KR20050060602A - Antimicrobial peptides with reduced hemolysis and methods of their use - Google Patents

Abstract

The present invention relates to antimicrobial peptides consisting of cyclic short peptides (less than 10 amino acid residues) with unique patterns of aromatic and cationic moieties, which exhibit a wide range of antimicrobial activity but have less hemolytic action.

Description

ANTIMICROBIAL PEPTIDES WITH REDUCED HEMOLYSIS AND METHODS OF THEIR USE}

The present invention relates to the field of antibiotic peptides. In particular, the present invention relates to cyclic short peptides (less than 10 amino acid residues) having a wide range of antimicrobial activities and having unique patterns of aromatic and cationic residues. Particularly compact peptides of the invention have less hemolytic action and superior efficacy compared to other long peptides with aromatic and cationic moieties.

The emergence of bacterial strains resistant to conventional antibiotics has led to an active search for new therapeutic agents, including various antimicrobial peptides of animal origin. Antimicrobial peptides are recognized to play an important role in the intrinsic host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals, and are known to possess effective antibiotic activity against bacteria, fungi and even some viruses. . Antimicrobial peptides impair membrane preservation by allowing> 95% fractions to bind lipids and easily distribute into the phospholipid bilayer. These peptides form small, transient conductance increases in planar lipid bilayers that partially negate the cytoplasmic membrane potential gradients of bacteria. The protective function of antimicrobial peptides in host defense has been demonstrated by convincing evidence in Drosophila. In other words, reducing the expression of antimicrobial peptides in Drosophila dramatically reduced survival after microbial chanllange. The function of this peptide in mammals is shown by incomplete bacterial killing of lungs and small mice in cystic fibrosis (CF) patients. Antimicrobial peptides found in mammals are classified into cysteine rich defensins (α- and β-defensins) and various cathelicidin families. Cathelicidins include various antimicrobial sequences in the C-terminal domain, highly conserved signal sequences, and proregions (“catelins”). Many cathelicidins contain a characteristic elastase cleavage site between the anionic catelin domain and the cationic C-terminal peptide domain. Proteolytic processing at this site was observed in neutrophils of cattle and pigs and was required for bactericidal activity. Catericidins are classified into three groups based on amino acid composition and structure. The first group includes amphoteric α-helix peptides such as LL-37, CRAMP, SMAP-29, PMAP-37, BMAP-27, and BMAP-28. The second group includes Arg / Pro-rich or Trp-rich peptides including Bac5, Bac7, PR-39, and indolisidine. The third group includes Cys containing peptides such as protegrins. Since antimicrobial peptides are generally low molecular weight molecules (<5 kDa) that possess a wide range of activities and constitute an important part of host defense against microbial infection, these peptides are the starting point for designing low molecular weight antibiotic compounds. It is also known that these peptides tend to fold into amphoteric structures with clusters of hydrophobic and charged regions that are closely related to membrane degradation activity. Although these peptides often exhibit a wide range of antimicrobial activities, they are hemolytic against human red blood cells to varying degrees, severely limiting their therapeutic potential. It is the primary purpose of this work to first design antimicrobial peptides having activity against clinically important bacterial strains with synthetic modifications of the primary structure, and secondly to obtain some information on the important structural characteristics of the peptides. Studies have shown that the production of synthetic analogs with modified primary and / or secondary structures can improve the activity or reduce toxicity of natural peptide antibiotics. The present invention relates generally to antibiotics and to novel antimicrobial peptides and derivatives thereof having a wide range of activities, including tryptophan rich sequences that exhibit antimicrobial activity but have reduced hemolytic performance. In short, the present invention relates to the design of antibiotic peptides with a broad range of antimicrobial activities against Gram positive and Gram negative bacteria, protozoa, fungi and enveloped virus HIV-1.

