JP2007055983A - Prophylactic/ameliorant for disease caused by pathogenic bacterium penetrated into cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new prophylactic/ameliorant for diseases caused by pathogenic bacteria. <P>SOLUTION: A substance such as a peptide composed of an amino acid sequence represented by sequence number 2, Z-VAD fmk (benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone) being a caspase inhibitor, PI3P(phosphatidylinositol-3-phosphate), U0126(1.4-diamino-2,3-dicyano-1,4-bis(2-amino-phenylthio)butadiene) being an MEK inhibitor, Ac-L-Ala-NH<SB>2</SB>(Acetyl-L-alanine amide)being an amino acid derivative, etc., has low toxicity to host cells and controls existence of bacterium penetrated to host cells. Consequently, a substance such as a peptide having an amino acid sequence represented by sequence number 1, a caspase inhibitor, PI3P, MEK inhibitor, an amino acid derivative, etc., or a composition obtained by combining two or more of theses substances is useful for preventing/ameliorating diseases caused by a pathogenic bacterium. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a preventive / ameliorating agent for diseases caused by pathogenic bacteria that have entered cells.

  Eukaryotic cells have a function of degrading and digesting pathogenic bacteria that have entered cells by autophagy. Induction of autophagy in cells can suppress the survival of pathogenic bacteria in cells (see, for example, Non-Patent Document 1). Therefore, diseases caused by pathogenic bacteria that have entered cells, particularly intracellular parasitic bacteria. It is thought to be useful as a preventive / ameliorating agent for diseases caused by.

However, since autophagy may be involved in cell death, the use of an autophagy-inducing agent for diseases caused by pathogenic bacteria may promote host cell death and may have side effects.
Cell 119,753-766, 2004

  Accordingly, an object of the present invention is to provide a novel preventive / ameliorating agent useful for diseases caused by pathogenic bacteria.

As a result of diligent efforts to solve the above-mentioned problems, the present inventor has found that rapamycin (Cell 119, 753-766, 2004), which is known as an autophagy inducer, has survived BCG (Bacillus Calmette-Guerin) that has invaded host cells A peptide having the amino acid sequence shown in SEQ ID NO: 1 (RSTDLPGLKAATHYTITIRGV) (RSTDLPGLKAATHYTITIRGVC (SEQ ID NO: 2); hereinafter referred to as “TNIII”), while suppressing and inducing cell death against most host cells. , Caspase inhibitor Z-VAD fmk (Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone), PI3P (phosphatidylinositol-3-phosphate), MEK (Mitogen-activated protein (MAP) kinases) inhibitor U0126 ( Substances such as 1.4-diamino-2,3-dicyano-1,4-bis (2-amino-phenylthio) butadiene) and the amino acid derivative Ac-L-Ala-NH ₂ (Acetyl-L-alanine amide) Suppresses the survival of BCG infected with host cells, but It found that weak toxicity against, and have completed the present invention.

That is, the preventive / ameliorating agent according to the present invention is for a disease caused by a pathogenic bacterium that has entered a cell, and has 1 or several peptides in the peptide having the amino acid sequence shown in SEQ ID NO: 1, 1 or 2 or more substances such as peptides, caspase inhibitors, PI3P, MEK inhibitors, amino acid derivatives, etc., comprising an amino acid sequence substituted, deleted, or added as an active ingredient. Examples of the caspase inhibitor include Z-VAD fmk. The MEK inhibitor is, for example, U0126. Examples of the amino acid derivative include Ac-L-Ala-NH ₂ . Examples of diseases caused by pathogenic bacteria that have entered the cells include Mycobacterium tuberculosis.

