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WO2010043046A1 - Inhibiteurs peptidiques de la sécrétion de type iii - Google Patents

Inhibiteurs peptidiques de la sécrétion de type iii Download PDF

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Publication number
WO2010043046A1
WO2010043046A1 PCT/CA2009/001473 CA2009001473W WO2010043046A1 WO 2010043046 A1 WO2010043046 A1 WO 2010043046A1 CA 2009001473 W CA2009001473 W CA 2009001473W WO 2010043046 A1 WO2010043046 A1 WO 2010043046A1
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Prior art keywords
peptide
gram negative
negative bacteria
amino acid
peptide inhibitor
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James Mahony
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McMaster University
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McMaster University
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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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • 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

Definitions

  • the present disclosure relates to novel peptide inhibitors and methods and uses thereof.
  • a number of Gram negative bacteria cause diseases in humans.
  • Gram negative bacteria include E. coli 0157, Salmonella enterica serovar Typhimurium, Shigella dysenteriae, Pseudomonas aeruginosa, Yersinia enterocolitca, Chlamydia pneumoniae and Chlamydia trachomatis. All of these bacteria have in common a protein secretion apparatus, called type III secretion system (T3SS), which allows the bacteria to inject a family of effector proteins into susceptible host cells.
  • T3SS type III secretion system
  • the T3SS is a virulence factor required for infection of host cells, and for host cell invasion.
  • the T3SS or injectosome is composed of approximately 25 proteins that form the secretion apparatus which resembles a hypodermic syringe. Some of the structural proteins of the injectosome are genetically conserved across the different bacteria and these conserved proteins perform essential functions. Two of these conserved proteins are called translocator proteins which form a pore or translocon in the host cell membrane through which the effector proteins from the bacteria are secreted. These two translocator proteins (i.e. PopB and PopD in Pseudomonas, SipB/C in Shigella, CopB/D in Chlamydia) interact in a specific way to form a hexameric protein complex or pore in the host cell membrane. However, the specific regions of the translocator proteins and the manner in which these regions interact with each other is not known.
  • the present inventor investigated the binding regions or interactive domains of translocator proteins, which are part of the type III secretion system (T3SS) in Gram negative bacteria. Specifically, the present inventor investigated and mapped the interactive domains of the T3SS translocator proteins PopD and PopB of Pseudomonas aeruginosa. The inventor then developed and synthesized novel peptide inhibitors of type III secretion using amino acid sequence data from the interactive domains of type III secretion translocator proteins. The inventor further determined that the novel peptide inhibitors inhibited type III secretion in Gram negative bacteria, inhibited subsequent infection of cells, and are not cytotoxic. The novel peptide inhibitors are used to inhibit and prevent type III secretion, to inhibit infection from Gram negative bacteria, to inhibit cell invasion of Gram negative bacteria and to treat or prevent infection from Gram negative bacteria.
  • T3SS type III secretion system
  • the present disclosure includes novel isolated peptide inhibitors of type III secretion in Gram negative bacteria.
  • Gram negative bacteria comprises the amino acid sequence: RAFESRNGALQVANTVIQSFVQMANASVQVR (SEQ ID NO: 1) or a variant thereof.
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprises the amino acid sequence: QMSFDAGFMKDVLQLIQQYTQSHNQAWRAAA (SEQ ID NO: 2) or a variant thereof.
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprises a fragment of the PopD translocator sequence:
  • composition comprising one or more peptide inhibitors disclosed herein.
  • kit comprising the peptide inhibitors or compositions comprising one or more of the peptide inhibitors disclosed herein.
  • Another aspect of the present disclosure is a method of inhibiting or preventing type III secretion in Gram negative bacteria comprising administering an effective amount of one or more of the peptide inhibitors disclosed herein or the compositions comprising one or more of the peptide inhibitors disclosed herein to a cell or subject in need thereof.
  • the present disclosure also includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to inhibit or prevent type III secretion in Gram negative bacteria in a cell or subject in need thereof.
  • An additional aspect of the present disclosure is a method of inhibiting infection from Gram negative bacteria comprising administering an effective amount of one or more of the peptide inhibitors disclosed herein or the compositions comprising one or more of the peptide inhibitors disclosed herein to a cell or subject in need thereof.
  • the present disclosure also includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to inhibit infection from Gram negative bacteria in a cell or subject in need thereof.
  • Another aspect of the present disclosure is a method of inhibiting cell invasion of Gram negative bacteria comprising administering an effective amount of one or more of the peptide inhibitors disclosed herein or the compositions comprising one or more of the peptide inhibitors disclosed herein to a cell or subject in need thereof.
  • the present disclosure also includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to inhibit cell invasion of Gram negative bacteria in a cell or subject in need thereof.
  • An additional aspect of the present disclosure is a method of treating or preventing infection from Gram negative bacteria comprising administering an effective amount of one or more of the peptide inhibitors disclosed herein or the compositions comprising one or more of the peptide inhibitors disclosed herein to a cell or subject in need thereof.
  • the present disclosure also includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to treat or prevent infection from Gram negative bacteria in a cell or subject in need thereof.
  • Figure 1A is a Pepscan analysis of PopD peptide library and soluble PopB binding.
