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WO2008032777A1 - Peptide antibactérien et son utilisation - Google Patents

Peptide antibactérien et son utilisation Download PDF

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Publication number
WO2008032777A1
WO2008032777A1 PCT/JP2007/067824 JP2007067824W WO2008032777A1 WO 2008032777 A1 WO2008032777 A1 WO 2008032777A1 JP 2007067824 W JP2007067824 W JP 2007067824W WO 2008032777 A1 WO2008032777 A1 WO 2008032777A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
acid sequence
peptide
nls
lifr
Prior art date
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Ceased
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PCT/JP2007/067824
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English (en)
Japanese (ja)
Inventor
Tetsuhiko Yoshida
Nahoko Kobayashi
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Toagosei Co Ltd
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Toagosei Co Ltd
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Priority to JP2008534381A priority Critical patent/JP5218843B2/ja
Publication of WO2008032777A1 publication Critical patent/WO2008032777A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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

Definitions

  • the present invention relates to an antimicrobial oligopeptide or polypeptide (hereinafter referred to as "antibacterial peptide") consisting of independent peptide chains in a form that does not exist in nature, and its use, in particular the antibacterial peptide.
  • antibacterial peptide an antimicrobial oligopeptide or polypeptide
  • the present invention relates to an antibacterial agent (composition) as a main component.
  • Antibacterial peptides generally have a broad antibacterial spectrum and are thought to be resistant to the emergence of drug-resistant bacteria. Therefore, antibacterial peptides can be used for the prevention and treatment of bacterial infectious diseases in humans and animals, or for foodstuffs and other items. It is expected to be used for the purpose of imparting antibacterial properties. To date, many antimicrobial peptides have been isolated from various animals and plants. For example, various natural antibacterial peptides are described in JP-A-2000-63400 and JP-A-2001-186887.
  • the present inventors have succeeded in developing an artificial antibacterial peptide by an approach completely different from the approach of isolating what exists as an antibacterial peptide in nature.
  • NLS nuclear localization signal sequence
  • an antibacterial peptide having only NLS as an amino acid sequence involved in antibacterial properties (hereinafter referred to as “NLS antibacterial peptide”). Although it has a broad antibacterial spectrum, its antibacterial activity against gram-negative bacteria is generally lower than that against gram-positive bacteria.
  • the present invention was developed to solve the above-mentioned problems related to existing NLS antibacterial peptides, and an object thereof is to provide an improved NLS antibacterial peptide that is superior in antibacterial activity against gram-negative bacteria. . It is a further object to provide an antibacterial agent (pharmaceutical composition) containing such an improved antibacterial peptide as an active ingredient. Another object of the present invention is to provide a method derived from an improved antibacterial peptide that can be used for various antibacterial applications (eg, DNA and other polynucleotides) and a method.
  • the antibacterial peptide provided by the present invention is a peptide developed by an approach different from the conventionally known antibacterial peptide, and utilizes an amino acid sequence different from a polypeptide that exists in nature as an antibacterial peptide. Is an antibacterial peptide.
  • the present inventor analyzed various amino acid sequences for the above purpose, and focused on a receptor protein identified as a leukemia inhibitory factor rec (to be referred to as LIFR hereinafter). .
  • the partial amino acid sequence (the EMBO Journal), Vol. 20, No. 7, 2001, pp. 1692- contained in the cytokine binding domain (CBD), which is a partial domain. 1703; FEBS Letters, 579, 2005, pp. 4317-4323; and The Journal of Biological Chemistry, 278, 26, 200 3 Pp. 23285-23294) and NLS have been found to be able to exert high antibacterial activity against gram-negative bacteria, and the present invention has been completed.
  • CBD cytokine binding domain
  • the non-naturally-occurring artificially designed antimicrobial peptide disclosed herein is a partial amino acid sequence consisting of at least 5 consecutive amino acid residues in the peptide chain, and at least An NLS-related amino acid sequence consisting of one unit of nuclear translocation sequence (NLS) or an amino acid sequence obtained by partially modifying the NLS;
  • NLS nuclear translocation sequence
  • X and X are independently or commonly S or F, and X is T or S.
  • X is L or I
  • X is Y, H, Q, L or T
  • X is ⁇ or S.
