WO2008032783A1 - Peptide antibactérien artificiel et utilisation de celui-ci - Google Patents

Peptide antibactérien artificiel et utilisation de celui-ci Download PDF

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Publication number
WO2008032783A1
WO2008032783A1 PCT/JP2007/067837 JP2007067837W WO2008032783A1 WO 2008032783 A1 WO2008032783 A1 WO 2008032783A1 JP 2007067837 W JP2007067837 W JP 2007067837W WO 2008032783 A1 WO2008032783 A1 WO 2008032783A1
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Prior art keywords
amino acid
acid sequence
peptide
nls
antibacterial
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PCT/JP2007/067837
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English (en)
Japanese (ja)
Inventor
Nahoko Kobayashi
Tetsuhiko Yoshida
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Toagosei Co., Ltd.
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Priority to JP2008534385A priority Critical patent/JP5218844B2/ja
Publication of WO2008032783A1 publication Critical patent/WO2008032783A1/fr

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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal

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 comprising only NLS as an amino acid sequence involved in antibacterial properties (hereinafter referred to as “NLS antibacterial peptide”) " ) Shows a broad antibacterial spectrum, but generally has a lower antibacterial activity against Gram-negative bacteria than that against Gram-positive bacteria.
  • NLS antibacterial peptides with higher antibacterial activity are required so that superior effects can be exerted at lower concentrations than in the past.
  • the present invention was developed to solve the above-mentioned problems related to existing NLS antibacterial peptides, and has improved antibacterial activity and / or improved antibacterial activity against gram-negative bacteria.
  • the purpose is to provide antimicrobial peptides. It is a further object to provide an antibacterial agent (pharmaceutical composition) containing such an improved antibacterial peptide as an active ingredient.
  • Another object is to provide a method (eg, DNA or other polynucleotide) derived from an improved antibacterial peptide that can be used for various antibacterial applications 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.
  • CNTF ciliary neurotrophic factor
  • CNTF is a CNTF receptor complex (CNTFRs) consisting of multiple subunits (ie, leukemia inhibitory factor receptor- ⁇ called LIFR, a protein called gp 130, and a CNTF a receptor protein).
  • the D 1 motif that binds is a structural part that is considered to be involved in the recognition or binding of such CNTFRs and the biological activation of CNTFRs! /. This is for example the literature by Inoue et al. And the following literature:
  • the inventor of the present invention has been able to combine gram-positive bacteria with gram-positive bacteria by combining NLS with the amino acid sequence that constitutes the above-mentioned D1 motif. It was also found that an artificial antibacterial peptide capable of exhibiting high antibacterial activity can be synthesized.
  • the present inventor has found that not only the DTF motif of CNTF but also the above-mentioned Bazan, Inoue et al. And Di Marco et al. D-motif of other proteins belonging to the superfamily (cytokines such as CNTF and IL-6, which are typically 4-helix-bundle proteins with four ⁇ -helical parts) Similarly, the amino acid sequence (typically consisting of 8 or 9 consecutive amino acid residues) in combination with NLS has a high antibacterial activity against gram-positive and gram-negative bacteria. It was found that an artificial antibacterial peptide capable of exhibiting the above can be synthesized, and the present invention has been completed.
  • the non-naturally-occurring artificially designed antimicrobial peptide disclosed herein has (1) a partial amino acid sequence consisting of at least 5 consecutive amino acid residues in the peptide chain.
  • An NLS-related amino acid sequence consisting of at least one unit of a nuclear translocation sequence (NLS) or an amino acid sequence obtained by partially modifying the NLS, and (2) in the peptide chain A partial amino acid sequence close to the NLS-related amino acid sequence in the above, and an amino acid sequence (D1) constituting a D1 motif included in any protein belonging to the ⁇ -helical 'site force-in' superfamily or a part of D1
  • a D1-related amino acid sequence consisting of an amino acid sequence subjected to various modifications.
  • the total number of amino acid residues 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). An artificial peptide fragment produced by engineering production).
  • antibacterial peptide refers to an amino acid polymer having a plurality of peptide bonds exhibiting 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 (ie, amino acid sequence derived from NLS or D1 motif) means the predetermined amino acid sequence.
