JP6578546B2 - LT inhibitory tetravalent peptide and ETEC infection therapeutic agent - Google Patents

Description

  The present invention relates to a LT-inhibiting tetravalent peptide and a therapeutic agent for ETEC infection.

  Enterotoxigenic E.coli (ETEC) is the causative agent of infant diarrhea in developing countries, and the worldwide number of infectious diseases exceeds 200 million people annually and 40 infants under 5 years old die. It has been reported to reach 10,000 people. At the same time, it is a major pathogen of traveler's diarrhea in developed countries including Japan. ETEC infection is mainly caused by severe water-soluble diarrhea, and the main treatment is the administration of fluids and antibiotics to supplement water and electrolytes lost due to dehydration due to diarrhea. On the other hand, since ETEC is rapidly gaining resistance to antibiotics, the development of new therapeutic agents is urgently needed.

  The main pathogenic factor of ETEC is heat-labile enterotoxin (hereinafter referred to as “LT”) produced by ETEC. LT is a toxin of type AB5 and recognizes and binds to a receptor molecule present on the target cell with one molecule of A-subunit, which is a protein ADP-ribosylating enzyme, and then binds the A-subunit into the cell. It consists of a B-subunit (LTB) pentamer that plays a role in transport to 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 LT receptor present on the target cell is ganglioside GM1 (Galβ1-3GalNAcβ1-4 [NeuAcα2-3] Galβ1-4Glcβ1-1Cer), which is a kind of glycolipid. The LTB1 molecule specifically recognizes the terminal sugar chain of GM1, and thus the LTB 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. At this time, the LTB1 molecule has a pocket related to recognition of terminal galactose of GM1 and a pocket related to recognition of terminal sialic acid, and both pockets are simultaneously involved in recognition of GM1 (non-) Patent Document 1).

  On the other hand, the present inventors have developed a new technology for inhibiting strong interaction based on the cluster effect, a multivalent peptide library method, and enterohaemorrhagic E.coli (EHEC) is produced. A series of novel Stx inhibitory peptides have been developed targeting Shiga toxin (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).

  The present inventors have also developed a CT-inhibiting tetravalent peptide that inhibits the toxicity of cholera toxin (Cholera toxin: CT) produced by Vibrio cholerae based on the above-described techniques and knowledge established so far. Successful (Japanese Patent Application 2014-050828).

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

Protein Science (1994), 3: 166-175

  An object of the present invention is to provide a novel LT inhibitory peptide targeting LTB, which is responsible for binding of LT to a target cell, and to provide a therapeutic agent for ETEC infection.

In order to solve the above problems, the LT-inhibiting tetravalent peptide of the present invention is an LT-inhibiting tetravalent peptide that binds to heat-labile enterotoxin (LT) and inhibits toxicity, and includes three lysines (Lys).
Each of the four amino groups located at the ends of the molecular nucleus structure formed by bonding
It is characterized in that any one of -4 peptide motifs is bound directly or via a spacer.

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

  The therapeutic agent for ETEC infection of the present invention is characterized by containing the LT-inhibiting tetravalent peptide.

  According to the LT-inhibiting tetravalent peptide of the present invention, it can bind to LT with high binding affinity and effectively inhibit cytotoxicity. Therefore, the therapeutic agent for ETEC infection of the present invention containing this LT-inhibiting tetravalent peptide can improve symptoms such as diarrhea caused by LT.