  • An LIFR-related amino acid sequence comprising an amino acid sequence obtained by partially modifying the LIFR-derived amino acid sequence
  • the total number of amino acid residues of the peptide chain is 50 or less.
  • an artificially synthesized antibacterial peptide that does not exist in nature means an artificial chemical synthesis or biosynthesis (ie, a gene whose peptide chain is not independently present in nature). Peptide fragments produced by engineering production).
  • antibacterial peptide refers to an amino acid polymer having a plurality of peptide bonds that exhibit antibacterial activity against at least one kind of bacteria, and is not limited by the number of amino acid residues contained in the peptide chain. Polypeptides composed of oligopeptides having up to about 10 amino acid residues or more amino acid residues are also encompassed by the antimicrobial peptide in the present specification.
  • amino acid residue means a peptide chain unless otherwise specified.
  • amino acids are represented by one-letter code (in the sequence listing, three-letter code) based on the nomenclature related to amino acids indicated in the IUPAC-IUB guidelines.
  • a “partially modified amino acid sequence (modified amino acid sequence IJ)” with respect to a predetermined amino acid sequence means the predetermined amino acid sequence.
  • the result of a so-called conservative amino acid replacement in which one or more (typically 2 or 3) amino acid residues are conservatively substituted is 1 J (for example, ⁇ 3 ⁇ 4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) , ⁇ ⁇ ⁇ ⁇ ( ⁇ ⁇ 2 ⁇ 3) ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ( ⁇ 3 ⁇ 4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • the peptide disclosed herein can express favorable antibacterial properties by having an NLS-related amino acid sequence (International Publication No. WO03 / 91429) in the peptide chain. Furthermore, the peptides disclosed herein have a LIFR-related amino acid sequence specified as described above in the peptide chain. This can improve antibacterial activity (particularly antibacterial activity against gram-negative bacteria such as E. coli) compared to NLS antibacterial peptides.
  • a preferred peptide is substantially composed of the NLS-related amino acid sequence and the LIFR-related amino acid sequence.
  • the LIFR-related amino acid sequence is arranged adjacent to the C-terminal side of the NLS-related amino acid sequence.
  • the peptide having such a structure is composed only of the amino acid sequence that characterizes the present invention (NLS-related amino acid sequence and LIFR-related amino acid sequence IJ), it can be easily produced by chemical synthesis with a short peptide chain. it can.
  • the LIFR-related amino acid sequence includes the following amino acid sequence:
  • the NLS-related amino acid sequence includes the following amino acid sequences:
  • NLS-related amino acid sequences are short NLS (a and c) consisting of 7 amino acid residues or Although it is a modified NLS (b), it can exhibit high antibacterial activity.
  • the antimicrobial peptide of this embodiment has a short NLS-related amino acid sequence and thus has a short peptide chain and can be easily produced by chemical synthesis.
  • the present invention provides a method for producing the antimicrobial peptide disclosed herein. That is, this method is a method for producing a naturally occurring antibacterial peptide having antibacterial activity against at least one kind of bacteria,
  • NLS-related partial amino acid sequence consisting of at least 5 consecutive amino acid residues, consisting of at least one unit of nuclear translocation sequence (NLS) or an amino acid sequence with partial modification of the NLS Determine (select) the amino acid sequence;
  • X is L or I
  • X is Y, H, Q, L or T
  • X is N or S.
  • the force S can be suitably used to suitably produce the antibacterial peptide of the present invention having the characteristics described above.
  • a peptide chain having a total number of amino acid residues of 50 or less is designed as the peptide chain.
  • Peptides consisting of such short and / or peptide chains are easy to chemically synthesize and are relatively low molecular weight peptides, so that they are used as materials for preparing pharmaceutical compositions such as antibacterial agents. I like it.
  • the peptide is substantially composed of an NLS-related amino acid sequence and a LIFR-related amino acid sequence, and the LIFR-related amino acid sequence is arranged adjacent to the C-terminal side of the NLS-related amino acid sequence.
  • the peptide chain with such a structure is Since it consists of only the amino acid sequence that characterizes the invention, the peptide chain can be shortened and can be easily produced by chemical synthesis.
  • the present invention provides an antibacterial agent comprising at least one antibacterial peptide disclosed herein and a pharmaceutically acceptable carrier.
