WO2012118070A1 - Anti-gram-negative bacteria agent - Google Patents

Anti-gram-negative bacteria agent Download PDF

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WO2012118070A1
WO2012118070A1 PCT/JP2012/054905 JP2012054905W WO2012118070A1 WO 2012118070 A1 WO2012118070 A1 WO 2012118070A1 JP 2012054905 W JP2012054905 W JP 2012054905W WO 2012118070 A1 WO2012118070 A1 WO 2012118070A1
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gram
present
negative bacteria
peptide molecule
bacterial agent
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PCT/JP2012/054905
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French (fr)
Japanese (ja)
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栄策 良原
滋樹 光永
猪子　英俊
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学校法人 東海大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to a drug effective for the prevention / treatment of Gram-negative bacterial infection. More specifically, a drug capable of exerting a bactericidal action, a growth inhibitory action, and / or a drug excretion inhibitory action against Gram-negative bacteria by inhibiting the formation of a YaeT complex present in the outer membrane of Gram-negative bacteria. About.
  • Gram-negative bacteria such as multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii are the main causative agents of nosocomial infections, leading to serious pathological conditions in patients with reduced immunity There is. When these bacteria have become multidrug resistant, treatment of infectious diseases is difficult and has become a major problem. Gram-negative bacteria have two membranes, an inner membrane and an outer membrane. In order for antibiotics to work effectively, antibiotics must pass through the complex membrane structure of Gram-negative bacteria to reach the inside of the cell. There is. However, it is thought that the effect of antibiotics decreases due to drug-modifying enzymes, decreased permeability of the outer membrane, changes in targets, drug discharge by a drug discharge pump, etc., and multidrug resistance is acquired.
  • the multi-drug discharge pump has contributed greatly to the acquisition of multi-drug resistance of Pseudomonas aeruginosa. This pump positively transports and discharges the drug that has entered the inside of the bacterium using energy.
  • Multidrug evacuation pumps are classified into several families. Among them, the RND (resistance nodition division) family pumps, which are known to evacuate various antibiotics, are composed of three types of subunits. Yes. Pseudomonas aeruginosa has multiple RND type pumps, of which the main pump is the MexAB-OprM pump.
  • the present inventors focused on the MexAB-OprM pump of Pseudomonas aeruginosa and inhibited the drug efflux pump function of Pseudomonas aeruginosa by modifying the amino acid sequence of OprM, a subunit constituting the multidrug efflux pump. Then, a method for enhancing the efficacy of antibiotics was developed, and a drug having the effect and a method for screening the drug were proposed (Patent Document 1). However, if a mutation occurs in the target sequence, the effect of the method may be lost.
  • Non-patent Document 1 On the other hand, research on Gram-negative bacteria including Pseudomonas aeruginosa and Escherichia coli has been advanced, and it has been known that the YaeT complex is essential for ⁇ -barrel protein biosynthesis (Non-patent Document 1). The three-dimensional structure has also been elucidated (Non-patent Document 2).
  • YaeT is responsible for biosynthesis of outer membrane protein (OMP) by forming a complex with four lipoproteins YfgL, YfiO, NlpB and SmpA. In other words, if YaeT complex formation is inhibited, the transport of OMP including the drug efflux pump to the outer membrane can be prevented.
  • OMP outer membrane protein
  • the present inventors have blocked the formation of YaeT complex in the outer membrane of Gram-negative bacteria, thereby blocking the transport of outer membrane protein (OMP) necessary for the survival of the bacteria, and therefore (Patent Document 2). That is, the peptide molecule containing the amino acid sequence “LTLR” inhibits the binding between YaeT and YfgL, the peptide molecule having at least a part of the amino acid sequence “FIRLHP” inhibits the binding between YaeT and YfiO, Furthermore, it has been found that the binding inhibition effect is improved by amidating the C-terminus of these peptide molecules.
  • An object of the present invention is to provide an anti-gram-negative bacterial agent that exhibits a sufficient bactericidal action even when used alone without being combined with an antibiotic.
  • LTLR which is an amino acid sequence conserved in YfgL of many Gram-negative bacteria such as Pseudomonas aeruginosa
  • FSLR which is a corresponding sequence of Acinetobacter
  • a peptide molecule having an amino acid sequence in which at least two LSLRs corresponding to Escherichia coli are linked in series and amidated the C-terminus is sufficient against Gram-negative bacteria without using an antibiotic.
  • the present inventors have found that it exhibits a good bactericidal action, and have made the present invention.
  • the present invention provides the following formula (I): XXLR (I) Provided is an anti-gram-negative bacterial agent comprising a peptide molecule comprising at least two repeating units represented by the formula (XX represents LT, FS or LS) and amidated at the C-terminus.
  • the amidation is preferably an addition of a hydrophobic functional group to the carboxyl group at the C-terminal of the peptide by an amide bond.
  • the formation of YaeT complex of gram-negative bacteria is sufficiently inhibited even in the absence of conventional antibiotics, thereby bactericidal action, growth inhibitory action, and / or drug. Since the excretion inhibitory action is exerted, the problem of multidrug resistance due to mutation of the outer membrane protein is fundamentally solved.
  • the anti-gram-negative bacterial agent of the present invention effectively inhibits the formation of the YaeT complex without using in combination with conventional antibiotics. It exhibits a drug excretion inhibiting action.
  • Gram-negative bacteria is used in a commonly used meaning. That is, it is a general term for bacteria whose staining with crystal violet is decolored in Gram staining.
  • Representative gram-negative bacteria include E. coli (Escherichia coli), Salmonella, Pseudomonas, Helicobacter and other proteobacteria and cyanobacteria.
  • gonococci include digestive disorders such as Pseudomonas aeruginosa, Acinetobacter, and Haemophilus influenzae that cause respiratory damage, such as Escherichia coli and Mirabilis sp. Examples include inducing Helicobacter pylori and Gertonel, and examples of cocci include Neisseria meningitidis, Catalaris, and Neisseria gonorrhoeae.
  • the YaeT complex in Gram-negative bacteria is composed of four lipoproteins, YfgL, YfiO, NlpB, and SmpA, gathered on YaeT, which is a membrane protein. Among them, in order for the YaeT complex to biosynthesize OMP, it is essential that YfgL and YfiO bind to each other, and OMP is not biosynthesized by inhibiting their binding to YaeT.
  • the present inventors compared the amino acid sequences of the binding sites of various gram-negative bacteria YfgL to YaeT, examined highly conserved regions, and based on the amino acid sequences of these regions, various peptide molecules As a result of synthesis and examination, it was found that a peptide molecule containing the amino acid sequence “LTLR” competes with the binding between YaeT and YfgL and can effectively inhibit the binding (see Patent Document 2).
  • FIG. 2 shows the amino acid sequences of the region containing “LTLR” of Pseudomonas aeruginosa and the corresponding region of other Gram-negative bacteria (Acinetobacter and E. coli).
