JP5635779B2 - Stx toxicity-inhibiting peptide and therapeutic agent for diseases caused by Stx - Google Patents

Description

  The present invention relates to a Stx toxicity-inhibiting peptide.

  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.

  Furthermore, it is also known that Stx is composed of two families, Stx1 and Stx2.

Then, Stxl and Stx2 are both in _{A-B 5} type toxin receptor on B subunit cell _{membrane, Gb 3 (globotriaosylceramide: Galα (} 1-4) -Galβ (1-4) -Glcβ1- Incorporated into cells by binding to Ceramide, and B subunit pentamers arranged in a radial symmetry form Gb3 sugar chain (globo trisaccharide: Galα (1-4) -Galβ (1-4 ) With the elucidation of specifically recognizing -Glcβ1-), a method for selectively inhibiting the binding between the B subunit and the receptor has attracted attention, and studies from various viewpoints have been promoted. I came.

  The inventors of this application have established a screening method using a multivalent peptide library based on the knowledge that there is a cluster effect in the binding of Stx1 and Stx2 B subunits to 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. Okay, Patent Document 1 specifies a Stx2 inhibitory peptide having a high binding affinity with Stx2 by exerting a cluster effect with the B subunit of Stx2 by making the peptide library multivalent. . The reason why the present inventors have focused on peptides as Stx2 inhibitors is that synthesis is relatively easy and generally safe for application as a drug.

  However, since the screening method of Patent Document 1 specifies peptides by deriving amino acid selectivity from the binding between an object and a library, generally, the toxic inhibitory effect of the specified peptides is also observed. It was difficult to predict. And in patent document 1, although examination about Stx2 toxicity inhibition peptide is made | formed, concrete examination was not made about Stx1 toxicity inhibition peptide.

  Since Stx1 is the same as Shiga toxin produced by Shigella dysenteriae Type I, if a Stx1 toxicity inhibitor is developed, it is an effective therapeutic agent for not only enterohemorrhagic Escherichia coli infection but also dysentery It is thought that it can be. In fact, in Asia, 91 million people suffer from bacterial dysentery annually, of which 410,000 people die, so the development of Stx1 toxicity inhibitors is urgent, but at present, Stx1 toxicity that can withstand clinical application Inhibitors have not been developed.

  On the other hand, as pointed out in Patent Document 1, it is Stx2 that causes more serious complications among Stx1 and Stx2, and clinically, it is considered important to develop a Stx2 toxicity inhibitor. be able to.

  Therefore, if a peptide that exhibits a toxic inhibitory effect on both Stx1 and Stx2 can be found, various diseases caused by Stx1 and Stx2 can be effectively treated by using this peptide as an active ingredient of a drug. Can be thought of as

WO2006 / 001542

  The present invention has been made in view of the circumstances as described above, and provides a peptide that exhibits an excellent toxicity inhibitory action on both Stx1 and Stx2, and a therapeutic agent for diseases caused by Stx1 and Stx2. The challenge is to do.

In order to solve the above problems, the Stx toxicity-inhibiting peptide of the present invention and the therapeutic agent for diseases caused by Stx are characterized by the following.
<1> The Stx toxicity-inhibiting peptide of the present invention has a binding property to Stx1 and Stx2, and is a peptide that inhibits the cytotoxicity of Stx1 and Stx2, and is a molecule formed by binding three lysines (Lys) The peptide of SEQ ID NO: 1 is bound to each of the four amino groups located at both ends of the core structure, either directly or via a spacer.
<2> In the Stx toxicity-inhibiting peptide of the present invention, the spacer has a hydrocarbon chain having 4 to 10 carbon atoms.
<3> The therapeutic agent of the present invention is a therapeutic agent for diseases caused by Stx1 and Stx2, and contains the Stx toxicity-inhibiting peptide of <1> or <2>.
<4> The therapeutic agent of the present invention is a therapeutic agent for diseases caused by Stx1 and Stx2, that is, enterohemorrhagic Escherichia coli infection or dysentery.

  According to the Stx toxicity-inhibiting peptide of the present invention, the cytotoxicity of Stx1 and Stx2 can be inhibited. Therefore, a drug containing this Stx toxicity-inhibiting peptide is used for diseases caused by Stx1 and 2, such as enterohemorrhagic E. coli infection (Stx1-producing enterohemorrhagic E. coli infection, Stx2-producing enterohemorrhagic E. coli infection, Stx1 and Stx2 both toxin-producing enterohemorrhagic Escherichia coli infections) and dysentery are extremely effective.