The present invention is directed to the design of cyclic short peptides with improved serum compatibility and reduced hemolytic activity. In addition, the peptides of the present invention exhibit a wide range of antimicrobial activities against gram positive, gram negative bacteria and multidrug resistant pathogens. Antimicrobial peptides of the invention generally consist of less than 10 amino acid residues and comprise the amino acid sequence (A ₁ X ₁ A ₂ ) _N. Wherein A ₁ represents arginine, lysine, valine or isoleucine, X ₁ represents tryptophan, phenylalanine or proline, A ₂ represents arginine, lysine, valine or isoleucine, N represents 1, 2 or 3, topology Is annular or linear.

The present invention provides several advantages. First, the peptides of the invention are useful for spanning cell membranes with a relatively small number of amino acids residues of less than 10 and very dense. Second, the best peptides obtained from the present invention exhibit a fairly wide range of activity against antibiotic resistant bacteria, along with activity against medically important fungi. In addition, these peptides possess antitoxin activity and synergize with conventional antibiotics. Most important in the present invention is the very low hemolysis to human erythrocytes and improved serum compatibility compared to natural protegrin and indolisidine analogs.

US Patent No. 6303575; US Patent No. 6180604; US Patent No. 5821224; US Patent No. 5547939; US Patent No. 5534939; Like the tryptophan rich peptides disclosed in US Pat. No. 5,533,525 and US Pat. No. 5,353,616, these natural peptides with amino acid sequences all have 10 or more residues and rapidly proteolyze in vivo. Indolisidine analogs are generally represented by the amino acid sequence ILPWKWPWWPWX, where X represents one or two selected amino acids (US Pat. No. 5,439,939). The above-described tryptophan-rich peptide is a peptide of the present invention in that the antimicrobial peptide of the present invention possesses a cyclic short peptide (less than 10 amino acids) and has a wide range of antimicrobial activity, improved selectivity, and low hemolytic activity. Is different. Cyclicization of linear antimicrobial peptides may exhibit several advantages in terms of selectivity and stability. For example: (i) Unfolded peptides may form aggregates due to hydrophobic interactions, resulting in nonspecific adsorption and low solubility to normal mammalian cells. Constraints on unfolded structures can result in limited exposure of hydrophobic stretches of amino acids to limit hydrophobic interactions. In addition, these constraints can enhance the role of electrostatic interactions upon initial binding with negatively charged target membranes, thereby substantially increasing the selectivity for bacteria versus mammalian cells. (ii) For the protease to bind and degrade, the peptide must provide a site of degradation in the extended structure. Thus, circularizing short peptides may limit the possibility of being affected for protease activity due to the rigid, restricted structure. (iii) As cyclization appears to affect only activity against Gram-negative bacteria, further studies in parallel could aid in the design of bacterial specific soluble peptides. The peptide of the present invention can be represented by the amino acid sequence (A ₁ X ₁ A ₂ ) _N. Wherein A ₁ represents arginine, lysine, valine or isoleucine, X ₁ represents tryptophan, phenylalanine or proline, A ₂ represents arginine, lysine, valine or isoleucine, N represents 1, 2 or 3, topology Is annular or linear.

Examples of bactericidal peptides derived from the present invention are as follows:

Round-K F I

Linear-K F I

Ring-R F I

Linear-R F I

Round-R F V

Linear-R F V

Ring-K F R

Linear-K F R

Annular-K W V

Linear-K W V

Ring-K W I

Linear-K W I

Annular-K W R

Linear-K W R

Annular-R W V

Linear-R W V

Ring-K W R R W I

Linear-K W R R W I

Ring-K W R R W V

Linear-K W R R W V

Ring-K W I K W R

Linear-K W I K W R

Round-K W V K W I

Linear-K W V K W I

Round-K W I K W I

Linear-K W I K W I

Round-K W I K W I K W I

Linear-K W I K W I K W I

Round-K F I K F I K F I

Linear-K F I K F I K F I

Ring-K W R R W V R W I

Linear-K W R R W V R W I

Ring-I W R V W R R W K

Linear-I W R V W R R W K

Ring-K F R R F V R F I

Linear-K F R R F V R F I

Ring-K P R R P V R P I

Linear-K P R R P V R P I

Ring-K W I R W V R W I

Linear-K W I R W V R W I

The aforementioned peptides can be amidated or esterified at C-terminal amino acids or acetylated at N-terminal amino acids. In addition, one or more amino acids of the peptide may be altered to the corresponding D-amino acid.