It is a figure which shows the GM1 binding activity of WT-LTB and E51A-LTB. 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 a multivalent type peptide library screening. From the top, the structure of each library and the results of screening using them are shown. The position number in the figure indicates the position from the N-terminal side of the degenerate position of each library. It is the schematic which showed the structure of the tetravalent peptide synthesize | combined on the cellulose sheet. (A) is a figure which shows the structure of the peptide library part which each tetravalent peptide has, and the positional information on a sheet | seat. X in the motif shown on the right in the figure indicates the amino acid shown at each position. In the figure, MA in I-5 indicates a compound that lacks the peptide library portion and retains all other structures. (B) is a view showing the binding of each sheet to ¹²⁵ I-labeled WT-LTB or ¹²⁵ I-labeled E51A-LTB. It is a figure which shows the effect of AAR-tet and MA-tet on the morphological change of the CHO cell by LT treatment. It is a figure which shows the effect of AAR-tet with respect to the cAMP production effect | action of LT. The amount of cAMP produced (0.18 nmol / mg of protein) when the Caco-2 cells cultured and differentiated in a 96-well plate are stimulated with 1.0 μg / ml LT is indicated as 100% (n = 5). (A) is the figure which showed the structure of the peptide library part which each tetravalent peptide has, and the positional information on a sheet | seat. X in the motif shown on the right in the figure indicates the amino acid shown at each position. In the figure, A-1 indicates a compound having a base GGRHRRR motif, and G-10 MA indicates a compound that lacks the peptide library portion and retains all other structures. (B) shows the binding of each sheet to ¹²⁵ I-labeled WT-LTB or ¹²⁵ I-labeled E51A-LTB. It is a figure which shows the effect (n = 3) of NNR-tet, ENR-tet, and GGR-tet on the morphological change of the CHO cell by LT treatment. It is a figure which shows the effect of NNR-tet, ENR-tet, and GGR-tet with respect to the cAMP production effect | action of LT. The cAMP production (0.18 nmol / mg of protein) is shown as 100% when the Caco-2 cells cultured and differentiated in a 96-well plate are stimulated with 1.0 μg / ml LT (GGR-tet is n = 3, others n = 5). (A) (B) is a figure which shows that a water | moisture content retention is caused in a mouse | mouth intestinal tract by LT. It is a figure which shows the inhibitory effect of GGR-tet with respect to the water retention by LT.

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

The LT-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: Ala-Ala-Arg-Arg-His-His-His (AARRHHH)
Sequence number 2: Asn-Asn-Arg-His-Arg-Arg-Arg (NNRHRRR)
Sequence number 3: Glu-Asn-Arg-His-Arg-Arg-Arg (ENRHRRR)
Sequence number 4: Gly-Gly-Arg-His-Arg-Arg-Arg (GGRHRRR)
Any one of them is bound.

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

Is exemplified by a tetravalent peptide in which four of any one of the peptide motifs of SEQ ID NOs: 1-4 are incorporated in the XXXX portion located at the end of the molecular nucleus structure composed of three lysines (Lys) The In the above chemical formula 1, the position where the peptide motif is incorporated is described as “XXXX” for convenience.

  Furthermore, among the peptide motifs of SEQ ID NOs: 1-4, the LT inhibitory tetravalent peptide having the peptide motif of SEQ ID NO: 4 (GGRHRRR) strongly inhibits the increase in cAMP production that is closely related to the onset of diarrhea. Is particularly preferred.

  Hereinafter, the LT-inhibiting tetravalent peptide having the peptide motif of SEQ ID NO: 1 is “AAR-tet”, the LT-inhibiting tetravalent peptide having the peptide motif of SEQ ID NO: 2 is “NNR-tet”, and having the peptide motif of SEQ ID NO: 3 The LT-inhibiting tetravalent peptide may be described as “ENR-tet”, and the LT-inhibiting tetravalent peptide having the peptide motif of SEQ ID NO: 4 may be described as “GGR-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 any peptide motif of SEQ ID NOs: 1-4. In the case of binding a spacer, any specific molecule and length may be used as long as the inhibitory activity against LT toxicity is not impaired. The spacer preferably has, for example, a chain length of about 4 to 10 carbon atoms having an amino acid at the terminal, and the LT 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 LT-inhibiting tetravalent peptide of the present invention preferably has a modified molecule at each end of the peptide motif of SEQ ID NOs: 1-4.

Further, 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 motif of SEQ ID NOs: 1-4 Is also considered to bind hydrophobic molecules. For example, when an LT-inhibiting tetravalent peptide or a therapeutic agent containing it 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. Thus, the LT inhibitory tetravalent peptide of the present invention has an increased inhibitory activity against LT 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 LT-inhibiting tetravalent peptide of the present invention.