  • the part of “LTLR” of “LTLR” in Pseudomonas aeruginosa is replaced with “FS” in Acinetobacter and “LS” in E. coli, but “LR” and the adjacent “G” are preserved in three types of bacteria. Has been.
  • a peptide molecule containing at least two repeating units consisting of “LTLR” and the corresponding “FSLR” or “LSLR” has a significantly superior bactericidal effect as compared with a peptide molecule containing simply “LTLR”.
  • the bactericidal effect has been found to be effective across species, albeit to varying degrees.
  • the repeating unit constituting the anti-gram-negative bacterial agent of the present invention is “LTLR”, “FSLR”, or “LSLR”, and the number of repeating units is at least two.
  • the upper limit of the number of repeating units is not particularly limited, but is preferably 20 or less, more preferably about 10 or less, in order to exert the effect through the cell membrane.
  • the repeating unit constituting the anti-gram-negative bacterial agent of the present invention may be directly bonded or may be bonded via a linker. Although it does not specifically limit as a linker, Various amino acids etc. can be mentioned. For example, “G” conserved adjacent to “LTLR” is preferably used as a linker.
  • the further aspect of the anti-gram negative bacteria agent of this invention is following formula (I): XXLR (I) (Wherein XX represents LT, FS or LS), the polypeptide consists of a polypeptide molecule in which at least two repeating units are represented, the repeating units are linked via a linker, and the C-terminal is amidated.
  • the above peptide molecule constituting the anti-gram-negative bacterial agent of the present invention is characterized in that its C-terminal is amidated.
  • the amidation is carried out by coupling of a carboxyl group at the C-terminal of the peptide with an amino group-containing compound, and the amino group-containing compound preferably has a hydrophobic functional group.
  • the amino group-containing compound used for amidation is not particularly limited, and examples thereof include ammonia, aliphatic or aromatic amines, amino acids, and the like.
  • Amino acids include aromatic amino acids such as tryptophan, phenylalanine, and tyrosine. Family amino acids, branched amino acids such as valine, leucine and isoleucine, and aliphatic amino acids such as alanine.
  • the bactericidal action is remarkably improved by amidating the C-terminus.
  • the present inventors consider that the membrane permeability is improved because the negative charge at the C-terminal is canceled out by the amide bond.
  • amino group-containing compound used for amidation in the present invention those having a hydrophobic functional group are particularly preferred.
  • a peptide molecule (LTLRGLTLLR- ⁇ NA) in which ⁇ -naphthylamine ( ⁇ NA) is amide-bonded to the C-terminal has a markedly improved bactericidal action compared to a peptide molecule (LTLRGLTLLR-NH 2 ) having an amino group bonded thereto.
  • the survival rate of Pseudomonas aeruginosa was 95% in the presence of 10 ⁇ M LTLRGLTLR- ⁇ NA, but the survival rate at 100 ⁇ M was 0%.
  • the bactericidal action of the peptide molecule is further improved by introducing a hydrophobic environment in the vicinity of the C-terminus of the polypeptide comprising the XXLR repeating unit. Therefore, instead of amidating the C-terminus of the peptide molecule constituting the anti-gram-negative bacterial agent of the present invention, a compound having a hydrophobic group, such as an aliphatic or aromatic higher alcohol, is esterified to the C-terminal carboxyl group. Even if it is, it is thought that a high bactericidal action is exhibited.
  • the anti-gram negative bacterial agent of the present invention may be one obtained by subjecting the peptide molecule to various chemical modifications other than those described above, as long as the effects of the present invention are not inhibited.
  • the interaction site between YaeT, YfgL, and YfiO targeted in the present invention is present in the periplasm between the outer and inner membranes of the fungus (see FIG. 1). Therefore, in order for the drug (peptide molecule) of the present invention to reach the action site from the outside, it is necessary to pass through the outer membrane.
  • the anti-gram-negative bacterial agent of the present invention is thought to be able to pass through the outer membrane by itself because the C-terminal of the peptide molecule is amidated and the negative charge at the C-terminal disappears. Therefore, the anti-gram-negative bacterial agent of the present invention exhibits a sufficient bactericidal action even if it is not co-administered with a drug that improves the permeability of the outer membrane such as polymyxin B, colistin and other antibiotics having a cell membrane alteration effect. And can be used as a remedy for Gram-negative bacterial infections.
  • the bactericidal action was further improved when the anti-gram-negative bacterial agent of the present invention and an antibiotic (with or without membrane alteration) were administered simultaneously.
  • the amidated peptide molecule constituting the anti-gram negative bacterial agent of the present invention passes through the outer membrane to reach the YaeT complex formation site, and the membrane is weakened by inhibiting biosynthesis of the outer membrane protein. It is considered to be more sensitive to antibiotics and have a great bactericidal effect.
  • a further aspect of the present invention is an anti-gram negative bacterial agent characterized by containing the above peptide molecule and an antibiotic (regardless of the presence or absence of membrane alteration).
  • Antibiotics are not particularly limited, and those conventionally used in general can be used.
  • cephalosporin antibiotics such as cephalexin, cefotaxime, etc., fluoroquinolone antibiotics ofloxacin, ciprofloxacin, etc. And the like, and amicomine, which is an aminoglycoside antibiotic, and meropenem, which is a carbapenem antibiotic.
  • the method for measuring the viable count of Gram-negative bacteria is as follows. A part of the bacteria cultured overnight at 37 ° C. is added to fresh LB medium, and the culture is continued at the same temperature. When the turbidity (absorbance at 600 nm) of the bacterium reaches 0.7 to 0.8, the microbial solution is added to the LB medium to prepare a microbial solution diluted 100 to 200 times and used for the experiment.
  • a solution obtained by adding 60 to 80 ⁇ L of the bacterial solution and a peptide solution or the like to a final volume of 100 ⁇ L is cultured at 37 ° C. with stirring for a certain time (3 hours). After culturing, the bacterial solution is diluted with a PBS solution, and the diluted solution is plated on an LB agar plate and cultured at 37 ° C. overnight. The viable cell count is obtained by counting the number of colonies growing on the plate the next day.
  • Example 1 The peptide molecule “LTLRGLTLR-NH 2 ” was added to the above experimental system at various concentrations and cultured. Under each condition, the viable cell count of Pseudomonas aeruginosa was measured, and how the viable cell count was changed by the addition of the peptide was measured as compared to the viable cell count when the peptide was not added. The results are shown in the graph of FIG.
  • Example 2 In Example 1, the survival rate was measured when an antibiotic (OFLX: 0.1 ⁇ g / mL) was added in addition to the peptide molecule.
  • the results are shown in FIG. FIG. 4A is the same data as FIG. 3A, and is shown here as a reference for viewing the effect of OFLX addition. It is clear that the bactericidal ability was further improved by coexisting antibiotics.