  • Figure 1B is a graph demonstrating that Peptide 8003 inhibits
  • Figure 1C is a graph demonstrating inhibition of Pseudomonas aeruginosa by various peptides, and inhibition of a multi-drug resistant (MDR) Pseudomonas aeruginosa by Peptide 8003.
  • MDR multi-drug resistant
  • Figure 2 is a micrograph demonstrating that Peptide 8003 inhibits Chlamydia trachomatis infection of HeLa cells.
  • Figure 3 is a micrograph demonstrating that Peptide 8003 inhibits Chlamydia pneumoniae infection of HeLa cells.
  • Figure 4A is a diagram showing the sequence alignment of
  • Figure 4B is a diagram showing the sequence alignment of
  • Figure 5 is a graph demonstrating that Peptide 8003 reduces the number of Chlamydia trachomatis bacteria in the genital tract of mice.
  • Figure 6 is a graph demonstrating that Peptide 8003 is not cytotoxic to HeLa cells.
  • the present inventor investigated the binding regions or interactive domains of translocator proteins in the type III secretion system (T3SS) of Gram negative bacteria. Specifically, the present disclosure describes the investigation and mapping of the binding regions or interactive domains on the T3SS translocator proteins PopD (SEQ ID NO: 3) and PopB of Pseudomonas aeruginosa.
  • the present disclosure includes novel peptide inhibitors of type III secretion developed using the amino acid sequence data from the interactive domains of T3SS translocator proteins.
  • the present disclosure also demonstrated that the novel peptide inhibitors inhibited type III secretion in Gram negative bacteria, inhibited subsequent infection of cells as shown in Figures 1-3, and are not cytotoxic (Figure 6).
  • novel peptide inhibitors reduced infection from Gram negative bacteria of cells in an animal model ( Figure 5).
  • the novel peptide inhibitors included in the present disclosure are used to inhibit and prevent type III secretion, to inhibit infection from Gram negative bacteria, to inhibit cell invasion of Gram negative bacteria and to treat or prevent infection from Gram negative bacteria.
  • the present disclosure includes novel isolated peptide inhibitors of type III secretion in Gram negative bacteria.
  • the present disclosure also includes a composition comprising one or more of the peptide inhibitors.
  • Peptide 8003 comprises the amino acid sequence: RAFESRNGALQVANTVIQSFVQMANASVQVR (SEQ ID NO: 1) and Peptide 8006 comprises the amino acid sequence:
  • the present disclosure includes an isolated peptide inhibitor of type III secretion in Gram negative bacteria.
  • peptide refers to a sequence of amino acids.
  • oligopeptide amino acids
  • polypeptide amino acids
  • the peptide inhibitor described herein may comprise a sequence of approximately 10 to 300 amino acids in length, or 10 to 100 amino acids in length or suitably 20 to 50 amino acids in length.
  • amino acid includes all of the naturally occurring amino acids as well as modified amino acids.
  • peptide inhibitor or peptide inhibitors includes those produced from chemical synthesis and also includes peptide inhibitors produced synthetically to have the same primary structure or composition and the same function and/or activity of the peptide inhibitors disclosed herein.
  • Proteins produced from recombinant DNA technology includes for example, peptide inhibitors produced from recombinant DNA technology.
  • isolated as used herein means a macromolecule such as a peptide that has been identified, separated, recovered from its natural environment, and/or has been produced synthetically.
  • isolated includes a peptide inhibitor, which is substantially free of chemical precursors, or other chemicals when produced synthetically from chemical synthesis, and also includes a peptide inhibitor, which is substantially free of cellular material or culture media when produced synthetically from recombinant techniques.
  • type III secretion refers to a function or activity that is part of the type III secretion system (T3SS), which is a protein secretion apparatus present in Gram negative bacteria responsible for virulence of cells, including cell invasion and/or infection of cells.
  • T3SS type III secretion system
  • Gram negative bacteria includes all types of Gram negative bacteria, including, without limitation, Pseudomonas aeruginosa, Chlamydia trachomatis, Chlamydia pneumoniae, Salmonella, Shigella, E. coli 0157, Salmonella, Shigella, Listeria, and/or Yersinia.
  • Gram negative bacteria also includes Gram negative bacteria that are multi-drug resistant.
  • multi-drug resistant or “antibiotic resistant bacteria” or “resistant bacteria” includes, for example, types and strains of Gram negative bacteria that do not respond to treatment with antibiotics.
  • Another embodiment of the present disclosure is an isolated peptide inhibitor of type III secretion in Gram negative bacteria, wherein the peptide inhibitor comprises interactive domains of translocator proteins of type III secretion in Gram negative bacteria.
  • interactive domains refers to binding regions of amino acid sequences in translocator proteins of the type III secretion system (T3SS) present in Gram negative bacteria that bind to and/or interact with each other to form a pore or translocon in a cell membrane.
  • translocator proteins refers to proteins of the type III secretion system (T3SS) present in Gram negative bacteria which interact with each other via interactive domains to form a pore or translocon in a cell membrane through which bacterial proteins are secreted into the cell.
  • translocator proteins include PopD and PopB of Pseudomonas aeruginosa.