  According to the present invention, it is possible to provide a novel preventive / ameliorating agent useful for diseases caused by pathogenic bacteria.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Preventive / improving agent according to the present invention ==
As mentioned above, substances such as TNIII, caspase inhibitor Z-VAD fmk, PI3P, MEK inhibitor U0126, and amino acid derivative Ac-L-Ala-NH ₂ In particular, substances such as TNIII, ME0 inhibitor U0126, and amino acid derivative Ac-L-Ala-NH ₂ were found to have no cytotoxicity. Therefore, a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted or added, caspase inhibitor, PI3P, MEK inhibitor, amino acid A substance such as a derivative or a composition in which two or more of these substances are combined is considered to be useful for the prevention / amelioration of diseases caused by pathogenic bacteria.

Examples of the caspase inhibitor include Z-VAD fmk, Ac-WEHD-CHO (acetyl-Trp-Glu-His-Asp-aldehyde), Ac-DEVD-CMK (acetyl-Asp-Glu-Val-Asp-chloromethylketone). ), Boc-D-FMK (benzyloxycarbonyl-Asp (OMe) fluoromethylketone), Z-YVAD-FMK (Z-Tyr-Val-Ala-Asp (OMe) -CH ₂ F), Z-WEHD-FMK (Z-Trp -Glu (OMe) -His-Asp (OMe) -CH ₂ F), Z-LEHD-FMK (Z-Leu-Glu (OMe) -His-Asp (OMe) -CH ₂ F), Z-IETD-FMK A known substance such as (Z-Ileu-Glu (OMe) -Thr-Asp (OMe) -CH ₂ F) can be used. Examples of the MEK inhibitor include U0125 (1,4-Diamino-2,3-dicyano-1,4-bis (phenylthio) butadiene), U0126, PD98059 (2′-Amino-3′-methoxyflavone). A known substance such as can be used.

Examples of the amino acid derivatives include Ac-L-Ala-NH ₂ , Ac-L-Met-NH ₂ , Ac-L-Val-NH ₂ , Ac-L-Leu-NH ₂ , Ac-L-Gln- Amino acid (especially L-amino acid) derivatives such as NH ₂ (where the N-terminus is protected by a known protecting group such as an acetyl group and the C-terminus is amidated) can be used.

  Diseases to which the preventive / ameliorating agent according to the present invention is applied include pathogenic bacteria (particularly intracellular parasitic bacteria) that have invaded cells, for example, BCG (Bacille Calmette-Guerin), Mycobacterium tuberculosis, group A streptococci, yellow Diseases caused by staphylococci, pathogenic Escherichia coli, Haemophilus influenzae, pneumococci, pneumococci, Clostridium botulinum, pathogenic Escherichia coli, Shigella, Legionella, Listeria, gingivalis, staphylococci For example, tuberculosis, pharyngitis, tonsillitis, pneumonia, scarlet fever, fulminant hemolytic streptococcal infection, poisoning, bacterial dysentery, periodontal disease, botulism, sepsis, meningitis, inflammation Enteric disease, vasculitis, type IV allergic disease, staphylococcal burn-like skin syndrome, MRSA infection, Ritter disease, blistering impetigo in neonates, arthritis, purulent disease, enteritis, bacteremia And eye infections.

== Peptides used in the present invention ==
The peptide having the amino acid sequence shown in SEQ ID NO: 1 or the peptide consisting of the amino acid sequence in which one or several amino acids are substituted, deleted or added in the peptide is not cytotoxic and is not Any peptide that can suppress the survival of the invading bacteria may be used. For example, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or two peptides for enhancing the action of the peptide Peptide TNIII prepared by adding Cys to the C-terminus of the peptide or a peptide in which a tag (for example, His tag, GST tag, etc.) is bound to these peptides.

  The peptide used in the present invention can be synthesized organically based on the sequence information. In addition, a polynucleotide encoding the peptide used in the present invention is inserted into an expression vector having an appropriate enhancer / promoter, the recombinant vector is introduced into bacteria such as Escherichia coli and Salmonella, yeast, animal cells, The peptide that is expressed and produced may be purified using conventional methods. In addition, mRNA obtained by in vitro transcription of this recombinant vector is translated by an in vitro translation system using rabbit reticulocyte extract, Escherichia coli S30 extract, malt extract, wheat germ extract, etc. The synthesized peptide may be purified.

