WO2007064903A1 - Cathelicidines du poulet tronquées et optimisées ('fowlicidines') et procédés d'utilisation de celles-ci - Google Patents

Cathelicidines du poulet tronquées et optimisées ('fowlicidines') et procédés d'utilisation de celles-ci Download PDF

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WO2007064903A1
WO2007064903A1 PCT/US2006/046022 US2006046022W WO2007064903A1 WO 2007064903 A1 WO2007064903 A1 WO 2007064903A1 US 2006046022 W US2006046022 W US 2006046022W WO 2007064903 A1 WO2007064903 A1 WO 2007064903A1
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fowlicidin
peptide
peptides
seq
lps
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PCT/US2006/046022
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English (en)
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Zhang Guolong
Xiao Yanjing
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The Board Of Regents For Oklahoma State 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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention generally relates to antimicrobial peptides.
  • the invention provides optimized truncated versions of chicken cathelicidins ("fowlicidins").
  • Antimicrobial peptides also known as natural antibiotics
  • Antimicrobial peptides comprise a large group of molecules that are capable of killing a broad spectrum of pathogens with similar activities against both antibiotic-susceptible and -resistant bacterial strains and extremely low risks of developing resistance (Zasloff, M. Nature 2002, 415:389). More desirably, many have capacity to bind bacterial endotoxin and neutralize bacterium-induced inflammatory response. Because of the dual capability to kill bacteria and neutralize endotoxins, these antimicrobial peptides hold great promise as a new class of antimicrobial and anti-sepsis agents (Finlay, B.B., and R.E. Hancock. Nature Reviews Microbiology 2004, 2: 497).
  • Van Kijk et al. disclose a novel chicken cathelicidin-like antimicrobial protein. However, a detailed structure-function analysis of the protein is not provided, and no testing or optimization of the protein sequence was undertaken.
  • the present invention is based on the discovery of antibiotic cathelicidins from chickens ("fowlicidins”), and elucidation of the structural and functional relationships of their amino acid sequences to their bactericidal properties. Accordingly, the invention provides novel peptides whose primary sequence is based on or derived from that of the fowlicidins, but optimized with respect to antibiotic and toxicity properties, hi general, the optimized peptides are truncated versions of the full length fowlicidins. The optimized, truncated peptides retain excellent bactericidal properties but display reduced toxicity to mammalian cells. In some cases, conservative amino acid substitutions are introduced into the sequences of the peptides.
  • the amino acid sequence of the peptide is selected from the group consisting of W-P-L-V-I-R-T-V-I-A-G-Y-N-L-Y-R-A-I-K-K (SEQ ID NO: 9) ; W-P-L-V-P-V-A-I-N-T-V-A-A-G-I-N-L-Y-K-A-I-K-K (SEQ ID NO: 66) ;
  • W-P-L-V-I-R-T-V-I-A-G-L-N-L-Y-R-A-I-K-K-K (SEQ ID NO: 11) ; W-P-L-V-I-R-T-V-I-A-G-L-N-L-L-R-A-I-K-K-K (SEQ ID NO: 12) ; W-P-L-V-I-R-T-V-I-A-G-L-N-L-L-R-A-I-K-K-K (SEQ ID NO: 13) ; W-P-L-V-I-R-T-V-I-A-G-G-Y-N-L-Y-R-A-I-K-K-K (SEQ ID NO: 14); W-P-L-V-I-R-T-V-I-A-P-Y-N-L-Y-R-A-I-K-K-K (SEQ ID NO: 16); and
  • the present invention provides a method of treating a bacterial infection in a patient in need thereof.
  • the method comprises the step of administering to the patient a peptide in a quantity sufficient to treat the bacterial infection, the peptide having amino acid sequence:X 0 -X r P-L- V-X 2 - 1-X 3 -T-V-X 4 -A-X 5 -X 3 -X 7 -N-L-X 8 -X 9 -A-I-X 10 -X 1 ,-
  • X 0 the tripeptide KRF or is absent;
  • X 1 W or L;
  • X 2 the tripeptide PVA or is absent;
  • X 3 R or N;
  • X 4 I or A;
  • X 5 G or P;
  • X 6 G or is absent;
  • X 7 Y, L or I;
  • X 8 Y or L;
  • X 9 R or K;
  • X 10 R, K or absent;
  • X n R, K or absent;
  • X 12 R, K or absent.
  • the amino acid sequence of the peptide is selected from the group consisting of
  • W-P-L-V-I-R-T-V-I-A-G-L-N-L-L-R-A-I-K-K-K (SEQ ID NO: 12) ; W-P-L-V-I-R-T-V-I-A-G-L-N-L-L-R-A-I-K-K-K (SEQ ID NO: 13) ;
  • the carboxyterminus of the peptide is amidated.
  • the bacterial infection is caused by antibiotic resistant bacteria.
  • the invention provides a method of killing or damaging a bacterium.
  • the amino acid sequence of the peptide is selected from the group consisting of W-P-L-V-I-R-T-V-I-A-G-Y-N-L-Y-R-A-I-K-K-K (SEQ ID NO: 9) ;
  • W-P-L-V-P-V-A-I-N-T-V-A-A-G-I-N-L-Y-K-A-I-K-K-K (SEQ ID NO: 66) ; W-P-L-V-I-R-T-V-I-A-G-L-N-L-Y-R-A-I-K-K-K (SEQ ID NO: 11) ; W-P-L-V-I-R-T-V-I-A-G-L-N-L-L-R-A-I-K-K-K (SEQ ID NO: 12) ;
  • the carboxyteraiinus of the peptide is amidated.
  • the bacterial infection is caused by antibiotic resistant bacteria.
  • the invention further provides a peptide having amino acid sequence L-V-Q-R-G-R-F-G-R-F-L-R-K-I-R-R-F-R (SEQ ID NO: 43) or
  • R-R-F-R-P-K-V-T-I-T-I-Q-G-S-A-R-F (SEQ ID NO: 44).
  • the carboxyterminus of the peptide is amidated.
  • the invention further provides a method of treating a bacterial infection in a patient in need thereof.
  • the method comprises the step of administering to the patient a peptide in a quantity sufficient to treat the bacterial infection, the peptide having amino acid sequence
  • R-R-F-R-P-K-V-T-I-T-I-Q-G-S-A-R-F (SEQ ID NO: 44).
  • the carboxyterminus of the peptide is amidated.
  • the bacterial infection is caused by antibiotic resistant bacteria.
  • the present invention also provides a method of killing or damaging a bacterium.
  • the method comprises the step of contacting the bacterium with a peptide in a quantity sufficient to kill or damage the bacterium, the peptide having amino acid sequence
  • FIG. 1 Multiple sequence alignment of chicken fowlicidins with representative mammalian cathelicidins.
  • Fowlicidins are aligned with classic cathelicidins (human LL37, mouse CRAMP, porcine Protegrin-1, and bovine Indolicidin) and a group of distantly related neutrophilic granule proteins (NGP) in the pig (pNGP), cow (bNGP), rat (rNGP), and rabbit
  • classic cathelicidins human LL37, mouse CRAMP, porcine Protegrin-1, and bovine Indolicidin
  • NGP neutrophilic granule proteins
  • the continuous genomic contig containing chicken fowlicidins was obtained by annotation of three whole-genome shotgun sequences (AADNOl 005055, AADNOl 005056, and AADNOl 081708) and additional genomic sequences obtained by PCR and vectorette PCR (see “Experimental Procedures").
  • the direction of transcription of each gene is indicated by the arrow, hi Panel A, the chicken cathelicidin cluster consists of three fowlicidin genes each containing four exons (E) shown as solid rectangles.
  • CAKV gene Located between fowlicidin- 1 and fowlicidin-2 genes is chicken CamKV gene, which is homologous to vesicle-associated, calmodulin kinase-like kinase (NP_076951).
  • Panel B relative position of each gene is indicated by a solid rectangle or vertical line.
  • chicken fowlicidins similar to neutrophilic granule proteins (NGP), are located closely adjacent to an evolutionarily conserved gene (KLHLl 8), but a NGP -like protein is missing in the dog or human genome.
  • Figure 3 Phylogenetic analysis of cathelicidins.
  • the tree was constructed by the neighbor-joining method based on the proportion difference (p-distance) of aligned amino acid sites of the full-length peptide sequences. A total of 1000 bootstrap replicates were used to test the reliability of each branch. Numbers on the branches indicate the percentage of
  • FIG. 4A-C Antibacterial properties of fowlicidins.
  • E. coli ATCC 25922 was incubated with peptides in 10 mM phosphate buffer, pH 7.4 at 37 0 C and surviving bacteria were plated onto trypticase soy agar plates and quantitated as CFU/ml following overnight incubation.
  • A dose-dependent killing of E. coli by fowlicidins and an ovine cathelicidin (SMAP -29). Bacteria were exposed to indicated peptide concentrations for 2 h followed by quantitative CFU assays. The MIC 50 value is indicated as a dotted line.
  • B Time-dependent killing of E. coli by peptides at MIC 50 concentrations.
  • FIG. 5A-C Cytolytic activities of fowlicidins.
  • A hemolytic activity of fowlicidins and SMAP-29 to chicken erythrocytes.
  • B hemolytic activity of fowlicidins and SMAP-29 to human erythrocytes.
  • C cytotoxicity of fowlicidins and SMAP-29 to MDCK cells. Cells were incubated with serially diluted peptides for 24 h in serum-free medium, followed by measurement of the viability of cells by an alamarBlue dye-based, colorimetric method.
  • the EC 50 values are indicated as dotted lines. Data shown are means ⁇ SEM of 2-3 independent experiments.
  • FIG. 6A-D Neutralization of lipopolysaccharide (LPS) by fowlicidins.
  • A LPS binding by fowlicidins.
  • Chromogenic Limulus amebocyte lysate assay was used to evaluate the binding of fowlicidins to LPS from E. coli Ol 11 :B4.
  • the EC 50 value is indicated as a dotted line. Data shown are means ⁇ SEM of three independent experiments.
  • B Hill plot of LPS binding by fowlicidins showing the binding affinity. The plot was graphed from the means in panel
  • C Blockage of LPS-induced IL-l ⁇ gene expression by fowlicidins.
  • D Blockage of LPS-induced CCL-2/MCP-1 gene expression by fowlicidins.
  • RAW264.7 cells were pretreated for 30 min with increasing concentrations (1, 5, and 20 ⁇ M) of peptides, and then stimulated with 100 ng/ml LPS or left untreated for 4 h.
  • Total RNA was isolated from cells and subjected to real time RT-PCR. Data shown are a representative of two independent experiments with similar results.
  • Figure 7 Solution structure of fowlicidin-1. A ribbon stereo-diagram of the restrained minimized average structure of fowlicidin-1.
  • FIG 8A and B Helical wheel projections of the central helical regions (residues 6-23) of fowlicidin-1 (A) and its substitution mutant, fowlicidin-1 -K 7 L 12 K 14 L 16 K 18 (B).
  • the representation shows the amphipathic structure of the helical region. Charged residues are indicated in black background, and polar uncharged residues are in gray background. The mutated residues are circled. Notice a significant enhancement in amphipathicity of the mutant peptide relative to the native peptide.
  • Figure 9A and B LPS-binding isotherms of the deletion (A) and substitution mutants (B) of fowlicidin-1.
  • the EC50 value indicated by a dotted line in each panel, was defined as the peptide concentration that inhibited LPS-mediated procoagulant activation by 50%.
  • A ⁇ fowlicidin-1 (1-26); O, fowlicidin-1 (8-26); ⁇ , fowlicidin-1 (1-15); A, fowlicidin-1 (5-26); ⁇ , fowlicidin-1 (1-23); •, fowlicidin-1 (8-26) + fowlicidin-1 (1-15).
  • panel B ⁇ fowlicidin-1 (1-26); A, fowlicidin-1 -Ll 6; and T, fowlicidin-1 -KLKLK. Data shown are means ⁇ SEM of three independent experiments.
  • FIG. 10 Alignment of representative linear ⁇ -helical antimicrobial peptides demonstrating the conservation of a kink induced by glycine near the center.