It is a figure which shows the toxicity inhibitory effect of PPR-tet and PPP-tet with respect to Stx1. It is a schematic diagram which illustrates the screening method of Stx1 toxicity inhibition peptide. It is a figure which shows the inhibitory effect with respect to Stxl cell cytotoxic activity. Five types of peptide motifs were examined. It is a figure which shows that MMA-tet has couple | bonded specifically with the site 1 of STX1B subunit. It is a figure which shows the inhibitory effect with respect to Stx2 cell disorder activity. Five types of peptide motifs were examined. It is a figure which shows the result of the infection experiment using a mouse | mouth.

  An embodiment of the present invention will be described. Hereinafter, the term “Stx” includes both Stx1 and Stx2.

  The Stx toxicity-inhibiting peptide of the present invention inhibits the cytotoxicity of Stx1 and Stx2 by binding to Stx1 and Stx2 with a strong binding affinity. Specifically, the Stx toxicity-inhibiting peptide of the present invention has SEQ ID NO: 1 as a peptide motif on each of four amino groups located at both ends of a molecular nucleus structure formed by binding three lysines (Lys). Met-Met-Ala-Arg-Arg-Arg-Arg-Arg (hereinafter sometimes referred to as MMARRRR).

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

In the example, the XXXXXX portion has MMARRRR (SEQ ID NO: 1). In addition, in the above chemical formula 1, a form in which each of the four amino groups located at both ends of the molecular nucleus structure has a spacer is exemplified, but the MMARRRR is directly attached to each of the four amino groups without using the spacer. (SEQ ID NO: 1) can also be bound. In the case of providing a spacer, it is sufficient that it does not impair the Stx toxicity inhibitory activity, and the specific molecule and length are not limited. 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 Stx1 toxicity inhibitory activity is particularly an amino hexanoic acid [NH] represented by “U” in the above chemical formula 1. _{2 - (CH 2) 5 -COOH} ] are preferred. The amino acid is exemplified by alanine (A), and binds to the above-mentioned MMARRRR (SEQ ID NO: 1).

  Furthermore, the Stx toxicity-inhibiting peptide of the present invention preferably has a modified molecule at each end of the four peptide motifs, MMARRRR (SEQ ID NO: 1).

  In addition, although the peptide illustrated by the above chemical formula 1 has MA (Met-Ala) at the terminal, this is an example of what was introduced at the time of screening in Examples described later, The above MA of Chemical Formula 1 is not necessarily required in the Stx toxicity-inhibiting peptide of the present invention.

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, as a modified molecule at each terminal of MMARRRR (SEQ ID NO: 1), Hydrophobic molecules are considered. For example, when a Stx toxicity-inhibiting peptide or a drug containing the same is orally administered, the terminal NH ₂ can be protected with an acetyl group for the purpose of stabilization for suppressing degradation by proteases in the digestive tract. This increases Stx toxicity inhibitory activity. Thus, the modified molecule at the end of the peptide motif can be appropriately selected.

  A known method can be appropriately employed as a method for producing the Stx toxicity-inhibiting peptide of the present invention. For example, it can be produced by a method such as using a peptide synthesizer.

  As described above, the Stx toxicity-inhibiting peptide of the present invention is a tetravalent peptide having four peptide motifs, MMARRRR (SEQ ID NO: 1), and has strong binding affinity for Stx1 and Stx2 due to the cluster effect. Therefore, according to the agent containing the Stx toxicity-inhibiting peptide of the present invention, diseases caused by Stx1 and 2, for example, enterohemorrhagic E. coli infection (Including Stx1-producing enterohemorrhagic Escherichia coli infection, Stx2-producing enterohemorrhagic Escherichia coli infection, Stx1, Stx2 both toxin-producing enterohemorrhagic Escherichia coli infection), and dysentery can be effectively treated.

  Hereinafter, as an embodiment of the present invention, a therapeutic agent for a disease containing the above Stx toxicity-inhibiting peptide will be described. Here, description about Stx toxicity inhibition peptide itself is abbreviate | omitted.