  Here, when preparing an expression vector, a fusion polynucleotide obtained by fusing a polynucleotide encoding a tag such as a His tag or a GST tag and a polynucleotide encoding a peptide used in the present invention is inserted into the vector and fused. More preferably, it is expressed as a protein. This is because the target peptide can be easily purified using the tag. The tag can be removed at the final stage, and only the peptide used in the present invention can be purified by HPLC (high-performance (-speed) liquid chromatography) or the like.

  In addition, in order to secrete the peptide used in the present invention to the outside of the cell, a signal peptide may be added, and a recombinant vector capable of expressing such a fusion peptide is, as described above, an appropriate enhancer / It can be produced by inserting a polynucleotide encoding a fusion peptide into an expression vector having a promoter.

  In addition, cells (recombinant cells) containing the recombinant vector include, for example, animal vectors, virus vectors such as electroporation method, microinjection method, lipofection method, adenovirus, retrovirus, etc. It can be prepared by introducing by a virus infection method using, or a transfection method using calcium.

== Use of the preventive / ameliorating agent according to the present invention ==
When the preventive / ameliorating agent according to the present invention is administered to a human or a non-human vertebrate for the prevention or amelioration of a disease caused by a pathogenic bacterium that has entered a cell, the affected area to be introduced However, it may be administered orally or parenterally such as intravenously or intraperitoneally. In addition, when two or more substances among substances such as TNIII, caspase inhibitor, PI3P, MEK inhibitor, and amino acid derivative are administered, they may be administered to the same site or different sites.

  When the above preventive / ameliorating agent is administered orally, it may be a tablet, capsule, granule, powder, syrup, etc. When it is administered intraperitoneally or intravenously, it is an injection or infusion. A preparation such as an agent may be used. In addition, the said formulation is manufactured by a conventional method using the conventionally used formulation additive (For example, an excipient | filler, binder, a lubricant, a disintegrating agent, a corrigent, a solvent, a stabilizer, etc.). be able to.

  Hereinafter, it demonstrates in detail using an Example.

[Example 1]
Macrophage-derived Raw264.7 cells (6 × 10 ⁶ cells), which are phagocytic cells, were cultured and adhered in a 10 cm dish containing DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum), and BCG ( 6 × 10 ⁷ pieces; manufactured by Nippon BCG Co., Ltd.) was added and the mixture was infected at 37 ° C. for 1 hour. Thereafter, the cells were washed 4 times with PBS to remove extracellular BCG, and the cells were collected with a scraper. This was suspended in DMEM containing 2% FBS and used as a BCG-infected cell solution (5 to 10 × 10 ⁵ cells / mL) in the following experiment. Raw 264.7 cells were treated in the same manner without addition of BCG and used as a control non-infected cell solution (5 to 10 × 10 ⁵ cells / mL) in the following experiments.

  Rapamycin (50 μg / mL; manufactured by Wako Pure Chemical Industries) which is an mTOR (mammalian target of Rapamycin) inhibitor, U0126 (2 μM; manufactured by Calbiochem) which is a MEK inhibitor, or rapamycin (50 μg / mL) and U0126 (2 μM) BCG-infected cell solution with or without BCG-infected cell solution (control) or non-infected cell solution (uninfected) at 10 μg / mL human plasma fibronectin (purified from human plasma; Fukai et al J Biol Chem 266: 8807-8813, 1991) inoculated into each well of a coated 6-well plate by incubating with a solution (FN in PBS) at 37 ° C for 1 hour and incubated at 37 ° C for 1 or 2 days did. Thereafter, the cells were collected with a scraper and centrifuged (2000 rpm × 5 minutes). After centrifugation, the supernatant is removed, and 1 mL of RIPA1 / 2 solution (0.05% SDS, 0.5% TritonX-100, 5 mM Tris-HCl, 75 mM NaCl, 2.5 mM EDTA) is added and allowed to stand at room temperature for 10 minutes. Was dissolved. After centrifuging the cell lysate (3000 rpm × 5 minutes), the supernatant was removed, and the precipitated BCG was washed 3 times with PBS.