  • Putatively mature fowlicidin-1 sequence is aligned with representative cathelicidins (mouse CRAMP, rabbit CAP 18, bovine BMAP34 and BMAP28 , sheep SMAP34 and SMAP29, and porcine PMAP37) as well as three insect peptides (fruit fly cecropin A 1 , a putative porcine cecropin P 1 , and honey bee melittin).
  • Dashes are inserted to optimize the alignment and conserved residues are shaded.
  • each peptide aligned has an a-helix N-terminal to the conserved glycine (boxed) near the center, followed by either a helical or unstructured tail.
  • CRAMP which has a kink at GIy 11 instead of GIy 18 .
  • SEQ ID NOS. for the peptides are given in Table X.
  • FIG. 11 Schematic drawing of the distribution of functional determinants of fowlicidin-1 (SEQ ID NO: 2). Note that the C-terminal helix from GIy 16 to He 23 is indispensable for antibacterial, cytolytic, and LPS-binding activities, whereas the three residues (Val 5 -Pro 7 ) in the N-terminal unstructured region constitute the core of the second determinant that is critically involved in cytotoxicity and LPS binding, but less significant in the bactericidal activity. The N-terminal helix (Leu 8 - Ala 15 ) also presumably facilitates the interactions of the
  • FIG. 12 Antibacterial activity of fowlicidin-2 and its analogs.
  • Mid-log phase bacteria (5 x 10 5 CFU/ml) were incubated overnight with serial twofold dilutions of peptides in the assay medium containing 20% TSB, 25 mM NaHCO 3 , and 1 mM NaH 2 PO 4 in the presence or absence of 100 mM NaCl.
  • the MIC value of individual peptides against each bacterial strain was determined as the peptide concentration that gave no visible bacterial growth after overnight incubation. The experiments were repeated at least three times and the ranges of MICs were presented.
  • Figure 13 Effect of serum on the antibacterial activity of fowlicidin-2 and its analogs. The antibacterial activity was measured by the radial diffusion assay using S. aureus ATCC
  • FIG. 14A-F Permeabilization of E. coli cytoplasmic membrane of fowlicidin-2 and its analogs.
  • E. coli ML-35p was incubated with each peptide and 1.5 mM of a chromogenic substrate (ONPG) in the presence and absence of 100 mM NaCl. All peptides were used at 0.5 MIC, except for fowlicidin-2(19-31), which was applied at 16 ⁇ M.
  • ONPG chromogenic substrate
  • FIG. 15 A-C Cytotoxicity of fowlicidin-2 and its analogs. Hemolytic activity was evaluated by incubating individual peptides in serial two-fold dilutions with freshly isolated human erythrocytes in the presence (A) or absence of 10% FBS (B) at 37 0 C for 2 h, followed by measuring the released hemoglobin at 405 nm.
  • C toxicity to human Caco-2 cells. Peptides at 100 ⁇ M were incubated with Caco-2 cells at 37 0 C for 24 h, and cell viabilities were measured by an Alamar-blue based, colorimetric method. Data shown are means ⁇ SEM of 2-3 independent experiments.
  • FIG 16A-C Neutralization of LPS by fowlicidin-2 and its analogs.
  • RAW 264.7 cells were pretreated with or without three different concentrations of peptides (1, 5, and 20 ⁇ M) for 30 min, followed by stimulation with 0.1 ⁇ g/ml LPS at 37 0 C for 4 h.
  • Total RNA was isolated, and the gene expression levels of IL-IB, CCL2/MCP-1, and CCL3/MIP-la were evaluated by real-time PCR. Data shown are means ⁇ SEM of two independent experiments.
  • Figure 17 A and B Tertiary structure of fowlicidin-3 in 50% TFE.
  • A Ribbon diagram of the minimized average structure of fowlicidin-3.
  • B Sequence alignment of fowlicidin-1 (SEQ ID NO: 2) and fowlicidin-3 (SEQ ID NO: 18). Dashes are created to maximize the alignment, and the total amino acid residue numbers are also indicated. Vertical bars connecting sequences denote identities, whereas colons mean similarities. The conserved glycine is shaded.
  • FIG. 18 Permeabilization of bacterial cytoplasmic membrane by fowlicidins.
  • E coli ML-35p, a lactose permease-deficient strain with constitutive production of ⁇ -galactosidase in the cytosol, was diluted to 2.5-5 x 10 7 CFU/ml and incubated with 1 ⁇ M of fowlicidin-3 or -1 in 10 mM sodium phosphate, pH 7.4, in the presence and absence of 100 mM NaCl at 37 0 C.
  • FIG. 19A and B Effect of serum on the antibacterial activity of fowlicidins by radial diffusion assay.
  • A One ⁇ g of fowlicidin-3 and -1, diluted in 0.01% acetic acid with and without 50% human or chicken serum, was added to the wells of the underlay gel containing S. aureus ATCC 25923 (4 x 10 5 CFU/ml). After overnight incubation, bacterial clearance zones were recorded, and relative antibacterial activities (%) in the presence of serum were calculated relative to the activities without serum.
  • panel B open bars represent no serum controls, whereas striped and solid bars are 50% human and chicken serum, respectively.
  • Figure 20 A and B Toxicity of fowlicidins to MDCK cells (A) and human erythrocytes (B) in the presence and absence of 10% FBS. 50% effective concentrations (EC 50 ) were indicated as dotted lines in both panels. Data shown are means ⁇ SEM of 2-4 independent experiments.
  • Figure 21 A and B Inhibition of LPS-induced expression of IL-l ⁇ and CCL3/MIP-la in
  • RAW264.7 cells RAW264.7 cells.
  • Cells were pretreated for 30 min with and without fowlicidin-3 (0.5, 2.5, and 10 ⁇ M) or fowlicidin-1 (2.5 and 10 ⁇ M) in duplicate, followed by stimulation for another 4 h with 100 ng/ml LPS.
  • Total RNA was then isolated and subjected to real-time RT-PCR analysis. Data shown are means ⁇ SEM of two independent experiments.
  • Figure 22 Toxicity of fowlicidin-1 analogs to human Caco-2 epithelial cells.
  • FIG. 23 Rational design of fowlicidin-1 analogs.
  • the amino acid sequences of exemplary peptides based on fowlicidin-1 are depicted.
  • the SEQ ID NOS. for the peptides are given in Table X.
  • the present invention provides novel, optimized antibiotic peptides, the amino acid sequences of which are derived from or based on three newly identified and characterized chicken cathelicidins ("fowlicidins").
  • the fowlicidins are denominated fowlicidin-1, fowlicidin-2 and fowlicidin-3.
  • the initial identification and characterization of the three chicken fowlicidins is described herein, as is the development of optimized forms of the originally identified, full length peptides.
  • the optimized versions are truncated forms of the original sequences that display significantly lower toxicity than the parent peptides, and yet retain excellent antibiotic activity. In some cases, amino acid substitutions are also introduced.
  • the optimized peptides are modified forms of fowlicidin-1 and/or fowlicidin-3, the primary sequences of which are similar (see Figures 1 and 17B, and Table X).
  • This category of truncated, optimized peptide has the general formula:
  • the tripeptide may or may not be present at this position in the peptide);
  • X 3 R or N;
  • X 4 I or A;
  • X 5 G or P;
  • X 6 G or is absent;
  • X 7 Y, L or i;
  • X 8 Y or L;
  • X 9 R or K;
  • X 10 R 3 K or absent;
  • X 11 R, K or is absent; and
  • X 12 R, K or is absent.
  • the antibiotic peptides are truncated variants of fowlicidin-2 (see Tables VI and X), and have sequences corresponding to amino acids 1-
  • the optimized antibiotic peptides of the invention are truncated versions (peptide fragments) of the three original fowlicidins that are identified herein.
  • the amino acid sequences of the optimized, truncated antibiotic peptides correspond directly to foreshortened portions or segments of the primary amino acid sequence of the original sequences of the fowlicidin peptides, this need not be the case.
  • the truncated sequences may be further modified.
  • the amino acid sequence of the original i.e. "natural" or "native" or
  • parent or 'wildtype is the amino acid sequence of the fowlicidin as it occurs in nature.
  • Those of skill in the art will recognize that such natural sequences of the three fowlicidins may also display variability, e.g. due to genotypic variations between chickens (for example, between breeds of chickens), or among individual chickens due to spontaneous mutations, etc. All such native sequences of fowlicidins 1-, 2- and 3- as isolated from nature are intended to be comprehended by the present invention, and may serve as the basis for the truncated, optimized peptides of the invention.
  • the sequences of the truncated, optimized peptides of the invention are "based on” or “derived from” or "from” such original sequences.
  • sequence of an optimized peptide is the result of modifications of the original, native sequences, e.g. by truncation of the original sequence, by amino acid substitutions, deletions, insertions, or chemical modification.
  • the amino acid sequence of a truncated, optimized antibiotic peptide as disclosed herein may be altered somewhat from that of a native fowlicidin on which it is based and still be suitable for use in the present invention. While certain specific variations are described, e.g. in Formula 1 where X 1 may be W or L, other variations may also occur to those of skill in the art. For example, certain conservative amino acid substitutions may be made without having a deleterious effect on the ability of the peptide to function as an antibiotic and without increasing toxicity, and in fact may lead to an increase in antibiotic activity and/or a decrease in toxicity. The resulting peptide may be referred to as a "conservative variant".
  • substituted sequences will be at least about 50% identical to the corresponding sequence in the native protein, preferably about 60 to 70, or even 70 to 80, or 80 to 90% identical to the wild type sequence, and preferably about 95 to about 100% identical.
  • certain other modifications e.g. chemical modifications
  • the carboxyl terminus of the peptides maybe amidated; reactive groups maybe sufonylated, lipidated, etc.; or L-amino acids of these sequences can be changed to or substituted with D-amino acids.
  • other variations of the sequences disclosed herein may also be carried out, e.g.
  • amino acid sequences of the peptides of the invention need not contain the precise number of residues as the exemplary optimized peptides disclosed herein. Certain deletions or additions may be tolerated, so long as the resulting peptide is bactericidal and of sufficiently low toxicity.
  • Chimeric polypeptides that contain more than one (i.e. multiple) antibiotic peptide sequence within a polypeptide are also envisioned.
  • the multiple peptides may be in tandem within a single, linear polypeptide chain, and may be separated, for example, by spacer peptides, many examples of which are known in the art.
  • the structure of such a chimera may be branched, or a combination of linear and branched.
  • the peptides that make up the chimera may be the same or different.
  • the optimized peptides of the invention are "antimicrobial” or “antibiotic” or “bactericidal”.
  • antibiotic or “bactericidal” we mean that the peptides exhibit a minimum inhibitory concentration in the low micromolar concentration range ( ⁇ 10 ⁇ M) when measured by standard broth microdilution assay as recommended by the Clinical and Laboratory Standards Institute (CLSI).
  • the peptides are non-toxic or of low toxicity.
  • non-toxic and/or of "low toxicity we mean that lysis of 50% of erythrocytes or kiling of 50% mammalian cells occurs at a concentration of >50 ⁇ M peptide.
  • the antimicrobial peptides of the invention maybe used in a variety of ways. As their designation suggests, they may be used as antimicrobial agents, e.g. as bactericidal agents, in order to kill or damage bacteria. Suitable scenarios for such a use of the antimicrobial peptides include but are not limited to: treatment of established bacterial infections (for example in bacterial hosts or potential bacterial hosts such as humans, other mammals, or other living organisms that are susceptible bacterial infections); or prophylactically for the prevention of bacterial infections in such hosts (e.g. the antimicrobial peptides may be administered to individuals whose immune systems are compromised and who may be susceptible bacterial infections); or administered topically to areas of a host that are susceptible to infection, e.g.
  • a wide variety of bacterial infections may be treated or prevented by administration of the antimicrobial peptides of the present invention.
  • bacteria include but are not limited to: coliform bacteria such as Escherichia coli; Salmonella specis, e.g. S. typhimurium, S. enteritidis, and S. choleraesuis; Klebsiella species, e.g. K. pneumoniae;
  • Pseudomonas species e.g. P. aeruginosa
  • Listeria species e.g. L. monocytogenes
  • Staphylococcus species e.g. S. aureus
  • Mycobacterium species e.g. M. tuberculosis Enterococcus species, e.g. E.faecatis
  • Campylobacter species e.g. C. jejuni, C. coli, and C. fetus
  • Clostridium species e.g. C. perfringens, C. difficile, C. tetani, and C.