  And the production method of LT 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 LT-inhibiting tetravalent peptide of the present invention is a tetravalent peptide having four of any one of the peptide motifs of SEQ ID NOs: 1-4, and strongly binds to LT by the cluster effect. It exhibits affinity and can effectively inhibit LT cytotoxicity.

  And since the LT inhibitory tetravalent peptide of this invention has LT inhibitory activity, the chemical | medical agent containing this LT inhibitory tetravalent peptide is a therapeutic agent which can treat ETEC infections, such as diarrhea resulting from LT. It becomes.

  The therapeutic agent for ETEC infection of the present invention can be prepared and stored by mixing with any pharmaceutically acceptable carrier, excipient or stabilizer in addition to the LT-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 drug for treating ETEC infection formulated in this way can be administered systemically, for example, orally, locally, or intravenously, depending on the symptoms, but LT 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 LT-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 LT-inhibiting tetravalent peptide of the present invention, which can significantly enhance the therapeutic effect upon oral administration.

  The dose of the therapeutic agent for ETEC infection of the present invention can be determined according to the patient's body weight, symptoms, etc., for example, about 5 to 100 mg / Kg per dose, about 5 to 10 mg as a guideline. / Kg or so can be used as a guide.

  The therapeutic agent for ETEC infection of the present invention contains an LT-inhibiting tetravalent peptide and can be effectively used for the treatment of symptoms such as diarrhea caused by LT. The therapeutic agent for ETEC infection of the present invention targeting LT is not expected to be toxic to ETEC itself, so there is no problem of resistance, and it is expected that it can overcome the problem of drug resistance to currently rapidly spreading antibiotics. The Furthermore, the peptidic compound (peptide motif) incorporated in the LT-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 LTB and LTB mutant used for screening In screening, a large amount of LTB in a state of being immobilized on beads is required. Thus, a large amount of recombinant LTB (WT-LTB) in which a His-tag was introduced on the C-terminal side of the LTB gene was prepared using an E. coli expression system and immobilized on the beads using Ni-beads. A part was eluted from the beads with imidazole, and after buffer exchange, soluble WT-LTB was prepared.

  On the other hand, in creating an LTB mutant having a mutation at the receptor binding site, the terminal galactose binding site of GM1 was targeted. Focusing on Glu51, which is thought to be involved in the binding of GM1 to the terminal galactose, based on the crystal structure analysis of LTB that has already been clarified, a large amount of E51A-LTB, in which this amino acid is replaced with Ala, is similarly produced. Prepared.

  The activity of the obtained recombinant LTB was evaluated by examining specific binding to GM1 using the ELISA method. Specifically, phosphatidylcholine: GM1 (or asialo-GM1) = 1: 0.005 (μg / plate, GM1 is 0.5%) is coated on an ELISA plate, and after combining each concentration of recombinant LTB, it is washed and remains. The LTB was detected using specific antibody.

  The results are shown in FIG. As shown in FIG. 1, it was confirmed that WT-LTB strongly bound to GM1 in a concentration-dependent manner. On the other hand, almost no binding activity is seen against asialo GM1 (AGM1) from which terminal sialic acid has been removed from GM1, that is, WT-LTB specifically recognizes GM1 depending on terminal sialic acid. Indicated. The amount of E51A-LTB bound to GM1 was attenuated to about 1/2 that of WT-LTB at a concentration of 1 μg / ml. In addition, binding to asialo GM1 (AGM1) was hardly observed as in the wild type.

  From the above, it was confirmed that E51 certainly plays an important role in LTB receptor recognition, that is, it can be an excellent target site in the development of LT inhibitors.

<Example 2> Identification of E51-specific binding motif using multivalent peptide library method In Example 1, it was confirmed that E51 plays an important role in receptor recognition of LTB. The multivalent peptide library was screened using as an index the higher binding activity to WT-LTB than E51A-LTB.