  • the antibacterial peptide comprising the NLS-related amino acid sequence and the LIFR-related amino acid sequence provided by the present invention can exhibit high antibacterial activity against gram-positive bacteria as well as gram-negative bacteria. Therefore, the antibacterial agent disclosed here is a suitable antibacterial agent for antibacterial (bactericidal or bacteriostatic) purposes of gram-negative bacteria.
  • Antibacterial agents based on antibacterial peptides with a total number of amino acid residues of 50 or less are preferred! Such an antibacterial agent containing a short chain length // peptide (that is, a relatively low molecular weight antibacterial peptide) is easy to handle and can be an antibacterial agent suitable for use in vivo and / or in vitro.
  • the antibacterial peptide used is preferably one in which at least one amino acid residue is amidated. Amidation of the carboxyl group of an amino acid residue (typically the C-terminal amino acid residue of the peptide chain) can improve the structural stability (eg protease resistance) of the antimicrobial peptide.
  • the present invention also relates to a non-naturally-occurring artificially designed polynucleotide comprising a nucleotide sequence encoding any of the antimicrobial peptides disclosed herein and / or a nucleotide sequence complementary to the sequence. (E.g., polynucleotides substantially composed of these sequences).
  • polynucleotide refers to a polymer (nucleic acid) in which a plurality of nucleotides are linked by phosphodiester bonds, and is not limited by the number of nucleotides. Various lengths of DNA and RNA fragments are encompassed by the polynucleotides herein.
  • an artificially designed polynucleotide that does not exist in nature refers to chemical synthesis or biosynthesis (ie, production based on genetic engineering) in which the nucleotide chain (full length) alone does not exist in nature. Polynucleotides that are artificially synthesized by!
  • polynucleotides disclosed herein include the amino acid sequence indicated by any of the sequence numbers in the present specification, or a sequence IJ) in which the sequence is partially modified. And / or a polynucleotide comprising or substantially consisting of a nucleotide sequence complementary to the sequence.
  • the antimicrobial peptide disclosed herein is an artificially designed peptide that does not exist in nature, and is a relatively short chain-length polypeptide or oligopeptide having an NLS-related amino acid sequence and an LIFR-related amino acid sequence. is there.
  • the antimicrobial peptides disclosed here exist in close proximity (particularly preferably adjacent) to the NLS and the partial amino acid sequence contained in the LIFR CBD, which have never been suggested to be combined in the past.
  • Peptide fragments are substances that are clearly distinguished from various naturally occurring polypeptides (peptide chains).
  • the peptide disclosed herein may be a primary structure in which an NLS-related amino acid sequence and an LIFR-related amino acid sequence are incorporated, or a primary structure consisting only of the two sequences.
  • the ratio of NLS-related amino acid sequences and LIFR-related amino acid sequences to the total number of amino acid residues constituting the peptide chain is 50% or more, and this ratio is preferably 70% or more. More preferably 90% by number or more.
  • all amino acid residues are preferably L-type amino acids, but as long as the antibacterial activity is not lost, part or all of the amino acid residues are substituted with D-type amino acids. It may be.
  • individual amino acid residues constituting the peptide chain are subjected to various modifications (for example, the force of the C-terminal amino acid, the amidation of the loxyl group, the acylation of the amino terminal of the N-terminal amino acid). It may be For example, an N-terminal amino acid residue of a peptide chain is acylated (acetylated). According to the bacterial peptide, both high antibacterial activity and low hemolysis can be realized.
  • the chain length (number of amino acid residues) of the antibacterial peptide disclosed herein is not particularly limited because it may vary depending on the length of the NLS-related amino acid sequence and LIFR-related amino acid sequence contained, Those having a total number of amino acid residues of 50 or less (for example, 17 to 50) are preferred, and those having 30 or less (for example, 17 to 30) amino acid residues are more preferred.
  • the peptide conformation (three-dimensional structure) is not particularly limited as long as it exhibits antibacterial properties in the environment in which it is used, but it is difficult to become an immunogen (antigen).
  • a straight or helix type is preferred.
  • Such a peptide is difficult to construct an epitope.
  • the antibacterial peptide applied to the pharmaceutical composition is linear and has a relatively low molecular weight (typically 17-30 amino acid residues, for example, the number of amino acid residues: 19-20) are preferred.