  • the peptides of the invention may be used to combat bacteria that are resistant to conventional antibiotics, such as MRSA (methicillin-resistant Staphylococcus aureus), VRSA (Vancomycin-resistant S. aureus), VRE (Vancomycin-Resistant Enterococcus),
  • MRSA methicillin-resistant Staphylococcus aureus
  • VRSA Vancomycin-resistant S. aureus
  • VRE Vancomycin-Resistant Enterococcus
  • Penicillin-Resistant Enterococcus Penicillin-resistant Streptococcus pneumoniae, isoniazid/rifampin-resistant Mycobacterium tuberculosis and other antibiotic-resistant strains of E. coli, Salmonella, Campylobacter, and Streptococci.
  • antibiotic-resistant or “drug-resistant” or “multidrug-resistant” or by other similar terms that are well understood in the art.
  • While both Gram-negative and Gram-positive bacterial infections may be treated with the peptides of the invention, certain of the optimized peptides are especially active against one or the other category of bacteria, e.g. fowlicidin-l(l-15) is more active against Gram-negative than Gram-positive bacteria. Such selectivity may advantageous when the identity of the bacterial infection being treated is known.
  • antibiotic peptides of the invention may be treated with the antibiotic peptides of the invention.
  • diseases or conditions include but are not limited to cystic f ⁇ brosis-associated chronic respiratory infections, inflammatory bowel diseases (particularly Crohn's disease), acne, and catheter- related infections, and others, hi addition, the antibiotic peptides of the invention may be administered prophylactically to patients who are at risk for developing bacterial infections, e.g. those with compromised immune systems due to, for example, HIV infection, chemotherapy, etc.
  • the present invention also provides new compositions for use in administration to patients (generally humans or mammals).
  • the compounds included one or more of the antimicrobial peptides of the invention.
  • the compositions include one or more substantially purified antimicrobial peptides as described herein, and a pharmacologically suitable carrier, hi some embodiments, a single type of antimicrobial peptide may be administered. In other embodiments a "cocktail" or mixture of different antimicrobial peptides maybe administered.
  • the preparation of such compositions for use as antimicrobial agents is well known to those of skill in the art.
  • compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders, pastes, ointments, suppositories, and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared.
  • the preparation may also be emulsified, or incorporated into nanoparticles, micorparticles, biodegradable polymers such as polylactide (PLA) and its copolymers with glycolide (PLGA), etc.
  • the active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof.
  • the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added.
  • the composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration.
  • the final amount of antimicrobial peptides in the formulations may vary. However, in general, the amount in the formulations will be from about 1-99%, weight/volume.
  • the antimicrobial peptide compositions (preparations) of the present invention may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, inhalation, orally, intranasally, by ingestion of a food product containing the antimicrobial peptide, topically, as eye drops, via sprays, incorporated into dressings or bandages (e.g. lyophilized forms may be included directly in the dressing), etc.
  • the mode of administration is topical or orally or by injection.
  • the compositions may be administered in conjunction with other treatment modalities such as substances that boost the immune system, various chemotherapeutic agents, other antibiotic agents, and the like.
  • the present invention also provides a method of killing or damaging bacteria.
  • the method involves contacting the bacteria with the antimicrobial peptides of the invention.
  • the bacteria will be killed outright, and signs or symptoms of bacterial colonization or infection will be completely eradicated.
  • those of skill in the art will recognize that much benefit can be derived even if all bacteria in a population are not killed outright.
  • the ability of the bacteria to carry out metabolic reactions may be slowed or otherwise attenuated by exposure to the antimicrobial peptides, or the reproductive potential of the bacteria may be decreased. All such lessening of the bacteria's ability to flourish in an environment in which they would typically establish colonies and persist may be of benefit to a host organism in need of treatment with the antimicrobial peptides of the invention.
  • treatment of bacterial host organisms or potential bacterial host organisms is contemplated (e.g. humans and other mammals, so that veterinary uses are also included), other uses of the antimicrobial peptides of the invention will also occur to those of skill in the art.
  • the treatment of surfaces for food preparation or of edible substances that might otherwise become colonized by bacteria use in cleansing products such as soaps, detergents, lotions, etc.
  • cleansing products such as soaps, detergents, lotions, etc.
  • Cationic antimicrobial peptides comprise a large group of gene-encoded molecules that have been discovered in virtually all species of life, playing a critical role in innate host defense and disease resistance (1-4).
  • Cathelicidins are most abundantly present in the granules of phagocytic cells and also to a lesser extent in many other cell types such as mucosal epithelial cells and skin keratinocytes (7-9).
  • most cathelicidin precursors are proteolytically cleaved to release the cathelin domain and the C-terminal mature peptides with antimicrobial activities, although the unprocessed or differentially processed forms are often found in the biological fluids where cathelicidins are expressed (8,9).
  • the physiological role of the cathelin domain or uncleaved precursors remains elusive, but is more likely to be involved in immune modulation other than just bacterial killing (10,11).
  • cathelicidins are actively involved in various phases of host defense.
  • Certain cathelicidins are found to chemoattract and activate a variety of immune cells, inhibit NADPH oxidase, kill activated lymphocytes, and promote angiogenesis and wound healing (1,8,9).
  • NADPH oxidase oxidase-catalyzed oxidase-catalyzed oxidase-cataly-like cells
  • kill activated lymphocytes and promote angiogenesis and wound healing (1,8,9).
  • aberrant expression of cathelicidins are often associated with various disease processes. For example, LL-37/hCAP-18 deficiency correlates with recurrent skin infections in atopic dermatitis patients (12) and chronic periodontal disease in morbus Kostmann patients (13).
  • cathelicidin gene CRAMP
  • mice resulted in a loss of protection against skin infection by Group A Streptococcus (14) or oral infection with murine enteric pathogen Citrobacter rodentium (15).
  • cathelicidins conferred enhanced protection against experimental infections (16-20).
  • cathelicidins kill bacteria appears to be mediated through physical interactions with negatively charged microbial membrane phospholipids, followed by membrane disruption (3,21). Many cathelicidins exhibit LPS-binding activity, and the binding affinity is often positively correlated with their antibacterial activity (7).
  • cathelicidins have been discovered in a range of mammalian species (8,9). Li contrast to the vast majority of "classic” cathelicidins, Pl 5 in rabbits (22) and neutrophilic granule protein (NGP) 1 in mice (23) are distantly related to classic cathelicidins with less homology in the cathelin domain. Hagfish was also found recently to contain two cathelicidin-like sequences (24). However, the evolutionary relationship of these cathelicidins remains uncertain.
  • the missing first intron sequence of the fowlicidin-3 gene i.e., the gap between AADNOl 005055 and AADNOl 005056, was cloned from chicken genomic DNA by PCR using primers (Forward: 5'-GCTGTGGACTCCTACA ACCAAC-3'(SEQ ID NO: 50; Reverse: 5'-TTGAGGTTGTGCA GGGAGCTGA-3'(SEQ ID NO: 51) located in two flanking exons. All PCR products were recovered from agarose gel, ligated into pGEM T-Easy Vector (Promega, USA), and sequenced from both directions.
  • YRAIKKK (SEQ ID NO: 2) and -2 (LVQRGRFGRFLRKIRRFRPKVTITIQGSARF (SEQ ID NO: 17) were chemically synthesized by SynPep (Dublin, CA), and a sheep cathelicidin, SMAP-29 (RGLR RLGRKIAHGVKKYGPTVLRIIRIA-NH2 (SEQ ID NO: 56) was synthesized by Bio-Synthesis (Lewisville, TX) by the standard solid-phase synthesis method. AU peptides were purified to >95% purity through reverse phase-HPLC.
  • Bacterial Culture - Gram-negative bacteria ⁇ Escherichia coli ATCC 25922, E. coli O157:H7 ATCC 700728, Salmonella typhimurium ATCC 14028, Klebsiella pneumoniae ATCC 13883, and Pseudomonas aeruginosa ATCC 27853), and Gram-positive bacteria (L. monocytogenes ATCC 19115 and Staphylococcus aureus ATCC 25923) were purchased from either ATCC (Manassas, VA) or MicroBiologics (St.
  • MIC 90 Minimum inhibitory concentration of individual peptides against each bacterial strain was determined as the lowest concentration that reduced bacterial growth by 90%.
  • fowlicidin-1 (0.1 ⁇ M), fowlicidin-2 (0.16 ⁇ M), and SMAP-29 (0.1 ⁇ M) at MIC 90 concentrations were incubated separately with E. coli ATCC 25922 at 37 0 C in 10 mM sodium phosphate buffer, pH 7.4. The reaction was stopped by addition of ice-cold PBS at 0, 5, 10, 15, 20, 30, and 60 min and plated immediately for counting viable bacteria.
  • fowlicidin-1 (0.1 ⁇ M) and fowlicidin-2 (0.16 ⁇ M) were incubated separately with E.
  • aeruginosa ATCC 27853 in 1% MHB this strain was grown in 10% cation-adjusted MHB with peptides in the presence and absence of 100 mM NaCl. After overnight incubation at 37 0 C, the MIC value of each peptide is determined as the lowest concentration that gave no visible bacterial growth.
  • Hemolysis Assay The hemolytic activities of fowlicidins were determined essentially as described (38,39). Briefly, fresh human and chicken blood were collected, washed twice with PBS, and diluted to 0.5% in PBS with and without addition of 10% FBS, followed by dispensing 90 ⁇ l into 96-well plates.
  • the effective concentration (EC 50 ) was defined as the peptide concentration that caused 50% lysis of erythrocytes.
  • Cytotoxicity Assay The cytotoxic effect of fowlicidins on mammalian cells was measured by using the alamarBlue dye (Biosource), which has been shown to be equivalent to the classic MTT-based assay (40). Madin-Darby canine kidney (MDCK) epithelial cells were purchased from ATCC and maintained in DMEM with 10% FBS. MDCK cells were seeded into 96-well plates at 1.5 x 10 5 /well.
  • the cells were washed once with DMEM, followed by addition of 90 ⁇ l of fresh DMEM with or without 10% FBS, together with 10 ⁇ l of serially diluted peptides in 0.01% acetic acid in duplicate. After incubation for 18 h, 10 ⁇ l of alamarBlue dye was added to cells for 6 h at 37 0 C in a humidified 5% CO 2 incubator. The fluorescence of dye was read with excitation at 545 nm and emission at 590 nm.
  • Percent cell death was calculated as [1 - (F peptide - F b a ckgroun d y( F ace tic ac i d - ⁇ back gr ound )] x l °0 >
  • F peptide is the fluorescence of cells exposed to different concentrations of peptides
  • F acetic acid is the fluorescence of cells exposed to 0.01 % acetic acid only
  • F background is the background fluorescence of 10% alamarBlue dye in cell culture medium without cells.
  • Cytotoxicity (EC 50 ) of individual peptides was defined as the peptide concentration that caused 50% cell death.
  • LPS Binding Assay The binding of LPS to fowlicidins was measured by the kinetic chromogenic Limulus amebocyte lysate assay (Kinetic-QCL 1000 kit; BioWhittaker,
  • Percent LPS binding was calculated as ⁇ [ ⁇ D1 - ⁇ D2 + ⁇ D3]/ ⁇ D1 ⁇ x 100, where ⁇ D1 represents the difference in the absorbance between 10 and 16 min for the sample containing LPS only, ⁇ D2 represents the difference in the absorbance between 10 and 16 min for the samples containing LPS and different concentrations of peptides, and ⁇ D3 represents the difference in the absorbance between 10 and 16 min for the samples containing different concentrations of peptides with no LPS. Hill plot was graphed as described (37,41) by plotting log 10 fowlicidin concentrations against log 10 [F 1 Z(1.0 -F 1 )], where F 1 was the fractional inhibition of LPS binding activity.
  • the first-strand cDNA of each sample was then used as a template for subsequent real-time PCR amplification by using QuantiTect® SYBR Green qRT-PCR Kit (Qiagen) and MyiQ® Real-Time PCR Detection System (Bio-Rad).