Example 1 Design, Synthesis, Purification and Characterization of Peptides

Peptide analogs and their names are shown in Table 1. All amino acids are represented by a one letter amino acid code.

   Name Amino Acid Sequence  Pac 301 CKWRRWI (SEQ ID NO: 1) Pac 301 L KWRRWI (SEQ ID NO: 1) Pac 302 C KWRRWV (SEQ ID NO: 2) Pac 302 L KWRRWV (SEQ ID NO: 2) Pac 303 C KWIKWR (SEQ ID NO: 3) Pac 303 L KWIKWR (SEQ ID NO: 2) Pac 304 C KWVKWI (SEQ ID NO: 4) Pac 304 L KWVKWI (SEQ ID NO: 4) Pac-305 C KWIKWI (SEQ ID NO: 5) Pac-305 L KWIKWI (SEQ ID NO: 5) Pac-521 C KWIKWIKWI (SEQ ID NO: 6) ) Pac-521 L KWIKWIKWI (SEQ ID NO: 6) Pac-522 C KFIKFIKFI (SEQ ID NO: 7) Pac-522 L KFIKFIKFI (SEQ ID NO: 7) Pac-525 C KWRRWVRWI (SEQ ID NO: 8) Pac-525 L KWRRWVRWI (SEQ ID NO: 8 ) Pac-526 C IWRVWRRWK (SEQ ID NO: 9) Pac-526 L IWRVWRRWK (SEQ ID NO: 9) Pac-527 C KFRRFVRFI (SEQ ID NO: 10) Pac-527 L KFRRFVRFI (SEQ ID NO: 10) Pac-528 C KPRRPVRPI (SEQ ID NO: 11 ) Pac-528 L KPR R P V R P I (SEQ ID NO: 11) Pac-529 C K W I R W V R W I (SEQ ID NO: 12) Pac-529 L K W I R W V R W I (SEQ ID NO: 12)  C: cyclic; L: linear

Manually FALc (N- (9-fluoroenyl) methoxycarbonyl) on PAL resin (5- (4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy) -valeric acid-MBHA resin) All linear peptides were synthesized by solid phase peptide synthesis using chemistry. Dendritic Fmoc protecting groups were removed with 20% piperidine / DMF. The coupling reaction time was about 1 to 1.5 hours and was examined by the ninhydrin test. The crude peptide was degraded from the resin by mixing with 95% TFA digestion mixture for 1-1.5 hours. The crude peptide was purified by reverse phase high pressure liquid chromatography. The RP-HPLC preparative column was a semi-preparative C18 reversed phase column. The mobile phase for elution was a mixture of acetonitrile and DI H ₂ O mixed in different proportions using the planned gradient (Table 1). The detection wavelength was set to 225 nm and 280 nm, and the elution flow rate was 4 ml / min. The major peptide product was characterized by FAB-MS (High Speed Atomic Impact Mass Spectrometry) to determine the molecular weight of each peptide. Purity of each peptide was analyzed by analytical RP-HPLC.

Example 2: Detection of Peptide Activity in Vitro

In general, the in vitro antimicrobial activity of antimicrobial agents was tested using standard NCCLS bacterial inhibition assays or minimum inhibitory concentration (MIC) tests. The MIC value is the lowest concentration of peptide whose growth is inhibited and reduced by visually identifiable test organisms. Test organisms used for MIC analysis are shown in Table 2 below.