  • Another embodiment of the present disclosure is an isolated peptide inhibitor of type III secretion in Gram negative bacteria, wherein the peptide inhibitor comprises interactive domains of translocator proteins of type III secretion in Gram negative bacteria and the translocator proteins are PopB and PopD of Pseudomonas aeruginosa.
  • Gram negative bacteria comprises a fragment of the PopD translocator sequence:MIDTQYSLAATQAAIPSEPIAPGAAGRSVGTPQAAADLPQVPAAR ADRVELNAPRQVLDPVRMEAAGSELDSSVELLLILFRIAQKARELGVLQRDN ENQSIIHAQKAQVDEMRSGATLMIAMAVIAGVGALASAWGSLGALKNGKAI SQEKTLQKNIDGRNELIDAKMQALGKTSDEDRKIVGKVWAADQVQDSVALR AAGRAFESRNGALQVANTVIQSFVQMANASVQVRQGESQASAREGEVNAT IGQSQKQKVEDQMSFDAGFMKDVLQLIQQYTQSHNQAWRAAAGW (SEQ ID NO: 3) or a variant thereof wherein the fragment or variant inhibits type III secretion.
  • the isolated peptide inhibitors of type III secretion in Gram negative bacteria disclosed herein include peptide inhibitors with amino acid sequences in SEQ ID NO:1 and/or SEQ ID NO:2 or variants thereof.
  • variants as used herein includes modifications, substitutions, additions, derivatives, analogs, fragments or chemical equivalents of the amino acid sequences disclosed herein that perform substantially the same function as the peptide inhibitors disclosed herein in substantially the same way.
  • the variants have the same function of being useful to inhibit type III secretion in Gram negative bacteria, to prevent type III secretion in Gram negative bacteria, to inhibit infection from Gram negative bacteria, to inhibit cell invasion of Gram negative bacteria and/or to treat or prevent infection from Gram negative bacteria.
  • Variants of the peptide inhibitors disclosed herein also include, without limitation, conservative amino acid substitutions.
  • a "conservative amino acid substitution” as used herein is one in which one amino acid residue is replaced with another amino acid residue without abolishing the desired function or activity of the peptide inhibitors disclosed herein.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • conserved amino acid substitutions involve replacing one or more amino acids of the polypeptides of the disclosure with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting variant should be functionally equivalent. Changes which result in production of a chemically equivalent or chemically similar amino acid sequence are included within the scope of the disclosure. If the peptide inhibitors of the present disclosure are made using recombinant DNA technology, variants of the peptide inhibitors may be made by using polypeptide engineering techniques such as site directed mutagenesis, which are well known in the art for substitution of amino acids. For example, a hydrophobic residue, such as glycine can be substituted for another hydrophobic residue such as alanine.
  • alanine residue may be substituted with a more hydrophobic residue such as leucine, valine or isoleucine.
  • a negatively charged amino acid such as aspartic acid may be substituted for glutamic acid.
  • a positively charged amino acid such as lysine may be substituted for another positively charged amino acid such as arginine.
  • the phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such polypeptide displays the requisite activity.
  • a variant of the peptide inhibitor having the amino acid sequence in SEQ ID NO: 1 comprises one or more conservative amino acid substitutions of the amino acid sequence in SEQ ID NO: 1.
  • a variant of the peptide inhibitor having the amino acid sequence in SEQ ID NO: 2 comprises one or more conservative amino acid substitutions of the amino acid sequence in SEQ ID NO: 2.
  • a variant of the peptide inhibitor having the amino acid sequence in SEQ ID NO: 3 comprises one or more conservative amino acid substitutions of the amino acid sequence in SEQ ID NO: 3.
  • Variants of the peptide inhibitors of the present disclosure also include additions and deletions to the amino acid sequences disclosed herein.
  • variants of the peptide inhibitors of the present disclosure also include analogs thereof.
  • analog includes any active agent capable of performing the function of the peptide inhibitors disclosed herein, and may include peptide mimetics and the like.
  • active refers to molecules in a conformation suitable for performing substantially the same functions as the peptide inhibitors disclosed herein in substantially the same way.
  • Peptide mimetics include synthetic structures that may serve as substitutes for peptides in interactions between molecules (see Morgan and
  • Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but are designed to retain the desired structural and functional features and thus may be suitable substitutes of the peptide inhibitor analog disclosed in the present disclosure.
  • Peptide mimetics also include molecules incorporating peptides into larger molecules with other functional elements (e.g., as described in WO
  • Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367), and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to an isolated peptide of the disclosure.
  • Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic.
  • the mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states.
  • the mimetics can also include mimics of inhibitor peptide secondary structures. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.
  • Variant peptide inhibitors of the present disclosure also include derivatives thereof.
  • derivatives refers to a peptide having one or more residues chemically derivatized by reaction of a functional side group.
  • derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p- toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O- alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine.
  • derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3- methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • a derivative of a polypeptide also optionally includes polypeptides comprising forms of amino acids that are oxidized.
  • Variant peptide inhibitors of the present disclosure also include fragments thereof.
  • fragment as used herein means a portion of a polypeptide that contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the entire length of the reference polypeptide.