  In addition to the Stx toxicity-inhibiting peptide, the therapeutic agent for diseases caused by Stx1 and Stx2 is prepared and stored by mixing with any pharmaceutically acceptable carrier, excipient or stabilizer. 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. 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 (less than about 10 residues) polypeptide; serum albumin , Gelatin, or protein such as immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, including lucose, mannose, or dextrin, and other carbohydrates such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn − Protein complexes) or TWEEN (trade name), PLURONICS (trade name), and nonionic surfactants such as polyethylene glycol (PEG).

  In addition, the drug administered into the body must be 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, LUPRON DEPOT (trade name) (injectable globules of lactic acid-glycolic acid copolymer and leuprolide acetate), poly- (D) -3-hydroxy Butyric acid and the like.

  The therapeutic agent thus formulated can be systemically administered, for example, orally, locally, or intravenously, depending on the type and symptoms of the disease. The dose can be determined according to the patient's weight, symptoms, etc., and can be about 5 to 100 mg / Kg per dose, for example, about 5 to 10 mg / Kg as a guide.

  According to the therapeutic agent of the present invention, the Stx2-producing enterohemorrhagic Escherichia coli infection is extremely effective against Stx1-producing enterohemorrhagic Escherichia coli infection and dysentery, and more closely related to the severity of symptoms. , Stx1 and Stx2 both toxin-producing enterohemorrhagic Escherichia coli infections can be treated.

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

<Example 1>
(1) Since Stx1 and Stx2 have a homology close to 60% at the amino acid level, among the Stx2 inhibitory peptides identified in Patent Document 1, the effect of a particularly strong inhibitory peptide on Stx1 was examined.

  Specifically, the following formula

Each of the four amino groups located at both ends of the molecular nucleus structure formed by bonding three lysines (Lys) represented by the following peptide to the XXXXXX moiety via a spacer:
Sequence number 2: Pro-Pro-Arg-Arg-Asn-Arg-Arg (PPRRNRR)
Sequence number 3: Pro-Pro-Pro-Arg-Arg-Arg-Arg (PPPRRRRR)
The effect on Stx1 was examined using a tetravalent peptide to which is bound.

  Hereinafter, for convenience, a tetravalent peptide having the peptide motif of SEQ ID NO: 2 is referred to as “PPR-tet”, and a tetravalent peptide having the peptide motif of SEQ ID NO: 3 is referred to as “PPP-tet”.

In addition, MA (Met-Ala) at the end of the above chemical formula 1 is introduced at the time of screening in order to confirm the amino acid sequence, and the spacer length is optimized to Stx. Specifically, amino hexanoic acid [NH ₂ — (CH ₂ ) ₅ —COOH] was used as a spacer.
(2) The results are shown in FIG. As shown in FIG. 1, it was confirmed that PPR-tet and PPP-tet have a low toxicity inhibitory effect on Stx1.

  Thus, the reason why PPR-tet and PPP-tet, which have been confirmed to be Stx2 inhibitory, has a low inhibitory effect on Stx1, is that there is a region with slightly different surface charges on the receptor binding surface of the B subunit of Stx1 and Stx2. This is probably because it exists.

<Example 2>
(1) Based on the result of Example 1, identification of the Stx1 toxicity-inhibiting peptide was attempted by the following method.

  In order to develop a Stx1 toxicity-inhibiting peptide, first, which binding site is targeted is a problem. The globo trisaccharide binding site is known to be composed of site 1, site 2 and site 3 of the Stx1B subunit. In addition, Stx1B subunits are arranged in a radially symmetrical manner to form pentamers, but site 1 and site 2 are present at the edge of B subunit, and site 3 is located at a position near the center of B subunit. It is known that

The inventors of the present invention have found that site 1 contributes most to the binding between Stx1 and globotrisaccharide (Gb3), and this binding involves the hydrophobicity of the aromatic ring of phenylalanine 30 present at site 1. Focusing on the fact that the interaction is important, a mutant of F30A (Phe30 is replaced with Ala) was created using site 1 as the target site of the Stx1 toxicity-inhibiting peptide, and the same multivalent peptide library as in Patent Document 1 The method was used to screen for Stx1 toxicity-inhibiting peptides.
(2) Although the screening method is the same as that described in Patent Document 1, specifically, the screening was performed using the above multivalent peptide library. The outline is shown in FIG.

  The XXXX part of the multivalent peptide library shown in Chemical Formula 1 and FIG. 2 represents a degenerate position consisting of a mixture of 19 amino acids other than Cys.