  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. Further, “XXXX” in FIG. 2 indicates the library portion, and X (degenerate position) indicates that the synthesis was performed using a mixture of 19 amino acids other than Cys.

  As the primary library, screening was carried out using two kinds of multivalent peptide libraries whose library part was XXXRXXX, XXXKXXX (6 degenerate positions). First, each LTB-immobilized bead (LTB amount: 200 μg) and multivalent peptide library (100 μg) were incubated in 300 μl of PBS at 4 ° C. for 18 hours. After washing the beads, peptides bound to LTB were eluted with 30% acetic acid, and after recovery, amino acid sequencing was performed sequentially from the N-terminus. 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 using wild-type LTB is divided by the value obtained when using LTB-E51A, and the ratio is calculated. Was corrected to be 19. Therefore, if there is no difference in the selectivity of each amino acid between wild-type LTB and LTB-E51A, the value is all 1. As an index of selectivity, when this value exceeded 1.2, it was judged that strong selectivity for wild-type LTB was observed. As a result, it was shown that the selectivity of basic amino acids was strong at all 6 degenerate positions (FIG. 3).

  Therefore, a secondary library with the RXR motif at positions 3-5 was created and screened in the same way, but Arg was still selected at positions 6 and 7, but Ala was selected at positions 1 and 2. It was shown that (Figure 3 lower part).

<Example 3> Identification of novel LTB-binding motif by multivalent peptide sheet screening method Based on the results of Example 2, a series of tetravalent peptide libraries with known sequences were prepared using multivalent peptide sheet synthesis technology. Prepared on a sheet.

  A schematic diagram of the polyvalent peptide sheet synthesis technique is shown in FIG. This method is proposed by the present inventors in Patent Document 3, and a tetravalent peptide with a known sequence having a nuclear structure that can be identified with the multivalent peptide library shown in FIG. This is a method of spot synthesis at several hundred levels. A more rigorous motif identification was attempted by screening spot-synthesized peptides using the binding ability of radiolabeled WT-LTB and E51A-LTB as an index.

  In the synthesis, a spot peptide synthesizer manufactured by Intavis AG was used. 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 extension reaction was performed so as to give the subsequent sequences equally to the four amino groups formed. . By this operation, the tetravalent peptide synthesized on the cellulose sheet can be considered to have a nuclear structure that can be identified with the nuclear structure of the multivalent peptide library shown in FIG. 2 (FIG. 4).

  Each peptide library synthesized on the sheet has a basic amino acid cluster immobilized at position 4-7 (RHHH or HKHHH), and Ala on the amino terminal side (position 1-3). XAA, AXA, AAX, XA, and AX (X introduced 19 types of amino acids other than Cys sequentially) were introduced.

  FIG. 5 (A) shows the structure of the peptide library part possessed by each tetravalent peptide spot-synthesized on the sheet by the above operation and positional information on the sheet.

Furthermore, after blocking the sheet on which the tetravalent peptide compound was synthesized with 5% skim milk powder, ¹²⁵ I-labeled WT-LTB (1 μg / ml) or ¹²⁵ I-labeled E51A-LTB (1 μg / ml) ), And after incubation at 4 ° C. for 1 hour, washed, and the radioactivity bound on the sheet was detected with BAS3000 (FIG. 5B), and quantitative analysis was performed (Tables 1 and 2).

  When blotting with labeled WT-LTB and blotting with labeled E51A-LTB, the density (PSL) of each spot is quantified, and the total PSL value of each spot is 92, which is the number of spots. Was corrected and displayed. That is, if there is no difference in binding between the compounds, all values are 1.0. In addition, as an index indicating binding specificity, the value obtained by dividing the corrected binding amount to WT-LTB (WT-LTB binding force) by the corrected binding amount to E51A-LTB (E51A-LTB binding force) WT-LTB / E51A-LTB (binding ratio) was calculated, and this value was corrected and displayed so that the total was 92. Furthermore, as an indicator that the binding strength to WT-LTB and the binding ratio are both excellent, the value obtained by multiplying the corrected binding amount to WT-LTB and the corrected binding ratio (WT-LTB binding) Force x binding ratio). In Tables 1 and 2, they are displayed in descending order of the value of WT-LTB binding force × binding ratio (Table 1: Ranking 1-60, Table 2: Ranking 61-92).

