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WO2013114363A2 - Agents antimicrobiens - Google Patents

Agents antimicrobiens Download PDF

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Publication number
WO2013114363A2
WO2013114363A2 PCT/IL2013/050083 IL2013050083W WO2013114363A2 WO 2013114363 A2 WO2013114363 A2 WO 2013114363A2 IL 2013050083 W IL2013050083 W IL 2013050083W WO 2013114363 A2 WO2013114363 A2 WO 2013114363A2
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WIPO (PCT)
Prior art keywords
genes
peptide
gene
seq
amino acid
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PCT/IL2013/050083
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English (en)
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WO2013114363A8 (fr
WO2013114363A3 (fr
Inventor
Rotem Sorek
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Yeda Research And Development Co.Ltd.
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Publication of WO2013114363A2 publication Critical patent/WO2013114363A2/fr
Publication of WO2013114363A8 publication Critical patent/WO2013114363A8/fr
Publication of WO2013114363A3 publication Critical patent/WO2013114363A3/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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci

Definitions

  • the present invention in some embodiments thereof, relates to antimicrobial agents, uses thereof and methods of identifying same.
  • Antibiotic resistance can result in severe adverse outcomes, such as increased mortality, morbidity and medical care costs for patients suffering from common infections, once easily treatable with antibiotics (Am. J. Infect. Control 24 (1996), 380- 388; Am. J. Infect. Control 27 (1999), 520-532; Acar, J. F. (1997), Clin. Infect. Dis. 24, Suppl 1, S17-S18; Cohen, M. L. (1992), Science 257, 1050-1055; Cosgrove, S. E. and Carmeli, Y. (2003), Clin. Infect. Dis. 36, 1433-1437; Holmberg, S. D. et al. (1987), Rev. Infect. Dis.
  • Microbes bacteria, archaea, fungi and viruses
  • Such compounds can be small molecule antibiotics, such as the ones produced by various Streptomyces species [Waive, Arch Microbiol. 2001 Nov;176(5):386-90], or proteinacious antibiotics, often known as bacteriocins [Riley & Wertz, Annu Rev Microbiol. 2002;56: 117-37] or antimicrobial peptides (AMPs).
  • AMPs antimicrobial peptides
  • Proteins that target bacteria have a broad medical and biotechnological application spectrum. They can be used as direct antibiotics for human and veterinary medicine [Gillor 2005, Curr Pharm Des. 2005;11(8): 1067-75], as growth enhancers in livestock [Brashears, 2003. J. Food Prot. 66, 748-754], as food preservatives [Delves- Broughton, Antonie Van Leeuwenhoek. 1996 Feb;69(2): 193-202], as genes engineered into probiotic bacteria [Gillor 2005, Curr Pharm Des. 2005;l l(8): 1067-75], as killers of phytopathogenic bacteria for crop management [Penyalver 2000, Eur. J. Plant Pathol. 106, 801-810], etc. In addition, they may serve as effective anti-microbial agents against antibiotic resistant organisms.
  • E.coli Escherichia coli
  • an isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 97-113, wherein the isolated peptide has antimicrobial activity.
  • isolated polynucleotide comprising a nucleic acid sequence encoding the polypeptide of the present invention.
  • an anti-microbial composition comprising a carrier and as an active ingredient an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-113.
  • an anti-microbial composition comprising a carrier and as an active ingredient an isolated polynucleotide comprising a nucleic acid sequence which encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-113.
  • the method comprising administering to the subject a therapeutically effective amount of the antimicrobial composition of the present invention, thereby treating the infection.
  • a solid support coated with an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-113.
  • a method of killing a microbe comprising contacting the microbe with an isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-113, thereby killing the microbe.
  • antimicrobial compositions for treating an infection.
  • a method of identifying a gene which encodes a product having putative antimicrobial activity comprising:
  • the gene is closer than 15 genes up or downstream from a gene that encodes a transporter polypeptide; (ii) the genes encode polypeptides which comprise an N terminal signal peptide;
  • the gene is closer than 15 genes upstream or downstream from a gene that encodes a peptidase
  • the gene is closer than 15 genes up or downstream from a gene that encodes a phage-, plasmid- or transposon- related gene.