  • Example 3 In Example 1, the survival rate when the bacteria were replaced with Acinetobacter was measured. The results are shown in FIG. As described in Example 1, the peptide molecule “LTLRGLTLLR-NH 2 ” which is an anti-gram-negative bacterial agent of the present invention showed an excellent bactericidal action against Pseudomonas aeruginosa (FIG. 5 (a)), but Acinetobacter It was also found that a sufficient bactericidal action was exhibited (FIG. 5 (b)).
  • Example 4 The peptide molecule “LTLRGLTLLR-NH 2 ” used in Example 1 was replaced with another peptide molecule “FSLRGFSLR-NH 2 ”, and the survival rates of Pseudomonas aeruginosa and Acinetobacter were measured.
  • a peptide molecule having the same “FSLR” as the amino acid sequence of Acinetobacter was used, an excellent bactericidal action against Acinetobacter was observed (FIG. 6 (a)).
  • this peptide molecule was also confirmed to exert a bactericidal action against Pseudomonas aeruginosa (FIG. 6B).
  • the anti-gram-negative bacterial agent of the present invention comprising a peptide molecule having two or more repeating units of “XXLR (XX: LS, FS or LS)” exhibits an excellent bactericidal action alone.
  • XXLR XX: LS, FS or LS

Abstract

The purpose of the present invention is to provide an anti-gram-negative bacteria agent exerting sufficient bactericidal action even when used alone and not used along with an antibiotic. The present invention provides an anti-gram-negative bacteria agent containing at least two repeating units represented by formula (I) which is XXLR, wherein the C-terminal of the repeating units contains an amidated peptide molecule. (In the formula, XX represents LT, FS or LS.) The repeating units may be linked via a linker. Preferably, the repeating units are selected from LTLR and FSLR.

Description

抗グラム陰性菌剤Anti-gram negative bacteria agent
　本発明は、グラム陰性菌感染症の予防／治療に有効な薬剤に関する。より詳細には、グラム陰性菌の外膜に存在するＹａｅＴ複合体の形成を阻害することにより、グラム陰性菌に対する殺菌作用、増殖阻害作用、及び／又は薬剤排出阻害作用を発揮することのできる薬剤に関する。 The present invention relates to a drug effective for the prevention / treatment of Gram-negative bacterial infection. More specifically, a drug capable of exerting a bactericidal action, a growth inhibitory action, and / or a drug excretion inhibitory action against Gram-negative bacteria by inhibiting the formation of a YaeT complex present in the outer membrane of Gram-negative bacteria. About.
　多剤耐性の緑膿菌（Ｐｓｅｕｄｏｍｏｎａｓ　ａｅｒｕｇｉｎｏｓａ）やアシネトバクター・バウマニ（Ａｃｉｎｅｔｏｂａｃｔｅｒ　ｂａｕｍａｎｎｉｉ）等のグラム陰性菌は院内感染症の主要起因菌であり、免疫力が低下した患者を重篤な病態に至らしめる場合がある。これらの菌が多剤耐性化したものは感染症の治療が難しく大きな問題となっている。グラム陰性菌は内膜と外膜という二つの膜を備えており、抗生物質が有効に作用するためには、抗生物質はグラム陰性菌の複雑な膜構造を通過して細胞内部に到達する必要がある。ところが、薬剤修飾酵素、外膜の透過性の低下、標的の変化、薬剤排出ポンプによる薬剤の排出などによって抗生物質の効果が低下し、多剤耐性を獲得するものと考えられている。 Gram-negative bacteria such as multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii are the main causative agents of nosocomial infections, leading to serious pathological conditions in patients with reduced immunity There is. When these bacteria have become multidrug resistant, treatment of infectious diseases is difficult and has become a major problem. Gram-negative bacteria have two membranes, an inner membrane and an outer membrane. In order for antibiotics to work effectively, antibiotics must pass through the complex membrane structure of Gram-negative bacteria to reach the inside of the cell. There is. However, it is thought that the effect of antibiotics decreases due to drug-modifying enzymes, decreased permeability of the outer membrane, changes in targets, drug discharge by a drug discharge pump, etc., and multidrug resistance is acquired.
　緑膿菌の多剤耐性獲得に大きく貢献しているのが多剤排出ポンプである。このポンプは、菌内部に入った薬剤をエネルギーを使って積極的に細胞外に輸送、排出する。多剤排出ポンプは幾つかのファミリーに分類され、それらの中で、様々な抗生剤を排出することが知られているＲＮＤ（ｒｅｓｉｓｔａｎｃｅ　ｎｏｄｕｌａｔｉｏｎ　ｄｉｖｉｓｉｏｎ）ファミリーのポンプは３種類のサブユニットから構成されている。緑膿菌には複数のＲＮＤ型ポンプが存在するが、そのうちで主要なポンプはＭｅｘＡＢ－ＯｐｒＭポンプである。 The multi-drug discharge pump has contributed greatly to the acquisition of multi-drug resistance of Pseudomonas aeruginosa. This pump positively transports and discharges the drug that has entered the inside of the bacterium using energy. Multidrug evacuation pumps are classified into several families. Among them, the RND (resistance nodition division) family pumps, which are known to evacuate various antibiotics, are composed of three types of subunits. Yes. Pseudomonas aeruginosa has multiple RND type pumps, of which the main pump is the MexAB-OprM pump.
　本発明者等は、緑膿菌のＭｅｘＡＢ－ＯｐｒＭポンプに着目し、当該多剤排出ポンプを構成するサブユニットであるＯｐｒＭのアミノ酸配列を改変することにより、緑膿菌の薬剤排出ポンプ機能を阻害し、抗生剤の薬効を増強する方法を開発し、当該効果を奏する薬剤並びに当該薬剤をスクリーニングする方法を提案した（特許文献１）。しかしながら、標的配列において変異が生ずると、当該方法の効果が失われる可能性があった。 The present inventors focused on the MexAB-OprM pump of Pseudomonas aeruginosa and inhibited the drug efflux pump function of Pseudomonas aeruginosa by modifying the amino acid sequence of OprM, a subunit constituting the multidrug efflux pump. Then, a method for enhancing the efficacy of antibiotics was developed, and a drug having the effect and a method for screening the drug were proposed (Patent Document 1). However, if a mutation occurs in the target sequence, the effect of the method may be lost.