  After washing, the precipitated BCG was suspended in PBS to 1 mL, and 200 μL was injected into each well of a 96-well plate. Then, 20 μL of 10% Alamar Blue (manufactured by Biosouce) was added to each well, stirred, and incubated at 37 ° C. for 2 hours. After the incubation, the number of BCG that survived in the cells was evaluated by measuring the fluorescence intensity using a fluorescence plate reader (Ex530 nm, Em590 nm; manufactured by Perseptive Biosystems). The result is shown in FIG.

  As shown in FIG. 1, it was revealed that infected cells treated with mTOR inhibitor rapamycin, MEK inhibitor U0126, and rapamycin + U0126 had a significantly reduced number of BCG compared to the control. .

[Example 2]
Next, instead of rapamycin or U0126, TNIII (80 μg / ml), caspase inhibitor Z-VAD fmk (100 μM; manufactured by Sigma), or TNIII (80 μg / ml) and Z-VAD fmk (100 μM) The number of BCG surviving in the cells was evaluated in the same manner as in the method described in Example 1 except that the cells were incubated at 37 ° C. for 1 day or 2 days. The result is shown in FIG.

  As shown in FIG. 2, it was revealed that TNIII and the caspase inhibitor Z-VAD fmk also significantly reduced the number of BCG compared to the control.

[Example 3]
Next, the following experiment was conducted in order to examine the presence or absence of the cytotoxic action of each substance (such as rapamycin, U0126, TNIII, and Z-VAD) on macrophages.

  Inoculate 200 μl of the BCG-infected cell solution or non-infected cell solution prepared in Example 1 in a 96-well plate, add rapamycin to a final concentration of 25 or 50 μg / mL, and culture at 37 ° C. for 1 day did. As a control, the same experiment was performed using a BCG-infected cell solution and a non-infected cell solution cultured without adding rapamycin.

  After culturing, the number of viable cells was evaluated using Cell Counting Kit (manufactured by Dojindo) according to the MTT (3- (4,5-dimethlthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay method ( Absorbance (measurement wavelength 450 nm, reference side 655 nm) was measured using a microplate reader (BioRad). The result is shown in FIG.

Furthermore, in 96-well plates were seeded suspension of Raw264.7 cells (DMEM containing 2% FBS) and ^{(1 × 10 4 / 200μl /} well) by 200 [mu] l, U0126 in place of rapamycin (2μM), TNIII (40 or 80 μg / mL) or Z-VAD (100 μM) was added and incubated at 37 ° C. for 1, 2 or 3 days, and cytotoxicity test was performed on Raw264.7 cells. The result is shown in FIG.

  As shown in FIG. 3, most of the host cells treated with rapamycin caused cell death, whereas as shown in FIG. 4, it was found that U0126 and TNIII had no damaging effect on macrophages. Further, as shown in FIG. 4, it was found that Z-VAD fmk has a damaging action on macrophages but its action is weaker than rapamycin. Therefore, MEK inhibitors, TNIII, caspase inhibitors, etc. have no toxicity to normal cells and can suppress the survival and proliferation of cells invaded by pathogenic bacteria. It is thought to be useful for the prevention and improvement of diseases caused by pathogenic bacteria.

[Example 4]
Next, instead of rapamycin, U0126, etc., the amino acid derivative Ac-L-Ala-NH ₂ (20 mM; manufactured by Bachem) or PI3P (10 μM; manufactured by Sigma) is used for one day at 37 ° C. In the same manner as described in Example 1, the number of BCG surviving in the cells was evaluated. The result is shown in FIG.

As shown in FIG. 5, it was revealed that the amino acid derivatives Ac-L-Ala-NH ₂ and PI3P also decreased the number of BCG compared to the control.