  • the amino acid sequence consists of the sequences selected from the group as set forth in SEQ ID NOs: 97-113.
  • the isolated peptide comprises at least one naturally occurring amino acid.
  • the isolated peptide comprises a synthetic amino acid.
  • the isolated peptide is attached to a cell penetrating agent.
  • the attached is covalently attached.
  • the cell penetrating agent is a peptide agent.
  • the isolated peptide is attached to a sustained-release enhancing agent.
  • the sustained-release enhancing agent is selected from the group consisting of hyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), polyethylene glycol (PEG), glyme and polyisopropylacrylamide.
  • the isolated polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 199-212.
  • the carrier is a pharmaceutically acceptable carrier.
  • the anti-microbial composition is formulated for topical application.
  • the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 114-212.
  • the contacting is effected in vivo.
  • the contacting is effected ex vivo.
  • the microbe comprises a bacteria.
  • the isolated polynucleotide is operably linked to a promoter.
  • the promoter is a plant-specific promoter.
  • FIG. 1 is a graph illustrating the growth of Enterococcus fecalis bacteria in the presence of the peptide having the sequence as set forth in SEQ ID NO: 97.
  • the present invention in some embodiments thereof, relates to antimicrobial agents, uses thereof and methods of identifying same.
  • AMPs Gene-encoded antimicrobial peptides
  • the present inventor has now devised a novel algorithm to narrow down the list of candidate peptides based on clonability.
  • the present inventor has reduced a master list comprising more than 15,000 candidate peptides to a list comprising less than 100 peptides, each of which having a much higher probability of comprising antimicrobial properties than those peptides only appearing in the master list.
  • an isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-113, wherein the isolated peptide has antimicrobial activity.
  • the peptide has a sequence which is at least 90 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113. According to another embodiment, the peptide has a sequence which is at least 91 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113. According to another embodiment, the peptide has a sequence which is at least 92 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113. According to another embodiment, the peptide has a sequence which is at least 93 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113.
  • the peptide has a sequence which is at least 94 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113. According to another embodiment, the peptide has a sequence which is at least 95 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113. According to another embodiment, the peptide has a sequence which is at least 96 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113. According to another embodiment, the peptide has a sequence which is at least 97 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113.
  • the peptide has a sequence which is at least 98 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113. According to another embodiment, the peptide has a sequence which is at least 99 % identical to any one of the sequences as set forth in SEQ ID NOs: 1-113.
  • Tables 1 and 2 provide the sequences of the peptides of the present invention
  • Table 1 providing peptides that were identified from known gene coding microbial DNA
  • Table 2 provides peptides that were identified from intergenic regions of microbial DNA.
  • NC_011658 819033 819239 207 68 55 168
  • NC_008554 2990792 2991019 228 75 108 210
  • the peptide has an amino acid sequence as set forth in SEQ ID NO: 85.
  • the peptide has an amino acid sequence as set forth in SEQ ID NO: 97.
  • antimicrobial activity refers to an ability to suppress, control, inhibit or kill microorganisms, such as bacteria and archae.
  • the antimicrobial activity may comprise bactericidal or bacteriostatic activity, or both.
  • peptide refers to a polymer of natural or synthetic amino acids, encompassing native peptides (either degradation products, synthetically synthesized polypeptides or recombinant polypeptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are polypeptide analogs.
  • polypeptides are produced as complex precursors, from which fragments of peptides are removed (processed) at some point during protein maturation, resulting in a mature form of the polypeptide that is different from the primary translation product.
  • a “mature protein” refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
  • a “precursor protein” refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may include, but are not limited to, intracellular or extracellular localization signals. "Pre” in this nomenclature generally refers to the signal peptide.
  • the form of the translation product with only the signal peptide removed but no further processing yet is called a "propeptide".
  • the present invention contemplates the mature form of the peptides of the present invention, and where relevant, the propeptide form or the precursor form of the peptides.
  • the skilled artisan is able to determine, depending on the species in which the proteins are being expressed and the desired intracellular location, if higher expression levels or higher antimicrobial activity might be obtained by using a gene construct encoding just the mature form of the protein, the mature form with a signal peptide, or the proprotein (i.e., a form including propeptides) with a signal peptide.
  • the peptides of the present invention consist of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-113.