  • NLS-related amino acid sequence for constituting the antibacterial peptide of the present invention it is conventionally possible to select any one of native NLS discovered from various organisms and viruses and use the sequence as it is. it can.
  • Specific examples include NLS force S shown in SEQ ID NO: 1 to SEQ ID NO: 81, respectively.
  • Those having a high content of basic amino acid residues are preferred.
  • it is preferable that 40% or more (more preferably 50% or more) of amino acid residues are basic amino acid residues (lysine and / or arginine).
  • NLS comprising one unit with about 5 to 25 amino acid residues is preferred.
  • RRMKWKK SEQ ID NO: 1
  • RVHPY QR SEQ ID NO: 2
  • PKKKRKV SEQ ID NO: 4
  • GKKRSKA SEQ ID NO: 5
  • RGRRR RQR SEQ ID NO: 7
  • RKKRRQRRR SEQ ID NO: 20
  • PRRRK Peptides containing 1 unit or 2 units or more of an NLS-related amino acid sequence composed of 5 amino acid residues or more as in 26) are preferred.
  • one unit is 4 amino acid residues or less, such as RKRR (SEQ ID NO: 27)
  • an amino acid sequence having a total of 5 amino acid residues or more in combination with the same or different NLS may be designed. That is, an NLS-related amino acid sequence containing 2 units or more (typically 2 units, 3 units, or 4 units) of NLS in which one unit is 4 amino acid residues or less may be designed.
  • RKRR SEQ ID NO: 27
  • the sequence is linked to a two-unit tandem! A sequence consisting of 8 amino acid residues (RKRRRKRR) is the NLS-related amino acid sequence.
  • force S is selected as the NLS.
  • an NLS for constructing an antibacterial peptide from an available information source such as a database, it is preferable to select an NLS rich in basic amino acid residues.
  • NLS in which 40% or more, preferably 50% or more, particularly preferably 70% or more of the total number of amino acid residues is an arginine residue and / or lysine residue may be a suitable candidate.
  • an NLS-related amino acid sequence so as to include 2 units or 3 units or more of NLS, it is preferably designed so that these NLSs are arranged adjacent to each other in the peptide chain.
  • a preferred example of such a modified sequence is a sequence in which one or several amino acid residues are conservatively substituted. Further, there may be mentioned a sequence in which one or several (typically about 2 or 3) abasic amino acid residues are substituted with basic amino acid residues.
  • a sequence in which one or several (typically about 2 or 3) abasic amino acid residues are substituted with basic amino acid residues For example, the sequence RKKKR KV (SEQ ID NO: 82) in which proline, which is the N-terminal amino acid residue of PKKKRKV (SEQ ID NO: 4), which is a typical NLS consisting of 7 amino acid residues, is substituted with a basic amino acid residue (eg, arginine). ) Or an arrangement 1!
  • the peptide disclosed herein includes a partial amino acid sequence included in the LIFR site force-in-binding 'domain (CBD) possessed by various biological species. As a related amino acid sequence.
  • the peptide disclosed here has the following sequence as a LIFR-related amino acid sequence: DFX TX X X X LKWX
  • X in the sequence is T or S, X is L or I, X is Y, H, Q, L or T,
  • X is N or S.
  • SEQ ID NO: 84- Each sequence shown by 88 is mentioned.
  • the amino acid sequence shown in SEQ ID NO: 84 is a partial amino acid sequence contained in CBD1 (cytokine binding domain 1), which is one of the cytoforce-in binding domains of human-derived LIFR.
  • CBD1 is known as a domain involved in cytoforce-in binding such as CNTF (Ciliary neurotrophic factor), and this partial amino acid sequence is thought to be involved in CNTF binding and signaling (FEBS Letters (F EBS Letters), 579, 2005, pp. 4317-4323).
  • CNTF Central neurotrophic factor
  • the position of the NLS-related amino acid sequence and the LIFR-related amino acid sequence in the peptide chain is not particularly limited as long as preferable antibacterial activity can be exhibited.
  • the peptide chain should be designed so that the NLS-related amino acid sequence is arranged near the N-terminus and the LIFR-related amino acid sequence is arranged on the C-terminal side. It is particularly preferable that the NLS-related amino acid sequence and the LIFR-related amino acid sequence are continuously arranged in tandem as viewed from the N-terminal side of the peptide chain.