  First, an affinity column fixed with wild-type Stx1B subunit and F30A (about 1 mg) having a mutation at site 1 is prepared, and as a primary screening, about 500 μg of the library is added and fully bound. I let you. After that, wash well with PBS etc., wash out the unbound library, finally elute the library remaining on the column with 30% acetic acid, dry the collected fraction, and analyze amino acid sequence Went.

  As a result, for the amino acid obtained at each degenerate position, a numerical result was obtained as to which amino acid was selected with what strength.

  That is, when using wild-type Stx1B subunit, the value of each amino acid at each degenerate position obtained as described above was obtained as described above when mutant type: F30A was used. Dividing by the value of the corresponding amino acid at the corresponding degenerate position, as a result, the number of times that the amino acid was selected in the wild-type Stx1B subunit compared to F30A was obtained as a numerical result. Then, it was standardized so that all 19 amino acid values would be 19. At this time, the value is 1 if there is no difference in selectivity between the amino acids. When this value exceeded 1.3, it was judged that strong selectivity was observed. The results are shown in the lower part of 1st-library in [Table 1].

  In primary cleaning, arginine (R) was strongly selected at degenerate positions: 5 to 7, so that the 2nd-libraries shown in the above [Table 1] were prepared using this arginine (R) as the locking position, Screening was performed. By introducing the locking position, the binding to the Stx1B subunit as a whole library increases, so that a more specific motif can be easily obtained. Using this 2nd-library, the peptide motif that binds to the wild type STX1B-subunit and the peptide motif that binds to the mutant STX1B-subunit of site 1 were determined in the same manner as described above.

  As a result, as shown in the lower part of 2nd-libraries in [Table 1] above, degenerate positions: 5 to 7 were identified as arginine (R). In degenerate positions: 1 to 3, methionine (M) and alanine (A) were strongly selected.

As a result, the following four
SEQ ID NO: 1 Met-Met-Ala-Arg-Arg-Arg-Arg (MMARRRR)
Sequence number 4: Met-Ala-Ala-Arg-Arg-Arg-Arg (MAARRRRR)
Sequence number 5: Ala-Met-Ala-Arg-Arg-Arg-Arg (AMARRRRR)
Sequence number 6: Ala-Ala-Ala-Arg-Arg-Arg-Arg (AAARRRRR)
The peptide motif was identified.

<Example 3>
(1) Each of the above peptide motifs: MMARRRR (SEQ ID NO: 1), MAARRRRR (SEQ ID NO: 4), AMARRRRR (SEQ ID NO: 5), and AAARRRRR (SEQ ID NO: 6) are formed by binding three lysines (Lys). four amino groups located at both ends of the molecular core structure, bound via a spacer, with the peptide motif described above were each examined binding affinity to STX1B subunits (K _D value). Hereinafter, the tetravalent peptides having the peptide motifs of SEQ ID NOS: 1 and 4-6 are referred to as “MMA-tet”, “MAA-tet”, “AMA-tet”, and “AAA-tet”, respectively. As a control, also with a compound MA-tet only core structure without a peptide motif, it was examined in the same manner the binding affinity (K _D value). Measurement of K _D values were performed using the BIAcore ^TM system (BIAcore, Uppsala, Sweden). In this system, STX1B subunits are immobilized on a sensor chip, and the amount of binding is measured by flowing various concentrations of tetravalent peptides. Analysis was carried out by BIAEVALIATION 3.0 (BIAcore), and calculates the maximum binding amount (RUmax) and K _D values.

In Chemical Formula 1, MA (Met-Ala) is bonded to the terminal, but this was introduced at the time of screening, and is not necessarily required in the Stx1 toxicity-inhibiting peptide of the present invention. Further, the specific configuration and length of the spacer are not limited to this embodiment.
(2) The results are shown in [Table 2]. The lower the _KD value, the higher the binding affinity.

As shown in [Table 2], it was confirmed that MMA-tet, MAA-tet, AMA-tet and AAA-tet all showed very high binding affinity to the Stx1B subunit. On the other hand, no binding was observed with MA-tet. This indicates that MMA-tet, MAA-tet, AMA-tet, and AAA-tet bind strongly to the STX1B subunit in a peptide motif-dependent manner. Note that RU _MAX (AU) indicates the maximum amount of binding.

<Example 4>
(1) Since the binding affinity of MMA-tet, MAA-tet, AMA-tet, and AAA-tet to the STX1B subunit was confirmed in Example 3, the inhibitory effect of each of the peptides on STX1 cytotoxicity investigated.

