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Anti-endotoxic, antimicrobial cationic peptides, the encoding DNA and methods of use thereof

a cationic peptide, anti-endotoxic technology, applied in the field of anti-endotoxic, anti-microbial cationic peptides, can solve the problems of limited amount of these peptides that can be isolated, defensin has been cloned, and no successful expression has been demonstrated

Inactive Publication Date: 2008-01-10
THE UNIV OF BRITISH COLUMBIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides antimicrobial cationic peptides that can inhibit the growth of a variety of microorganisms, including gram negative bacteria, gram positive bacteria, fungi, protozoa, parasites, and viruses. These peptides can be used alone or in combination with other antibiotics to treat bacterial infections. The invention also provides methods for inhibiting the growth of eukaryotic cells and treating disorders associated with cell proliferation. The peptides can accelerate wound healing and are effective in treating respiratory and pulmonary disorders. The invention also includes transgenic non-human animals and fish expressing the peptides.

Problems solved by technology

However, only limited amounts of these peptides can be isolated from the host species.
The gene for human defensin has been cloned and sequenced, but no successful expression has been demonstrated, as yet.
Furthermore, production of these peptides using peptide synthesis technology produces peptides in limited amounts and is expensive when scaled up or when many variant peptides must be produced.
Also, structural analysis is difficult without specific incorporation of 15N and 13C tagged amino acids which is prohibitively expensive using amino acid synthesis technology.
In addition, the antimicrobial activity of magainin appears to result in the disruption of the membrane functions of Paramecium caudatum.
Resistance to such a generally destructive mechanism may prove difficult for some microbial pathogens, as compared with the more specific mechanisms of the currently used antibiotics.

Method used

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  • Anti-endotoxic, antimicrobial cationic peptides, the encoding DNA and methods of use thereof
  • Anti-endotoxic, antimicrobial cationic peptides, the encoding DNA and methods of use thereof
  • Anti-endotoxic, antimicrobial cationic peptides, the encoding DNA and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Synthesis of Novel Cationic Antimicrobial Peptides

[0084] Cationic peptides were synthesized at the University of British Columbia service facility. The amino acid sequence of the peptides are shown in Table 1.

TABLE 1Peptide Amino Acid Sequences and CharacteristicsSEQ%IDPeptideAmino Acid SequenceLengthChargeHydrophobicityNO:CEMAKWKLFKKIGIGAVLKVLTTGL28+6641PALKLTKCP-29KWKSFIKKLTTAVKKVLVGLP26+6502ALISCM-1:KWKSFIKKLTSAAKKVVTTAK27+7563PLALISCM-2:KWKSFIKKLTKAAKKVVTTAK26+9544KPLIVCM-3:KWKKFIKSLTKSAAKTVVKTA27+9525KKPLIVCM-4:KWKLFKKIGIGAVLKVLKVLI32+7666TTGLPALKLTLKCM-5:KLFKKIGIGAVLKVLKVLTTG30+6657LPALKLTLKCM-6:KWKFKKIGIGAVLKVLKVLTT31+7638GLPALKLTLKCM-7:KLWKLFKKIGIGAVLKVLKVL33+7669TTGLPALKLTLKCPα1:KWKSFIKKLTSAAKKVTTAAK25+84410PLTKCPα2:KWKKFIKKIGIGAVLKVLTTG30+96011LPALKLTKKCPα3:KKWKKFIKKIGIGAVLTTPGA23+85712KK

example 2

Susceptibility Testing

[0085] A method which employed polypropylene microtiter trays in a broth microdilution assay was developed. Theses studies showed that several of the peptides had good antimicrobial activity (Table 2).

TABLE 2MIC (μg / ml) of the antimicrobial peptidesagainst a variety of bacteriaPeptideP. aeruginosaE. coliS. epidermisS. aureusK147CEMA32343CP-296251911CM151216432CM2315>64>64CM35116>64>64CM444488CM516411>6464CM68283232CM782484-8CPα1≧641.5—>64>64CPα262—1616CPα3≧644—>64>64

[0086] The data in this table represent an average of three separate experiments.

example 3

Synergy with Conventional Antibiotics

[0087] It has been shown that some peptides demonstrated synergy with conventional antibiotics. To test whether the peptides of the invention synergize with various antibiotics, the following method was employed. Fractional Inhibitory Concentration (FIC) was used to determine synergy of peptides combined with antibiotics (e.g., carbenicillin / ciprofloxacin) against Pseudomonas aeruginosa. The following methodology was followed:

[0088] (1) Determination of MIC of cationic peptides (MIC A): [0089] 100 μl Mueller Hinton broth (MHB) was added per well [0090] 12.5 μl of 10× test concentration peptide was added per well to get final peptide concentration of e.g., 128, 64, 32, . . . 0.025 μg / ml from row 1 to 11; [0091] 10 μl of 10−4 dilution of overnight bacterial culture was added per well.

[0092] (2) Determination of MIC of Carbenicillin / Ciprofloxacin (MIC B): [0093] 100 μl MHB was added per well; [0094] 100 μl of 2× test concentration of antibiotic w...

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Abstract

A novel class of cationic peptides having antimicrobial activity is provided. Exemplary peptides of the invention include KWKSFIKKLTSAAKKVVTTAKPLALIS (SEQ ID NO:3) and KGWGSFFKKAAHVGKHVGKAALTHYL (SEQ ID NO:15). Also provided are methods for inhibiting the growth of bacteria utilizing the peptides of the invention. Such methods are useful for the treatment of respiratory infections, such as in cystic fibrosis patients. Such methods are further useful for accelerating wound healing.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of 10 / 823,425 filed Apr. 12, 2004, which is a divisional application of U.S. Ser. No. 09 / 908,139 filed Jul. 17, 2001, now issued as U.S. Pat. No. 6,818,407; which is a divisional application of U.S. application Ser. No. 09 / 143,124 filed Aug. 28, 1998, now issued as U.S. Pat. No. 6,288,212. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this applicationBACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates generally to antimicrobial peptides and specifically to antimicrobial cationic peptides useful for overcoming antibiotic resistance and effective as therapeutics for pathologies resulting from microbial infections. [0004] 2. Description of Related Art [0005] In 1981, the self-promoted uptake hypothesis was first proposed to explain the mechanism of action of polycationic antibiotics in Pse...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/711A61K38/16A61P31/04A61K38/00C07K14/47
CPCA61K31/711C07K14/4723A61K38/00A61P31/04Y02A50/30
Inventor HANCOCK, ROBERT E. W.GOUGH, MONISHA A.PATRZYKAT, ALEKSANDERWOODS, DONALDJIA, XIAOYAN
Owner THE UNIV OF BRITISH COLUMBIA