  • the antibacterial peptides shown in the examples described later do not mean that the suitable peptides that are preferred specific examples of the antibacterial peptides disclosed herein are limited to those having these amino acid sequences.
  • antimicrobial peptides disclosed herein those having a relatively short peptide chain can be easily produced according to a general chemical synthesis method. For example, it is possible to adopt a deviation or deviation from a conventionally known solid phase synthesis method or liquid phase synthesis method! /.
  • a solid phase synthesis method using Boc (t-butyloxycarbonyl) or! /, Or Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group for the amino group is preferred.
  • the desired amino acid sequence and modified (C-terminal amidation, etc.) moiety can be obtained by a solid phase synthesis method using a commercially available peptide synthesizer (for example, available from PerSeptive Biosystems, Applied Biosystems, etc.). It is possible to synthesize peptide chains.
  • a commercially available peptide synthesizer for example, available from PerSeptive Biosystems, Applied Biosystems, etc.
  • This recombinant vector is introduced into a predetermined host cell (for example, yeast, insect cell, plant cell, animal (mammalian) cell) by a general technique, and the host cell or a tissue containing the cell under a predetermined condition. And cultivate individuals.
  • a predetermined host cell for example, yeast, insect cell, plant cell, animal (mammalian) cell
  • the target polypeptide can be expressed and produced in the cell.
  • the target antimicrobial peptide can be obtained by isolating and purifying the polypeptide from the host cell (in the medium if secreted).
  • This recombinant vector is introduced into a predetermined host cell (for example, a yeast, an insect cell, a plant cell, or a mammalian cell) by a general technique, and the host cell or a tissue or individual containing the cell under a predetermined condition. Is cultured. As a result, the target polypeptide can be expressed and produced in the cell.
  • the target antimicrobial peptide can be obtained by isolating and purifying the polypeptide from the host cell (in the medium if secreted).
  • the method for constructing the recombinant vector, the method for introducing the constructed recombinant vector into the host cell, etc. can be applied as it is if the method conventionally used in the field is used as it is. It is not a characterization! /, So a detailed explanation is omitted.
  • a fusion protein expression system can be used to efficiently produce large quantities in a host cell. That is, a gene (DNA) encoding the amino acid sequence of the target antibacterial peptide is chemically synthesized, and the synthesized gene is expressed in an appropriate fusion protein expression vector (for example, Novagen Corp. is also provided! /, PET series). And GST (Glutathione S-transferase) fusion protein expression vectors (such as the pGEX series) provided by Amersham Biosciences. A host cell (typically E. coli) is transformed with the vector. Culturing the obtained transformant Prepare the fusion protein of interest. The protein is then extracted and purified.
  • fusion protein expression vector for example, a gene (DNA) encoding the amino acid sequence of the target antibacterial peptide is chemically synthesized, and the synthesized gene is expressed in an appropriate fusion protein expression vector (for example, Novagen Corp. is also provided! /, PET series).
  • GST Glutathione S-transfer
  • the obtained purified fusion protein is cleaved with a predetermined enzyme (protease), and the released target peptide fragment (designed antibacterial peptide) is recovered by a method such as affinity chromatography.
  • a predetermined enzyme protease
  • affinity chromatography a method such as affinity chromatography.
  • a cage DNA for cell-free protein synthesis system ie, synthetic gene fragment containing nucleotide sequence encoding amino acid sequence of antibacterial peptide
  • various compounds ATP, RNA polymerase, Amino acids
  • Cell-free protein synthesis systems are described in, for example, Shimizu et al. (Shimizu et al., Nature Biotechnology, 19, 751-755 (2001), Madin et al., Proc. Natl. Acad. Sci. USA, 97 (2), 559-564 (2000)).
  • the target antimicrobial peptide can be easily produced by the cell-free protein synthesis system according to the amino acid sequence.
  • the antibacterial peptide of the present invention based on the Pure System (registered trademark) of Post Genome Research Institute in Japan.
  • a single-stranded or double-stranded polynucleotide containing a nucleotide sequence encoding the antimicrobial peptide of the present invention and / or a nucleotide sequence complementary to the sequence can be easily produced (synthesized) by a conventionally known method. it can. That is, by selecting a codon corresponding to each amino acid residue constituting the designed amino acid sequence IJ, a nucleotide sequence corresponding to the amino acid sequence of the antimicrobial peptide can be easily determined and provided. Once the nucleotide sequence is determined, a desired nucleoside is obtained using a DNA synthesizer or the like.