  As a result, AARRHHH (SEQ ID NO: 1), which has the highest value of WT-LTB binding force × binding ratio, is a new LTB, which is an index indicating that both binding strength to WT-LTB and binding ratio are excellent. It was selected as a binding motif and used for the following studies. That is, the peptide motif consisting of this AARRHHH (SEQ ID NO: 1) was incorporated into four portions of XXXX in the multivalent peptide library shown in FIG. 2 to synthesize a tetravalent peptidic compound, AAR-tet.

<Example 4> Evaluation of LTB binding ability of AAR-tet The binding activity of AAR-tet synthesized in Example 3 to WT-LTB was examined using Biacore manufactured by GE Healthcare Japan. Specifically, the Kd value and RUmax (maximum resonance unit, AU is an arbitrary unit in the Biacore system) of AAR-tet for WT-LTB were calculated using Biacore. After immobilizing WT-LTB on the sensor chip using 10 μg / ml WT-LTB, each concentration of AAR-tet was bound, and the Kd value and RUmax were calculated from each sensorgram.

  The results are shown in Table 3.

  As shown in Table 3, MA-tet, which is a tetravalent peptide that has no motif in the XXXX part shown in FIG. 2 and consists only of the 3Lys nuclear structure, shows no binding, whereas AAR-tet It was revealed that it has a strong binding affinity for WT-LTB.

<Example 5> Inhibitory effect of AAR-tet on morphological change induction of CHO cells by LT
It is known that CHO cells undergo a remarkable change in shape by LT treatment and become spindle-shaped. This system is a simple and widely used LT activity evaluation system. Therefore, the effect of each peptide compound (AAR-tet, MA-tet) on the morphological change of CHO induced by LT treatment was examined. Treatment of CHO cells with 1 μg / ml LT in the presence or absence of each peptidic compound (AAR-tet: 5.6 μM, 18.5 μM, MA-tet: 51.4 μM) Changes were observed under a microscope. 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.

  The results are shown in FIG.

  As shown in FIG. 6, when AAR-tet coexists with MA-tet, the proportion of cells showing spindle shape decreases in a dose-dependent manner, and decreases to 40% or less at 18.5 μM. It was shown that. This is thought to be because AAR-tet inhibited the action of LT on CHO by binding to the receptor binding site of LTB.

<Example 6> Inhibitory effect of AAR-tet on cAMP production in Caco-2 cells by LT
When LT is taken up by intestinal epithelial cells via a receptor, the A subunit is carried into the cytoplasm via retrograde transport, and activated by ADP-ribosylating Gs coupled to adenylate cyclase, As a result, intracellular cAMP concentration increases, A kinase is activated, Cl channels are released through phosphorylation, Cl ions flow out, and a large amount of water flows out according to osmotic pressure, causing diarrhea. Are known.

  Therefore, we decided to investigate the inhibitory effect of AAR-tet on cAMP increase by LT using Caco-2, a human colon cancer-derived epithelial cell line.

  After incubating Caco-2 cells in the Confluent state with or without AAR-tet and MA-tet (AAR-tet: 5.6 μM, 18.5 μM, MA-tet: 51.4 μM) for 30 minutes at 37 ° C., 1.0 After adding μg / ml LT and incubating at 37 ° C. for 5 hours, a cell lysate was prepared. The amount of cAMP produced in each lysate was measured using PerkinElmer's cAMP Alpha Screen Kit.

  The results are shown in FIG.

  As shown in FIG. 7, a significant increase in cAMP was confirmed 5 hours after LT treatment. It was confirmed that MA-tet does not inhibit cAMP production by LT treatment at all, whereas AAR-tet suppresses cAMP production in a dose-dependent manner and inhibits to 60% at 18.5 μM. This indicates that AAR-tet has an excellent LT inhibitory ability.