  • one or more (typically 2 or 3) amino acid residues conservatively replaced by a conservative amino acid replacement For example, a sequence in which a basic amino acid residue is substituted with a basic amino acid residue), or one or more (generally about 2 to 3) amino acid residues are added to a given amino acid sequence ( ⁇ In this specification, the sequences etc. included in the description are included in the “partially modified sequence (modified amino acid arrangement IJ)”. It is a typical example included.
  • the peptide disclosed here 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 D1-related amino acid sequence identified in the peptide chain as described above. This can improve antibacterial activity (especially antibacterial activity against gram-negative bacteria such as E. coli) compared to NLS antibacterial peptides.
  • a preferred embodiment of the antimicrobial peptide is substantially composed of the NLS-related amino acid sequence and the D1-related amino acid sequence, and the D1-related amino acid sequence is adjacent to the C-terminal side or the N-terminal side of the NLS-related amino acid sequence. It is characterized by being arranged.
  • the strength and the composition it is possible to improve the antibacterial activity (especially antibacterial activity against gram-negative bacteria) with the power S.
  • the D1-related amino acid sequence includes the following amino acid sequences:
  • X is any force of a hydrophobic amino acid residue (eg, any of L, V, A, I, P),
  • X is F or W
  • X is E or Q or S
  • X is K or R or Q or A
  • X is K or R or Q or A
  • X is L or K or V
  • X is W or M or R or L or E
  • X is G
  • the D1-related amino acid sequence may be any one of CNTF, IL2, 1-3, and 1-4 that are derived from humans or animals. 01 It is characterized by comprising an amino acid sequence (D1) constituting a motif or an amino acid sequence obtained by partial modification of D1.
  • D1 amino acid sequence
  • a peptide comprising an amino acid sequence as described above may have antibacterial activity equal to or higher than that of Gram-positive bacteria against Gram-negative bacteria.
  • the NLS-related amino acid sequence includes the following amino acid sequences:
  • NLS-related amino acid sequences are short NLS consisting of 7 amino acid residues (a and c) or modified NLS (b), but 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, as another aspect, 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,
  • amino acid sequence consisting of at least 5 consecutive amino acid residues, and comprising an NLS-related amino acid sequence consisting of at least one unit of nuclear translocation sequence (NLS) or an amino acid sequence obtained by partially modifying the NLS To make a decision (to make a choice),
  • NLS nuclear translocation sequence
  • the D1-related amino acid sequence consisting of the amino acid sequence (D1) that constitutes the D1 motif provided by any protein species belonging to the ⁇ -helicanoin-site force-in 'superfamily or the amino acid sequence obtained by partially modifying the D1 motif Determining (selecting), designing a peptide chain having the determined (selected) NLS-related amino acid sequence and the determined (selected) D1-related amino acid sequence in close proximity to each other,
  • the artificial antibacterial peptide of the present invention having the characteristics described above.
  • a D1-related amino acid sequence one of the D1 motifs included in human or animal-derived CNTF, IL-2, IL-3, or IL-4 It is possible to use an amino acid sequence (Dl) or an amino acid sequence in which the Dl has been partially modified with a force S.
  • 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 NLS-related amino acid sequence and the D1-related amino acid sequence are substantially composed, and the D1-related amino acid sequence is arranged adjacent to the C-terminal side or the N-terminal side of the NLS-related amino acid sequence.
  • the peptide chain is designed as follows. Since the peptide chain having such a structure is composed only of the amino acid sequences that characterize the present invention (NLS-related amino acid sequence and D1-related amino acid sequence), the peptide chain can be shortened and can be easily produced by chemical synthesis. it can.
  • 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 D1-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 various 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!
  • amino acid sequence indicated by any of the sequence numbers in the present specification or the sequence IJ) in which the sequence is partially modified are encoded. 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 includes an NLS-related amino acid sequence and a D1-related amino acid sequence (typically at least 8 consecutive amino acid residues). And a relatively short chain polypeptide or oligopeptide.