  • a polynucleotide (single strand) corresponding to the tide sequence can be easily obtained. Further, the obtained double-stranded DNA can be obtained by employing various enzymatic synthesis means (typically PCR) using the obtained single-stranded DNA as a cage.
  • various enzymatic synthesis means typically PCR
  • the polynucleotide provided by the present invention may be in the form of DNA or RNA (such as mRNA).
  • DNA can be provided in double-stranded or single-stranded form. When provided as a single strand, it may be a coding strand (sense strand) or a non-coding strand (antisense strand) of a complementary sequence.
  • the polynucleotide provided by the present invention is used as a material for constructing a recombinant gene (expression cassette) for production of antimicrobial peptides in various host cells or in a cell-free protein synthesis system. Can be used.
  • nucleotide sequence that encodes a peptide having an amino acid sequence that has been partially modified (or a peptide comprising the amino acid sequence) and / or a nucleotide sequence that is complementary to the sequence (or consists essentially of the sequence) A) non-naturally-occurring artificially designed polynucleotides are provided.
  • the antibacterial peptide of the present invention exhibits effective antibacterial activity against gram-negative bacteria as well as gram-positive bacteria, and preferably has a relatively broad antibacterial spectrum. For this reason, it can be suitably used as the main component of the antibacterial agent. For example, it can be used for the purpose of treating bacterial infections, disinfecting wound surfaces, preventing eye diseases, cleaning the mouth (gargle), preserving foods, maintaining freshness, deodorizing, sterilizing furniture or sanitary equipment surfaces, or bacteriostatically.
  • the carrier or secondary component (typically pharmaceutically acceptable depending on the application) included in the antibacterial agent may vary depending on the use and form of the antibacterial agent.
  • Water typically distilled water, saline and other buffers
  • various organic solvents such as ethanol, glycol dimethacrylate, glycol dimethacrylate, glycol dimethacrylate, glycol dimethacrylate, glycol dimethacrylate, glycol dimethacrylate, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol, glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol glycol dimethoxylate, glycerin, ol dimethoxymethyl methacrylate,
  • the antibacterial agent since it is used for injection and the like, it can be dissolved in physiological saline or an appropriate buffer solution (for example, PBS) immediately before use to prepare a lyophilized product or a granulated product for preparing a drug solution.
  • the carrier contained in the antibacterial agent may vary depending on the form of the antibacterial agent.
  • the preparation method itself does not characterize the present invention as long as it is in accordance with a conventionally known method, and therefore detailed description is omitted.
  • Comprehensive Medicinal Chemistry supervised by Corwin Hansch, published by Pergamon Press (1990) can be cited as a detailed information source regarding the treatment.
  • the antibacterial agent provided by the present invention can be used in a method and dose depending on its form and purpose.
  • the antimicrobial peptides containing the NLS-related amino acid sequences and LIFR-related amino acid sequences disclosed herein have high antimicrobial activity even in systems where organic substances such as relatively high concentrations of cations, salts (eg sodium chloride) or serum are present. Can be maintained. Therefore, the antibacterial agent disclosed herein is particularly suitable for use in a system (place) where cations, salts, serum, and the like are present.
  • the antibacterial agent provided by the present invention can be administered to a patient as a liquid agent by intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal injection, or ileum. Alternatively, solid forms such as tablets can be administered orally.
  • sanitary ware surface disinfection (sterilization) or food preservatives spray a liquid containing a relatively large amount (for example, 1 to 100 mg / ml) of an antimicrobial peptide directly on the surface of the object.
  • a liquid containing a relatively large amount for example, 1 to 100 mg / ml
  • the surface of the object may be wiped with force or with a cloth or paper wetted with the solution.
  • the antibacterial peptides disclosed herein are the bacteria causing the infection (eg Gram-positive bacteria such as Staphylococcus aureus, pathogenic E. coli etc. High antibacterial activity against gram-negative bacteria). Therefore, the antibacterial peptide of the present invention is useful as a main component of the antibacterial agent.
  • the polynucleotide encoding the antimicrobial peptide of the present invention can be used as a material used for so-called gene therapy.