  • Three common proinflammatory cytokines and chemokines namely interleukin (IL)-IB, CC chemokine ligand 2 (CCL2)/MCP-1 , and CCL3/MIP-la, were selected. All primers were designed to expand at least an intron sequence (Table T).
  • the PCR reaction was set up in a total volume of 15 ⁇ l containing 0.4 ⁇ M of each primer and 0.2 ⁇ g of the first-strand cDNA. PCR cycling conditions were as follows: 95 0 C for 10 min, followed by 50 cycles of 95 0 C for 15 sec, 55 0 C for 30 sec, and 72 0 C for 30 sec.
  • Gene expression levels were quantified by the comparative ⁇ ⁇ C T method as described (42) by using ⁇ -actin as an internal standard for normalization.
  • the ⁇ C T value was determined by subtracting the C ⁇ value of each sample from that of ⁇ -actin in the corresponding sample.
  • the ⁇ ⁇ C T values were further calculated by subtracting the highest mean ⁇ C T value as an arbitrary constant from all other ⁇ C T values.
  • Relative gene expression levels were calculated using the formula 2 "A ⁇ Ct .
  • the presence of contaminating genomic DNA was determined by including a no-reverse transcriptase control and signal generated by primer dimers was determined through no-template controls. Melting curve analysis (55-95 0 C) was performed and confirmed no visible nonspecific amplification of any PCR products from genomic DNA or primer dimers.
  • fowlicidins 1-3 Three putative cathelicidin peptide sequences were subsequently deduced and termed fowlicidins 1-3. Because the N-teraiinal sequence including the start codon of fowlicidin-2 was missing in GenBank, a genome walking approach known as vectorette PCR was performed by using chicken genomic DNA as previously described (30,31). As a result, a 1.8-kb upstream sequence of the fowlicidin-2 gene was obtained following two rounds of vectorette PCR (data not shown). The missing N-terminal peptide sequence of fowlicidin-2 was predicted by GenomeScan (29) based on its homology with the other two fowlicidins.
  • the chicken genome contains three cathelicidin genes, namely fowlicidins 1-3, encoded by expressed sequence tags (EST) and whole genome shotgun sequences (WGS) as indicated.
  • Each fowlicidin gene consists of four exons (E) separated by three introns (I). The sizes of the first and last exons are given according to the coding sequences without the 5'- and 3 '-untranslated regions being counted. Note that the first three exons are 100% identical between fowlicidin- 1 and fowlicidin-3, except for exon 4, which encodes different mature sequences.
  • fowlicidins and classic cathelicidins are drastically diverged at the C-terminus ( Figure 1). Similar to classic cathelicidins, fowlicidins 1-3 are positively charged at the C-terminus due to the presence of an excess number of cationic residues (R and K). The preferred cleavage site for elastase in the processing and maturation of bovine and porcine cathelicidins (34,35) also appears to be conserved in the chicken. Therefore, mature fowlicidins 1-3 are predicted to be devoid of cysteines and composed of 26, 32, and 29 amino acid residues with a net charge of +8, +10, and +7, respectively ( Figure 1).
  • AADNOl 081708 contains a part of the last exon sequence of the fowlicidin- 1 gene, and AADN01005055 and AADN01005056 encode the majority of the fowlicidin-2 and -3 genes ( Figure 2A).
  • the fowlicidin- 1 gene was cloned from chicken genomic DNA by PCR using the primers located in the first and last exons, whereas the missing first intron sequence of the fowlicidin-3 gene, i.e., the gap between AADN01005055 and AADNOl 005056, was cloned directly by PCR with primers located in two flanking exons
  • FIG. 2A The 5'-end of the fowlicidin-2 gene was cloned by two rounds of vectorette PCR as described in the previous section. Structural organizations of three fowlicidin genes were obtained by comparing their cDNA with genomic DNA sequences. As shown in Table II, all three genes are organized similarly with four exons separated by three introns. The first three exons encode the signal and cathelin pro-sequences, whereas the last exon primarily encodes the mature sequences. Such structures are surprisingly identical to the mammalian cathelicidin genes, a clear indication of significant conservation during evolution.
  • fowlicidin-1 and -2 are separated 2.4 kb from each other by a gene homologous to the C-terminal end of vesicle-associated, calmodulin kinase-like kinase (CamKV) (GenBank accession no. NP_076951). Chromosomal location of the chicken, cathelicidin gene cluster was further revealed by using the Map Viewer Program. AADNOl 005055 and
  • AADNOl 005056 were found to locate on the p arm of chromosome 2 that are less than 3.5 Mb from the proximal end in the current chicken genome assembly (Build 1.1) released on July 1, 2004, but AADNO 1081708 remains unmapped. Comparative and Evolutionary Analyses of Vertebrate Cathelicidins - Identification of three chicken fowlicidins provides an excellent opportunity to study the evolutionary relationship of mammalian cathelicidins. The physical locations of the cathelicidin gene clusters across several phylogenetically distant vertebrate species were examined.
  • the cathelicidin genes are located in the syntenic regions flanking an evolutionarily conserved gene, Kelch-like 18 (KLHLl 8) (NP_071737) across rodents, dogs, and humans, clearly indicating that cathelicidins in mammals and birds share a common ancestor. It is noteworthy that the chicken CamKV gene is absent in syntenic regions in mammals ( Figure 2B).
  • Fowlicidins To test antibacterial properties of chicken cathelicidins, putatively mature fowlicidin-1 and -2 were synthesized commercially by the standard solid phase synthesis method and purified to >95% purity. A reference strain of E. coli ATCC 25922 was tested by using the colony counting assay as previously described
  • fowlicidin-1 and -2 displayed a MIC 90 of 65 nM and 180 nM, respectively, against E. coli.
  • SMAP-29 which is the most potent cathelicidin that has been reported thus far (7)
  • both fowlicidin-1 and -2 showed comparable antibacterial potency, implying the promising therapeutic potential of these two chicken cathelicidins.
  • both fowlicidins showed a rapid killing of E. coli with the maximum killing occurring at 30 min at MIC 90 concentrations ( Figure 4B), reinforcing the notion that both fowlicidins kill bacteria most likely through physical membrane disruption.
  • fowlicidin-1 and -2 maintained their activities up to 150 mM NaCl ( Figure 4C), implying their potential for systemic therapeutic applications.
  • no obvious synergistic effect of two fowlicidin peptides in killing E. coli was observed when applied together (data not shown).
  • both peptides were equally effective against antibiotic-resistant bacterial strains, such as multidrug-resistant S. typhimurium DTl 04 and methicilin-resistant S. aureus (MRSA) (Table III).
  • Fowlicidin-3 also showed similar antibacterial activities against Gram-positive and Gram-negative bacteria to fowlicidin-1 (data not shown), but was omitted for further functional analyses because of its high homology with fowlicidin-1 ( Figure 1).
  • Cytotoxicity ofFowlicidins To evaluate the hemolytic activity of fowlicidins against red blood cells, freshly isolated human and chicken erythrocytes were incubated with fowlicidins, together with SMAP29 as a positive reference. Hemolysis was monitored by measuring the absorbance at 405 ran for released hemoglobin as described (38,39).
  • RAW264.7 macrophage cells were stimulated with LPS in the presence of different concentrations of peptides, followed by evaluation of proinflammatory cytokine/chemokine gene expression by real time reverse transcriptase-PCR.
  • Fowlicidin-1 and -2 when applied up to 20 ⁇ M in the absence of LPS, did not alter gene expression. However, they blocked LPS-induced expression of IL-I ⁇ and CCL-2/MCP-1 in a dose-dependent fashion ( Figures 6C and 6D). The same trend also occurred with CCL-3/MIP-la for both peptides (data not shown).
  • Three chicken cathelicidins consist of linear cationic sequences at the C-termini, which are expected to be freed from the cathelin domain to become biologically active. Indeed, putatively mature fowlicidins possess potent antibacterial activities (Figure 4 and Table III). Although valine on the fourth exon of fowlicidins ( Figure 1) is likely to be the processing site for chicken elastase-like protease as in the case of bovine and porcine cathelicidins (34,35), the protease and exact cleavage site for fowlicidins need to be experimentally confirmed.
  • NGPs were also identified in rats, pigs and cows that are highly homologous to P 15s in rabbits (22) and NGP in mice (23) ( Figure 1). These NGP-like proteins appear to be evolutionarily conserved only in glires and ungulates, but not in dogs and primates. In spite of relatively low homology in the cathelin domain with the majority of other mammalian cathelicidins, NGPs share similar tissue expression pattern (22,23), chromosomal location (Figure 2B), gene structure (data not shown), and antimicrobial activities (22) to classic mammalian cathelicidins, and therefore, clearly belong to the cathelicidin family.
  • ungulates have multiple cathelicidins, whereas most other mammals have one or very few members.
  • fowlicidin-1 and -3 are apparently a result of gene duplication, because of significant homology across the entire open reading frame ( Figure 1).
  • the intron sequences of these two cathelicidin genes are highly similar (data not shown).
  • Fowlicidin-2 also shares significant homology in the first three exons with fowlicidin-1 and -3, but diverged greatly in the last exon encoding the mature sequence ( Figure 1).
  • alignment of the last intron and exon nucleotide sequences of three fowlicidin genes revealed approximately 45% identity (data not shown).
  • NGPs are all negatively charged, as opposed to classic cathelicidins with cationic sequences. Therefore, it will be interesting to study the processing and biological roles of these NGPs. Because of the existence of two cathelicidins (NGP and CRAMP) in mice as opposed to a single cathelicidin (LL-37/hCAP-18) in humans, extrapolation of the data from CRAMP-deficient mouse to the human system needs to be more prudent.
  • fowlicidins Unlike many cationic antimicrobial peptides that are inactivated at physiological concentrations of salt, fowlicidins maintained antibacterial activity in the presence of high NaCl ( Figure 4C and Table III). Salt-independent killing of bacteria of fowlicidins may offer an attractive therapeutic option for cystic fibrosis and Crohn's disease, both of which are associated with aberrant local expression or inactivation of antimicrobial peptides (49-51).
  • Cathelicidins are a major family of animal antimicrobial peptides with hallmarks of a highly conserved prosequence (cathelin domain) and an extremely variable, antibacterially active sequence at the C-terminus [1-3]. The exact microbicidal mechanism for this family of antimicrobial peptides is not clearly understood. However, it is generally believed that the electrostatic interaction between the C-terminal cationic peptides with anionic lipids followed by membrane permeabilization is mainly responsible for killing prokaryotic cells. Because of such a non-specific membrane-lytic mechanism, many cathelicidins kill a variety of bacteria at low micromolar concentrations with much less chance of developing resistance
  • fowlicidins 1-3 Three novel chicken cathelicidins have been identified [14-16], which are called fowlicidins 1-3 herein. All three fowlicidins share little similarity with mammalian cathelicidins in the C-terminal sequence [16]. Putatively mature fowlicidin-1, a linear peptide of 26 amino acid residues, is broadly active against a range of Gram-negative and Gram-positive bacteria with a similar potency to SMAP-29 [16]. However, fowlicidin-1 also displayed considerable cytotoxicity toward human erythrocytes and mammalian epithelial cells with 50% lysis in the range of 6-40 ⁇ M [16].
  • Fowlicidin-1 was shown to be composed of an ⁇ -helical segment with a slight kink near the center and a flexible unstructured region at the
  • Fowlicidin-1 was synthesized using the standard solid-phase method by SynPep (Dublin, CA) and its analogs were synthesized by either Sigma Genosys (Woodlands, TX) or Bio-Synthesis (Lewisville, TX).
  • the peptides were purified through RP-HPLC and purchased at >95% purity. The mass and purity of each peptide were further confirmed by 15% Tris-Tricine polyacrylamide gel electrophoresis (data not shown) and by MALDI-TOF mass spectrometry (not shown) using the Voyager DE-PRO instrument (Applied Biosystems, Foster City, CA) housed in the Recombinant DNA/Protein Resource Facility of Oklahoma State University.
  • MRE Mean residue ellipticity
  • NMR experiments for fowlicidin-1 were performed as previously described [43, 44]. Briefly, NMR data were acquired on a 11.75T Varian UNITYplus spectrometer (Varian, Palo Alto, CA), operating at 500 MHz for 1 H, with a 3-mm triple-resonance inverse detection probe.