  • the isolated peptide inhibitors of type III secretion in Gram negative bacteria disclosed herein include peptide inhibitors with amino acid sequences that are substantially or essentially identical to the amino acid sequences of SEQ ID NO:1 and/or SEQ ID NO:2.
  • substantially identical or “essentially identical” as used herein means an amino acid sequence that, when optimally aligned, for example using the methods described herein, share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a second amino acid sequence.
  • sequence identity refers to the percentage of sequence identity between two polypeptide and/or nucleotide sequences.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues at corresponding amino acid positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are the same length.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. MoI. Biol. 215:403.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389- 3402.
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • the default parameters of the respective programs e.g., of XBLAST and NBLAST
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • a PAM120 weight residue table When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted. [0054] The percentage of identity between two polypeptide sequences, the amino acid sequences of such two sequences are aligned, for example using the Clustal W algorithm (Thompson, JD 1 Higgins DG, Gibson TJ, 1994, Nucleic Acids Res. 22(22): 4673-4680.), together with BLOSUM 62 scoring matrix (Henikoff S. and Henikoff J. G., 1992, Proc.
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprising an amino acid sequence that is substantially or essentially identical to the amino acid sequence in SEQ ID NO: 1 may be determined by percent (%) sequence identity.
  • Gram negative bacteria comprises 90% sequence identity with the amino acid sequence: RAFESRNGALQVANTVIQSFVQMANASVQVR (SEQ ID NO: 1).
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprises 95% sequence identity with the amino acid sequence: RAFESRNGALQVANTVI QSFVQM ANASVQ VR (SEQ ID NO: 1).
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprises all or part of the amino acid sequence: RAFESRNGALQVANTVIQSFVQMANASVQVR (SEQ ID NO: 1) and/or a variant thereof.
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprises the amino acid sequence: RAFESRNGALQVANTVIQSFVQMANASVQVR (SEQ ID NO: 1).
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprising an amino acid sequence that is substantially or essentially identical to the amino acid sequence in SEQ ID NO: 2 may be determined by percent (%) sequence identity.
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprises 90% sequence identity with the amino acid sequence: QMSFDAGFMKDVLQLIQQYTQSHNQAWRAAA (SEQ ID NO: 2).
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprises 95% sequence identity with the amino acid sequence: QMSFDAGFMKDVLQLIQQYTQSHNQAWRAAA (SEQ ID NO: 2).
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprises all or part of the amino acid sequence: QMSFDAGFMKDVLQLIQQYTQSHNQAWRAAA (SEQ ID NO: 2) and/or a variant thereof.
  • the peptide inhibitor of type III secretion in Gram negative bacteria comprises the amino acid sequence: QMSFDAGFMKDVLQLIQQYTQSHNQAWRAAA (SEQ ID NO: 2).
  • the disclosure includes a peptide inhibitor of type III secretion comprising a fragment of a sequence shown in Figures 4A and/or 4B (SEQ ID NOS: 4-5) or a variant thereof.
  • Another aspect of the present disclosure is an isolated nucleotide encoding a peptide inhibitor and/or variant disclosed herein.
  • a nucleotide sequence that encodes a peptide inhibitor and/or variant of the present disclosure may be deduced by a person skilled in the art using computer nucleic acid prediction programs and/or mRNA codon- amino acid tables that are readily available in the art.
  • isolated nucleotide refers to a nucleotide or nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
  • An "isolated nucleic acid” is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.
  • nucleic acid is intended to include DNA and RNA and can be either double stranded or single stranded.
  • the nucleic acid sequences contemplated by the present disclosure include isolated nucleotide sequences encoding a peptide inhibitor and/or variant disclosed herein.
  • peptide inhibitors may be developed and identified using the methods described herein.
  • peptide inhibitors may be developed by mapping binding regions or interactive domains of translocator proteins, identifying candidate peptide inhibitors, and producing candidate peptide inhibitors.
  • the interactive domains of translocator proteins are mapped using an overlapping peptide library map.
  • the interactive domains of translocator proteins are mapped using Pepscan analysis.
  • candidate peptide inhibitors are identified or selected by evaluating the degree of binding or interaction of various amino acid regions within the interactive binding domains on translocator proteins.
  • the amino acid sequence of candidate peptide inhibitors is extended to include an approximate length of 30 to 35 amino acids to improve binding affinity.
  • candidate proteins any proteins that are known to interact to form bi- or tri- molecular complexes that are functionally important either as an holoenzyme or scaffolding complex would be candidate proteins for the development of peptide inhibitors.
  • candidate peptide inhibitors may be produced synthetically.
  • candidate peptide inhibitors may be produced synthetically using solid-phase chemistry or solid phase chemical synthesis.
  • candidate peptide inhibitors may be produced synthetically using recombinant protein molecules to act as a carrier molecule for the peptide inhibitor.
  • a "fusion protein" (carrier molecule plus peptide inhibitor at one end) may be produced as a recombinant protein in, for example, without limitation, yeast or bacteria.
  • yeast or bacteria may be produced in large fermenters.
  • the peptide inhibitor may be produced as a GST (glutathione S-transferase) peptide construct.
  • the peptide inhibitor may be produced by TEV cleavage from a recombinant GST protein or other protein construct with purification of the cleaved peptide.