  Specifically, the experimental conditions were Vero cells highly sensitive to STX, and cultured for 3 days in the presence of 1 pg / ml STX1 and tetravalent peptides at each concentration, and the ratio of viable cells was measured. did.

Moreover, the inhibitory effect with respect to the cytotoxic activity by STX1 was examined also about PPP-tet.
(2) As a result, as shown in FIG. 3, it was confirmed that MMA-tet significantly inhibits STX1 cytotoxicity in a concentration-dependent manner. Specifically, the cell viability was 100% at a peptide concentration of 100 μg / ml. The IC50 value was 25 μg / ml.

  On the other hand, the inhibitory effect decreased with AMA-tet and PPP-tet, and the IC50 values were 100 μg / ml and 130 μg / ml, respectively. In MAA-tet and AAA-tet, the inhibitory effect was further reduced, and the IC50 values were 250 μg / ml and 300 μg / ml, respectively. Furthermore, it was revealed that MMA-tet exhibits STX1 toxicity inhibitory activity 5 times or more stronger than the STX2 inhibitory peptide PPP-tet identified so far.

  As described above, MMA-tet, MAA-tet, AMA-tet, and AAA-tet all have binding affinity for the STX1B subunit. Among them, MMA-tet is more STX1 than other peptides. The cytotoxic inhibitory effect of was markedly significant. That is, it was shown that there is not necessarily a correlation between the binding affinity for STX1 (Table 2) and the cytotoxic inhibitory effect (FIG. 3).

  From the comparison of the inhibitory activities of MMA-tet, AMA-tet and MAA-tet shown in FIG. 3, both Met (M) of SEQ ID NO: 1, which is a peptide motif of MMA-tet, are both necessary for inhibitory activity. However, it has been clarified that the second Met (M) plays a more important role in the STX1 toxicity inhibitory activity than the first Met (M).

  From the above, it was shown that a drug containing MMA-tet is effective as a therapeutic agent for diseases caused by Stx1, for example, enterohemorrhagic Escherichia coli infection and dysentery.

<Example 5>
(1) Based on the result of Example 4, the binding specificity of MMA-tet to STX1 was examined by ELISA.

Specifically, MMA-tet at each concentration was immobilized on an ELISA plate, and the wild-type STX1 and each F30A having a mutation at site 1 of the STX1B subunit were bound to each experimental condition. After washing, the amount of each specifically bound B subunit was quantified using a specific antibody.
(2) The results are shown in FIG. The vertical axis represents the absorbance of light at 490 nm. The binding activity of MMA-tet to F30A was reduced to about 1/10 compared to binding to wild-type STX1. From this, it was confirmed that MMA-tet specifically binds to site 1 of the STX1B subunit.

<Example 6>
As described in the above-mentioned Patent Document 1, the present inventor has previously proposed a globo trisaccharide moiety having a carbosilane dendrimer as a core structure as a compound that binds to the STXB subunit and inhibits its toxicity. For example, a compound having six globo trisaccharide moieties is named SUPER TWIG (1) 6 or the like. In Patent Document 1, mutation is introduced into each of site 1, site 2 and site 3 of the STX2B subunit, and each mutant is compared with SUPER TWIG (1) 6 to compare the binding affinity with SUPER TWIG (1) 6. Based on this finding, the peptide library method targeting STX2B site 3 was used to bind STX2B subunit to STX2B. Peptide motifs (PPP-tet, PPR-tet, etc.) that exhibit binding affinity have been determined, and their toxicity inhibitory effects have been studied.

In Examples 4 and 5, since the STX1 toxicity inhibitory activity of MMA-tet and the specific binding property of STX1B subunit to site 1 were confirmed, MMA-tet and MAA- were newly added in this example. We examined the binding affinity and STX2 toxicity inhibitory activity for STX2B subunit by tet, AMA-tet and AAA-tet.
(1) in a manner similar to the binding affinity in Example 3, it was examined MMA-tet and MAA-tet, AMA-tet, binding affinity for STX2B-subunit by AAA-tet the _{(K D} value). Further, PPP-tet, PPR-tet , and as a control, for the compound MA-tet only core structure without a peptide motif, were examined in the same manner the binding affinity (K _D value). The results are shown in [Table 3].

  As shown in [Table 3], binding affinity for STX2B subunit was confirmed for all of MMA-tet, MAA-tet, AMA-tet, and AAA-tet. This indicates that MMA-tet, MAA-tet, AMA-tet and AAA-tet bind strongly to the STX2B subunit in a peptide motif-dependent manner.