In addition, a cytotoxicity test was performed on uninfected cells in the same manner as in Example 3 except that Ac-L-Ala-NH ₂ was used instead of rapamycin and the cells were incubated at 37 ° C. for 1 or 2 days. (FIG. 6) It was found that Ac-L-Ala-NH ₂ also showed no damaging action on macrophages. As a result, amino acid derivatives, PI3P, etc. are not toxic to normal cells, and can suppress the survival and proliferation of cells invaded by pathogenic bacteria, resulting in pathogenic bacteria invading cells. It is thought that it is useful for prevention and improvement of diseases.

In one Example of this invention, it is a figure which shows the result of having investigated the influence which rapamycin, U0126, or rapamycin and U0126 has on the survival of BCG which infected the cell. In one Example of this invention, it is a figure which shows the result of having investigated the influence which TNIII, Z-VAD fmk, or TNIII and Z-VAD fmk gives to the survival of BCG which infected the cell. In one Example of this invention, it is a figure which shows the result of having investigated the cytotoxic effect of rapamycin with respect to a macrophage. In one Example of this invention, it is a figure which shows the result of having investigated the cytotoxic effect of each substance (U0126, TNIII, Z-VAD etc.) with respect to a macrophage. In one embodiment of the present invention, Ac-L-Ala-NH 2, or is a diagram showing the results of examining the effect on survival of BCG which PI3P infects a cell. In one embodiment of the present invention, showing the results of examining the cytotoxic effects of Ac-L-Ala-NH ₂ on macrophage.

Claims (16)

A preventive / ameliorating agent for diseases caused by pathogenic bacteria that have invaded cells,
A preventive / ameliorating agent comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1 as an active ingredient.   The preventive / ameliorating agent according to claim 1, wherein the peptide is a peptide having an amino acid sequence in which one or several amino acids are substituted, deleted, or added.   The preventive / ameliorating agent according to claim 1 or 2, further comprising a caspase inhibitor.   The preventive / ameliorating agent according to claim 3, wherein the caspase inhibitor is Z-VAD fmk (Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone).   The preventive / ameliorating agent according to any one of claims 1 to 4, wherein the pathogenic bacterium is Mycobacterium tuberculosis. A preventive / ameliorating agent for diseases caused by pathogenic bacteria that have invaded cells,
A preventive / improving agent comprising a caspase inhibitor as an active ingredient.   The preventive / ameliorating agent according to claim 6, wherein the pathogenic bacterium is Mycobacterium tuberculosis.   The preventive / ameliorating agent according to claim 6 or 7, wherein the caspase inhibitor is Z-VAD fmk (Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone). A preventive / ameliorating agent for diseases caused by pathogenic bacteria that have invaded cells,
An improving agent containing PI3P (phosphatidylinositol-3-phosphate) as an active ingredient.   The preventive / ameliorating agent according to claim 9, wherein the bacterium is Mycobacterium tuberculosis. A preventive / ameliorating agent for diseases caused by pathogenic bacteria that have invaded cells,
A preventive / ameliorating agent containing a MEK (Mitogen-activated protein (MAP) kinases) inhibitor as an active ingredient.   The preventive / ameliorating agent according to claim 11, wherein the bacterium is Mycobacterium tuberculosis.   The preventive / ameliorating agent according to claim 11 or 12, wherein the MEK inhibitor is U0126 (1.4-diamino-2,3-dicyano-1,4-bis (2-amino-phenylthio) butadiene). . A preventive / ameliorating agent for diseases caused by pathogenic bacteria that have invaded cells,
A preventive / improving agent containing an amino acid derivative as an active ingredient.   The preventive / ameliorating agent according to claim 14, wherein the bacterium is Mycobacterium tuberculosis. The preventive / ameliorating agent according to claim 14 or 15, wherein the amino acid derivative is Ac-L-Ala-NH ₂ (Acetyl-L-alanine amide).

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