<Example 7> Identification of novel LTB binding motif by multivalent peptide sheet screening method The present inventors have developed a CT-inhibiting tetravalent peptide that inhibits the toxicity of cholera toxin (Cholera toxin: CT) produced by Vibrio cholerae (Japanese Patent Application 2014-050828). Since CT B-subunit (CTB) and LTB have very high homology, it is based on GGRHRRR (SEQ ID NO: 4) found as one of the CT inhibitory tetravalent peptide motifs. We tried to identify a novel LTB binding motif.

  Specifically, as in Example 3, the screening of the multivalent peptide library is based on the fact that the binding activity to WT-LTB is higher than that of E51A-LTB. A series of tetravalent peptide libraries with known sequences were prepared on a sheet.

Position 3-6 (RHRR) of GGRHRRR (SEQ ID NO: 4) was immobilized, and 19 amino acids other than Cys were introduced with other positions as X (degenerate position). GGRHRRR (SEQ ID NO: 4) corresponds to position number A-1 in FIG. Further, a compound in which the peptide library portion was deleted and all other structures were conserved (designated as MA; corresponding to position number G-10 in FIG. 8A) was also synthesized at the same time. After blocking the sheet on which this tetravalent peptide compound was synthesized with 5% skim milk powder, ¹²⁵ I-labeled WT-LTB (1 μg / ml) or ¹²⁵ I-labeled E51A-LTB (1 μg / ml) After washing at 4 ° C. for 1 hour, washing was performed, and the radioactivity bound on the sheet was detected with BAS3000 (FIG. 8B), and quantitative analysis was performed (Tables 4 and 5).

  When blotting with labeled WT-LTB and when blotting with labeled E51A-LTB, the density (PSL) of each spot was quantified, and the total PSL value of each spot was the number of spots 75 (MA is The display was corrected so that That is, if there is no difference in binding between the compounds, all values are 1.0. In addition, as an index indicating binding specificity, the value obtained by dividing the corrected binding amount to WT-LTB (WT-LTB binding force) by the corrected binding amount to E51A-LTB (E51A-LTB binding force) WT-LTB / E51A-LTB (binding ratio) was calculated, and this value was corrected and displayed so that the total was 75. Furthermore, as an indicator that the binding strength to WT-LTB and the binding ratio are both excellent, the value obtained by multiplying the corrected binding amount to WT-LTB and the corrected binding ratio (WT-LTB binding) Force x binding ratio). In Tables 4 and 5, the WT-LTB binding force × binding ratio values are displayed in descending order (Table 4: Ranking 1-60, Table 5: Ranking 61-75).

  As shown in Table 4 and Table 5, the value of WT-LTB binding force x coupling ratio, which is an index indicating that both the binding strength to WT-LTB and the binding ratio are excellent, is 1.65 or more. In addition, NNRHRRR (SEQ ID NO: 2) and ENRHRRR (SEQ ID NO: 3) were selected as novel LTB binding motifs and used for the following studies. That is, each of NNRHRRR (SEQ ID NO: 2) and ENRHRRR (SEQ ID NO: 3) is incorporated into four XXXX parts in the multivalent peptide library shown in FIG. 2, a tetravalent peptide compound, NNR-tet and ENR-tet was synthesized and used in Example 8 below.

<Example 8> Evaluation of LTB binding ability of NNR-tet, ENR-tet and GGR-tet
The binding activity of NNR-tet and ENR-tet to WT-LTB was examined using Biacore manufactured by GE Healthcare Japan. At the same time, the same study was performed on a compound (GGR-tet) having a tetravalent motif GGRHRRR (SEQ ID NO: 4) as a base. Specifically, using GE Healthcare Japan Co., Ltd. Biacore, NNR-tet, ENR-tet and GGR-tet Kd values for WT-LTB and RUmax (maximum resonance unit, AU is arbitrary in Biacore system unit). After immobilizing WT-LTB on the sensor chip using 10 μg / ml WT-LTB, each concentration of compound was bound, and the KD value and RUmax were calculated from each sensorgram.