  • the peptides of this aspect of the present may comprise modifications or additions which render the peptides even more stable while in a body or more capable of penetrating into cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
  • Trp, Tyr and Phe may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • polypeptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L- amino acids (stereoisomers).
  • Tables 3 and 4 below list naturally occurring amino acids (Table 3) and non- conventional or modified amino acids (Table 4) which can be used with the present invention.
  • Non-conventional amino acid Code Non-conventional amino acid Code
  • amino acids of the peptides of the present invention may be substituted either conservatively or non-conservatively.
  • conservative substitution refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non- naturally occurring amino or a peptidomimetics having similar steric properties.
  • side-chain of the native amino acid to be replaced is either polar or hydrophobic
  • the conservative substitution should be with a naturally occurring amino acid, a non- naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).
  • amino acid analogs synthetic amino acids
  • a peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.
  • the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.
  • non-conservative substitutions refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties.
  • the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
  • non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid.
  • Those non-conservative substitutions which fall under the scope of the present invention are those which still constitute a peptide having anti-bacterial properties.
  • N and C termini of the peptides of the present invention may be protected by function groups.
  • Suitable functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference.
  • Preferred protecting groups are those that facilitate transport of the compound attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the compounds.
  • Hydroxyl protecting groups include esters, carbonates and carbamate protecting groups.
  • Amine protecting groups include alkoxy and aryloxy carbonyl groups, as described above for N-terminal protecting groups.
  • Carboxylic acid protecting groups include aliphatic, benzylic and aryl esters, as described above for C-terminal protecting groups.
  • the carboxylic acid group in the side chain of one or more glutamic acid or aspartic acid residue in a peptide of the present invention is protected, preferably with a methyl, ethyl, benzyl or substituted benzyl ester.
  • N-terminal protecting groups include acyl groups (-CO-R1) and alkoxy carbonyl or aryloxy carbonyl groups (-CO-0-R1), wherein Rl is an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or a substituted aromatic group.
  • acyl groups include acetyl, (ethyl)-CO-, n-propyl-CO-, iso-propyl-CO-, n-butyl-CO-, sec-butyl-CO-, t-butyl-CO-, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoyl phenyl-CO-, substituted phenyl-CO-, benzyl-CO- and (substituted benzyl)-CO-.
  • alkoxy carbonyl and aryloxy carbonyl groups include CH3-0-CO-, (ethyl)-O-CO-, n-propyl-O-CO-, iso-propyl-O-CO-, n-butyl-O-CO-, sec-butyl-O-CO-, t-butyl-O-CO-, phenyl-O- CO-, substituted phenyl-O-CO- and benzyl-O-CO-, (substituted benzyl)- 0-CO-.
  • one to four glycine residues can be present in the N-terminus of the molecule.
  • the carboxyl group at the C-terminus of the compound can be protected, for example, by an amide (i.e., the hydroxyl group at the C-terminus is replaced with -NH 2,
  • ester i.e. the hydroxyl group at the C-terminus is replaced with
  • R2 an d R3 are independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or a substituted aryl group.
  • R2 and R3 taken together with the nitrogen atom, R2 and R3 can form a C4 to C8 heterocyclic ring with from about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur.
  • suitable heterocyclic rings include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl.
  • C-terminal protecting groups include -NI3 ⁇ 4, -NHCH3, -N(CH3)2, -NH(ethyl), -N(ethyl)2 , -N(methyl) (ethyl), -NH(benzyl), -N(C1-C4 alkyl) (benzyl), -NH(phenyl), -N(C1-C4 alkyl) (phenyl), -OCH3, -O-(ethyl), -O-(n-propyl), -O-(n-butyl), -O-(iso-propyl), -0-(sec- butyl), -O-(t-butyl), -O-benzyl and -O-phenyl.
  • the peptides of the present invention may be attached (either covalently or non- covalently) to a penetrating agent.
  • penetrating agent refers to an agent which enhances translocation of any of the attached peptide across a cell membrane.
  • the penetrating agent is a peptide and is attached to the antimicrobial peptide (either directly or non-directly) via a peptide bond.
  • peptide penetrating agents typically have an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
  • peptide penetrating agents include those set forth in SEQ ID NOs: 213-215.