  • the antibacterial peptides disclosed here are completely suggested to be combined in the past. /, NA! /, NLS amino acid sequence and CNTF or other ⁇ -helical 'site force-in' superfamily (typically a 4-helix-bundle protein with four ⁇ -helix parts, site force-in '
  • ⁇ -helical 'site force-in' superfamily typically a 4-helix-bundle protein with four ⁇ -helix parts, site force-in '
  • the peptide disclosed here may be a primary structure in which an NLS-related amino acid sequence and a D1-related amino acid sequence are incorporated, or a primary structure consisting of only the two sequences.
  • the ratio of the NLS-related amino acid sequence and the D1-related amino acid sequence to the total number of amino acid residues constituting the peptide chain is 50% by number or more, and this ratio is preferably 70% by number or more. More than 90% by number is more preferable.
  • 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. As long as the antibacterial property is not lost, the individual amino acid residues constituting the peptide chain may be modified in various ways (for example, the force of the C-terminal amino acid, the amidation of the loxyl group, the acylation of the amino group of the N-terminal amino acid (for example, ))).
  • the chain length (number of amino acid residues) of the antimicrobial peptide disclosed herein is not particularly limited because it may vary depending on the length of the NLS-related amino acid sequence and D1-related amino acid sequence contained therein. Those having a radical number of 50 or less (for example, 15 to 50) are preferred, and those having 30 or less (for example, 15 to 20) 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 15-30 amino acid residues, for example, the number of amino acid residues: Those of 15 to 20) are preferred.
  • NLS-related amino acid sequence for constituting the antibacterial peptide of the present invention any one of the native NLSs that have been conventionally discovered from various organisms and viruses is selected, and the sequence thereof is selected. It can be used as it is. 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. For example, 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.
  • 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 can be linked to a 2-unit tandem! / A sequence consisting of 8 amino acid residues (RKRRRKRR) can be used as an NLS-related amino acid sequence.
  • 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. Also, one or several (typically around 2 or 3) non-bases And a sequence in which a basic amino acid residue is substituted with a basic amino acid residue.
  • 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).
  • PKKKRKV (SEQ ID NO: 4), the 6th amino acid residue “lysine” from the N-terminal side is substituted with “arginine” in the same manner as the 1! 3 ⁇ 43 ⁇ 43 ⁇ 4 ⁇ ⁇ (SEQ ID NO: 83) as a preferred example. Can be mentioned.
  • the peptide disclosed herein has an amino acid sequence constituting a D1 motif of a protein belonging to the ⁇ helical 'cytokine' superfamily or a modified amino acid sequence thereof as a D1-related amino acid sequence.
  • D1-related amino acid arrangement IJ that is, the amino acid sequence constituting the D1 motif
  • human-derived CNTF described as SEQ ID NO: 84 Human CNTF
  • D1 motif-constituting amino acid sequences provided in various proteins (site force ins) belonging to the ⁇ helical 'site force in' superfamily can be employed.
  • SEQ ID NOs: 85 to 111 describe D1 motif-constituting amino acid sequences derived from various suitable proteins that can be selected and used as D1-related amino acid sequences. That is, ⁇ ⁇ ⁇ IJ No. 85 is a rat-derived CNTF (Rat ciliary neurotrophic factor),
  • IJ No. 86 is a chicken-derived GPA (Chicken growth promoting activity)
  • SEQ ID NO: 87 is a human-derived LIF (Human leukemia inhibitory factor)
  • SEQ ID NO: 88 is a mouse-derived LIF (Mouse leukemia inhibitory factor)
  • SEQ ID NO: 89 is human-derived OM (Human oncostatin M),
  • SEQ ID NO: 90 is mouse-derived CT l (Mouse cardiotrophin_l),
  • SEQ ID NO: 91 is human-derived IL 6 (Human interleukin_6),
  • SEQ ID NO: 92 is human-derived ILll (Human interleukin_ll),
  • SEQ ID NO: 93 is monkey-derived OM (Simian oncostatin M)
  • SEQ ID NO: 94 is mouse-derived IL 6 (Mouse interleukin_6),
  • SEQ ID NO: 95 is human-derived IL 2 (Human interleukin_2)
  • SEQ ID NO: 96 is human-derived IL 3 (Human interleukin_3),
  • SEQ ID NO: 97 is human-derived IL 4 (Human interleukin_4)
  • SEQ ID NO: 98 is human-derived IL 5 (Human interleukin_5)