  The results are shown in Table 6.

  As shown in Table 6, MA-tet, which is a tetravalent peptide consisting only of the 3Lys molecular nucleus structure, does not show any binding, whereas both compounds have high binding affinity for WT-LTB. In particular, it became clear that NNR-tet showed the highest binding affinity.

<Example 9> Inhibitory effect of NNR-tet, ENR-tet and GGR-tet on induction of morphological change of CHO cells by LT As in Example 5, each peptidic effect on CHO morphological change induced by LT treatment The effects of the compounds (NNR-tet, ENR-tet, GGR-tet) were examined. Changes in cell morphology after treatment of CHO cells with 1 μg / ml LT in the presence or absence of peptide compounds (NNR-tet, ENR-tet, GGR-tet) at various concentrations and incubation at 37 ° C. for 7 hours, Observed under a microscope. 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.

  The results are shown in FIG.

  As shown in FIG. 9, when NNR-tet, ENR-tet, or GGR-tet coexisted, the proportion of cells having a spindle shape decreased in a dose-dependent manner. At 17 μM, NNR-tet was 56%. It was confirmed that ENR-tet showed 47% inhibitory effect and GGR-tet showed 51% inhibitory effect. On the other hand, MA-tet was confirmed to have an inhibitory effect of only 18% even when 51.4 μM was used.

<Example 10> Inhibitory effect of NNR-tet, ENR-tet and GGR-tet on cAMP production in Caco-2 cells by LT As in Example 6, Caco-2 which is a human colon cancer-derived epithelial cell line Using cells, the inhibitory effect of NNR-tet, ENR-tet and GGR-tet on cAMP increase by LT was examined.

  The results are shown in FIG.

  As shown in FIG. 10, a significant increase in cAMP was confirmed 5 hours after LT treatment. MA-tet did not inhibit cAMP production by LT treatment at all, whereas NNR-tet and ENR-tet suppressed cAMP production in a dose-dependent manner, and both showed a 20% inhibitory effect at 17 μM. On the other hand, GGR-tet showed a significant inhibitory effect in a dose-dependent manner, and showed a 69% inhibitory effect at 17 μM.

  The results of Examples 9 and 10 show that all of NNR-tet, ENR-tet and GGR-tet can be effective LT inhibitors, and in particular, GAMP-tet is closely related to the development of diarrhea. It is considered that it can be the most effective LT inhibitor because it potently inhibits the increase.

<Example 11> Effect of GGR-tet on water retention in mouse intestine caused by LT As shown in FIGS. 11A and 11B, it is known that water retention in mouse intestine is caused by LT. Yes. Therefore, we examined the inhibitory effect of water retention by LT by administering GGR-tet to the mouse intestine.

  Specifically, the intestinal tract of a mouse (ICR mouse (female, 5-6 weeks)) was ligated under anesthesia, LT (0.3 μg) and GGR-tet (300 μg) were administered, and after 8 hours, the intestinal tract was removed. The water content per 1 cm of the intestinal tract was evaluated as the water retention amount FA.

  The results are shown in FIG. As shown in FIG. 12, it was revealed that GGR-tet has a 90% inhibitory effect on significant water retention by LT. This result indicates that GGR-tet can be a therapeutic agent (ETEC infection therapeutic agent) that inhibits the individual toxicity of LT.

Claims (3)

An LT-inhibiting tetravalent peptide that binds to heat-labile enterotoxin (LT) and inhibits toxicity, the following molecular nucleus structure formed by combining three lysines (Lys) ,
LT inhibitory tetravalent, characterized in that any one of the peptide motifs of SEQ ID NOs: 1-4 is bound to each of four amino groups located at the ends of SEQ ID NO: 1-4 directly or via a spacer peptide. The LT-inhibiting tetravalent peptide according to claim 1 , wherein NH _{2 at} the terminal of each peptide motif is acetylated.   A therapeutic agent for ETEC infection, comprising the LT-inhibiting tetravalent peptide according to claim 1 or 2.