　一方、緑膿菌や大腸菌などを含むグラム陰性菌の研究が進んでおり、β－バレルプロテイン生合成にＹａｅＴ複合体が必須であることが知られるに至り（非特許文献１）、当該ＹａｅＴの立体構造も解明されてきている（非特許文献２）。ＹａｅＴは、ＹｆｇＬ、ＹｆｉＯ、ＮｌｐＢ及びＳｍｐＡなる４つのリポタンパク質と複合体を形成することにより、外膜タンパク質（ＯＭＰ）の生合成を司っている。言い換えれば、ＹａｅＴの複合体形成を阻害すれば薬剤排出ポンプを含むＯＭＰの外膜への輸送を阻止することができると考えられる。 On the other hand, research on Gram-negative bacteria including Pseudomonas aeruginosa and Escherichia coli has been advanced, and it has been known that the YaeT complex is essential for β-barrel protein biosynthesis (Non-patent Document 1). The three-dimensional structure has also been elucidated (Non-patent Document 2). YaeT is responsible for biosynthesis of outer membrane protein (OMP) by forming a complex with four lipoproteins YfgL, YfiO, NlpB and SmpA. In other words, if YaeT complex formation is inhibited, the transport of OMP including the drug efflux pump to the outer membrane can be prevented.
　本発明者等は、上記の事情に鑑み、グラム陰性菌の外膜におけるＹａｅＴ複合体の形成を阻害することにより、菌の生存に必要な外膜タンパク質（ＯＭＰ）の輸送を遮断する方法及びそのための薬剤を開発した（特許文献２）。即ち、アミノ酸配列「ＬＴＬＲ」を含むペプチド分子が、ＹａｅＴとＹｆｇＬとの結合を阻害すること、アミノ酸配列「ＦＩＲＬＨＰ」の少なくとも一部を有するペプチド分子が、ＹａｅＴとＹｆｉＯとの結合を阻害すること、更には、これらのペプチド分子のＣ末端をアミド化することにより結合阻害効果が向上することを見出した。 In view of the above circumstances, the present inventors have blocked the formation of YaeT complex in the outer membrane of Gram-negative bacteria, thereby blocking the transport of outer membrane protein (OMP) necessary for the survival of the bacteria, and therefore (Patent Document 2). That is, the peptide molecule containing the amino acid sequence “LTLR” inhibits the binding between YaeT and YfgL, the peptide molecule having at least a part of the amino acid sequence “FIRLHP” inhibits the binding between YaeT and YfiO, Furthermore, it has been found that the binding inhibition effect is improved by amidating the C-terminus of these peptide molecules.
　しかしながら、上記のペプチド分子単独ではグラム陰性菌に対する殺菌作用は十分ではなく、完全に殺菌するためには抗生剤と併用することが必要であった。 However, the above peptide molecule alone is not sufficient for bactericidal action against Gram-negative bacteria, and it was necessary to use it together with an antibiotic to completely sterilize it.
国際公開第ＷＯ２００７／０９１３９５号パンフレットInternational Publication No. WO2007 / 091395 Pamphlet 国際公開第ＷＯ２０１０／１４７１４５号パンフレットInternational Publication No. WO2010 / 147145 Pamphlet
　本発明は、抗生剤と併用せず単独で用いた場合でも十分な殺菌作用を示す抗グラム陰性菌剤を提供することを目的とする。 An object of the present invention is to provide an anti-gram-negative bacterial agent that exhibits a sufficient bactericidal action even when used alone without being combined with an antibiotic.
　本発明者等は、前記課題を解決すべく鋭意検討を行った結果、緑膿菌など多くのグラム陰性菌のＹｆｇＬに保存されているアミノ酸配列であるＬＴＬＲ、アシネトバクターの対応する配列であるＦＳＬＲ、又は大腸菌の対応する配列であるＬＳＬＲを少なくとも２つ直列に結合させたアミノ酸配列を有し、そのＣ末端をアミド化したペプチド分子が、抗生剤と併用しなくてもグラム陰性菌に対して十分な殺菌作用を発揮することを見出し、本発明をなすに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that LTLR, which is an amino acid sequence conserved in YfgL of many Gram-negative bacteria such as Pseudomonas aeruginosa, FSLR, which is a corresponding sequence of Acinetobacter, Alternatively, a peptide molecule having an amino acid sequence in which at least two LSLRs corresponding to Escherichia coli are linked in series and amidated the C-terminus is sufficient against Gram-negative bacteria without using an antibiotic. The present inventors have found that it exhibits a good bactericidal action, and have made the present invention.
　即ち本発明は、下記式（Ｉ）：
　ＸＸＬＲ　　　（Ｉ）
（但し、ＸＸはＬＴ、ＦＳ又はＬＳを示す）で表される繰り返し単位を少なくとも２個含み、そのＣ末端をアミド化したペプチド分子を含んでなる抗グラム陰性菌剤を提供する。前記アミド化は、ペプチドＣ末端のカルボキシル基への疎水性官能基のアミド結合による付加であるのが好ましい。 That is, the present invention provides the following formula (I):
XXLR (I)
Provided is an anti-gram-negative bacterial agent comprising a peptide molecule comprising at least two repeating units represented by the formula (XX represents LT, FS or LS) and amidated at the C-terminus. The amidation is preferably an addition of a hydrophobic functional group to the carboxyl group at the C-terminal of the peptide by an amide bond.
　本発明の抗グラム陰性菌剤によれば、従来の抗生物質が存在しなくても、グラム陰性菌のＹａｅＴ複合体形成を十分に阻害し、それにより殺菌作用、増殖阻害作用、及び／又は薬剤排出阻害作用が発揮されるため、外膜タンパク質の変異による多剤耐性化の問題が根本的に解決される。 According to the anti-gram-negative bacterial agent of the present invention, the formation of YaeT complex of gram-negative bacteria is sufficiently inhibited even in the absence of conventional antibiotics, thereby bactericidal action, growth inhibitory action, and / or drug. Since the excretion inhibitory action is exerted, the problem of multidrug resistance due to mutation of the outer membrane protein is fundamentally solved.