  • CPP cell penetrating peptide
  • CPPs may be used in order to enhance intracellular penetration.
  • CPPs may included short and long versions of TAT (YGRKKRR - SEQ ID NO: 213 and YGRKKRRQRRR - SEQ ID NO: 214) and PTD (RRQRR- SEQ ID NO: 215).
  • TAT YGRKKRR - SEQ ID NO: 213 and YGRKKRRQRRR - SEQ ID NO: 214)
  • PTD RRQRR- SEQ ID NO: 215
  • the disclosure is not so limited, and any suitable penetrating agent may be used, as known by those of skill in the art.
  • the peptides of the present invention may also comprise non-amino acid moieties, such as for example, hydrophobic moieties (various linear, branched, cyclic, polycyclic or hetrocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptides; non- peptide penetrating agents; various protecting groups, especially where the compound is linear, which are attached to the compound's terminals to decrease degradation.
  • non-amino acid moieties such as for example, hydrophobic moieties (various linear, branched, cyclic, polycyclic or hetrocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptides; non- peptide penetrating agents; various protecting groups, especially where the compound is linear, which are attached to the compound's terminals to decrease degradation.
  • Chemical (non-amino acid) groups present in the compound may be included in order to improve various physiological properties such; decreased degradation or clearance; decreased repulsion by various cellular pumps, improve immunogenic activities, improve various modes of administration (such as attachment of various sequences which allow penetration through various barriers, through the gut, etc.); increased specificity, increased affinity, decreased toxicity and the like.
  • the antimicrobial peptides of the present invention are attached to a sustained-release enhancing agent.
  • Exemplary sustained- release enhancing agents include, but are not limited to hyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), polyethylene glycol (PEG), glyme and polyisopropylacrylamide.
  • Attaching the amino acid sequence component of the peptides of the invention to other non-amino acid agents may be by covalent linking, by non-covalent complexion, for example, by complexion to a hydrophobic polymer, which can be degraded or cleaved producing a compound capable of sustained release; by entrapping the amino acid part of the peptide in liposomes or micelles to produce the final peptide of the invention.
  • the association may be by the entrapment of the amino acid sequence within the other component (liposome, micelle) or the impregnation of the amino acid sequence within a polymer to produce the final peptide of the invention.
  • the peptides of the invention may be linear or cyclic (cyclization may improve stability). Cyclization may take place by any means known in the art. Where the compound is composed predominantly of amino acids, cyclization may be via N- to C- terminal, N-terminal to side chain and N-terminal to backbone, C-terminal to side chain, C-terminal to backbone, side chain to backbone and side chain to side chain, as well as backbone to backbone cyclization. Cyclization of the peptide may also take place through non-amino acid organic moieties comprised in the peptide.
  • the peptides of the present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
  • Synthetic peptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing. Recombinant techniques may also be used to generate the peptides of the present invention.
  • a polynucleotide encoding the peptide of the present invention is ligated into a nucleic acid expression vector, which comprises the polynucleotide sequence under the transcriptional control of a cis-regulatory sequence (e.g., promoter sequence) suitable for directing constitutive, tissue specific or inducible transcription of the polypeptides of the present invention in the host cells.
  • a cis-regulatory sequence e.g., promoter sequence
  • prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of some embodiments of the invention.
  • yeast transformed with recombinant yeast expression vectors containing the coding sequence include, but are not limited to; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence.
  • virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • Mammalian expression systems can also be used to express the polypeptides of some embodiments of the invention.
  • bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).
  • yeast a number of vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. Application No: 5,932,447.
  • vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • the expression of the coding sequence can be driven by a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter to TMV [Takamatsu et al. (1987) EMBO J. 3:17-311] can be used.
  • plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J.
  • the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed antimicrobial peptide.
  • the expression of a fusion protein or a cleavable fusion protein comprising the antimicrobial peptides of some embodiments of the invention and a heterologous protein can be engineered.
  • Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein.
  • the antimicrobial can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265: 15854-15859].
  • polypeptides of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • the peptides of the present invention can also be synthesized using in vitro expression systems. These methods are well known in the art and the components of the system are commercially available.
  • the peptides of the present invention comprise anti-microbial properties they may be used to kill microbes.