  • the NMR sample of fowlicidin-1 consisting of 4 mM in water containing 50% deuterated TFE (TFE-d3, Cambridge Isotope Laboratories) and 10% D 2 O, was used to record spectra at 10, 20, 30, and 35 0 C. The spectra acquired at 35 0 C were determined to provide the optimal resolution of overlapping NMR resonances.
  • NOESY experiments were performed with 200, 300, 400 and 500 ms mixing times. A mixing time of 200 ms was used for distance constraints measurements. The NOE cross-peaks were classified as strong, medium, weak and very weak based on an observed relative number of contour lines.
  • TOCSY spectra were recorded by using MLEV- 17 for isotropic mixing for 35 and 100 ms at a Bl field strength of 7 KHz.
  • NOE-derived distance restraints were classified into four ranges: 1.8-2.7, 1.8-3.5, 1.8-4.0 and 1.8-5.0 A, according to the strong, medium, weak and very weak NOE intensities.
  • Upper distance limits for NOEs involving methyl protons and non-stereospecifically assigned methylene protons were corrected appropriately for center averaging [45].
  • a distance of 0.5 A was added to the upper distance limits only for NOEs involving the methyl proton after correction for center averaging [46].
  • the distance restraints were then used to create initial peptide structures starting from extended structures using the program CNS (version 1.1) [47] .
  • CNS uses both a simulated annealing protocol and molecular dynamics to produce low energy structures with the minimum distance and geometry violations.
  • default parameters supplied with the program were used with 100 structures for each CNS run.
  • the final round of calculations began with 100 initial structures and 20 best structures with the lowest energy were selected and analyzed with MOLMOL [48] and PROCHECK-NMR [19]. Structure figures were generated by using MOLMOL.
  • the structures of fowlicidin-1 analogs were further modeled by using Modeller [20], based on the parent peptide.
  • E. coli ATCC 25922 and S. enterica serovar Typhimurium ATCC 14028 Two representative Gram-negative bacteria (E. coli ATCC 25922 and S. enterica serovar Typhimurium ATCC 14028) and two Gram-positive bacteria (L. monocytogenes ATCC 19115 and S. aureus ATCC 25923) were purchased from ATCC (Manassas, VA) and tested separately against fowlicidin-1 and its analogs by using a modified broth microdilution assay as described [16, 21].
  • the hemolytic activity of fowlicidin-1 and its mutants were determined essentially as described [13, 22]. Briefly, fresh anti-coagulated human blood was collected, washed twice with PBS, diluted to 0.5% in PBS, and 90 ⁇ l were dispensed into 96-well plates. Serial 2-fold dilutions of peptides were added in duplicate to erythrocytes and incubated at 37 0 C for 2 h. Following centrifugation at 800 x g for 10 min, the supernatants were transferred to new 96-well plates and monitored by measuring the absorbance at 405 nm for released hemoglobin. Controls for 0 and 100% hemolysis consisted of cells suspended in PBS only and in 1% Triton X-100, respectively.
  • Percent hemolysis was calculated as [(A 405nm , peptide - A 40SiVm, PBS) / (A 4 o 5nm , 1 % T rit o n x-ioo -A 4 osnm, PBS)] x 100.
  • EC 50 of the hemolytic activity was defined as the peptide concentration that caused 50% lysis of erythrocytes. Cytotoxicity assay
  • LPS The binding of LPS to fowlicidin-1 and its analogs was measured by a kinetic chromogenic Limulus amebocyte lysate assay (Kinetic-QCL 1000 kit; BioWhittaker, Walkersville, MD) as previously described [21, 25]. Briefly, 25 ⁇ l of serially diluted peptide were added in duplicate into 25 ⁇ l of E. coli Ol 11:B4 LPS containing 0.5 endotoxin units/ml and incubated for 30 min at 37 0 C, followed by incubation with 50 ⁇ l of the amoebocyte lysate reagent for 10 min.
  • Kinetic-QCL 1000 kit BioWhittaker, Walkersville, MD
  • the absorbance at 405 nm was measured at 10 and 16 min after addition of 100 ⁇ l of chromogenic substrate, Ac-Ile-Glu-Ala-Arg-p-nitroanilide.
  • Percent LPS binding was calculated as [( ⁇ D 1 - ⁇ D2 + ⁇ D3)/ ⁇ D 1 ] x 100, where ⁇ D 1 represents the difference in the optical density between 10 and 16 min for the sample containing LPS only, ⁇ D2 represents the difference in the optical density between 10 and 16 min for the samples containing LPS and different concentrations of peptides, and ⁇ D3 represents the difference in the optical density between 10 and 16 min for the samples containing different concentrations of peptides with no LPS.
  • EC 50 of the LPS-binding activity was defined as the peptide concentration that inhibited LPS-mediated procoagulant activation by 50%.
  • fowlicidin-1 is primarily an ⁇ -helical peptide consisting of a helical segment from Leu 8 to Lys 25 and a disordered region near the N-terminus from Arg 1 to Pro 7 .
  • No unambiguous long range NOEs for the first four N-terminal residues were observed , indicative of their extremely flexible nature.
  • a closer examination revealed that the long helix of fowlicidin-1 is further composed of two short, but perfect, ⁇ -helical segments (Leu 8 -Ala 15 and Arg ⁇ -Lys 25 ) with a slight bend between GIy 16 and Tyr 20 , due to the presence of GIy 16 ( Figure 7).
  • the central helical region (residues 6-23) of fowlicidin-1 is highly hydrophobic, containing only two cationic residues (Arg 11 and Arg 21 ) and two uncharged polar residues
  • C-terminal helix (residues 16-23), but not the last three lysines, is a critical determinant of cytotoxcity.
  • fowlicidin-1 (5-26) maintained a similar lytic activity, whereas fowlicidin-1 (8-26) only caused minimal 20% lysis of human red blood cells at 360 ⁇ M, the highest concentration tested (data not shown), suggesting possible presence of another cytotoxicity determinant in the N-terminal unstructured segment between residues 5-7. Consistent with these results, a significant > 10-fold reduction in killing MDCK cells was also observed with fowlicidin-1 (8-26) (Table V). Because of the fact that two peptide analogs, fowlicidin-1 (1-15) and fowlicidin-1 (8-26) each containing one cytolytic determinant, had substantially reduced toxicity, it is likely that the two lytic sites
  • amphipathic helix has a stronger binding affinity and permeability toward erythrocyte membranes than to epithelial membranes, perhaps due to the difference in the lipid composition of the two host cell types.
  • Binding and disrupting anionic LPS the major outer membrane component of Gram-negative bacteria, is often the first step for antimicrobial peptides to interact with bacteria and permeabilize membranes [10].
  • cathelicidins including human LL-37/hCAP-18 [21, 23], rabbit CAP-18 [24], and sheep SMAP-29 [25] have been shown to bind and neutralize LPS with EC 50 at low micromolar concentrations.
  • fowlicidin-1 has at least two LPS binding sites [16].
  • Residues 5-7 is clearly involved in LPS binding and may constitute the core region of one LPS-binding site, because fowlicidin-1 (8-26) showed a >15-fold reduction in binding to LPS relative to fowlicidin-1 (5-26), which had a similar affinity for LPS to the full-length peptide.
  • the other LPS-binding site is likely located in the C-terminal short helix between residues 16-23, because deletion of that region [fowlicidin-1 (1-15)] resulted in a >25-fold reduction in LPS binding, as compared to fowlicidin-1 (1-23) Figure 9A, Table V).
  • Cathelicidins are highly conserved from birds to mammals in the prosequence, but are extremely divergent in the C-terminal mature sequence [1-3]. Cathelicidin-like molecules have also been found in the hagfish, the most ancient extant jawless fish with no adaptive immune system [26]. With the finding that fowlicidin-1 adopts an ⁇ -helix, it is now evident that at least one cathelicidin in ⁇ -helical conformation is present in each of the fish, bird, and mammalian species examined. This suggests that, in addition to the prosequence, cathelicidins appear to be conserved in the mature region structurally and presumably functionally as well.
  • fowlicidin-1 is primarily composed of two short ⁇ -helical segments connected by a slight kink caused by GIy 16 near the center ( Figure 7). Interestingly, such a helix-hinge-helix structural motif is not uncommon for cathelicidins.
  • fowlicidin-1 structurally more resembles melittin, a helical peptide found in honey bee venom that has a curved hydrophobic helix with positively charged residues located primarily at the C-terminal end [32] ( Figure 10).
  • melittin displays considerable antibacterial and hemolytic activities.
  • An attempt to reduce hydrophobicity and enhance amphipathicity of the helical region of fowlicidin-1 to make fowlicidin-l-K 7 L 12 K 14 L 16 K 18 led to a dramatically increased toxicity particularly toward erythrocytes with a minimum change in the antibacterial activity against certain bacteria (Table ). This is consistent with an earlier conclusion that an amphipathic helix is more essential for interactions with zwitteronic lipid membranes on eukaryotic cells than for anionic lipids on prokaryotic cells [33].
  • the ⁇ -helix before the kink at Glyl 6 is likely to be involved in membrane penetration as well, because the minimum length required for a helical peptide to traverse membranes and exert antimicrobial and lytic activities is about 11-14 residues [34].
  • Trp6 is known to have a preference to be inserted into lipid bilayers at the membrane- water interface [35, 36]. Because of such membrane-seeking ability, inclusion of tryptophan often renders peptides with higher affinity for membranes and more potency against bacteria [37, 38]. It is not known why tryptophan is not significantly involved in the antibacterial activity of fowlicidin- 1.
  • the N-terminal helix of many cathelicidins plays a major role in LPS binding and bacterial killing, while the C-terminal segment is either dispensable for antimicrobial activity or more involved in cytotoxicity [12, 25, 39, 40].
  • the C-terminal helix after the kink of fowlicidin-1 is more important in killing bacteria than the N-terminal helix.
  • Such a sharp difference in the distribution of functional domains along the peptide chain between fowlicidin-1 and other cathelicidins is probably because of a more pronounced hydrophobic nature of the helix and the presence of an additional highly flexible segment at the N-terminus of fowlicidin-1.
  • Fowlicidin-1-L16 and fowlicidin-1 -K 7 L 12 K 14 L 16 K 18 also had a more pronounced reduction in antibacterial activity than in toxicity, therefore with reduced clinical potential.
  • fowlicidin-1 (8-26) with the N-terminal toxicity determinant (residues 5-7) deleted and the C-terminal antibacterial domain (residues 16-23) left unaltered, had a slight reduction in MIC against bacteria, but with > 10-fold reduction in toxicity toward mammalian epithelial cells and negligible toxicity toward erythrocytes (Table V). Coupled with its smaller size, this peptide analog may represent a safer and more attractive therapeutic candidate than the parent peptide. Given the fact that fowlicidin-1 is broadly effective against several common bacterial strains implicated in cystic fibrosis, including S.
  • Zanetti M (2004) Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 75, 39-48.
  • MOLMOL a program for display and analysis of macromolecular structures. J MoI Graph 14, 51-55.
  • EXAMPLE 3 The Central Kink Region of ⁇ -Helical Fowlicidin-2 is Critically Involved in Antibacterial and Lipopolysaccharide-Neutralizing Activities _ With rapid emergence of antibiotic-resistant pathogens, there is an unprecedented urgency to develop new antimicrobials with a less likelihood of gaining resistance. Cationic antimicrobial peptides, an integral component of innate immunity found in virtually all species of life, are capable of killing a variety of pathogens with a similar efficiency against the strains that are resistant to conventional antibiotics (1,2). Because of nonspecific membrane-lytic activities of most cationic peptides, it is conceivable that bacteria are much more difficult to develop resistance against these peptides than conventional antibiotics (2-4).
  • Cathelicidins a major family of vertebrate antimicrobial peptides, are receiving a particular attention as novel antimicrobials, due to simple structures (devoid of cysteines in most cases) and high antibacterial efficacy relative to other families of cationic peptides (11-14).