Test strain used to measure MIC          Organism source  Bacillus substilis ATCC 6633 Staphylococcus aureus ATCC 9144 Staphylococcus epidermidis ATCC 12228 Staphylococcus aureus ATCC 29737 Bacillus pumilus ATCC 14884 Bacillus cereus ATCC 11778 Pseudomonas aeruginosa ATCC 27853 Staphylococcus aureus ATCC 29213 E. coli ATCC 25922

In brief, the culture in which the test organism was incubated overnight was diluted to obtain an inoculum containing about 10 ⁵ colonies in Meuller-Hinton Broth (MHB). 50 μl volumes of dilutions of two serially diluted peptides from the peptide mother liquor were prepared in 96-well microtiter plates, and then all wells were inoculated with diluted cultures of test organisms. After 18 hours of incubation at 37 ° C., the results were analyzed for turbidity (cell growth). MIC values for each peptide are shown in Table 3 and FIG. 1. The MIC value is performed three times in each case (FIG. 2) and the median value is confirmed. From these results, the inventors confirmed that Pac521-530 exhibited maximum antimicrobial activity against gram positive and gram negative bacteria. Pac521-530 also had greater bactericidal activity than Pac 301-310.

MIC value (μg / ml) of synthetic peptides against E. coli and S. aureus Name E. coli S. aureus Pac 301 C> 64> 64Pac 301 L 16 8Pac 303 C> 64> 64Pac 303 L 24 16Pac-305 C> 64> 64Pac-305 L 24 16Pac-521 C 64 64Pac-521 L 8 16Pac-522 C 64 64Pac-522 L 8 4Pac-525 C 64 64Pac-525 L 2 4Pac-526 C 64 64Pac-526 L 4 4Pac-527 C 64 64Pac-527 L 4 4Pac-528 C 64 64Pac-528 L 2 4Pac-529 C 64 64Pac-529 L 8 4 C: cyclic; L: Linear

Example 3: Membrane Permeation Analysis

The outer membrane permeation activity of the peptide variant was determined by 1-N-phenylnaphthylamine (NPN) uptake assay using intact cells of E. coli. NPN shows weak fluorescence in aqueous environment but strong fluorescence in hydrophobic environment. Since NPN is hydrophobic, the outer membrane permeability can be measured directly. E. coli absorbs little or no NPN under normal conditions. In the presence of a penetrant compound (EDTA, polymyxin B, neomycin or antimicrobial peptide), NPN is distributed to the bacterial outer membrane to increase fluorescence. Briefly, incubate at 37 ° C. with shaking using 1 ml of overnight culture to inoculate 50 ml of medium. After propagation to OD ₆₀₀ = 0.4-0.6, the cells were precipitated by rotation (3500 rpm, 10 min). Wash and resuspend in buffer at OD ₆₀₀ = 0.5. Record OD ₆₀₀ . 1 ml of cells (OD ₆₀₀ = 0.5) is added to the cuvette and measured for 2-5 seconds. Add 20 [mu] l NPN 0.5 mM (shake and mix) and measure for 2-5 seconds. Add 10 μl of antibiotic at 100 × predetermined final concentration (shake and mix) and measure until reaching maximum (1-5 min). Therefore, the fluorescence varies depending on the concentration of the peptide. Peptide concentrations reaching 50% of the maximum increase in NPN uptake were recorded as P ₅₀ . Indeed, all peptides can interact with the membrane as demonstrated by NPN uptake assays, which are shown in Table 5 and FIG. 2.

Ability to permeate NPN through the outer membrane of E. coli and promote its absorption Peptide P ₅₀ (μg / ml)  Pac 301 L 8 Pac 305 L 16 Pac 309 L 8 Pac 521 L 2 Pac 525 L 2 Pac 529 L 4

Example 4: Characterization of the Trp Residue Environment

Because of the sensitivity of tryptophan to the polarity of the environment, tryptophan has been used for polarity and binding studies. Fluorescence emission spectra were recorded on an LS-55 fluorometer [Perkin-Elmer]. Measurements were made at 300-450 nm in 1 nm increments using 5 mm quartz cells at 25 ° C. The excitation and emission slit widths were both set to 5 nm and the excitation wavelength to 280 nm. In phosphate buffer, a series of antimicrobial peptides showed maximum release at 357 nm. In the presence of SDS, these peptides exhibited 8 nm blue shifted maximum emission wavelength with increasing intensity. This result suggests that the tryptophan side chain has moved to a more hydrophobic environment. The results of the study are presented in FIG. 3.