  • a peptide inhibitor obtained by: mapping interactive domains of translocator proteins, identifying a candidate peptide inhibitor and producing the candidate peptide inhibitor.
  • identifying or “identified” means candidate peptide inhibitors that are selected on the basis of the degree of binding or interaction of amino acid regions on translocator proteins.
  • candidate as used herein in reference to a peptide inhibitor includes peptide inhibitors that comprise interactive domains of translocator proteins of the type III secretion system present in Gram negative bacteria.
  • Another aspect of the present disclosure includes peptide inhibitors disclosed herein suitably formulated into pharmaceutical compositions for administration to mammals in a biologically compatible form suitable for administration in vivo.
  • biologically compatible form suitable for administration in vivo means a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects.
  • another embodiment of the present disclosure includes a composition comprising one or more peptide inhibitors and a pharmaceutically acceptable carrier or diluent, or mixtures thereof.
  • the composition comprises Peptide 8003 and a pharmaceutically acceptable carrier or diluent, or mixtures thereof.
  • the composition comprises Peptide 8006 and a pharmaceutically acceptable carrier or diluent, or mixtures thereof.
  • the composition comprises a peptide inhibitor comprising a fragment of the amino acid sequence of SEQ ID NO: 3 or variant thereof and a pharmaceutically acceptable carrier or diluent, or mixtures thereof.
  • the composition comprises: Peptide 8003, Peptide 8006 and a pharmaceutically acceptable carrier or diluent, or mixtures thereof.
  • the composition comprises: Peptide 8003, Peptide 8006, a peptide inhibitor comprising a fragment of the amino acid sequence of SEQ ID NO: 3 or variant thereof and a pharmaceutically acceptable carrier or diluent, or mixtures thereof.
  • compositions containing the peptide inhibitors described herein can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle, including for example, a carrier or diluent.
  • a pharmaceutically acceptable vehicle including for example, a carrier or diluent.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20 th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles, carriers or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient.
  • Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.
  • compositions of the present disclosure may comprise a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition.
  • suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1 (2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes.
  • DOTMA N-(1 (2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride
  • DOPE diolesylphosphotidyl-ethanolamine
  • compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the subject.
  • the composition may be in the form of a pharmaceutically acceptable salt which includes, without limitation, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol, histidine, procaine, etc.
  • the peptide inhibitors or compositions comprising one or more of the peptide inhibitors may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
  • the peptide inhibitors or compositions comprising one or more of the peptide inhibitors disclosed in the present disclosure may be administered for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, transepithelial, intrapulmonary, aerosol, topical, transdermal, buccal, nasal, rectal, intrathecal, sublingual, or oral administration, and the pharmaceutical compositions may be formulated accordingly.
  • Parenteral administration may occur by continuous infusion over a selected period of time.
  • KITS KITS
  • kits comprising one or more of the peptide inhibitors disclosed herein or the compositions comprising one or more of the peptide inhibitors disclosed herein and instructions for use thereof.
  • the kit can also include ancillary agents.
  • the kit can include an instrument for injecting the peptide inhibitors or compositions of the present disclosure into a subject, such as a syringe; a vessel for storing or transporting the peptide inhibitors or compositions; and/or pharmaceutically acceptable carriers or diluents, or mixtures thereof.
  • Another aspect of the present disclosure is a method of inhibiting type III secretion in Gram negative bacteria comprising administering an effective amount of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to a cell or subject in need thereof.
  • the present disclosure also includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to inhibit type III secretion in Gram negative bacteria in a cell or subject in need thereof.
  • Another aspect of the disclosure includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for the manufacture of a medicament to inhibit type III secretion in Gram negative bacteria in a cell or subject in need thereof.
  • a further aspect of the present disclosure includes one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for use in inhibiting type III secretion in Gram negative bacteria in a cell or subject in need thereof.
  • inhibit or “inhibiting” a function or activity, such as type III secretion in Gram negative bacteria, infection from Gram negative bacteria, or cell invasion from Gram negative bacteria, is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest.
  • inhibitors in the context of the present disclosure, are intended to have a broad meaning and include peptide inhibitors which directly or indirectly act on type III secretion and/or the T3SS in Gram negative bacteria, infection from Gram negative bacteria or cell invasion from Gram negative bacteria and/or reduce function or activity of type III secretion and/or T3SS in Gram negative bacteria, or infection from Gram negative bacteria or cell invasion from Gram negative bacteria.
  • inhibitors which directly or indirectly act on type III secretion and/or the T3SS in Gram negative bacteria, infection from Gram negative bacteria or cell invasion from Gram negative bacteria and/or reduce function or activity of type III secretion and/or T3SS in Gram negative bacteria, or infection from Gram negative bacteria or cell invasion from Gram negative bacteria.
  • inhibitorting type III secretion or “inhibit type III secretion” means reducing type III secretion as compared to otherwise same conditions, and includes reduction accomplished by directly or indirectly acting on the type III secretion system in Gram negative bacteria.
  • the term "subject" as used herein refers to any member of the animal kingdom, preferably avian or mammal.
  • the mammal is a cow, sheep, horse, swine, pig, goat or koala bear.