  • many cathelicidins hi addition to a broad spectrum of antimicrobial activities, many cathelicidins have capacity to directly bind lipopolysaccharide (LP S)4 and lipoteichoic acid in vitro and neutralize Gram-negative and Gram-positive bacteria-induced septic shock in vivo (15-17). Thus these cathelicidins may also represent excellent therapeutic candidates for sepsis treatment.
  • Example 1 describes the identification of three novel chicken cathelicidins, namely fowlicidins 1-3, with highly potent antibacterial and LPS-neutralizing activities (18).
  • fowlicidin-2 exhibited similar antibacterial efficacy with slightly less lytic activity to host cells (18).
  • NMR nuclear magnetic resonance
  • CD spectroscopy To determine the secondary structure of fowlicidin-2 and its analogs, circular dichroism (CD) spectroscopy was performed with a Jasco-715 spectropolarimeter using a 0.1 -cm path length cell over the 180-260 run range as we previously described (19). The spectra were acquired at 25 0 C every 1 nm with a 2-s averaging time per point and a 1-nm band pass. Peptides (10 ⁇ M) were measured in 50 mM potassium phosphate buffer, pH 7.4, with or without different concentrations of trifluoroethanol (TFE) (0%, 10%, 20%, 40%, and 60%) or SDS micelles (0.25% and 0.5%).
  • TFE trifluoroethanol
  • Mean residue ellipticity was expressed as [ ⁇ ], ⁇ (deg.cm 2 .dmor 1 ).
  • NMR experiments were performed with an 11.75 T Varian UNITYplus spectrometer (Varian, Palo Alto, CA, USA), operating at 499.96 MHz, with a 3-mm triple-resonance inverse detection probe.
  • Fowlicidin-2 (3 mM) was used to record TOCSY and NOESY experiments at 25 0 C in 50% deuterated TFE (TFE-d3)/50% H 2 O essentially as described (19).
  • a series of ID experiments were conducted from 5-35 0 C with 5 0 C increments. Water peak suppression was obtained by low-power irradiation of the H2O resonance.
  • a total of 256 increments of 4K data points were recorded for the 2D experiments.
  • AU data sets were obtained in the hypercomplex phase-sensitive mode.
  • TOCSY spectra were recorded by using MLEV- 17 for isotropic mixing for 100 ms at a Bl field strength of 8 KHz.
  • NOESY experiments were performed with mixing times of 100, 300, and 400 ms
  • TFE methylene peak was considered a reference for the chemical shift values.
  • Varian software, VNMR 6.1C, on a Silicon Graphics Octane workstation was used for data processing, and Sparky 3 software (Goddard, T.D. and D. G. Kneller, University of California, San Francisco, CA) was used for data analysis.
  • Structure calculation - Proton resonance assignments were made using 2D TOCSY for intra-residue spin systems and NOESY spectra for inter-residue connectivities as described (21). A total of 174 distance constraints obtained from the NOESY spectrum were used for structure calculations. NOE peak were classified as intra-residue, sequential, medium, and long ranges.
  • NOE cross peak intensities were classified as strong (1.8-2.7 A), medium (1.8-3.5 A), weak (1.8-4.0 A), and very weak (1.8-5.0 A).
  • Upper distance limits for NOEs involving methyl protons and non-stereospecifically assigned methylene protons were corrected appropriately by adding 1 A to the constraints for center averaging (22).
  • the obtained distance restraints were then used to create initial peptide structures starting from extended structures using the program CNS version 1.1 (23), which uses both a simulated annealing protocol and molecular dynamics to produce low energy structures with minimum distance and geometry violations. Default parameters were used to generate 100 structures for the initial CNS run.
  • a second round of calculations generated 20 structures, from which 10 with fewer or no restriction violations and the lowest energy were selected and analyzed with Sybyl 7.1 (Tripos, St. Louis, MO). Surface accessibilities of fowlicidin-2 was generated using GRASP (24).
  • Antibacterial assay Three representative Gram-negative bacteria (Escherichia coli ATCC 25922 and Salmonella enterica serovar Typhimurium ATCC 14028, and Klebsiella pneumoniae ATCC 13883) and four Gram-positive bacteria (Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC 25923, and two methicilin-resistant S. aureus ATCC BAA-39 and S. aureus ATCC 43300) were purchased from ATCC (Manassas, VA) or MicroBiologics (St. Cloud, MN). The antibacterial activities of fowlicidin-2 and its analogs were tested by using a modified broth microdilution assay in the bicarbonate buffer as described (25).
  • bacteria were cultured overnight in tryptic soy broth (TSB) at 37 0 C, diluted 1 :1000 in fresh subculture medium containing 20% TSB, 10% fetal bovine serum (FBS), 100 mM NaCl, 25 mM NaHCO 3 , and 1 mM NaH 2 PO 4 , and grown to the mid-log phase. Cells were then washed twice with 10 mM sodium phosphate buffer, pH 7.4, and resuspended to 5 x 10 5 colony-forming units (CFU)/ml in the assay medium containing 20%
  • TSB tryptic soy broth
  • FBS fetal bovine serum
  • CFU colony-forming units
  • TSB 25 mM NaHCO 3 , and 1 mM NaH 2 PO 4 (25). If necessary, 100 mM NaCl was incorporated to test the effect of salinity on antibacterial activity.
  • Bacteria (90 ⁇ l) were dispended into 96- well plates followed by addition in duplicate of 10 ⁇ l of peptides in serial two-fold dilutions in 0.01% acetic acid. The minimum inhibitory concentration (MIC) of each peptide against each bacterial strain was determined as the lowest concentration that gave no visible bacterial growth after overnight incubation at 37 0 C.
  • Bacteria (80 ⁇ l) were then dispended into a 96-well plate, followed by addition of each peptide (at 0.25, 0.5 or 1 MIC concentration) and 1.5 mM of a chromogenic substrate of ⁇ -galactosidase, p-nitrophenyl- ⁇ -D-galactopyranoside (ONPG) (Sigma, St. Louis, MO) to a total volume of 100 ⁇ l. Because the MIC of fowlicidin-2( 19-31) could not be determined even at the highest peptide concentration tested (>32 ⁇ M) by the modified broth microdilution method described above, the concentration of this analog used was 16 ⁇ M. The plate was incubated at 37 0 C for 2 h with shaking and reading every two minutes for monitoring the production of o-nitrophenol at 420nm. Negative controls contained no peptide but an equal volume of 0.01% acetic acid.
  • Hemolytic assay The hemolytic activity of fowlicidin-2 and its analogs were determined as described in Examples 1 and 2.
  • Cytotoxicity assay The toxicity of fowlicidin-2 and its analogs was evaluated with human colonic epithelial Caco-2 cells (ATCC) by using alamarBlue dye (Biosource) similarly as described in Examples 1 and 2.
  • the average solution structure of fowlicidin-2 indicated that it has two well-defined ⁇ -helices from Arg 6 to Arg 12 and from He 23 to GIy 27 , abend from Phel7 to Lys 20 , and a flexible region at the N-terminus from Leu 1 to Arg 4 .
  • a closer examination revealed that the N-terminal ⁇ -helix adopts a typical amphipathic structure, while the C-terminal ⁇ -helix is highly hydrophobic.
  • CD spectra showed that all peptide analogs have a higher degree of ⁇ -helicity than the parent peptide.
  • Three C-terminal deletion analogs namely fowlicidin-2(l-14), fowlicidin-2(l-l 5) and fowlicidin-2(l-18), have a gradual increase in the ⁇ -helical content ranging from 44% and 53% to 62%, suggesting that residues 15-18 in the central kink region of the parent peptide likely adopt an ⁇ -helical conformation without Pro 19.
  • the ⁇ -helical content of two N-terminal deletion analogs, fowlicidin-2(15-31) and fowlicidin-2( 19-31), were estimated to be 42% and 79%, respectively, consistent with the secondary structural contents predicted from the parent peptide.
  • Antibacterial activities offowlicidin-2 and its analogs - A modified broth microdilution assay was used to test the antibacterial activity of fowlicidin-2 and its analogs using a bicarbonate-based buffer as described (25).
  • Three representative Gram-negative bacteria E. coli ATCC 25922 and S. enterica serovar Typhimurium ATCC 14028, and K. pneumoniae
  • fo wlicidin-2( 1-14) and (1-18) with inclusion of additional positively charged residues in the central bending region of the parent peptide showed an graduate increase in antibacterial potency ( Figure 12), consistent with earlier observations that a simultaneous increase in cationicity and ⁇ -helicity is often directly correlated with the antibacterial efficacy (9,10).
  • Fowlicidin-2(1-18) with a concurrent increase of +3 in the net charge and 18% in the ⁇ -helical content exhibited 8- to 16-fold enhancement in bactericidal efficiency, as compared with fowlicidin-2(l-14).
  • fowlicidin-2(19 ⁇ 31) Relative to fowlicidin-2(19 ⁇ 31), fowlicidin-2(15-31) containing additional residues from Arg 15 to Arg 18 also demonstrated significant antibacterial activities, with only 2- to
  • Permeabilization of bacterial cytoplasmic membrane by fowlicidin-2 and its analogs -Electrostatic interaction with and subsequent disruption of bacterial membrane is a common mode of action for most antimicrobial peptides (2-4).
  • Cytotoxicity offowlicidin-2 and its analogs were tested for their toxicities to human erythrocytes and colonic epithelial Caco-2 cells as previously described (Example 1).
  • the full-length fowlicidin-2 peptide displayed a noticeable, dose-dependent hemolytic activity.
  • fowlicidin-2 lysed 80% and 100% erythrocytes in the presence ( Figure 15A) and absence of 10% FBS ( Figure 15B), respectively.
  • the full-length peptide showed 100% killing in the presence of 10% FBS and 30% killing without FBS, whereas all analogs lost their toxicity significantly.
  • Fowlicidin-2(1-14), fowlicidin-2(l-15) and fowlicidin-2(19-31) caused only a negligible degree of cell death, when 100 ⁇ M was used with and without serum (Figure 15C).
  • Fowlicidin-2(1-18) and fowlicidin-2(15-31) caused 15% and 55% of cell death at 100 ⁇ M without serum, respectively, whereas serum reduced the cytotoxicity of both peptides to minimal 8%.
  • fowlicidin-2(l-14) and fowlicidin-2(19-31) displayed no toxicity at all against either human erythrocytes or epithelial cells, it is obvious that neither the N- or C-terminal ⁇ -helix alone is sufficient in the interaction with and lysis of mammalian cell membranes. Therefore, apparent cytoxicity of the full-length fowlicidin-2 must be caused by the amino acid sequence in the central kink region.
  • Arg 15 -Arg 18 [fowlicidin-2(l-18) and fowlicidin-2(15-31)] exhibited a substantially reduced cytotoxicity, although such toxicity was higher than those of the peptide analogs containing the a-helix alone.
  • the obvious reduction in toxicity associated with fowlicidin-2(l-l 8) and fowlicidin-2(15-31) is likely due to the conformational differences of the four-amino acid segment (Arg 15 -Arg 18 ) in these two analogs as opposed in the full-length peptide.
  • RAW264.7 mouse macrophage cells N gene expression in RAW264.7 mouse macrophage cells (Example 1).
  • RAW264.7 cells were stimulated with 0.1 ⁇ g/ml of LPS for 4 in the presence or absence of three different concentrations of fowlicidin-2 and its analogs (1, 5, and 20 ⁇ M), and the expression levels of three common cytokine/ chemokine genes, including IL- 1 ⁇ , CCL-2/MCP- 1 , and CCL-3//MIP- 1 a, were measured by real time RT-PCR as described (Example 1).
  • [fowlicidin-2(19-31)] showed no obvious inhibition of LPS-induced inflammatory gene expression even at 20 ⁇ M, indicating that, in contrary to the N-terminal ⁇ -helix, the C-terminal helix iteself is not sufficient in LPS binding and neutralization.
  • the failure for fowlicidin-2(19-31) to interact with LPS is likely due to the fact that this segment is relatively hydrophobic with a net charge of +2, whereas the N-terminal 14 residues is amphipathic, with a net charge of +5.
  • ⁇ -helical antimicrobial peptides are composed of a predominant ⁇ -helix with a short central hinge sequence, due to the presence of a glycine (9,19).