  • a method of killing a microbe comprising contacting the microbe with the isolated peptides of the present invention.
  • the microbe may be for example a gram-positive or gram negative bacteria.
  • Gram-positive bacteria refers to bacteria characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids and are characterized by their blue-violet color reaction in the Gram-staining procedure.
  • Gram-positive bacteria include: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium haemophilium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium scro
  • Gram-negative bacteria refer to bacteria characterized by the presence of a double membrane surrounding each bacterial cell.
  • Representative Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella baciUiformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chalmydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophil
  • the term "contacting" refers to the positioning of the peptides of the present invention such that they are in direct or indirect contact with the bacterial cells.
  • the present invention contemplates both applying the peptides of the present invention to a desirable surface and/or directly to the bacterial cells.
  • Contacting surfaces with the peptides can be effected using any method known in the art including spraying, spreading, wetting, immersing, dipping, painting, ultrasonic welding, welding, bonding or adhering.
  • the peptides of the present invention may be attached as monolayers or multiple layers.
  • the present invention envisages coating a wide variety of surfaces with the peptides of the present invention including fabrics, fibers, foams, films, concretes, masonries, glass, metals, plastics, polymers, and like.
  • An exemplary solid surface that may be coated with the peptides of the present invention is an intracorporial or extra-corporial medical device or implant.
  • an “implant” as used herein refers to any object intended for placement in a human body that is not a living tissue.
  • the implant may be temporary or permanent.
  • Implants include naturally derived objects that have been processed so that their living tissues have been devitalized.
  • bone grafts can be processed so that their living cells are removed (acellularized), but so that their shape is retained to serve as a template for ingrowth of bone from a host.
  • naturally occurring coral can be processed to yield hydroxyapatite preparations that can be applied to the body for certain orthopedic and dental therapies.
  • An implant can also be an article comprising artificial components.
  • the present invention therefore envisions coating vascular stents with the peptides of the present invention.
  • Another possible application of the peptides of the present invention is the coating of surfaces found in the medical and dental environment.
  • Surfaces found in medical environments include the inner and outer aspects of various instruments and devices, whether disposable or intended for repeated uses. Examples include the entire spectrum of articles adapted for medical use, including scalpels, needles, scissors and other devices used in invasive surgical, therapeutic or diagnostic procedures; blood filters, implantable medical devices, including artificial blood vessels, catheters and other devices for the removal or delivery of fluids to patients, artificial hearts, artificial kidneys, orthopedic pins, plates and implants; catheters and other tubes (including urological and biliary tubes, endotracheal tubes, peripherably insertable central venous catheters, dialysis catheters, long term tunneled central venous catheters peripheral venous catheters, short term central venous catheters, arterial catheters, pulmonary catheters, Swan-Ganz catheters, urinary catheters, peritoneal catheters), urinary devices (including long term urinary devices, tissue bonding urinary devices, artificial urinary sphincters, urinary dilators), shunts (including ventricular or arterio-venous shunt
  • Surfaces found in the medical environment include also the inner and outer aspects of pieces of medical equipment, medical gear worn or carried by personnel in the health care setting. Such surfaces can include counter tops and fixtures in areas used for medical procedures or for preparing medical apparatus, tubes and canisters used in respiratory treatments, including the administration of oxygen, of solubilized drugs in nebulizers and of anesthetic agents. Also included are those surfaces intended as biological barriers to infectious organisms in medical settings, such as gloves, aprons and faceshields. Commonly used materials for biological barriers may be latex-based or non-latex based. Vinyl is commonly used as a material for non-latex surgical gloves. Other such surfaces can include handles and cables for medical or dental equipment not intended to be sterile. Additionally, such surfaces can include those non-sterile external surfaces of tubes and other apparatus found in areas where blood or body fluids or other hazardous biomaterials are commonly encountered.
  • Other surfaces related to health include the inner and outer aspects of those articles involved in water purification, water storage and water delivery, and those articles involved in food processing.
  • the present invention envisions coating a solid surface of a food or beverage container to extend the shelf life of its contents.
  • Surfaces related to health can also include the inner and outer aspects of those household articles involved in providing for nutrition, sanitation or disease prevention. Examples can include food processing equipment for home use, materials for infant care, tampons and toilet bowls.