グラム陰性菌の外膜及び内膜並びにＹａｅＴ複合体と外膜タンパク質との関係を説明する模式図である（非特許文献１から引用）。It is a schematic diagram explaining the outer membrane and inner membrane of Gram-negative bacteria, and the relationship between a YaeT complex and outer membrane protein (cited from nonpatent literature 1). 緑膿菌、アシネトバクター、大腸菌のＹｆｇＬの、ＹａｅＴとの結合部位のアミノ酸配列を比較する図である。It is a figure which compares the amino acid sequence of the binding site of YfgL of Pseudomonas aeruginosa, Acinetobacter, and Escherichia coli with YaeT. （ａ）ＬＴＬＲＧＬＴＬＲ－ＮＨ２の投与量と緑膿菌（ＰＡＯ１株）の生存率との関係を示すグラフである。（ｂ）一定濃度（１ｍＭ又は２ｍＭ）ＬＴＬＲＧＬＴＬＲ－ＮＨ２の存在下でのＨｅＬａ細胞及びＭＧ６３細胞の生存率を示すグラフである。(A) A graph showing the relationship between the dose of LTLRGLTL-NH 2 and the survival rate of Pseudomonas aeruginosa (PAO1 strain). (B) A graph showing the viability of HeLa cells and MG63 cells in the presence of a constant concentration (1 mM or 2 mM) LTLRGLTLLR-NH 2 . ＯＦＬＸ（０．１μｇ／ｍＬ）の存在下および非存在下での緑膿菌（ＰＡＯ１株）の生存率の濃度依存性を示すグラフである。It is a graph which shows the density | concentration dependence of the survival rate of Pseudomonas aeruginosa (PAO1 strain | stump | stock) in presence and absence of OFLX (0.1 microgram / mL). （ａ）ＬＴＬＲＧＬＴＬＲ－ＮＨ２の投与量と緑膿菌（ＰＡＯ１株）の生存率との関係を示すグラフである。（ｂ）ＬＴＬＲＧＬＴＬＲ－ＮＨ２の投与量とアシネトバクターの生存率との関係を示すグラフである。(A) A graph showing the relationship between the dose of LTLRGLTL-NH 2 and the survival rate of Pseudomonas aeruginosa (PAO1 strain). (B) A graph showing the relationship between the dose of LTLRGLTL-NH 2 and the survival rate of Acinetobacter. （ａ）ＦＳＬＲＧＦＳＬＲ－ＮＨ２の投与量とアシネトバクターの生存率との関係を示すグラフである。（ｂ）ＦＳＬＲＧＦＳＬＲ－ＮＨ２の投与量と緑膿菌（ＰＡＯ１株）の生存率との関係を示すグラフである。(A) A graph showing the relationship between the dose of FSLRGFSLR-NH 2 and the survival rate of Acinetobacter. (B) A graph showing the relationship between the dose of FSLRGFSLR-NH 2 and the survival rate of Pseudomonas aeruginosa (PAO1 strain).
　上記したように、本発明の抗グラム陰性菌剤は、従来の抗生剤と併用しなくてもＹａｅＴ複合体の形成を有効に阻害することにより、グラム陰性菌に対する殺菌作用、増殖阻害作用、及び／又は薬剤排出阻害作用を発揮することを特徴とする。 As described above, the anti-gram-negative bacterial agent of the present invention effectively inhibits the formation of the YaeT complex without using in combination with conventional antibiotics. It exhibits a drug excretion inhibiting action.
　本発明における「グラム陰性菌」なる用語は、一般に用いられている意味で使用される。即ち、グラム染色においてクリスタルバイオレットによる染色が脱色される細菌の総称である。代表的なグラム陰性菌としては、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）、サルモネラ菌、シュードモナス、ヘリコバクター等のプロテオバクテリア及びシアノバクテリアが含まれる。医学に関連して分類すると、桿菌としては、呼吸器系の傷害を惹起する緑膿菌、アシネトバクター、インフルエンザ菌など、泌尿器系の障害を惹起する大腸菌、ミラビリス変形菌など、消化器系の障害を惹起するヘリコバクター・ピロリ、ゲルトネル菌などが挙げられ、球菌としては、髄膜炎菌、カタラリス菌、淋菌が挙げられる。 In the present invention, the term “Gram-negative bacteria” is used in a commonly used meaning. That is, it is a general term for bacteria whose staining with crystal violet is decolored in Gram staining. Representative gram-negative bacteria include E. coli (Escherichia coli), Salmonella, Pseudomonas, Helicobacter and other proteobacteria and cyanobacteria. When classified in relation to medicine, gonococci include digestive disorders such as Pseudomonas aeruginosa, Acinetobacter, and Haemophilus influenzae that cause respiratory damage, such as Escherichia coli and Mirabilis sp. Examples include inducing Helicobacter pylori and Gertonel, and examples of cocci include Neisseria meningitidis, Catalaris, and Neisseria gonorrhoeae.
　グラム陰性菌におけるＹａｅＴ複合体は、膜タンパク質であるＹａｅＴに、ＹｆｇＬ、ＹｆｉＯ、ＮｌｐＢ及びＳｍｐＡなる４つのリポタンパク質が集合して構成されている。中でも、ＹａｅＴ複合体がＯＭＰの生合成を行うためには、ＹｆｇＬ及びＹｆｉＯが結合することが必須とされており、これらのＹａｅＴへの結合を阻害することによりＯＭＰは生合成されないことになる。 The YaeT complex in Gram-negative bacteria is composed of four lipoproteins, YfgL, YfiO, NlpB, and SmpA, gathered on YaeT, which is a membrane protein. Among them, in order for the YaeT complex to biosynthesize OMP, it is essential that YfgL and YfiO bind to each other, and OMP is not biosynthesized by inhibiting their binding to YaeT.
　本発明者等は、各種グラム陰性菌のＹｆｇＬのＹａｅＴへの結合部位のアミノ酸配列を比較し、高度に保存されている領域を検討し、それら領域のアミノ酸配列を基にして様々なペプチド分子を合成して検討した結果、アミノ酸配列「ＬＴＬＲ」を含むペプチド分子がＹａｅＴとＹｆｇＬとの結合と競合し、それらの結合を有効に阻害できることを見出した（特許文献２参照）。 The present inventors compared the amino acid sequences of the binding sites of various gram-negative bacteria YfgL to YaeT, examined highly conserved regions, and based on the amino acid sequences of these regions, various peptide molecules As a result of synthesis and examination, it was found that a peptide molecule containing the amino acid sequence “LTLR” competes with the binding between YaeT and YfgL and can effectively inhibit the binding (see Patent Document 2).
　図２において、緑膿菌の「ＬＴＬＲ」を含む領域、及び他のグラム陰性菌（アシネトバクター及び大腸菌）の対応する領域のアミノ酸配列を示す。緑膿菌における「ＬＴＬＲ」の「ＬＴ」の部分は、アシネトバクターでは「ＦＳ」、大腸菌では「ＬＳ」に置換されているが、「ＬＲ」及びそれに隣接する「Ｇ」は３種の菌で保存されている。 FIG. 2 shows the amino acid sequences of the region containing “LTLR” of Pseudomonas aeruginosa and the corresponding region of other Gram-negative bacteria (Acinetobacter and E. coli). The part of “LTLR” of “LTLR” in Pseudomonas aeruginosa is replaced with “FS” in Acinetobacter and “LS” in E. coli, but “LR” and the adjacent “G” are preserved in three types of bacteria. Has been.
　本発明においては、「ＬＴＬＲ」並びに対応する「ＦＳＬＲ」又は「ＬＳＬＲ」からなる繰り返し単位を少なくとも２個含むペプチド分子が、単に「ＬＴＬＲ」を含むペプチド分子に比較して格段に優れた殺菌効果を示すことを新たに見出した。驚くべきことに、その殺菌効果は、程度の差はあるものの、種を越えて有効であることも判明した。 In the present invention, a peptide molecule containing at least two repeating units consisting of “LTLR” and the corresponding “FSLR” or “LSLR” has a significantly superior bactericidal effect as compared with a peptide molecule containing simply “LTLR”. New headings to show. Surprisingly, the bactericidal effect has been found to be effective across species, albeit to varying degrees.