  • fowlicidin-2 consists primarily of two short ⁇ -helices (residues 6-12 and 23-27), connected by an excessive kink region (residues 13-20) induced by Pro 19 (not shown).
  • the N-terminal ⁇ -helix adopts a typical amphipathic structure, whereas the C-terminal helix is more hydrophobic.
  • the central kink region of fowlicidin-2 is highly positively charged containing six cationic residues.
  • Fowlicidin-1 is composed of two hydrophobic ⁇ -helices separately by a slight kink caused by glycine (19). Most of the cationic residues of fowlicidin-1 are instead concentrated at both ends (19). Such structural differences between fowlicidin-2 and other ⁇ -helical antimicrobial peptides suggest that fowlicidin-2 might be functionally different, with distinct antimicrobial spectra or immunomodulatory activities.
  • Cationicity, ⁇ -helicity and amphipathicity are among the most important physico-chemical parameters that dictate the functional properties of ⁇ -helical antimicrobial peptides (8-10). To a certain degree, these three parameters are often positively correlated with the antibacterial activity. Consistent with these observations, a gradual increase in cationicity, a-helicity and amphipathicity among all peptide analogs [fowlicidin-2(l-14), fowlicidin-2(l-15) and fowlicidin-2(l-18)] leads to a gradual enhancement in permeabilization of E. coli inner membranes (Figure 14), LPS neutralization (Figure 16) and antibacterial potency (Figure 12).
  • fowlicidin-2(l-14), fowlicidin-2(l-15), and fowlicidin-2( 19-31) lack cytotoxicity, their antibacterial and LPS-neutralizing activities are disappointing, indicative of little clinical utility.
  • relative of the parent peptide, fowlicidin-2(l-18) and fowlicidin-2(l 5-31) have a MIC of 2-4 ⁇ M against most bacteria tested, with a minimum reduction in bacterial killing activity (Figure 12), but cause no hemolysis and minimum 8% death to human Caco-2 cells at 100 ⁇ M in the presence of serum ( Figure 15).
  • Both fowlicidin-2(l-18) and fowlicidin-2(15-31) are further capable of blocking LPS-induced proinflammatory responses in RAW264.7 cells to a similar extent as the parent peptide ( Figure 16). Moreover, these two short peptide analogs kill bacteria irrespective of salt, and the antibacterial activity of fowlicidin-2(15-31) is not affected by serum ( Figure 12 and Figure 13). Therefore, both peptides represent better candidates with a significant improvement in therapeutic window and safety as compared with the parent peptide.
  • the in vivo toxicity and antibacterial and anti-LPS efficacy of fowlicidin-2(l-18) and fowlicidin-2(l 5-31) are currently under evaluation.
  • Example 1 19. Xiao, Y., Dai, H., Bommineni, Y. R., Soulages, J. L., Gong, Y. X., Prakash, O., and
  • Fowlicidin-3 is an ⁇ -Helical Cationic Host Defense Peptide with Potent Antibacterial and Lipopolysaccharide-Neutralizing Activities
  • Cationic antimicrobial peptides comprise a large group of small peptides with extremely diverse amino acid sequences but with conserved features in each family [1, 2].
  • these peptides are mostly produced by innate immune cells such as phagocytes, mucosal epithelial cells, and skin keratinocytes in vertebrates, capable of killing a broad range of bacteria, fungi, and viruses, including resistant strains [1, 2]. Because of non-specific membrane-lytic activities, antimicrobial peptides have a low tendency to develop resistance, a desirable feature as a new class of antimicrobial agents [1, 3, 4].
  • antimicrobial peptides Besides having direct microbicidal activities, antimicrobial peptides have increasingly been appreciated to play a profound role in regulating host immune responses to infections. Many peptides have been shown to be actively involved in binding and neutralization of LPS, chemotaxis of immune cells, regulation of dendritic cell differentiation, induction of angiogenesis and re-epithelialization, and modulation of cytokine and chemokine gene expression [5-7]. To better reflect the pleiotropic effects of antimicrobial peptides on various aspects of innate and adaptive immunity, these peptides have been proposed to be renamed as host defense peptides [6, 7], Both antimicrobial and immunomodulatory activities of these peptides are being harnessed and manipulated for therapeutic benefit. It is possible to employ these peptides for antimicrobial therapy without provoking detrimental proinflammatory responses [6-8].
  • Cathelicidins represent a major family of host defense peptides that have been identified in fish, birds, and mammals [9-11]. AU cathelicidins share a highly conserved
  • cathelin pro-sequence at the N-terminus with extremely variable C-terminal sequences having antimicrobial and immune regulatory activities [9-11].
  • fowlicidins 1-3 three chicken cathelicidins, namely fowlicidins 1-3, and found that putatively mature fowlicidin-1 and -2 are among the most efficacious cathelicidins that have been reported, with fowlicidin- 1 being slightly more potent than fowlicidin-2 in killing bacteria [12; Example 2] .
  • fowlicidin-3 a third chicken cathelicidin that is likely to have evolved from fowlicidin-1 by gene duplication [Example 2]. Similar to fowlicidin-1, putatively mature fowlicidin-3 peptide was found to be largely ⁇ -helical with a kink in the central region and a relatively flexible unstructured segment in the N-terminal region. Fowlicidin-3 is highly active against a broad range of bacteria in vitro, including antibiotic-resistant strains, but 4- to 6-fold less toxic to mammalian host cells than fowlicidin-1. Moreover, fowlicidin-3 is more potent than fowlicidin-1 in blocking LPS-induced proinflammatory responses. Therefore, fowlicidin-3 should be an attractive antibacterial and anti-sepsis drug.
  • Fowlicidin-1 and -3 were predicted to consist of 26 and 27 amino acid residues in the C-terminal regions of their precursors, respectively [Example I].
  • Fowlicidin-1 RVKRVWPLVIRTVIAGYNLYRAIKKK, SEQ ID NO: 2
  • -3 KRFWPLVPVAINTVAAGIN LYKAIRRK, SEQ ID NO: 18
  • Both peptides were purified to >95% purity by reverse phase high-performance liquid chromatography (RP-HPLC).
  • the secondary structure of fowlicidin-3 was determined on a Jasco-715 spectropolarimeter using a 0.1 -cm path length cell over the 180-260 nm range as described [Example 2]. NMR spectroscopy and tertiary structure calculations
  • Gram-negative bacteria ⁇ Escherichia coli ATCC 25922, S. enterica serovar Typhimurium ATCC 14028, S. enterica serovar Typhimurium DTl 04 ATCC 700408, and
  • Klebsiella pneumoniae ATCC 13883 and Gram-positive bacteria ⁇ Listeria monocytogenes ATCC 19115, S. aureus ATCC 25923, S. aureus ATCC BAA-39, and S. aureus ATCC 43300) were purchased from either ATCC (Manassas, VA) or MicroBiologics (St. Cloud, MN) and tested individually against fowlicidin-1 and -3. The MICs were determined by a standard broth microdilution assay as recommended by the Clinical and Laboratory
  • Cytoplasmic membrane permeabilization assay E, coli ML-35p, a lactose permease-deficient strain with constitutive production of ⁇ -galactosidase in the cytosol was used as described [17-19]. Briefly, mid log-phase bacteria were washed twice in 10 mM sodium phosphate buffer, pH 7.4, diluted to 0.03 OD600 (equivalent to 2.5-5 x 10 7 CFU/ml) in the same phosphate buffer containing 1% TSB with and without 100 niM of NaCl.
  • Radial diffusion assay [22] was used to study the effect of serum on the antibacterial activity of fowlicidins. Briefly, after solidification of the underlay gel containing 4 x 10 5 CFU/ml of a reference strain of S. aureus ATCC 25923, small wells ( ⁇ 3 mm in diameter) were punched. One ⁇ g of fowlicidin-1 or -3 was diluted to a total of volume of 4 ⁇ l in 0.01% acetic acid with or without 50% chicken or human serum and then added separately to the wells. After 3 h of diffusion at 37 0 C, the nutrient rich overlay gel was poured and incubated at 37 0 C overnight. The diameters of bacterial clearance zone were measured. Cytotoxicity assay
  • the toxicity offowlicidin-3 toward mammalian epithelial cells was evaluated by using MDCK cells (ATCC) and an alamarBlue dye (Biosource, Camarillo, CA) as described
  • Mouse macrophage RAW 264.7 cells were used to study the modulation of LPS-induced cytokine/chemokine gene expression by fowlicidin-3 in comparison with fowlicidin-1.
  • Cells were seeded in 12-well tissue culture plates with 5 x 10 5 cells/well in DMEM containing 10% FBS. After overnight incubation, cells were pretreated for 30 min with 0.5, 2.5, and 10 ⁇ M of fowlicidins in duplicate, followed by stimulation for 4 h with 100 ng/ml LPS from E. coli Ol 14:B4 (Sigma).
  • Total RNA was then isolated from cells using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
  • RT-PCR Quantitative real-time reverse transcriptase (RT)-PCR was used to analyze the expressions of three common proinflammatory genes, namely IL-IB, CCL2/MCP-1, and CCL3/MIP-la, using exon-spanning primers as described [Example I].
  • the first-strand cDNA from 1.5 ⁇ g of each RNA sample was synthesized in a reaction volume of 20 ⁇ l at 42 0 C for 30 min by using QuantiTect® Reverse Transcription Kit (Qiagen, Valencia, CA), which included removal of genomic DNA contamination prior to cDNA synthesis.
  • Real-time PCR was performed by using 0.2 ⁇ g of the first-strand cDNA, gene-specific primers, SYBR® Premix Ex TaqTM (Takara Bio, Japan), and MyiQ® Real-Time PCR Detection System (Bio-Rad, Hercules, CA) in a total volume of 10 ⁇ l.
  • PCR cycling conditions were as follows: 95 0 C for 30 sec, followed by 40 cycles of 95 0 C for 15 sec, 55 0 C for 30 sec, and 72 0 C for 30 sec.
  • the comparative ⁇ CT method was used to quantify the gene expression levels, where ⁇ -actin was used as an internal control for normalization [12]. Relative fold changes in gene expression were calculated using the formula 2- ⁇ Ct. Melting curve analysis (55-95 0 C) was performed and confirmed amplification of a single product in each case.
  • Circular dichroism (CD) spectroscopy was first performed to determine the secondary structure offowlicidin-3 in the presence of different concentrations of trifluoroethanol (TFE) and SDS.
  • TFE trifluoroethanol
  • Fowlicidin-3 was largely unstructured in phosphate buffer and began to transform into a typical ⁇ -helical conformation following addition of increasing concentrations of TFE.
  • Significant ⁇ -helical content (86%) with virtually no ⁇ -sheet structure was observed with fowlicidin-3 in 60% TFE (not shown).
  • fowlicidin-3 also exhibited 53% ⁇ -helical content in the presence of 0.25% SDS micelles (data not shown).
  • the energy minimized average structure of fowlicidin-3 was further calculated, showing a predominant ⁇ -helical structure extending from V 9 -R 25 with a relatively flexible
  • MICs multidrug-resistant Salmonella enterica serovar Typhimurium DT 104 and two methicillin-resistant Staphylococcus aureus (MRSA) strains tested.
  • MRSA methicillin-resistant Staphylococcus aureus
  • a MICs were determined as the lowest peptide concentration that gave no visible bacterial growth after overnight incubation in a standard CLSI broth microdilution assay using 100% Muller-Hinton broth. The experiments were repeated at least twice for each bacterial strain with similar values.
  • cationic host defense peptides including cathelicidins are membrane-lytic agents, killing bacteria by physical interaction with and disruption of bacterial cell membranes, although increasing evidence suggests the presence of intracellular targets for certain peptides [1, 16].
  • E. coli ML-35p a strain that contains a plasmid giving constitutive expression of ⁇ -galactosidase in the cytosol, was incubated with different concentrations of peptides for 1 h in the presence of a chromogenic substrate, p-nitrophenyl- ⁇ -D-galactopyranoside (ONPG) [17-19].
  • Physiological concentrations of salt prove to be inhibitory to antibacterial activities of many antimicrobial peptides, such as human cathelicidin LL-37 [18] and ⁇ - and ⁇ -defensins [20, 21].
  • many antimicrobial peptides such as human cathelicidin LL-37 [18] and ⁇ - and ⁇ -defensins [20, 21].