  • the surface is comprised in a biological tissue, such as for example, mammalian tissues e.g. the skin.
  • the microbes may be comprised inside a particular organism, (e.g. intracellularly or extracellularly) for example inside a mammalian body or inside a plant.
  • the contacting may be effected by administering the peptides per se or by transfecting the cells of the organism with a nucleic acid construct which comprises a nucleic acid sequence which encodes the peptides of the present invention.
  • Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
  • Constitutive promoters suitable for use with some embodiments of the invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).
  • Inducible promoters suitable for use with some embodiments of the invention include for example the tetracycline-inducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804) or pathogen-inducible promoters.
  • Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen.
  • the nucleic acid construct (also referred to herein as an "expression vector") of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
  • a typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
  • such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • the nucleic acid construct of some embodiments of the invention typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
  • Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
  • the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
  • the other upstream promoter elements determine the rate at which transcription is initiated.
  • the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed.
  • cell type-specific and/or tissue- specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
  • Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site.
  • enhancer elements derived from viruses have a broad host range and are active in a variety of tissues.
  • the SV40 early gene enhancer is suitable for many cell types.
  • Other enhancer/promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.
  • CMV cytomegalovirus
  • the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation.
  • Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
  • Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.
  • the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA.
  • a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
  • the vector may or may not include a eukaryotic replicon.
  • the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
  • the expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • IRS internal ribosome entry site
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205.
  • exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
  • viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the type of vector used by some embodiments of the invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
  • bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovirus Autographa calif ornica nucleopolyhedro virus (AcMNPV) as described in Liang CY et al., 2004 (Arch Virol. 149: 51-60).
  • HTLV-I human T cell leukemia virus type I
  • AcMNPV Autographa calif ornica nucleopolyhedro virus
  • Recombinant viral vectors are useful for in vivo expression of the antimicrobial peptides of the invention since they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
  • nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lenti viruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • the polynucleotides may be optimized for increased expression in the transformed organism.
  • the polynucleotides can be synthesized using preferred codons for improved expression.
  • the pre- and propeptide sequences may be needed.
  • the propeptide segments may play a role in aiding correct peptide folding.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4- dichlorophenoxyacetate (2,4-D).
  • Additional selectable markers include phenotypic markers such as beta-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al.
  • GFP green fluorescent protein
  • one type of organism which may be transfected with expression constructs encoding peptides of the present invention is a plant.
  • the present invention contemplates, a plant, transformed by the antimicrobial RNA or peptides of the present invention (or an offspring thereof) rendered resistant to a plant-pathogenic microorganism.
  • the plant (or plant cells thereof) are transformed with a nucleic acid construct comprising a nucleic acid sequence which encodes an antimicrobial gene product (RNA or peptide) of the present invention located under the control of a suitable promoter capable of functioning in plant cells.
  • the transformed plant of the present invention can express, in its body, the protein having an antimicrobial activity according to the present invention.
  • plant encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and plant cells, tissues and organs.
  • the plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantee, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroe
  • the expression vector usable in the method of transforming plant cells with the gene of the present invention include pUC vectors (for example pUC118, pUC119), pBR vectors (for example pBR322), pBI vectors (for example pBI112, pBI221), pGA vectors (pGA492, pGAH), pNC (manufactured by Nissan Chemical Industries, Ltd.).
  • virus vectors can also be used.
  • the terminator gene to be ligated may include a 35S terminator gene and a Nos terminator gene.
  • the present invention contemplates use thereof for treating infection in a mammalian subject.
  • the peptides are used to treat a topical infection (i.e. infection of the skin) and are provided in a topical formulation.
  • the peptides are used to treat an infection inside the body.
  • the peptides (or polynucleotides encoding same) may be provided ex vivo or in vivo.
  • the present invention contemplates contacting cells with the peptides (or with expression constructs that encode the peptides) per se or as part of a pharmaceutical composition.
  • compositions of the present invention are administered to a subject in need thereof in order to prevent or treat a bacterial infection.
  • the term "subject in need thereof” refers to a mammal, preferably a human subject.
  • treating refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a pathogen infection.