  • a gene encoding an antimicrobial peptide typically a DNA segment or RNA segment
  • the antimicrobial peptide according to the present invention is always present in a living body (cell).
  • Peptides can be expressed. Therefore, the polynucleotide (DNA segment, RNA segment, etc.) encoding the antibacterial peptide of the present invention is useful as a drug for preventing or treating bacterial infection in the above-mentioned patients.
  • the antimicrobial peptides disclosed herein can selectively exhibit antibacterial activity against bacteria that have extremely low toxicity to mammalian cells and tissues. Therefore, it is extremely useful as a drug for preventing bacterial infection of cultured organs. For example, by adding an antibacterial peptide of the present invention alone or an antibacterial agent comprising the peptide as one of the main components to the culture solution at an appropriate concentration, bacterial infection of organs or the like being cultured can be prevented.
  • the polynucleotide encoding the antimicrobial peptide of the present invention can be used as a material for gene therapy for cultured cells and cultured tissues.
  • a gene typically a DNA segment or RNA segment
  • the antimicrobial peptide according to the present invention can be expressed in (cell). Therefore, the polynucleotide (DNA segment, RNA segment, etc.) encoding the antimicrobial peptide of the present invention provided by the present invention is useful as a drug for preventing bacterial infection of cultured tissues.
  • each of the peptides of samples ;! to 7 has an NLS-related amino acid sequence arranged on the N-terminal side and an LIFR-related amino acid sequence arranged on the C-terminal side adjacent thereto. That is, the N-terminal arrangement IJ “: KKKRKV” in sample 1, the N-terminal arrangement “RIRKKLR” in samples 2 to 6, and the N-terminal arrangement “PKKKRKV” in sample 7 are! /, Both of which are NLS-related amino acids. All sequences adjacent to the C-terminal side of the sequence are LIFR-related amino acid sequences.
  • DFSTSTLYLKWN in samples 1, 2 and 7 is a partial amino acid sequence consisting of 12 amino acid residues from position 147 (Asp) to position 158 (Asn) contained in CB D1 of human-derived LIFR. is there.
  • DFSTSTIHLKWN in Sample 3 is the amino acid sequence of the corresponding site in the LIFR derived from Inu.
  • DFSTSTLQLKWN in Sample 4 is the amino acid sequence of the corresponding site of rat-derived LIFR.
  • DFFTSSLLLK WN in sample 5 is the amino acid sequence of the corresponding site of mouse-derived LIFR.
  • samples 8 to 11 are samples for comparison. That is, as shown, sample 8 is a peptide consisting only of LIFR-related amino acid sequences, and samples 9-1 1 is a peptide consisting only of an NLS-related amino acid sequence.
  • Each peptide described above (20 amino acid residues or less) was synthesized by a solid phase synthesis method (F modi) using a commercially available peptide synthesizer (PEP TIDE SYNTHESIZER 9050, manufactured by PerSeptive Biosystems).
  • PEP TIDE SYNTHESIZER 9050 commercially available peptide synthesizer
  • HATU Applied Biosystems product
  • the resin and amino acid used in the solid phase synthesis method were purchased from NOVA biochem.
  • the peptide chain is extended from the Fmoc-amino acid that binds to the resin by repeating the deprotection group reaction and the condensation reaction according to the synthesis program of the above peptide synthesizer, and the synthetic peptide having the desired chain length is obtained.
  • the synthesized peptide chain is transferred together with the resin to a centrifuge tube, and ethanediol 1.8 mL, m-taresol 0.6 mL, thioanisole 3.6 mL and trifluoroacetic acid 24 mL are added, and the mixture is added at room temperature. Stir for hours. Thereafter, the resin bound to the peptide chain was removed by filtration.
  • the column was run at a flow rate of 1.5 mL / min. Separation and purification was performed for 30 to 40 minutes.
  • the peptide eluted from the reverse phase column was detected at a wavelength of 220 nm using an ultraviolet detector (490E Detector: product of Waters), and was shown as a peak on the recording chart.
  • the molecular weight of each eluted peptide was measured using the Voyager DE RP (trademark) manufactured by PerS tive Biosystems, using MALD-TOF / MS (Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry). Analysis). As a result, it was confirmed that the target peptide was synthesized and purified.