  • the presence of 100 mM NaCl had little impact on membrane lysis with only a minimal delay in killing kinetics for fowlicidin-3 (Figure 18), consistent with our direct colony counting assay (data not shown).
  • These data suggested that, similar to fowlicidin-1 and -2 [Example 1], fowlicidin-3 kills bacteria in a salt-independent manner, in contrast with many other peptides whose activities are severely suppressed in the presence of salt [18, 20, 21].
  • Serum has been found to be another important inhibitory factor in bactericidal activities of many host defense peptides, probably due to the presence of certain salts, divalent cations, and peptide-binding proteins.
  • a radial diffusion assay [22] was performed with S. aureus ATCC 25923 and peptides diluted with and without 50% human or chicken serum. The results revealed that both fowlicidin-3 and -1 retained >80% antibacterial activity in either serum ( Figure 19A and B), implying their in vivo therapeutic potential for systemic applications. Evaluation of the toxicity offowlicidin-3 to mammalian cells
  • fowlicidin-3 is slightly more potent than fowlicidin-1 in killing many bacterial strains tested, but appears to be 4- to 6-fold less toxic to mammalian cells than fowlicidin-1. Inhibition of LPS-induced proinflammatory gene expression by fowlicidin-3
  • fowlicidin-1 and -2 were found to be able to bind LPS directly and suppressed LPS-induced cytokine gene expression [Example 1], we sought to determine whether fowlicidin-3 has a similar LPS-neutralizing activity.
  • Mouse macrophage RAW264.7 cells were stimulated for 4 h with 100 ng/ml LPS in the presence and absence of different concentrations of fowlicidins, followed by real-time RT-PCR analysis of the expressions of three common proinflammatory genes, including interleukin (IL)-IB, CC chemokine ligand 2 (CCL2)/MCP-1, and CCL3/MIP-la.
  • IL interleukin
  • CCL2 CC chemokine ligand 2
  • CCL3/MIP-la CCL3/MIP-la
  • the long helices of fowlicidin-1 and -3 are much less amphipathic, with no obvious segregation of hydrophobic residues from hydrophilic residues.
  • the ⁇ -helical region is highly hydrophobic in that fowlicidin-3 is composed of only one cationic (K 22 ) and two polar uncharged residues (N 12 , T 13 and N 19 ), whereas fowlicidin-1 consists of only two cationic (R 11 and R 21 ) and two polar uncharged residues (T 12 and N 18 ) (Figure 17B). Instead, positively charged residues are mostly concentrated at both tails (Figure 17B).
  • fowlicidin-3 is much less toxic to mammalian cells than fowlicidin-1. Because the cytotoxicity (EC 50 ) of fowlicidin-3 is at least 10-to 40-fold (in the presence of serum) higher than MICs against all bacterial strains tested, a therapeutic window clearly exists for fowlicidin-3 particularly for systemic applications. More desirably, fowlicidin-3 is highly potent in blocking LP S -induced proinflammatory gene expression. Collectively, fowlicidin-3 appears to have promising therapeutic potential for further development as a novel antimicrobial and antisepsis agent.
  • fowlicidin-1 is likely due to limited flexibility of the a-helix, which is a result of the physical hindrance caused by the side chain of a nearby tyrosine [Example 2].
  • fowlicidin-3 is devoid of aromatic residues adjacent to the conserved glycine ( Figure 17B), it will be important to examine the impact of further enhancing its flexibility on the functional properties. In fact, the flexibility of the hinge region has often been found to be positively correlated with an decrease in the toxicity of many ⁇ -helical peptides [23, 24].
  • MOLMOL a program for display and analysis of macromolecular structures. J MoI Graph 14, 51-55.
  • Fowlicidin-1 has been found to possess potent antibacterial activity against a broad range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains.
  • fowlicidin-1 (5-26) and fowlicidin-1 (6-26) showed identical MICs against all five bacterial strains tested, clearly suggesting that valine at position 5 is dispensable for the antibacterial activity.
  • Fowlicidin- 1(8-26) with an additional deletion of proline 7 displayed low antibacterial activities similar to fowlicidin- 1 (7-26), implying that proline 7 is not required for maintaining the antibacterial activity.
  • fowlicidin- 1(6-26) retaining potent antibacterial activity with a significantly reduced cytotoxicity, is a short peptide analog with enhanced therapeutic potential as an antimicrobial drug candidate.
  • Cationic antimicrobial peptides are naturally occurring antibiotics that are actively being explored as a new class of anti-infective agents.
  • LPS lipopolysaccharide
  • fowlicidin- 1 consists of a highly flexible, unstructured segment at the N-terminus from amino acid residues 1-8 and that the first five residues play a minimum role in antibacterial, LPS-binding, and cytotoxic activities (Example 2).
  • truncation mutants of fowlicidin-1 namely fowlicidin-1 (5-26), fowlicidin- 1(6-26), fowlicidin- 1(7-26) and fowlicidin- 1(8-26) (Table VIII), were chemically synthesized using the standard solid-phase method by Bio-Synthesis (Lewisville, TX). All peptides were purified to >95% purity by reverse phase high-performance liquid chromatography (RP-HPLC).
  • each peptide was further confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using the Voyager DE-PRO instrument (Applied Biosystems, Foster City, CA) housed in the Recombinant DNA/Protein Core Facility of Oklahoma State University. Lyophilized peptides were reconstituted in 0.01% acetic acid. The peptide concentrations were measured by UV absorbance at 280 nm in the presence of 6 M guanidine hydrochloride, based on the extinction coefficients for aromatic tryptophan and tyrosine residues present in peptides.
  • MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight
  • Gram-negative bacteria Salmonella enterica serovar Typhimurium DT104 ATCC 700408, and Klebsiella pneumoniae ATCC 13883
  • Gram-positive bacteria ⁇ Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC 25923, and S. aureus ATCC 43300
  • ATCC Manassas, VA
  • MicroBiologics St. Cloud, MN
  • the minimum inhibitory concentrations (MICs) were determined by a standard broth microdilution assay as recommended by the Clinical and Laboratory Standards Institute.
  • the toxicity was evaluated with human colonic epithelial Caco-2 cells (ATCC) by using alamarBlue dye (Biosource) as described (20,21). Briefly, cells were seeded into a 96-well plate at 5 x 10 4 cells/well in Dulbecco's modified Eagle's medium
  • DMEM fetal bovine serum
  • Percent cell death was calculated as [1 - (F peptide - F background )/ (F acetic acid - F background )] xlOO, where F peptide is the fluorescence of cells exposed to peptides, F acetic acid is the fluorescence of cells exposed to 0.01% acetic acid only, and F background is the background fluorescence of 10% alamarBlue dye in DMEM without cells. Cytotoxicity (EC 50 ) of individual peptides will be defined as the peptide concentration that causes 50% cell death.
  • the standard broth microdilution assay was used to test the activities of four fowlicidin analogs against three Gram-positive and two Gram-negative bacteria, including multidrug-resistant strains S. aureus ATCC 43300 and S. enterica serovar Typhimurium
  • fowlicidin- 1(5-26) and fowlicidin- 1(6-26) showed identical MICs against all five bacterial strains tested (Table DC), clearly suggesting that valine at position 5 (V5) is dispensable for the antibacterial activity.
  • Fowlicidin- 1(7-26) and fowlicidin- 1(8-26) also displayed similar antibacterial activities (Table TX), implying that P7 is not required for maintaining the antibacterial activity.
  • fowlicidin-1 (6-26) had substantially reduced toxicity to human colonic Caco-2 cells, relative to either the full-length peptide or fowlicidin-1 (5-26) ( Figure 22), showing a further enhancement in safety and therapeutic window.
  • fowlicidin-1 (6-26) retaining potent antibacterial activity with a significantly reduced cytotoxicity, is a short peptide analog with enhanced therapeutic potential as an antimicrobial drug candidate.
  • fowl-2(l-18) and fowl-2(15-31) analogs is also designed and compared for antibacterial, cytotoxic, and endotoxin-neutralizing activities.
  • Various deletion variants are also made to probe the minimal sequence required for antimicrobial activity of fowlicidins. C-terminal amidation of these short fragments is performed as well.

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Abstract

L'invention concerne des peptides antimicrobiens et des procédés pour l'utilisation de ceux-ci. Les peptides sont des versions tronquées optimisées de cathélicidines du poulet (des 'fowlicidines').
PCT/US2006/046022 2005-12-02 2006-12-01 Cathelicidines du poulet tronquées et optimisées ('fowlicidines') et procédés d'utilisation de celles-ci WO2007064903A1 (fr)

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CN105732792A (zh) * 2016-03-30 2016-07-06 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） 一种酵母表达的鸡Cathelicidin1抗菌肽及其制备方法与应用
CN105753959A (zh) * 2016-03-30 2016-07-13 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） 一种酵母表达的鸡Cathelicidin2抗菌肽及其制备方法与应用
CN107586790A (zh) * 2017-11-11 2018-01-16 河南牧翔动物药业有限公司 一种鸡Fowlicidins‑1抗菌肽在毕赤酵母中的分泌表达方法
CN115960197A (zh) * 2022-11-14 2023-04-14 北京大北农科技集团股份有限公司 猪源抗菌肽pamp-37-bt3及其应用

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Title
LYNN ET AL.: "Bioinformatic Discovery and Initial Characterisation of Nine Novel Antimicrobial Peptide Genes in the Chicken", IMMUNOGENETICS, vol. 56, 2004, pages 170 - 177, XP003013382 *
SANCHEZ ET AL.: "Overexpression and Structural Study of the Cathelicidin Motif of the Protegin-3 Precursor", BIOCHEMISTRY, vol. 41, 2002, pages 21 - 30, XP003013381 *
VAN DIJK ET AL.: "CMAP27, A Novel Chicken Cathelicidin-like Antimicrobial Protein", VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY, vol. 106, 2005, pages 321 - 327, XP004936120 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100221272A1 (en) * 2007-06-28 2010-09-02 University Of Saskatchewan Immunomodulatory compositions and methods for treating disease with modified host defense peptides
US9102754B2 (en) * 2007-06-28 2015-08-11 University Of Saskatchewan Immunomodulatory compositions and methods for treating disease with modified host defense peptides
US20160083443A1 (en) * 2007-06-28 2016-03-24 University Of Saskatchewan Immunomodulatory compositions and methods for treating disease with modified host defense peptides
US10301363B2 (en) 2009-02-13 2019-05-28 Universiteit Utrecht Holding B.V. Antimicrobial peptides based on CMAP27
US20120093844A1 (en) * 2009-02-13 2012-04-19 Universiteit Utrecht Holding B.V. Antimicrobial peptides based on cmap27
US9006174B2 (en) 2009-02-13 2015-04-14 Universiteit Utrecht Holding B.V. Antimicrobial peptides based on CMAP27
WO2010093245A1 (fr) * 2009-02-13 2010-08-19 Nederlandse Organisatie Voor Toegepast- Natuurwetenschappelijk Onderzoek Tno Peptides antimicrobiens à base de cmap27
US11186619B2 (en) 2009-02-13 2021-11-30 Universiteit Utrecht Holding B.V. Antimicrobial peptides based on CMAP27
EP3150627A1 (fr) * 2009-02-13 2017-04-05 Universiteit Utrecht Holding B.V. Peptides anti-microbiens basés sur cmap27
CN105732792A (zh) * 2016-03-30 2016-07-06 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） 一种酵母表达的鸡Cathelicidin1抗菌肽及其制备方法与应用
CN105753959A (zh) * 2016-03-30 2016-07-13 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） 一种酵母表达的鸡Cathelicidin2抗菌肽及其制备方法与应用
CN107586790A (zh) * 2017-11-11 2018-01-16 河南牧翔动物药业有限公司 一种鸡Fowlicidins‑1抗菌肽在毕赤酵母中的分泌表达方法
CN115960197A (zh) * 2022-11-14 2023-04-14 北京大北农科技集团股份有限公司 猪源抗菌肽pamp-37-bt3及其应用
CN115960197B (zh) * 2022-11-14 2023-06-30 北京大北农科技集团股份有限公司 猪源抗菌肽pamp-37-bt3及其应用

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