT-CTB / E51A-CTB values (binding ratio) and WT-CTB binding power × binding ratio values. The above results show that the third to sixth amino acids from the N-terminal side, RHRR, plays an essential role for E51-specific binding to WT-CTB, while the first, second, and seventh from the N-terminal. It shows that the second amino acid can be substituted with another amino acid. Then, RHRR in the GGRHRRR motif was fixed, and the first, second, and seventh amino acids from the N-terminal were sequentially spot-synthesized as tetravalent peptides with 19 kinds of amino acids except Cys. Investigation was performed (FIGS. 8A and 8B). Table 2 shows the results of the quantitative analysis. In Table 2, correction was displayed so that the total PSL value of each spot was 56, which is the number of spots. In addition, as an indicator of binding specificity, the value obtained by dividing the corrected binding amount to WT-CTB (WT-CTB binding force) by the corrected binding amount to E51A-CTB (E51A-CTB binding force) WT-CTB / E51A-CTB (binding ratio) was calculated, and this value was corrected and displayed so that the total was 56. Furthermore, as an indicator that the binding strength to WT-CTB and the binding ratio are both excellent, the value obtained by multiplying the corrected binding amount to WT-CTB and the corrected binding ratio (WT-CTB binding) Force x binding ratio). The results are displayed in order of strong WT-CTB binding force.

  As shown in Table 2, when the top 5 species (WT-CTB binding strength 1.25 or more) with high binding to WT-CTB were selected, all peptides had high WT-CTB binding strength x binding ratio. It became clear to show a value (above 1.48). That is, these peptides are excellent in both binding power and binding specificity to WT-CTB.

  Therefore, GNRHRRR and GGRHRRL were selected from these five types, and GNRHRRR was substituted with various amino acids for position 1Gly and position 7Arg, and for GGRHRRL, position 1Gly and position 2Gly, respectively. Performed (FIG. 9). Table 3 shows the results of the quantitative analysis. In Table 3, correction is displayed so that the total PSL value of each spot is 76, which is the number of spots. In addition, as an indicator of binding specificity, the value obtained by dividing the corrected binding amount to WT-CTB (WT-CTB binding force) by the corrected binding amount to E51A-CTB (E51A-CTB binding force) WT-CTB / E51A-CTB (binding ratio) was calculated, and this value was corrected and displayed so that the total was 76. Furthermore, as an indicator that the binding strength to WT-CTB and the binding ratio are both excellent, the value obtained by multiplying the corrected binding amount to WT-CTB and the corrected binding ratio (WT-CTB binding) Force x binding ratio). The results were displayed in descending order of the value of WT-CTB binding force × binding ratio.

As a result, we focus on 5 types (1.7 or more) from the highest values of WT-CTB binding force x binding ratio, which is an indicator of excellent binding strength and binding ratio to WT-CTB. did. These five peptides,
Sequence number 2: Tyr-Gly-Arg-His-Arg-Arg-Leu (YGRHRRL)
Sequence number 3: Gly-Asn-Arg-His-Arg-Arg-Gly (GNRHRRG)
Sequence number 4: Gly-Lys-Arg-His-Arg-Arg-Leu (GKRHRRL)
Sequence number 5: Arg-Gly-Arg-His-Arg-Arg-Leu (RGRHRRL)
Sequence number 6: Gly-Asn-Arg-His-Arg-Arg-Arg (GNRHRRR)
All showed high WT-CTB binding force (1.25 or more).

  <Example 6> Evaluation of CTB binding ability of YGR-tet and GNR-tet Among the five peptides focused on in Example 5, two types of YGRHRRL (SEQ ID NO: 2) and GNRHRRG (SEQ ID NO: 3) were newly developed as CTBs. It was selected as a binding motif and used for the following studies. That is, the motifs of YGRHRRL (SEQ ID NO: 2) and GNRHRRG (SEQ ID NO: 3) were incorporated into the four XXXX parts of the multivalent peptide library shown in FIG. 2 to synthesize tetravalent peptide compounds (hereinafter referred to as YGR). -tet and GNR-tet). As a negative control, a peptide compound, MA-tet, in which only the XXXX part of the multivalent peptide library shown in FIG. 2 was deleted and all other structures were retained was also synthesized.

  Then, the binding activity of YGR-tet and GNR-tet to WT-CTB and E51A-CTB was examined by ELISA (FIGS. 10 and 11). Specifically, ELISA plates are coated with various concentrations of YGR-tet (Fig. 10) or GNR-tet (Fig. 11), and after binding CTB (1.0 µg / ml) or CTB-E51A (1.0 µg / ml) After washing, the remaining WT-CTB or E51A-CTB was detected using an anti-CTB antibody.

  As a result, both YGR-tet and GNR-tet showed strong binding activity to WT-CTB in a concentration-dependent manner, but the binding activity to E51A-CTB was attenuated, especially GNR-tet It was confirmed that it does not bind to E51A-CTB. That is, it was revealed that YGR-tet and GNR-tet show excellent binding site specificity.

<Example 7> Inhibitory effect of YGR-tet and GNR-tet on morphological change induction of CHO cells by CT As in Example 4, the inhibitory effect of YGR-tet and GNR-tet on morphological change induction of CHO cells by CT As a result, it was clarified that all showed a stronger inhibitory effect than GGR-tet (FIG. 12).

<Example 8> Inhibitory effect of YGR-tet and GNR-tet on cAMP production in Caco-2 cells by CT When CT is taken in via a receptor on the intestinal epithelial cell, the cytoplasm passes through retrograde transport A subunit is carried in. A- subunit is ADP- ribosylation of G _s is a GTP-binding protein, activate adenylate cyclase by holding the activated state. As a result, the intracellular cAMP concentration increases, cAMP-dependent A kinase is activated, and is activated by phosphorylating ion channels. As a result, Cl ions flow out and a large amount of water flows out according to the osmotic pressure, causing diarrhea. Therefore, we decided to examine the inhibitory effect of YGR-tet and GNR-tet on cAMP production by CT using Caco-2 cells, a human colon cancer-derived cell line.

  Specifically, Caco-2 cells differentiated into epithelial cells were incubated at 37 ° C. for 30 minutes in the presence or absence of YGR-tet and GNR-tet (30,100 μg / ml), and then 1.0 μg / ml After adding CT and incubating at 37 ° C. for 3 hours, a cell lysate was prepared. The amount of cAMP produced in each lysate was measured using a PerkinElmer cAMP Alpha Screen Kit.

  As a result, a significant increase in cAMP (1.4 nmol / mg protein) was observed with CT treatment, while YGR-tet and GNR-tet both inhibited this cAMP production in a dose-dependent manner, particularly 100 μg / In ml, YGR-tet showed a decrease to 69% and GNR-tet to 54% (FIG. 13).

Claims (3)

  A CT-inhibiting tetravalent peptide that binds to cholera toxin and inhibits toxicity, wherein each of the four amino groups located at the end of the molecular nucleus structure formed by combining three lysines (Lys) has a sequence A CT-inhibiting tetravalent peptide, wherein any one of the peptide motifs of Nos. 1 to 6 is bound directly or via a spacer.   The CT-inhibiting tetravalent peptide according to claim 1, wherein the N-terminus is acetylated. A therapeutic agent for cholera comprising the CT-inhibiting tetravalent peptide according to claim 1 or 2.