  • the avian is a bird susceptible to infections with Gram negative bacteria.
  • the mammal is a human.
  • the human is a subject that is immunocompromised.
  • immunocompromised means a subject who has an immunodeficiency, and thus has an immune system that is impaired by disease or treatment of a disease as compared to a subject who is not immunocompromised.
  • an immunocompromised subject includes a subject having chronic obstructive pulmonary disease (COPD), a lung infection, or is infected with HIV.
  • COPD chronic obstructive pulmonary disease
  • terapéuticaally effective amount means a quantity sufficient to, when administered to the subject, including a mammal, for example a human, achieve a desired result, for example an amount effect to inhibit type III secretion in a subject.
  • Effective amounts of therapeutic may vary according to factors such as the disease state, age, sex, weight of the subject. Dosage or treatment regime may be adjusted to provide the optimum therapeutic response. In addition, a “treatment" regime of a subject with a therapeutically effective amount may consist of a single administration, or alternatively comprise a series of applications.
  • the length of the treatment period depends on a variety of factors, such as the severity of the infection or disease, the age of the subject, the concentration and the activity of the peptide inhibitors, or a combination thereof. It will also be appreciated that the effective dosage of the peptide inhibitors used for the treatment or prevention may increase or decrease over the course of a particular treatment or prevention regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.
  • the peptide inhibitors of the present disclosure may be administered before, during or after exposure to the bacteria.
  • a further aspect of the present disclosure is a method of preventing type III secretion in Gram negative bacteria comprising administering an effective amount of one or more of the peptide inhibitors disclosed herein or the compositions comprising one or more of the peptide inhibitors disclosed herein to a cell or subject in need thereof.
  • the present disclosure also includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to prevent type III secretion in Gram negative bacteria in a cell or subject in need thereof.
  • Another aspect of the disclosure includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for the manufacture of a medicament to prevent type III secretion in Gram negative bacteria in a cell or subject in need thereof.
  • a further aspect of the present disclosure includes one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for use in preventing type III secretion in Gram negative bacteria in a cell or subject in need thereof.
  • preventing type III secretion or “prevent type III secretion” as used herein means blocking virulence of cells via blockage and/or interference of the type III secretion system in Gram negative bacteria as compared to otherwise same conditions.
  • An additional aspect of the present disclosure is a method of inhibiting infection from Gram negative bacteria comprising administering an effective amount of one or more of the peptide inhibitors disclosed herein or the compositions comprising one or more of the peptide inhibitors disclosed herein to a cell or subject in need thereof.
  • the present disclosure also includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to inhibit infection from Gram negative bacteria in a cell or subject in need thereof.
  • Another aspect of the disclosure includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for the manufacture of a medicament to inhibit infection from Gram negative bacteria in a cell or subject in need thereof.
  • a further aspect of the present disclosure includes one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for use in inhibiting infection from Gram negative bacteria in a cell or subject in need thereof.
  • inhibiting infection means reducing infection from Gram negative bacteria of cells as compared to otherwise same conditions.
  • the inhibition of infection may be assessed using assays known to those skilled in the art. For example, inhibition of infection may be assessed by measuring bacterial counts, colonization, and replication of Gram negative bacteria and comparing to otherwise same conditions. Inhibition of infection may also be assessed for example, by evaluating symptoms of infection, including for example, inflammation, redness, heat and/or pain as compared to otherwise same conditions.
  • Another aspect of the present disclosure is a method of inhibiting cell invasion of Gram negative bacteria comprising administering an effective amount of one or more of the peptide inhibitors disclosed herein or the compositions comprising one or more of the peptide inhibitors disclosed herein to a cell or subject in need thereof.
  • the present disclosure also includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to inhibit cell invasion from Gram negative bacteria in a cell or subject in need thereof.
  • Another aspect of the disclosure includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for the manufacture of a medicament to inhibit cell invasion from Gram negative bacteria in a cell or subject in need thereof.
  • a further aspect of the present disclosure includes one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for use in inhibiting cell invasion from Gram negative bacteria in a cell or subject in need thereof.
  • inhibiting cell invasion means reducing migration of Gram negative bacteria into cells as compared to otherwise same conditions. Inhibition of cell invasion may be assessed using assays known to those skilled in the art including, but not limited to, in vitro invasion assays.
  • An additional aspect of the present disclosure is a method of treating or preventing infection from Gram negative bacteria comprising administering an effective amount of one or more of the peptide inhibitors disclosed herein or the compositions comprising one or more of the peptide inhibitors disclosed herein to a cell or subject in need thereof.
  • the present disclosure also includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein to treat or prevent infection from Gram negative bacteria in a cell or subject in need thereof.
  • Another aspect of the disclosure includes uses of one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for the manufacture of a medicament to treat or prevent infection from Gram negative bacteria in a cell or subject in need thereof.
  • a further aspect of the present disclosure includes one or more of the peptide inhibitors disclosed herein or a composition comprising one or more of the peptide inhibitors disclosed herein for use in treating or preventing infection from Gram negative bacteria in a cell or subject in need thereof.