　即ち、本発明の抗グラム陰性菌剤を構成する繰り返し単位は「ＬＴＬＲ」、「ＦＳＬＲ」又は「ＬＳＬＲ」であり、この繰り返し単位の数は少なくとも２個である。繰り返し単位数の上限は特に限定されないが、細胞膜を通過して効果を発揮させるためには、好ましくは２０個以下、より好ましくは１０個以下程度である。 That is, the repeating unit constituting the anti-gram-negative bacterial agent of the present invention is “LTLR”, “FSLR”, or “LSLR”, and the number of repeating units is at least two. The upper limit of the number of repeating units is not particularly limited, but is preferably 20 or less, more preferably about 10 or less, in order to exert the effect through the cell membrane.
　本発明の抗グラム陰性菌剤を構成する繰り返し単位は、直接結合されていてもよいが、リンカーを介して結合していてもよい。リンカーとしては、特に限定されないが、各種アミノ酸等を挙げることができる。例えば、前記「ＬＴＬＲ」に隣接して保存されている「Ｇ」をリンカーとするのが好ましい。 The repeating unit constituting the anti-gram-negative bacterial agent of the present invention may be directly bonded or may be bonded via a linker. Although it does not specifically limit as a linker, Various amino acids etc. can be mentioned. For example, “G” conserved adjacent to “LTLR” is preferably used as a linker.
　即ち、本発明の抗グラム陰性菌剤の更なる態様は、下記式（Ｉ）：
　ＸＸＬＲ　　　（Ｉ）
（但し、ＸＸはＬＴ、ＦＳ又はＬＳを示す）で表される繰り返し単位を少なくとも２個含み、当該繰り返し単位がリンカーを介して結合し、そのＣ末端をアミド化したポリペプチド分子からなる。 That is, the further aspect of the anti-gram negative bacteria agent of this invention is following formula (I):
XXLR (I)
(Wherein XX represents LT, FS or LS), the polypeptide consists of a polypeptide molecule in which at least two repeating units are represented, the repeating units are linked via a linker, and the C-terminal is amidated.
　また、本発明の抗グラム陰性菌剤を構成する上記のペプチド分子は、そのＣ末端をアミド化したものであることを特徴としている。当該アミド化は、ペプチドＣ末端のカルボキシル基とアミノ基含有化合物とのカップリングによって行われるが、当該アミノ基含有化合物としては、疎水性官能基を有しているものが好ましい。 Further, the above peptide molecule constituting the anti-gram-negative bacterial agent of the present invention is characterized in that its C-terminal is amidated. The amidation is carried out by coupling of a carboxyl group at the C-terminal of the peptide with an amino group-containing compound, and the amino group-containing compound preferably has a hydrophobic functional group.
　アミド化に用いられるアミノ基含有化合物としては、特に限定されないが、アンモニア、脂肪族又は芳香族アミン、アミノ酸等が挙げられ、アミノ酸としては、トリプトファン等の複素環式アミノ酸、フェニルアラニン、チロシン等の芳香族アミノ酸、バリン、ロイシン、イソロイシン等の分岐アミノ酸、アラニン等の脂肪族アミノ酸が含まれる。 The amino group-containing compound used for amidation is not particularly limited, and examples thereof include ammonia, aliphatic or aromatic amines, amino acids, and the like. Amino acids include aromatic amino acids such as tryptophan, phenylalanine, and tyrosine. Family amino acids, branched amino acids such as valine, leucine and isoleucine, and aliphatic amino acids such as alanine.
　本発明におけるペプチド分子では、そのＣ末端をアミド化することにより殺菌作用が格段に向上する。本発明者等は、Ｃ末端の負電荷がアミド結合によって打ち消されるために膜透過性が向上することが一因ではないかと考えている。 In the peptide molecule of the present invention, the bactericidal action is remarkably improved by amidating the C-terminus. The present inventors consider that the membrane permeability is improved because the negative charge at the C-terminal is canceled out by the amide bond.
　さらに、本発明におけるアミド化に用いられるアミノ基含有化合物としては、疎水性官能基を有しているものが特に好ましい。例えば、Ｃ末端にβナフチルアミン（βＮＡ）をアミド結合させたペプチド分子（ＬＴＬＲＧＬＴＬＲ－βＮＡ）では、アミノ基が結合したペプチド分子（ＬＴＬＲＧＬＴＬＲ－ＮＨ２）に比較しても、その殺菌作用は格段に向上し、緑膿菌の生存率は、１０μＭのＬＴＬＲＧＬＴＬＲ－βＮＡの存在下では９５％であったが、１００μＭでの生存率は０％であった。 Furthermore, as the amino group-containing compound used for amidation in the present invention, those having a hydrophobic functional group are particularly preferred. For example, a peptide molecule (LTLRGLTLLR-βNA) in which β-naphthylamine (βNA) is amide-bonded to the C-terminal has a markedly improved bactericidal action compared to a peptide molecule (LTLRGLTLLR-NH 2 ) having an amino group bonded thereto. The survival rate of Pseudomonas aeruginosa was 95% in the presence of 10 μM LTLRGLTLR-βNA, but the survival rate at 100 μM was 0%.
　このことは、前記ＸＸＬＲの繰り返し単位からなるポリペプチドのＣ末端近傍に疎水性環境を導入することにより、当該ペプチド分子の殺菌作用が更に向上することを示唆している。
　従って、本発明の抗グラム陰性菌剤を構成するペプチド分子のＣ末端をアミド化する代わりに、例えば脂肪族又は芳香族の高級アルコールといった疎水性基を有する化合物をＣ末端カルボキシル基にエステル結合させたとしても高い殺菌作用を発揮すると考えられる。
　なお、本発明の抗グラム陰性菌剤は、本発明の効果を阻害しない限り、ペプチド分子に前記以外の各種化学修飾を施したものであってもよい。 This suggests that the bactericidal action of the peptide molecule is further improved by introducing a hydrophobic environment in the vicinity of the C-terminus of the polypeptide comprising the XXLR repeating unit.
Therefore, instead of amidating the C-terminus of the peptide molecule constituting the anti-gram-negative bacterial agent of the present invention, a compound having a hydrophobic group, such as an aliphatic or aromatic higher alcohol, is esterified to the C-terminal carboxyl group. Even if it is, it is thought that a high bactericidal action is exhibited.
The anti-gram negative bacterial agent of the present invention may be one obtained by subjecting the peptide molecule to various chemical modifications other than those described above, as long as the effects of the present invention are not inhibited.