  Moreover, the binding affinity with respect to STX2B subunit was confirmed also about PPP-tet and PPR-tet on the same conditions. In the binding affinity, a significant difference from MMA-tet, MAA-tet, AMA-tet and AAA-tet was not confirmed.

On the other hand, no binding was observed with MA-tet. Note that RU _MAX (AU) indicates the maximum amount of binding.
(2) Cytotoxic inhibitory effect The cytotoxic inhibitory effect by STX2 by MMA-tet, MAA-tet, AMA-tet, AAA-tet was examined. The specific experimental conditions were the same as in Example 4, using Vero cells, culturing for 3 days in the presence of 1 pg / ml STX2 and each concentration of peptide, and measuring the ratio of viable cells.

  Moreover, the inhibitory effect with respect to the cytotoxic activity by STX2 was examined also about PPP-tet.

  Specifically, as shown in FIG. 5, it was confirmed that MMA-tet significantly inhibits STX2 cytotoxicity in a concentration-dependent manner. Taking the survival rate of 140% as the maximum effect, the IC50 value was 17 μg / ml.

  On the other hand, the inhibitory effect decreased with AMA-tet and PPP-tet, and the IC50 values were 62 μg / ml and 42 μg / ml, respectively. In MAA-tet and AAA-tet, the inhibitory effect was further reduced, and the IC50 values were 140 μg / ml and 130 μg / ml, respectively. That is, it was revealed that MMA-tet exhibits about 2.5 times stronger STX2 toxicity inhibitory activity than the STX2 inhibitory peptide PPP-tet identified so far.

Thus, it was revealed that MMA-tet exerts a superior toxicity inhibitory action than PPP-tet identified by targeting STX2B.
<Example 7>
In Examples 4 and 6, it was confirmed that MMA-tet has a strong inhibitory activity against cytotoxicity by STX1 and STX2. Based on these results, infection experiments were actually performed using mice.

As a mouse, low protein breeding C57BL / 6 (SPF, female) was used, and as an infecting bacterium, STEC strain N9-D6 (stx1 ⁺ , stx2 ⁺ ) was used at 7.5 × 10 ⁶ cfu. Then, Ac-MMA-tet (0.15 mg / mouse, 0.5 mg / mouse) was orally administered twice a day to mice infected on the 2nd day to the 5th day of infection. An acetyl group was modified at the end of MMA for the purpose of stabilizing in order to suppress degradation by protease in the digestive tract.

Specifically, it was divided into the following three groups.
Group 1 (0.15 mg / mouse group): Average body weight 20.3 g (6 animals)
Group 2 (0.5mg / mouse group): Average body weight 20.3g (6 animals)
Group 3 (control mouse group): average body weight 21.4g (8 mice)
The results are shown in FIG. As shown in FIG. 6, the control mouse group survived only 10 to 13 days, whereas group 1 (0.15 mg / mouse group) had 5 mice and group 2 (0.5 mg / mouse group). Then all six survived over 60 days. Therefore, it is considered that the administration of Ac-MMA-tet inhibited the toxicity of Stx1 and 2, and the disease of infected mice was improved, so that the survival of mice was maintained.

  That is, the drug containing MMA-tet is extremely effective for diseases caused by Stx1, such as Stx1-producing enterohemorrhagic Escherichia coli infection and dysentery, and more closely related to the severity of symptoms. Productive enterohemorrhagic E. coli infection, Stx1 and Stx2 both toxin-producing enterohemorrhagic E. coli infections have been shown to be treatable.

Claims (4)

  A peptide having binding properties to Stx1 and Stx2 and inhibiting the cytotoxicity of Stx1 and Stx2, comprising four amino groups located at both ends of a molecular nucleus structure formed by combining three lysines (Lys) A Stx toxicity-inhibiting peptide, wherein the peptide of SEQ ID NO: 1 is linked to each directly or via a spacer.   The Stx toxicity-inhibiting peptide according to claim 1, wherein the spacer has a hydrocarbon chain having 4 to 10 carbon atoms.   A therapeutic agent for a disease caused by Stx1 and Stx2, which comprises the Stx toxicity-inhibiting peptide of claim 1 or 2.   The therapeutic agent according to claim 3, wherein the disease caused by Stx1 and Stx2 is enterohemorrhagic Escherichia coli infection or dysentery.

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