Example 5: Secondary Structure of Peptides Measured by CD Spectroscopy

CD spectra were recorded on an AVIV 62DS spectropolarimeter after assaying with d-10-camphorsulfonic acid. All measurements were performed using a 1 mm path length cuvette at a peptide concentration of 30 μM in 10 mM sodium phosphate buffer pH7.2. One-UV spectra were collected in the range of 190-260 nm using 0.5 nm step size and 1 second average time. In the absence of phospholipids, Pac301-310 and Pac 521-530 were characterized by an unordered structure (Table 6). However, this structure is derived by adding SDS (FIG. 4).

  Structure in Peptide Phosphate Buffer Structure in SDS Pac 301 C Not tidy Pac 301 L Not tidy Slightly tidy Pac 305 C Not tidy Pac 305 L Not tidy Tidy Pac 522 C Not tidy Pac 522 L Not trimmed Slightly trimmed Pac 525 C Not trimmed Pac 525 L Not trimmed Slightly trimmed

Example 6: Erythrocyte Lysis

Hemolysis against human red blood cells (RBCs) was tested in Pac 521-530. RBCs containing EDTA were rinsed three times in PBS (800 g, 10 min) and resuspended in PBS. RBC was diluted to 10% with phosphate buffered saline and 50 μl was added to each Eppendorf tube. Peptides dissolved in PBS were added to 50 μl of 10% RBC solution and incubated for 1 hour at 37 ° C. (final RBC concentration, 5% v / v). Samples were centrifuged at 800 g for 10 min at OD ₅₄₀ . Various peptide concentrations were incubated with pretreated RBCs and percent hemolysis was measured. As a result, it was confirmed that Pac525 had substantially less hemolytic response to RBC than other antimicrobial peptides (Table 7 and FIG. 5).

  Dissolution rate (%) Dissolution rate (%) Dissolution rate (%) at 50 μg / ml at peptide 5 μg / ml Pac 301 L 0.85 6.8 14 Pac 305 L 0.74 7.2 15 Pac 524 L 0.82 7.3 15 Pac 525 L 0.81 7.2 14

Sequence list

(1) General Information:

   (iii) number of sequences: 12

(2) information for SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 6 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO 1:

       Lys Trp Arg Arg Trp Ile

       1 5

(2) Information about SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 6 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO: 2:

        Lys Trp Arg Arg Trp Val

        1 5

(2) Information about SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 6 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO: 3:

       Lys Trp Ile Lys Trp Arg

       1 5

(2) Information about SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 6 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) information about SEQ ID NO:

       Lys Trp Val Lys Trp Ile

       1 5

(2) Information about SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 6 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO: 5:

       Lys Trp Ile Lys Trp Ile

       1 5

(2) Information about SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 9 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO 6:

        Lys Trp Ile Lys Trp Ile Lys Trp Ile

        1 5

(2) Information about SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 9 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO 7:

        Lys Phe Ile Lys Phe Ile Lys Phe Ile

        1 5

(2) Information about SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 9 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO: 8:

        Lys Trp Arg Arg Trp Val Arg Trp Ile

        1 5

(2) Information about SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 9 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) information about SEQ ID NO:

        Ile Trp Arg Val Trp Arg Arg Trp Lys

        1 5

(2) information for SEQ ID NO: 10:

   (i) Sequence Features:

      (A) Length: 9 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO: 10:

        Lys Phe Arg Arg Phe Val Arg Phe Ile

        1 5

(2) information for SEQ ID NO:

   (i) Sequence Features:

      (A) Length: 9 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO: 11:

        Lys Pro Arg Arg Pro Val Arg Pro Ile

        1 5

(2) information for SEQ ID NO: 12:

   (i) Sequence Features:

      (A) Length: 9 amino acids

      (B) type: amino acid

      (C) strand form: unknown

      (D) topology: linear or annular

   (ii) Molecular Type: Peptide

   (xi) SEQ ID NO: 12:

        Lys Trp Ile Arg Trp Val Arg Trp Ile

        1 5

The short peptides (less than 10 amino acid residues) of the present invention exhibit a wide range of antimicrobial activity and less hemolytic action.