　本発明において標的としているＹａｅＴとＹｆｇＬ及びＹｆｉＯとの相互作用部位は菌の外膜と内膜の間にあるペリプラズムに存在する（図１参照）。従って、本発明の薬剤（ペプチド分子）が外部から作用部位に到達するためには、外膜を通過する必要がある。 The interaction site between YaeT, YfgL, and YfiO targeted in the present invention is present in the periplasm between the outer and inner membranes of the fungus (see FIG. 1). Therefore, in order for the drug (peptide molecule) of the present invention to reach the action site from the outside, it is necessary to pass through the outer membrane.
　本発明の抗グラム陰性菌剤は、ペプチド分子のＣ末端がアミド化され、Ｃ末端の負電荷が消失しているので、それ自身で外膜を通過できると考えられる。それゆえ、本発明の抗グラム陰性菌剤は、例えばポリミキシンＢ、コリスチン等の細胞膜変質作用を有する抗生剤といった外膜の透過性を向上させる薬剤と同時投与しなくても十分な殺菌作用を発揮し、グラム陰性菌感染症の治療薬として使用できる。 The anti-gram-negative bacterial agent of the present invention is thought to be able to pass through the outer membrane by itself because the C-terminal of the peptide molecule is amidated and the negative charge at the C-terminal disappears. Therefore, the anti-gram-negative bacterial agent of the present invention exhibits a sufficient bactericidal action even if it is not co-administered with a drug that improves the permeability of the outer membrane such as polymyxin B, colistin and other antibiotics having a cell membrane alteration effect. And can be used as a remedy for Gram-negative bacterial infections.
　しかしながら、本発明の抗グラム陰性菌剤と抗生剤（膜変質作用の有無に関わらず）とを同時投与すると、殺菌作用が更に向上することも確認された。
　本発明の抗グラム陰性菌剤を構成するアミド化ペプチド分子が外膜を通過してＹａｅＴ複合体形成部位に到達し、外膜タンパク質の生合成を阻害することにより膜が脆弱化し、同時投与した抗生剤に対して、より感受性となり、大きな殺菌効果を奏するものと考えられる。 However, it was also confirmed that the bactericidal action was further improved when the anti-gram-negative bacterial agent of the present invention and an antibiotic (with or without membrane alteration) were administered simultaneously.
The amidated peptide molecule constituting the anti-gram negative bacterial agent of the present invention passes through the outer membrane to reach the YaeT complex formation site, and the membrane is weakened by inhibiting biosynthesis of the outer membrane protein. It is considered to be more sensitive to antibiotics and have a great bactericidal effect.
　よって、本発明の更なる態様は、上記ペプチド分子と抗生剤（膜変質作用の有無によらない）とを含有することを特徴とする抗グラム陰性菌剤である。 Therefore, a further aspect of the present invention is an anti-gram negative bacterial agent characterized by containing the above peptide molecule and an antibiotic (regardless of the presence or absence of membrane alteration).
　抗生剤としては、特に限定されず、従来から一般に使用されているものが使用できる。例えば、上記した膜変質作用を持つ抗生剤に加えて、膜変質作用を持たないものとして、セファロスポリン系抗生剤であるセファレキシン、セフォタキシム等、フルオロキノロン系抗生剤であるオフロキサシン、シプロフロキサシン等、アミノグリコシド系抗生剤であるアミカミン等、カルバペネム系抗生剤であるメロペネム等が挙げられる。 Antibiotics are not particularly limited, and those conventionally used in general can be used. For example, in addition to the above-mentioned antibiotics having a membrane altering action, cephalosporin antibiotics such as cephalexin, cefotaxime, etc., fluoroquinolone antibiotics ofloxacin, ciprofloxacin, etc. And the like, and amicomine, which is an aminoglycoside antibiotic, and meropenem, which is a carbapenem antibiotic.
　以下に具体例を挙げて本発明を更に詳細に説明するが、本発明はこれらの実施例に限定されるものではない。
　以下に示す実施例等において、グラム陰性菌（緑膿菌又はアシネトバクター）の生菌数の測定方法は次の通りである。
　３７℃で一晩培養した菌の一部をフレッシュなＬＢ培地に添加して、同じ温度で培養を続ける。菌の濁度（６００ｎｍでの吸光度）が０．７～０．８に達したとき、その菌液をＬＢ培地に加えて１００～２００倍希釈した菌液を調製し、実験に供する。
　菌液６０～８０μＬとペプチド等の溶液を加えて最終的に１００μＬにした溶液を３７℃で攪拌しながら一定時間（３時間）培養する。培養後、菌液をＰＢＳ溶液で希釈し、その希釈液をＬＢ寒天プレートに播き、３７℃で一晩培養する。翌日プレートに生えたコロニーの数をカウントすることで生菌数を求める。 Hereinafter, the present invention will be described in more detail with specific examples, but the present invention is not limited to these examples.
In the following examples and the like, the method for measuring the viable count of Gram-negative bacteria (Pseudomonas aeruginosa or Acinetobacter) is as follows.
A part of the bacteria cultured overnight at 37 ° C. is added to fresh LB medium, and the culture is continued at the same temperature. When the turbidity (absorbance at 600 nm) of the bacterium reaches 0.7 to 0.8, the microbial solution is added to the LB medium to prepare a microbial solution diluted 100 to 200 times and used for the experiment.
A solution obtained by adding 60 to 80 μL of the bacterial solution and a peptide solution or the like to a final volume of 100 μL is cultured at 37 ° C. with stirring for a certain time (3 hours). After culturing, the bacterial solution is diluted with a PBS solution, and the diluted solution is plated on an LB agar plate and cultured at 37 ° C. overnight. The viable cell count is obtained by counting the number of colonies growing on the plate the next day.
（実施例１）
　上記の実験系にペプチド分子「ＬＴＬＲＧＬＴＬＲ－ＮＨ２」を種々の濃度で加えた条件で培養した。それぞれの条件下で、緑膿菌の生菌数を測定し、ペプチドを添加しないときの生菌数に比べて、ペプチド添加によって生菌数がどのように変化するかを測定した。結果を図３（ａ）のグラフに示した。 Example 1
The peptide molecule “LTLRGLTLR-NH 2 ” was added to the above experimental system at various concentrations and cultured. Under each condition, the viable cell count of Pseudomonas aeruginosa was measured, and how the viable cell count was changed by the addition of the peptide was measured as compared to the viable cell count when the peptide was not added. The results are shown in the graph of FIG.