  • treatment or “prevention” are approaches for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • “Palliating" a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
  • the phrase "treating or preventing infection from Gram negative bacteria” includes treating infection from Gram negative bacteria, preventing infection from Gram negative bacteria, decreasing the severity of infection from Gram negative bacteria, inhibiting Gram negative bacteria colonization, reducing shedding of Gram negative bacteria, and preventing Gram negative bacteria colonization or improving signs and symptoms related to infection from Gram negative bacteria.
  • the present disclosure also includes the treatment or prevention of any disease that is associated with an infection from Gram negative bacteria.
  • the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • HeLa cells, Pseudomonas aeruginosa, C. pneumonaie and other bacteria used in the study are obtained commercially from the American Type Culture Collection (ATCC).
  • Antibody for staining C. pneumoniae is obtained commercially from one of several suppliers.
  • Peptides were synthesized by the APTC Peptide Synthesis Facility at The Hospital for Sick Children, Toronto. All other common reagents were purchased from Sigma Chemicals in St. Louis.
  • Pepscan analysis was performed as described by Timmerman et al. (Journal of Molecular Recognition 2007, 20:
  • Candidate peptides were selected from the results of the Pepscan analysis based on signal strength obtained in the enzyme immunoassay. Signals greater than three-fold above background with readings above 300 fluorescent units were deemed to be significant. Readings generated for over 3000 individual peptides were aligned with the peptide sequence and the amino acid sequence with the highest weighted signal was selected as a candidate peptide.
  • Solid phase synthesis of peptides Solid phase peptide synthesis (SSPS) using F-moc blocking groups is commonly used by commercial suppliers of synthetic peptides (Carpino L.A. "1-Hydroxy-7- azabenzotriazole. An efficient peptide coupling additive".
  • Invasion assay An in vitro invasion assay for Pseudomonas aeruginosa was performed using standard methods as described by Ha and Jin (Infect. Immun. 2001 , 69: 4398-4406) and for Chlamydia pneumoniae as described by Coombes and Mahony (Ce//. Microbiol. 2002, 4:447-460). Preformed monolayers of confluent HeLa cells were used for invasion with Pseudomonas aeruginosa and Chlamydia pneumoniae. Briefly, an overnight culture of Pseudomonas aeruginosa was subcultured 1 :50 and after 3 hours cells were collected and a dilution of 1 :100 prepared.
  • Animal model An animal model of Chlamydia trachomatis in the genital tract of mice was used to evaluate peptide inhibitors of the present disclosure. Two concentrations of Peptide 8003 (low, 100 ⁇ M and high, 200 ⁇ M) were mixed with 107 ifu of C.
  • trachomatis serovar D in 30 ⁇ L PBS and injected into the vagina of female Balb/c mice on day 0 and genital tract swabs were collected on days 5, 7, 10, and 13. Swabs were rolled onto glass slides, fixed and stained for C. trachomatis using an FITC-conjugated monoclonal antibody.
  • Direct fluorescent antibody (DFA) scores are the mean counts (+/- SEM) of bacteria per high power field.
  • Adenylate kinase activity assay HeLa cells were treated with various concentrations of Peptide 8003 for 30 minutes and culture supernatants (0.05 ml_) were collected and tested for adenylate kinase activity measured as relative light units (RLU) using the ToxiLightTM assay (Lonza) according to the manufacturer's instructions. The detergent lysis aliquot shows maximum enzyme activity when cells are completely lysed by detergent.
  • Pepscan analysis was performed with 3000 peptides to map the interactive domains of Pseudomonas aeruginosa PopB and PopD.
  • the Pepscan analysis results show the enzyme immunoassay absorbance results for soluble recombinant PopB protein binding to immobilized overlapping peptides (3,000) of the PopD library (Fig 1A).
  • Candidate peptides from the interactive domains were synthesized by solid phase chemistry and tested for their ability to block HeLa cell invasion using an in vitro invasion assay. Two peptides that form the interactive domains of PopB and PopD were evaluated extensively in an in vitro cell invasion assay.
  • Peptide 8003 inhibits invasion of Pseudomonas aeruginosa in a dose-dependent fashion (Fig 1 B). Inhibition is seen at a peptide concentration as low as 25 uM and complete inhibition is seen at 200 uM.
  • a second peptide, designated as Peptide 8006 which is also part of the interactive domain gave partial inhibition (45%) at 100 uM, and 78% inhibition at 20OuM while an irrelevant peptide based on influenza A, designated as Peptide 8005, gave no inhibition (Fig 1C).
  • Peptide 8003 also inhibited a multi-drug resistant (MDR) Pseudomonas aeruginosa bacteria isolated from a patient with pneumonia by 90% (Fig 1C).
  • MDR multi-drug resistant
  • Peptide 8003 was tested in a Chlamydia trachomatis mouse model. Swabs from infected mice were examined under high power microscopy and counts of bacteria were determined. Peptide 8003 reduced infection in animals at a concentration of 100 ⁇ M as soon as 5 days following infection by approximately 25%. This 25% reduction in infection remained steady for up to 13 days post-infection. A dose of 200 ⁇ M of peptide did not impact infection until 13 days post infection, at which point an approximate 50% reduction in infection was observed (Fig 5).
