Modified apidaecin derivatives as antibiotic peptides

Abstract

This invention relates to modified antibiotic peptides, particularly for use in medicine. The invention further relates to composite and methods for destroying microorganisms, such as bacteria, viruses or fungi, and to methods for treating microbial infections. The object of the invention is to develop novel antibiotic peptides, particularly having enhanced antibiotic activity and an expanded spectrum of activity against other strains of bacteria, particularly gram-positive bacteria such as Staphylococcus aureus. According to the invention, the object is attained in a first aspect by a peptide according to claim 1.

Claims

1. A peptide for use as a medicament in the treatment of an infection with gram-positive bacteria comprising an amino acid sequence according to the general formula A or B: TABLE-US-00018 (SEQIDNO:122) X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16(Formula A) (SEQIDNO:94) X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18 (FormulaB) wherein the amino acid sequence according to Formula A or B has at least 80% amino acid sequence identity to the native Apidaecin 1b according to SEQ ID NO: 2 and wherein: X.sub.1 is selected from nonpolar amino acid residues, aromatic amino acid residues, positively charged amino acid residues, amino acid residues with a thiol group, and amino acid residues with a selenol group; X.sub.2, X.sub.3 and X.sub.5 are selected independently from each other from neutral and positively charged amino acid residues; X.sub.4 is selected from positively charged amino acid residues, amino acid residues with a thiol group and amino acid residues with a selenol group; X.sub.6 is selected from nonpolar amino acid residues with at least 2 C atoms in the side chain, aromatic amino acid residues, positively charged amino acid residues, amino acid residues with a thiol group, and amino acid residues with a selenol group; X.sub.7 is selected from tyrosine, positively charged amino acid residues, amino acid residues with a thiol group and amino acid residues with a selenol group; X.sub.8 is selected from nonpolar, aromatic amino acid residues with at least 2 and at most 8 C atoms in the side chain, positively charged amino acid residues, amino acid residues with a thiol group and amino acid residues with a selenol group; X.sub.9, X.sub.13, X.sub.14 and X.sub.16 are selected independently of each other from positively charged amino acid residues, amino acid residues with a thiol group, amino acid residues with a selenol group, nonpolar aromatic amino acid residues, and heteroaromatic amino acid residues; X.sub.10 is selected from neutral amino acid residues, positively charged amino acid residues, amino acid residues with a thiol group and amino acid residues with a selenol group; X.sub.11 is selected from proline, proline derivatives, positively charged amino acid residues, amino acid residues with a thiol group and amino acid residues with a selenol group; X.sub.12 is a positively charged amino acid residue; X.sub.17 is selected from positively charged amino acid residues; X.sub.18 is selected from nonpolar amino acid residues, positively charged amino acid residues, amino acid residues with a thiol group and amino acid residues with a selenol group, characterised in that at least one of the positions 2, 5 to 11, 13 to 16 and 18 of SEQ ID NO: 2 is modified, so that at least one of the following conditions applies to the peptide according to formula A or B: X.sub.2 is selected from nonpolar amino acid residues, positively charged amino acid residues, amino acid residues with a thiol group and amino acid residues with a selenol group, X.sub.10 is selected from lysine, -hydroxylysine, -N-methyllysine, allo-hydroxylysine, cysteine and selenol-cysteine, at least one of the residues selected from X.sub.5, X.sub.6, X.sub.7, X.sub.8, X.sub.9, X.sub.11, X.sub.13, X.sub.14, X.sub.16 and X.sub.18 is a positively charged residue, an amino acid residue with a thiol group or an amino acid residue with a selenol group, and/or X.sub.15 is selected from amino acid residues with a thiol group and amino acid residues with a selenol group.

2. The peptide according to claim 1, characterised in that at least one of the following positions of SEQ ID NO: 2 is modified, so that at least one of the following conditions applies to the peptide according to formula A or B: X.sub.2 is selected from nonpolar amino acid residues, positively charged amino acid residues, amino acid residues with a thiol group and amino acid residues with a selenol group, at least one of the residues selected from X.sub.5, X.sub.6, X.sub.7, X.sub.8, X.sub.9, X.sub.11, X.sub.13, X.sub.14, X.sub.16 and X.sub.18 is a positively charged residue, an amino acid residue with a thiol group or an amino acid residue with a selenol group, and/or X.sub.15 is selected from amino acid residues with a thiol group and amino acid residues with a selenol group.

3. The peptide according to claim 1, wherein at least one of the following conditions applies: X.sub.2 is arginine or glutamine, X.sub.5 is cysteine or arginine, at least one of the residues selected from X.sub.8, X.sub.13, X.sub.14, X.sub.18 is arginine, and/or X.sub.16 is a cysteine and optionally in addition X.sub.10 is an arginine.

4. The peptide according to claim 1 according to formula A, comprising an amino acid sequence according to the general formula C:
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17 (SEQ ID NO:123)(Formula C) wherein X.sub.17 is selected from positively charged amino acid residues.