  • ⁇ ⁇ 1 J ⁇ 99 is a human-derived GM—CSF (Human granulocyte / macrophage colony stimulating factor),
  • ⁇ ⁇ ⁇ 1 J number 100 is human-derived G-CSF (Human granulocyte colony stimulating factor)
  • ⁇ ⁇ ⁇ 1 J-number 101 is mouse-derived G-CSF (Mouse granulocyte colony stimulating factor)
  • SEQ ID NO: 102 is chicken-derived MGF (Chicken myelomonocytic growth factor)
  • SEQ ID NO: 103 is human-derived GH (Human growth hormone)
  • SEQ ID NO: 104 is human-derived PRL (Human prolactin),
  • SEQ ID NO: 105 is human-derived EPO (Human erythropoietin),
  • SEQ ID NO: 106 is human-derived IFN a (Human a interferon),
  • IJ No. 107 is mouse-derived CDF (Mouse cholinergic differentiation factor)
  • SEQ ID NO: 108 is human-derived CDF (Human cholinergic differentiation factor)
  • SEQ ID NO: 109 is mouse-derived IL 3 (Mouse interleukin_3)
  • SEQ ID NO: 1 10 is human-derived IFN / 3 (Human ⁇ interferon), and
  • SEQ ID NO: 1 1 1 is a mouse-derived IFN ⁇ (Mouse ⁇ interferon),
  • it may be an amino acid sequence obtained by partially modifying the D 1 motif amino acid sequence listed above.
  • a modified sequence one or several (typically 2 to 9) amino acid sequences consisting of 8 or 9 consecutive amino acid residues described in any of the above SEQ ID NOs: Examples include sequences in which about 3) amino acid residues have been deleted, appended, or similarly substituted.
  • D1-related amino acid sequences including partially modified amino acid sequences
  • amino acid sequences including partially modified amino acid sequences
  • X is any force of a hydrophobic amino acid residue (eg, any of L, V, A, I, P),
  • X is F or W; X is E or Q or S;
  • X is K or R or Q or A
  • X is K or R or Q or A
  • X is L or K or V
  • X is W or M or R or L or E
  • X is G
  • X force is preferable to force S, or X force 3 ⁇ 4
  • X and X are either R or K, respectively
  • the position of the NLS-related amino acid sequence and the D1-related amino acid sequence in the peptide chain is not particularly limited as long as preferable antibacterial activity can be exhibited.
  • the peptide chain is designed so that the NLS-related amino acid sequence is arranged near the N-terminal and the D1-related amino acid sequence is arranged on the C-terminal side, but the reverse arrangement is also possible.
  • the NLS-related amino acid sequence and the D1-related amino acid sequence are continuously (ie, adjacent) and arranged in tandem on the peptide chain.
  • the antimicrobial peptide shown in the Example mentioned later is a suitable specific example of the antimicrobial peptide disclosed here.
  • 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 containing the cell under a predetermined condition The individual is cultured.
  • a predetermined host cell for example, a yeast, an insect cell, a plant cell, or a mammalian cell
  • the individual is cultured.
  • 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. The obtained transformant is cultured to prepare the desired fusion protein. 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-transfera
  • the purified fusion protein obtained was cleaved with a predetermined enzyme (protease) and released.
  • the target peptide fragment (designed antibacterial peptide) is recovered by a method such as affinity chromatography.
  • a conventionally known fusion protein expression system for example, GST / His system provided by Amersham Bioscience
  • the antimicrobial peptide of the present invention can be produced.
  • 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
  • ATP ATP
  • RNA polymerase RNA polymerase
  • Amino acids can be used to synthesize target polypeptides in vitro using a so-called cell-free protein synthesis system.
  • 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 strand comprising a nucleotide sequence encoding an antimicrobial peptide of the present invention for example, an amino acid sequence described in the examples described later (peptides consisting of SEQ ID NO: 112-126) and / or a nucleotide sequence complementary thereto, or
  • a double-stranded polynucleotide can be easily produced (synthesized) by a conventionally known method, that is, by selecting a codon corresponding to each amino acid residue constituting the designed amino acid sequence, an antimicrobial peptide
  • the nucleotide sequence corresponding to the amino acid sequence is easily determined and provided, and once the nucleotide sequence is determined, the DNA sequence corresponding to the desired nucleotide sequence can be obtained using a DNA synthesizer or the like. Nucleotides (single strands) can be easily obtained.