  • Peptide 8003 inhibited type III secretion likely by disrupting or preventing the formation of the T3SS translocon.
  • Peptide 8003 inhibited three different Gram negative bacteria including Pseudomonas aeruginosa, Chlamydia trachomatis and Chlamydia pneumoniae, inhibited all three bacteria from invading epithelial cells, and was not cytotoxic even at high concentrations.
  • Peptide 8003 also reduced infection from Chlamydia trachomatis of cells in a mouse model.
  • the peptide inhibitors may also inhibit additional Gram negative bacteria that have a conserved T3SS and thus block the infection process.
  • the peptide inhibitors may be used in clinical medicine for the treatment of infections due to Gram negative bacteria.
  • bacteria such as Pseudomonas aeruginosa that are multi-drug resistant
  • the peptides described could provide an important adjunctive therapy for strains of P. aeruginosa that are resistant to antibiotics currently in use.
  • the approach employed in the present disclosure aimed at treating Gram negative bacteria by targeting or blocking T3SS has for example at least four apparent advantages.
  • First, the peptides were useful for several different types of Gram negative bacteria as demonstrated in the present disclosure.
  • Second, the peptides were useful for Gram negative antibiotic resistant bacteria as demonstrated above.
  • Third, the peptide should not induce or select for resistant bacteria as commonly occurs with antibiotics that are small molecules because the peptide interacts with a long stretch of amino acid residues within the interactive domain of the target protein.
  • the peptides were not cytotoxic.
  • the peptide inhibitors described herein may inhibit or block the function of the T3SS by preventing the formation of the translocon protein complex, or by disrupting an already formed translocon protein complex via competitive inhibition.
  • Cpn1019 C-terminus of Chlamydia pneumoniae CopD2
  • Cpn1020 C- terminus of Chlamydia pneumoniae CopD1

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Abstract

L’invention concerne des peptides qui inhibent la sécrétion de type III dans les bactéries à Gram négatif, des compositions comprenant lesdits inhibiteurs peptidiques, ainsi que leurs kits, procédés et utilisations. Spécifiquement, l’invention concerne des peptides qui comprennent des domaines interactifs de protéines de translocation, et notamment des inhibiteurs peptidiques qui comprennent des domaines interactifs des protéines de translocation PopD de Pseudomonas aeruginosa. Ces domaines interactifs médient la liaison entre PopD et PopB, une autre protéine de translocation. Les peptides qui comprennent ces domaines interactifs perturbent le développement des bactéries à Gram négatif, inhibent l’infection ultérieure des cellules, réduisent l’infection par des bactéries à Gram négatif dans un modèle animal, et ne sont pas cytotoxiques.
PCT/CA2009/001473 2008-10-16 2009-10-16 Inhibiteurs peptidiques de la sécrétion de type iii Ceased WO2010043046A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104143283A (zh) * 2014-07-31 2014-11-12 江南大学 一种革兰阴性菌肽聚糖结构模型教具
WO2018183850A3 (fr) * 2017-03-31 2020-03-19 Arizona Board Of Regents On Behalf Of The University Of Arizona Procédés, systèmes et compositions pour inhiber la virulence des pathogènes de la famille a/e

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WO2004019980A1 (fr) * 2002-08-31 2004-03-11 The Secretary Of State For Defence Vaccin contre yersinia comprenant un ou deux anticorps, l'un specifique de l'antigene yersinia pestis f1 et l'autre specifique de l'antigene yersina pestis v
US20060287252A1 (en) * 2004-02-09 2006-12-21 Satoshi Omura Substances k01-0509 and process for producing the same
CA2648406A1 (fr) * 2008-01-03 2009-07-03 Guy Tran Van Nhieu Derives d'elements de translocateur et leurs utilisations

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WO2004019980A1 (fr) * 2002-08-31 2004-03-11 The Secretary Of State For Defence Vaccin contre yersinia comprenant un ou deux anticorps, l'un specifique de l'antigene yersinia pestis f1 et l'autre specifique de l'antigene yersina pestis v
US20060287252A1 (en) * 2004-02-09 2006-12-21 Satoshi Omura Substances k01-0509 and process for producing the same
CA2648406A1 (fr) * 2008-01-03 2009-07-03 Guy Tran Van Nhieu Derives d'elements de translocateur et leurs utilisations

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FRANK, D.W. ET AL.: "Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV", THE JOURNAL OF INFECTIOUS DISEASES, vol. 186, no. 1, 1 July 2002 (2002-07-01), pages 64 - 73 *
OCHOA, T.J. ET AL.: "Lactoferrin disruption of bacterial type III secretion systems", BIOMETALS, vol. 17, no. 3, June 2004 (2004-06-01), pages 257 - 260 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104143283A (zh) * 2014-07-31 2014-11-12 江南大学 一种革兰阴性菌肽聚糖结构模型教具
WO2018183850A3 (fr) * 2017-03-31 2020-03-19 Arizona Board Of Regents On Behalf Of The University Of Arizona Procédés, systèmes et compositions pour inhiber la virulence des pathogènes de la famille a/e
US11484603B2 (en) 2017-03-31 2022-11-01 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods, systems, and compositions for inhibiting virulence of A/E family pathogens

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