　ＨｅＬａ細胞及びＭＧ６３細胞の生存率の測定
　ＬＴＬＲＧＬＴＬＲ－ＮＨ２を１ｍＭあるいは２ｍＭになるように、ＨｅＬａ細胞およびＭＧ６３細胞に添加した。それを２日間３７℃で培養した後に、ＭＴＴアッセイ方法によって生細胞数をカウントした。ペプチドを添加しない場合の生細胞数と比較して、その効果を調べた。その結果を図３（ｂ）に示す。 Measurement of the viability of HeLa cells and MG63 cells LTLRGLTL-NH 2 was added to HeLa cells and MG63 cells so as to be 1 mM or 2 mM. After culturing it at 37 ° C. for 2 days, the number of viable cells was counted by MTT assay method. The effect was examined in comparison with the number of viable cells when no peptide was added. The result is shown in FIG.
（実施例２）
　実施例１において、ペプチド分子に加えて抗生剤（ＯＦＬＸ：０．１μｇ／ｍＬ）を添加した場合の生存率を測定した。結果を図４（ｂ）に示す。図４（ａ）は図３（ａ）と同じデータであり、ここではＯＦＬＸ添加効果を見るための基準として記した。
　抗生剤を共存させることにより、殺菌能力が更に向上したことは明らかである。 (Example 2)
In Example 1, the survival rate was measured when an antibiotic (OFLX: 0.1 μg / mL) was added in addition to the peptide molecule. The results are shown in FIG. FIG. 4A is the same data as FIG. 3A, and is shown here as a reference for viewing the effect of OFLX addition.
It is clear that the bactericidal ability was further improved by coexisting antibiotics.
（実施例３）
　実施例１において、細菌をアシネトバクターに換えた場合の生存率を測定した。結果を図５に示す。実施例１で述べたように、本発明の抗グラム陰性菌剤であるペプチド分子「ＬＴＬＲＧＬＴＬＲ－ＮＨ２」は、緑膿菌に対する優れた殺菌作用を示したが（図５（ａ））、アシネトバクターに対しても十分な殺菌作用を示すことがわかった（図５（ｂ））。 (Example 3)
In Example 1, the survival rate when the bacteria were replaced with Acinetobacter was measured. The results are shown in FIG. As described in Example 1, the peptide molecule “LTLRGLTLLR-NH 2 ” which is an anti-gram-negative bacterial agent of the present invention showed an excellent bactericidal action against Pseudomonas aeruginosa (FIG. 5 (a)), but Acinetobacter It was also found that a sufficient bactericidal action was exhibited (FIG. 5 (b)).
（実施例４）
　実施例１で使用したペプチド分子「ＬＴＬＲＧＬＴＬＲ－ＮＨ２」を別のペプチド分子「ＦＳＬＲＧＦＳＬＲ－ＮＨ２」に換えて、緑膿菌及びアシネトバクターの生存率を測定した。
　アシネトバクターのアミノ酸配列と同じ「ＦＳＬＲ」を有するペプチド分子を用いた場合、アシネトバクターに対する優れた殺菌作用が観察された（図６（ａ））。一方、このペプチド分子は、緑膿菌に対する殺菌作用を発揮することも確認された（図６（ｂ））。 Example 4
The peptide molecule “LTLRGLTLLR-NH 2 ” used in Example 1 was replaced with another peptide molecule “FSLRGFSLR-NH 2 ”, and the survival rates of Pseudomonas aeruginosa and Acinetobacter were measured.
When a peptide molecule having the same “FSLR” as the amino acid sequence of Acinetobacter was used, an excellent bactericidal action against Acinetobacter was observed (FIG. 6 (a)). On the other hand, this peptide molecule was also confirmed to exert a bactericidal action against Pseudomonas aeruginosa (FIG. 6B).
　以上の実験結果から、「ＸＸＬＲ（ＸＸ：ＬＳ、ＦＳ又はＬＳ）」の繰り返し単位を２以上有するペプチド分子からなる本発明の抗グラム陰性菌剤は、単独で優れた殺菌作用を示し、当該作用は抗生剤の同時投与によって更に向上すること、また、本発明の抗グラム陰性菌剤の殺菌作用は種を越えて有効であることが明らかになった。 From the above experimental results, the anti-gram-negative bacterial agent of the present invention comprising a peptide molecule having two or more repeating units of “XXLR (XX: LS, FS or LS)” exhibits an excellent bactericidal action alone. Was further improved by simultaneous administration of antibiotics, and the antibacterial action of the anti-gram-negative bacterial agent of the present invention was found to be effective across species.

Claims (6)

  1. 下記式（Ｉ）：
    ＸＸＬＲ　　　（Ｉ）
    （但し、ＸＸはＬＴ、ＦＳ又はＬＳを示す）で表される繰り返し単位を少なくとも２個含み、そのＣ末端をアミド化したペプチド分子を含んでなる抗グラム陰性菌剤。     The following formula (I):
    XXLR (I)
    (However, XX represents LT, FS, or LS.) An anti-gram-negative bacterial agent comprising a peptide molecule comprising at least two repeating units represented by the formula and amidating the C-terminus thereof.
  2. 前記繰り返し単位の数が２～２０であることを特徴とする、請求項１に記載の抗グラム陰性菌剤。 The anti-gram-negative bacterial agent according to claim 1, wherein the number of repeating units is 2 to 20.
  3. 前記繰り返し単位がリンカーを介して結合していることを特徴とする、請求項１又は２に記載の抗グラム陰性菌剤。 The anti-gram-negative bacterial agent according to claim 1 or 2, wherein the repeating unit is bonded via a linker.
  4. 前記リンカーがＧであることを特徴とする、請求項３に記載の抗グラム陰性菌剤。 The anti-gram-negative bacterial agent according to claim 3, wherein the linker is G.
  5. 前記繰り返し単位が、ＬＴＬＲ又はＦＳＬＲであることを特徴とする、請求項１から４のいずれか一項に記載の抗グラム陰性菌剤。 The anti-gram-negative bacterial agent according to any one of claims 1 to 4, wherein the repeating unit is LTLR or FSLR.
  6. 抗生剤を更に含むことを特徴とする、請求項１から５のいずれか一項に記載の抗グラム陰性菌剤。 The anti-gram negative bacterial agent according to any one of claims 1 to 5, further comprising an antibiotic.
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JP2002519155A (en) * 1998-07-02 2002-07-02 スティヒティング・スケルトル・ティシュー・エンジニアリング・グループ・アムステルダム Bone cement with antimicrobial peptide
JP2007524420A (en) * 2004-02-27 2007-08-30 エイアイ２ リミテッド Treatment of bacterial infection
WO2010147145A1 (en) * 2009-06-16 2010-12-23 学校法人 東海大学 Anti-gram-negative bacteria agent

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002519155A (en) * 1998-07-02 2002-07-02 スティヒティング・スケルトル・ティシュー・エンジニアリング・グループ・アムステルダム Bone cement with antimicrobial peptide
JP2007524420A (en) * 2004-02-27 2007-08-30 エイアイ２ リミテッド Treatment of bacterial infection
WO2010147145A1 (en) * 2009-06-16 2010-12-23 学校法人 東海大学 Anti-gram-negative bacteria agent

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