  • the obtained single-stranded DNA can be used as a cage, and various enzymatic synthetic means (typically PCR) can be
  • 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.
  • Some of the polynucleotides provided by the present invention encode antimicrobial peptides of novel amino acid sequences.
  • the total number of amino acid residues constituting the peptide chain is 50 or less (preferably 30 or less, such as 15 to 30), and the amino acid sequence represented by any of SEQ ID NOs: 112 to 126 or the amino acid
  • a nucleotide sequence encoding a peptide having an amino acid sequence in which the sequence is partially modified (or a peptide comprising the amino acid sequence) and / or a nucleotide sequence complementary to the sequence (or substantially consisting of the sequence) Constructed) 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 bacteriostatic.
  • 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, saline and other buffers
  • various buffers and other fillers such as saline and other buffers
  • extenders such as saline and other buffers
  • binders such as binders, moisturizers, surfactants, excipients, dyes And fragrances.
  • the antibacterial agent there is no particular limitation on the form of the antibacterial agent.
  • typical for internal or external use examples include ointments, solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets and capsules.
  • physiological saline or an appropriate buffer solution for example, PBS
  • the carrier contained in the antibacterial agent may vary depending on the form of the antibacterial agent. The process of preparing various forms of drugs (compositions) using the antibacterial peptide (main component) and various carriers (subcomponents) as materials.
  • 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 sequence and the D1-related amino acid sequence disclosed herein have high antibacterial activity even in systems where organic substances such as cations, salts (eg sodium chloride) or serum are present at relatively high concentrations. 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 can exhibit high antibacterial activity against bacteria causing infection (eg, Gram positive bacteria such as Staphylococcus aureus, Gram negative bacteria such as pathogenic E. coli). Therefore, the antibacterial peptide of the present invention Chido is useful as a main component of antibacterial agents.
  • bacteria causing infection eg, Gram positive bacteria such as Staphylococcus aureus, Gram negative bacteria such as pathogenic E. coli. Therefore, the antibacterial peptide of the present invention Chido is useful as a main component of antibacterial agents.
  • 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.
  • the NLS-related amino acid sequence and the D1-related amino acid sequence are arranged adjacent to each other in the peptides of Samples 1 to 17; It is arranged on the end side.
  • KRKV modified NLS
  • the sequence adjacent to the sequence is a D1-related amino acid sequence.
  • the amino acid sequence of the native D1 motif of the origin protein (site force in) shown in the table is used as the D1-related amino acid sequence.
  • samples 18 to 21 are samples for comparison. That is, as shown, sample 18 is a peptide that only has a D1-related amino acid sequence, and samples 19-21 are peptides that consist only of an NLS-related amino acid sequence.
  • the amino group (one NH 3) of the N-terminal amino acid is acetylated (one NHCOCH 3).
  • Each peptide described above (all 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 a 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. Obtained. Specifically, 20% piperidine / dimethylformamide (DMF) (grade for peptide synthesis, product of Kanto Chemical Co., Ltd.) was used to cleave and remove Fmoc, the amino protecting group of amino acids, and washed with DMF. Fmoc—reaction of 4 eq each of amino acid (—OH) and washing with DMF were repeated. After all the peptide chain elongation reactions were completed, the Fmoc group was cleaved with 20% piperidine / DMF, and the reaction product was washed with DMF and methanol in this order.
  • DMF dimethylformamide
  • 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 obtained peptide precipitate was vacuum-dried and purified using a high-performance liquid chromatograph (Waters 600: manufactured by Watters). Specifically, pre-column (Nippon Waters Co., Ltd. product, Guard-Pak Delta-pak C 18 A 300) and C 18 reverse phase column (Nippon Waters Co., Ltd. product, XTerra (registered trademark) column, MSC 18, 5 01, 4.6 ⁇ 150 mm), and a mixed solution of 0.1% trifnoreo-acetic acid aqueous solution and 0.1% trifanolorecetate acetonitrile solution was used as an eluent.