1 (A) Bacillus substilis ATCC6633 treated with Pac-525 (() and Pac-524 ((); (B) Staphylococcus aureus ATCC9144; (C) E. coli ATCC25922; (D) Survival curve for Pseudomonas aeruginosa ATCC27853.

2 Pac-525 induced outer membrane permeation and was evaluated by NPN absorption analysis (0 μg / ml: ■; 1 μg / ml: ○; 2 μg / ml: □; 3 μg / ml: iii).

Fig. 3 Tryptophan fluorescence showing local environmental changes of Pac-525 induced by SDS.

4 Circular dichroism spectra of (A) Pac-525 and (B) Pac-526 in phosphate buffer (o) and 10 mM SDS (o) are shown.

5 This figure shows that hemolysis to human erythrocytes is very low. (●: melittin; ○: Pac-525; ▲: Pac-527).

SEQUENCE LISTING <110> Tzeng, Shiou-Ru Cheng, Jya-Wei <120> Antimicrobial Peptides with Reduced Hemolysis and Methods of Their Use <130> MR1035-1115 <140> US 10 / 247,476 <141> 2002-09-20 <160> 24 <170> PatentIn version 3.2 <210> 1 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 1 Lys Trp Arg Arg Trp Ile 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 2 Lys Trp Arg Arg Trp Ile 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 3 Lys Trp Arg Arg Trp Val 1 5 <210> 4 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 4 Lys Trp Arg Arg Trp Val 1 5 <210> 5 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 5 Lys Trp Ile Lys Trp Arg 1 5 <210> 6 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 6 Lys Trp Ile Lys Trp Arg 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 7 Lys Trp Val Lys Trp Ile 1 5 <210> 8 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 8 Lys Trp Val Lys Trp Ile 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 9 Lys Trp Ile Lys Trp Ile 1 5 <210> 10 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 10 Lys Trp Ile Lys Trp Ile 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 11 Lys Trp Ile Lys Trp Ile Lys Trp Ile 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 12 Lys Trp Ile Lys Trp Ile Lys Trp Ile 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 13 Lys Phe Ile Lys Phe Ile Lys Phe Ile 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 14 Lys Phe Ile Lys Phe Ile Lys Phe Ile 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 15 Lys Trp Arg Arg Trp Val Arg Trp Ile 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 16 Lys Trp Arg Arg Trp Val Arg Trp Ile 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 17 Ile Trp Arg Val Trp Arg Arg Trp Lys 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 18 Ile Trp Arg Val Trp Arg Arg Trp Lys 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 19 Lys Phe Arg Arg Phe Val Arg Phe Ile 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 20 Lys Phe Arg Arg Phe Val Arg Phe Ile 1 5 <210> 21 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 21 Lys Pro Arg Arg Pro Val Arg Pro Ile 1 5 <210> 22 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 22 Lys Pro Arg Arg Pro Val Arg Pro Ile 1 5 <210> 23 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 23 Lys Trp Ile Arg Trp Val Arg Trp Ile 1 5 <210> 24 <211> 9 <212> PRT <213> Artificial <220> <223> Artificial peptide <400> 24 Lys Trp Ile Arg Trp Val Arg Trp Ile 1 5

Claims (3)

(A ₁ X ₁ A ₂ ) A peptide having microbicidal activity which may be represented by _N , wherein A ₁ represents arginine, lysine, valine or isoleucine, X ₁ represents tryptophan, phenylalanine or proline, A ₂ represents arginine, lysine, valine or isoleucine, N represents 1, 2 or 3, the topology represents cyclic or linear and the disclosed peptides are amidated or esterified at C-terminal amino acids, or N-terminal A peptide that can be acetylated at an amino acid and wherein one or more amino acids of the peptide can be changed to the corresponding D-amino acid. A composition comprising the peptide of claim 1 in the form of a mixture with a carrier or excipient. A method of treating or preventing a microbial or viral infection in a subject comprising administering a therapeutically effective amount of the peptide of claim 1 to a patient in need thereof.