  • pharmaceutical composition refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to peptides of the present invention accountable for the intended biological effect. It will be appreciated that a polynucleotide encoding a peptide of the present invention may be administered directly into a subject (as is, or part of a pharmaceutical composition) where it is translated in the target cells i.e. by gene therapy. Accordingly, the phrase “active ingredient” also includes such polynucleotides.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier,” which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • peptides of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.
  • antibiotics e.g. rifampicin, chloramphenicol and spectinomycin.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • the preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • rectal compositions such as suppositories or retention enemas
  • conventional suppository bases such as cocoa butter or other glycerides.
  • the preparation of the present invention may also be formulated as a topical compositions, such as a spray, a cream, a mouthwash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment, a paste and a gel.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p. l].
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • the presently disclosed peptides were uncovered based on clonability of their encoding genes and further using an algorithm based on the size of the peptide, the closeness of the gene encoding the peptide to a gene encoding a transporter polypeptide, the peptide having an N terminal signal peptide and the closeness of the gene encoding the peptide to a gene encoding a processing enzyme such as a peptidase.
  • a method of identifying a gene which encodes a product having putative anti-microbial activity comprising:
  • the gene is closer than 15 genes upstream or downstream from a gene that encodes a transporter polypeptide
  • the gene is closer than 15 genes up or downstream from a gene that encodes a peptidase
  • the gene is closer than 15 genes up or downstream from a gene that encodes a phage-, plasmid- or transposon- related gene.
  • Tables 5 and 6 herein below provide data for each of the presently disclosed peptide and indicates on what basis the peptides were determined as being antimicrobial.
  • Read mapping for each microbial genome that was sequenced and finished, map the original reads back on the finished, assembled genomic sequence. This mapping could be done by a sequence alignment tool, such as BLAST [Altschul, J Mol. Biol. 1990 Oct. 5; 215(3):403-10] or mummer [Delcher, Nucleic Acids Res. 2002 Jun. 1; 30(l l):2478-83].
  • BLAST Altschul, J Mol. Biol. 1990 Oct. 5; 215(3):403-10
  • mummer Deslcher, Nucleic Acids Res. 2002 Jun. 1; 30(l l):2478-83.
  • the two mates should be positioned such that the distance between them is approximately the relevant insert size (usually 2-8 kb in case of plasmid-carried inserts or 30-40 kb in case of fosmid-carried inserts).
  • the two mates should also be positioned such that one of them lies on the forward strand and the other on the reverse strand. Clones for which both mates have unambiguous positioning on the genomic sequence are deemed "mapped clones" and taken into further analysis.
  • library refers to a set of clones containing DNA fragments randomly generated by fragmentation of a genome or large DNA fragment, inserted into a suitable plasmid vector and cloned into a suitable host organism, such as E. coli. Sequencing of clones in a library involves carrying out sequence reactions to sequence the beginning and the end of the DNA fragment inserted into each sequenced clone, also referred to as "end sequences", or "reads".
  • the genome or large DNA fragments may be from any eukaryote, including human, mammal, plant or fungus, or prokaryote, including bacteria, virus or archaea.
  • read refers to a sequence corresponding to stretches of nucleotide sequence of on average 200-1000 bp in length, acquired from a single end- sequencing event of a clone in a genomic library.
  • sister reads or "clone mates” refers to two reads that come from the beginning (forward read) and the end (reverse read) of the same cloned DNA fragment.
  • mapping refers to finding the correct position of a read on an already assembled genomic sequence.
  • clone mapping refers to finding the correct position of two sister reads on an already assembled genomic sequence.
  • shotgun sequencing refers the sequencing strategy whereby an entire genomic library is sequenced and assembled as described by the methods found at URL: www.worldwidewebjgidotdoedotgov/sequencing/strategy.
  • the advantage of shotgun sequencing is that a majority, if not all, of the genomic sequence will be represented by random clones about 5-20 times, depending on the number and the sizes of clones in the library.
  • clone coverage refers to the number of clones in a library that span a particular position in a genome. A position in the genome is considered as covered by a clone if it is found between the first position of the forward- strand clone mate and the last position of the reverse strand clone mate.
  • Low clone coverage refers to a particular position or a region having statistically significant under-representation of clones than expected by chance.
  • gap refers to a region of the genome or the large DNA fragment where there is an absence or low coverage.