  • a high-performance liquid chromatograph Waters 600: manufactured by Watters. Specifically, pre-column (Nippon Waters Co., Ltd. product, Guard-Pak Delta-pak C 18 A 300) and C 18 reverse phase column (Nippon Waters Co., Ltd. product, XTerra (registered trademark) column, MSC 18, 5 01, 4.6
  • the column was run at a flow rate of 1.5 mL / min. Separation and purification were 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 21) : 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 inoculated into MHB medium containing a predetermined concentration of peptide (number of test bacteria: about 1 ⁇ 10 6 cell S / mL). After inoculation, cultivation was started in a 37 ° C incubator, and the presence or absence of bacteria was examined by turbidity after 24 hours. No increase in turbidity due to bacteria at the time of measurement was observed! /, And the minimum peptide concentration (ie, drug concentration) was defined as MIC (unit: M) in this example. The results are shown in Table 2. In the results shown in the table, those with an inequality sign (>) were those in which the peptide did not dissolve at the numerical concentration, and accurate antibacterial activity was not required.
  • a drug solution (synthetic peptide of sample 1) 40 times the maximum test concentration in sterile distilled water, and the peptide concentration is 50, 25, 12.5, 6.25, 3.13, 1. 56 and 0.78 M cation-containing MHB medium (ie, DIFCO's product “Mueller Hinton Broth”) containing cation prepared as follows: Ca CI ⁇ 2 ⁇ 03. 68g Dissolve lOOmU of purified water (10 mg 'Ca 2+ / mU, then add 500 ml of it to Mueller Hinton broth lOOmL and dissolve MgCl ⁇ 6 ⁇ 08. 36 g in 10 OmL of purified water (10 mg'Mg 2+ / After mU, 250 L of each was added to 1 OOmL of Mueller Hinton broth).
  • high concentration cation-containing MHB a medium in which horse serum (filter sterilized by Nippon Biotest Co., Ltd.) was added to the above high concentration cation-containing MHB medium so that the volume ratio was 10% of the whole medium (hereinafter “high concentration cation-containing MHB”). "Serum medium”).
  • high concentration cation-containing MHB serum media were also prepared with peptide concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 ⁇ M.
  • the peptide according to the present invention has a high concentration of cation. It was confirmed that high antibacterial activity can be maintained even in the presence (including high concentrations of calcium and magnesium in this example). In addition, high antibacterial activity could be maintained even in the presence of serum. Therefore, the antibacterial peptide of the present invention is suitable for use in a system (for example, in blood) in which various cations such as serum or salts) are present in a relatively large amount.
  • the peptide of the present invention since the peptide of the present invention has 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 qui n'existe pas naturellement et est conçu artificiellement. Le peptide antibactérien comprend une séquence du signal de localisation nucléaire (NLS) ou une variante de la séquence de celui-ci et une séquence d'acides aminés partielle d'un motif D1 contenu dans une protéine appartenant à la superfamille des cytokines à hélice α ou une variante de la séquence de celle-ci.
PCT/JP2007/067837 2006-09-14 2007-09-13 Peptide antibactérien artificiel et utilisation de celui-ci WO2008032783A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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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Title
CHOW D.-C. ET AL.: "A structural template for gp130-cytokine signaling assemblies", BIOCHIMICA ET BIPHYSICA ACTA, vol. 1592, 2002, pages 225 - 235, XP002384264 *
DI MARCO A. ET AL.: "Identification of ciliary neutrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists", PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 9247 - 9252, XP002056938 *
NEGRO A. ET AL.: "Cloning and expression of human ciliary neutrophic factor", EUR. J. BIOCHEM., vol. 201, 1991, pages 289 - 294, XP003019982 *
WUESTEFELD T. ET AL.: "Lack of gp130 Expression Results in More Bacterial Infection and Higher Mortality During Chronic Cholestasis in Mice", HEPATOLOGY, vol. 42, no. 5, 2005, pages 1082 - 1090, XP003019981 *
YAMADA Y. ET AL.: "Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity", BIOORGANIC & MEDICAL CHEMISTRY, vol. 14, January 2006 (2006-01-01), pages 77 - 82, XP005168788 *

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