  • Antibacterial activity (minimum inhibitory concentration) against Gram-negative bacteria (E. coli IFO 3972) and Gram-positive bacteria (S. aureus FDA209P) for the synthetic peptides obtained above (samples;! To 11) : MIC) was determined by a liquid medium dilution method using a 96-well microplate.
  • each sample peptide at a concentration 40 times the maximum test concentration in sterile distilled water, and then adjust the peptide concentration to one in the range of 200 to 0 ⁇ 78 ⁇ .
  • Each medium (DIFCO product “Mueller Hinton Broth”) was prepared.
  • test cells on an agar plate (DIFCO product “Müller Hinton Agar”) cultured at 37 ° C. for 18 hours were sprinkled in a loop and suspended in sterile physiological saline.
  • 2 10 6113/111 and a correspondingly adjusted bacterial solution 5 were inoculated into each well of the microplate and inoculated into each MHB medium containing a predetermined concentration of peptide (number of test bacteria: approx. X 10 6 cell S / mL).
  • cultivation was started in a 37 ° C incubator, and the presence or absence of bacteria was examined by turbidity after 24 hours.
  • the antibacterial peptide according to the present invention can maintain high antibacterial activity even with MHB containing a relatively high concentration of cations. Therefore, the antibacterial peptide of the present invention is suitable for use in a system (for example, in blood) in which various cations (or salts) such as serum are present in a relatively large amount.
  • NLS related The force S adopts two kinds of sequences shown in Table 1 as amino acid sequences, other known NLS (see Sequence Listing), or modified sequences thereof.
  • the peptide of the present invention since the peptide of the present invention has a high antibacterial activity, it can be used as an active ingredient of bactericides and antibacterial agents used in various applications including pharmaceuticals and agricultural chemicals.

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Abstract

La présente invention concerne un peptide antibactérien non naturel et conçu de manière synthétique. Le peptide antibactérien comporte une séquence de signal de localisation nucléaire (NLS) ou une séquence variante de celle-ci et une séquence partielle d'acides aminés dérivée d'un récepteur du facteur inhibiteur de la leucémie (LIFR) ou une séquence variante de celle-ci.
PCT/JP2007/067824 2006-09-14 2007-09-13 Peptide antibactérien et son utilisation Ceased WO2008032777A1 (fr)

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Cited By (1)

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JPWO2013108871A1 (ja) * 2012-01-19 2015-05-11 昇一 城武 植物病原細菌用抗菌剤

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WO2003091429A1 (fr) * 2002-04-25 2003-11-06 Toagosei Co.,Ltd. Polypeptide microbicide et ses utilisations
JP2005120050A (ja) * 2003-10-20 2005-05-12 Toagosei Co Ltd 新規抗菌性ペプチドとその利用
WO2005049819A1 (fr) * 2003-10-29 2005-06-02 Toagosei Co., Ltd. Peptide antibacterien et utilisation associee
JP2006160640A (ja) * 2004-12-06 2006-06-22 Toagosei Co Ltd 抗菌ペプチド及びその利用
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WO2003091429A1 (fr) * 2002-04-25 2003-11-06 Toagosei Co.,Ltd. Polypeptide microbicide et ses utilisations
JP2005120050A (ja) * 2003-10-20 2005-05-12 Toagosei Co Ltd 新規抗菌性ペプチドとその利用
WO2005049819A1 (fr) * 2003-10-29 2005-06-02 Toagosei Co., Ltd. Peptide antibacterien et utilisation associee
JP2006160640A (ja) * 2004-12-06 2006-06-22 Toagosei Co Ltd 抗菌ペプチド及びその利用
WO2006088010A1 (fr) * 2005-02-15 2006-08-24 Toagosei Co., Ltd. Peptide antimicrobien et emploi dudit peptide

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GEARING P.D. ET AL.: "Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130", THE EMBO J., vol. 10, no. 10, 1991, pages 2839 - 2848, XP003019980 *
HE W. ET AL.: "The N-terminal cytokine binding domain of LIFR is required fo CNTGF binding and signaling", FEBS LETTERS, vol. 579, 2005, pages 4317 - 4323, XP005010803 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2013108871A1 (ja) * 2012-01-19 2015-05-11 昇一 城武 植物病原細菌用抗菌剤

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