  • the term, "finished” when used referring to a genome, or large DNA fragment, refers to when all or most gaps in the sequence have been closed following additional specific sequencing reactions, and assembly of the final consensus sequence is completed.
  • analysis of clone coverage may be effected in genomic DNA known to be protein coding based on gene-prediction software that detect open reading frames (ORFs)[see for example Sorek Nature Reviews Genetics, 11(1):9-16 (2010)].
  • ORFs open reading frames
  • these software are error prone, and frequently fail to detect protein- coding genes that have small sizes, i.e., peptides [Sorek Nature Reviews Genetics, 11(1):9-16 (2010)]. Therefore, the present invention further contemplates searching intergenic regions as well for areas that are not covered by any single clone.
  • Prediction of signal peptides may be carried out by using various software for example using the signal server (worldwidewebdotcbsdotdtudotdk/services/SignalP/).
  • Identification of sequences encoding peptidases may be carried out by examining the gene annotations in the NCBI reference genome.
  • the MEROPS database of peptidases (meropdotssangerdotacdotuk/) may be used to determine if a gene is a suspected peptidase.
  • Identification of sequences encoding transporters may be carried out by examining the gene annotations in the NCBI reference genome for genes annotated as "transporter”.
  • Identification of mobile element sequences may be carried out by examining the gene annotations in the NCBI reference genome for genes whose annotations include one or more of the words “plasmid”, “phage”, “transposase”, “transposon”, “integrase”, “recombinase”.
  • cell- free protein synthesis could be used to translate the DNA sequence of each gene into protein.
  • genes can be expressed in an eukaryotic or a prokaryotic expression system as mentioned above. Proteins can be applied on various pathogenic and non-pathogenic bacteria to determine the spectrum of activity and whether they have a bactericidal or bacteristatic effect. Minimal inhibitory concentration (MIC) can be determined by testing the growth inhibition activity of serial dilutions of the protein.
  • MIC minimal inhibitory concentration
  • selected genes can be cloned into a vector that contains a tightly regulated inducible promoter, such that the expression of the gene is induced only after a specific molecule ("inducer") was added to the growth media.
  • a vector that contains a tightly regulated inducible promoter such that the expression of the gene is induced only after a specific molecule ("inducer") was added to the growth media.
  • Such vectors could be inserted into E coli without killing it, as the gene product will only be expressed in the cell following induction. Growth of E. coli can be tested before and after induction to determine if the gene has a growth inhibition effect.
  • in vitro antimicrobial assays that can be used include, for example, the addition of varying concentrations of the antimicrobial composition to paper disks and placing the disks on agar containing a suspension of the pathogen of interest. Following incubation, clear inhibition zones develop around the discs that contain an effective concentration of the antimicrobial polypeptide (Liu et al. (1994) Plant Biology 91: 1888-1892, herein incorporated by reference). Additionally, micro spectrophotometrical analysis can be used to measure the in vitro antimicrobial properties of a composition (Hu et al. (1997) Plant Mol. Biol. 34:949-959 and Cammue et al. (1992) J. Biol. Chem.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • peptide was selected and synthesized in vitro (Peptide 2.0) in a crude, non-purified form.
  • the peptide was: MNRHEIGYVFYVKIYIMITLWIIIVEYSV (SEQ ID 97). Increasing concentrations of this peptide were incubated overnight with the pathogenic gram positive bacteria Enterococcus fecalis in quadruplicate, and optical density was measured the next morning.
  • Figure 1 shows that increasing concentration of the peptide result in decreased growth of the bacteria. Since the peptide was tested in a crude non-purified form, its actual inhibitory concentrations are probably much higher.

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Abstract

La présente invention concerne des compositions antimicrobiennes, comprenant un véhicule et en tant que substance active un polypeptide isolé comprenant une séquence d'acides aminés choisie dans le groupe constitué de SEQ ID NO: 1-113.
PCT/IL2013/050083 2012-01-30 2013-01-30 Agents antimicrobiens WO2013114363A2 (fr)

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Cited By (2)

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US10226537B2 (en) 2013-12-18 2019-03-12 The University Of Nottingham Transduction
US11672870B2 (en) 2013-12-18 2023-06-13 University Of Nottingham Transduction

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