5. The peptide according to claim 1, comprising one of the following amino acid sequences: TABLE-US-00019 (SEQ ID NO: 96) X.sub.1-W-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16, (SEQ ID NO: 97) X.sub.1-R-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16, (SEQ ID NO: 124) X.sub.1-X.sub.2-X.sub.3-X.sub.4-C-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16, (SEQ ID NO: 125) X.sub.1-X.sub.2-X.sub.3-X.sub.4-R-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16, (SEQ ID NO: 126) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-R-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16, (SEQ ID NO: 127) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-R-X.sub.14-X.sub.15-X.sub.16, (SEQ ID NO: 128) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-C-X.sub.15-X.sub.16, (SEQ ID NO: 129) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-C, (SEQ ID NO: 130) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16- X.sub.17-R, (SEQ ID NO: 105) X.sub.1-W-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-R-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16, (SEQ ID NO: 106) X.sub.1-W-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-R-X.sub.14-X.sub.15-X.sub.16, (SEQ ID NO: 131) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-R-X.sub.11-X.sub.12-R-X.sub.14-X.sub.15-X.sub.16, (SEQ ID NO: 108) X.sub.1-W-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-R-X.sub.11-X.sub.12-R-X.sub.14-X.sub.15-X.sub.16, wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 and X.sub.10 have the meanings given in claim 1.

6. The peptide according to claim 1, characterised in that X.sub.1 is selected from arginine, lysine, -hydroxylysine, homoarginine, 2,4-diaminobutyric acid, -homoarginine, D-arginine, arginal, 2-amino-3-guanidinopropionic acid, nitroarginine, N-methylarginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, 3-aminotyrosine, glycine, alanine, valine, isoleucine, leucine, methionine, N-methylleucine, tertbutyl glycine, cyclohexylalanine, -alanine, 1-amino-cylcohexyl carboxylic acid, N-methylisoleucine, norleucine, norvaline, N-methylvaline, cysteine, selenocysteine, phenylalanine, tryptophan, phenylglycine, homophenylalanine, 4-tertbutylphenylalanine, methyltryptophan, naphtylalanine, diphenylalanine, methylphenylalanine, phenyl-phenylalanine, benzoylphenylalanine, histidine, N-methylhistidine, 3,5-dinitrotyrosine, tyrosine, proline, -cyclohexylalanine, 3,4-cis-methanoproline, 3,4-dehydroproline, homoproline, mercaptoproline, thioproline, fluoroproline and pseudoproline.

7. The peptide according to claim 1, characterised in that X.sub.3, X.sub.2 and X.sub.5 are selected independently from each other from arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methylarginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, 3-aminotyrosine, asparagine, cysteine, selenocysteine, glutamine, serine, threonine, citrulline, N-methylserine, homoserine, allo-threonine, tyrosine, 3,5-dinitrotyrosine, histidine, N-methylhistidine, phenylalanine, tryptophan, phenylglycine, homophenylalanine, 4-tertbutylphenylalanine, methyltryptophan, naphtylalanine, diphenylalanine, methylphenylalanine, phenyl-phenylalanine, benzoylphenylalanine, -homoserine, proline, -cyclohexylalanine, 3,4-cis-methanoproline, 3,4-dehydroproline, homoproline, mercaptoproline, thioproline, fluoroproline and pseudoproline.

8. The peptide according to claim 1, characterised in that X.sub.4 is selected from arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, -homoarginine, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methylarginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, cysteine and selenocysteine.

9. The peptide according to claim 1, characterised in that X.sub.6 is selected from arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, -homoarginine, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methylarginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, valine, isoleucine, leucine, methionine, N-methylleucine, tertbutyl glycine, cyclohexylalanine, 1-amino-cylcohexyl carboxylic acid, N-methylisoleucine, norleucine, norvaline, N-methylvaline, phenylalanine, phenylglycine, homophenylalanine, 4-tertbutylphenylalanine, methyltryptophan, naphtylalanine, diphenylalanine, methylphenylalanine, phenyl-phenylalanine, benzoylphenylalanine, histidine, N-methylhistidine, tryptophan, tyrosine, cysteine, selenocysteine, proline, -cyclohexylalanine, 3,4-cis-methanoproline, 3,4-dehydroproline, homoproline, mercaptoproline, thioproline, fluoroproline and hydroxyproline.

10. The peptide according to claim 1, characterised in that X.sub.7 is selected from tyrosine, arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, -homoarginine, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methylarginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2-diaminopimelic acid, ornithine, sym-dimethyl arginine, asym-dimethyl arginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, cysteine and selenocysteine.

11. The peptide according to claim 1, characterised in that X.sub.8 is selected from arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl arginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, 3-aminotyrosine, cysteine, selenocysteine, valine, isoleucine, leucine, N-methylleucine, tertbutyl glycine, cyclohexylalanine, 1-amino-cylcohexyl carboxylic acid, N-methylisoleucine, norleucine, norvaline, N-methylvaline, phenylalanine, phenylglycine, homophenylalanine, 4-tertbutylphenylalanine, methyltryptophan, naphtylalanine, diphenylalanine, methylphenylalanine, phenyl-phenylalanine, benzoylphenylalanine, histidine, N-methylhistidine and tyrosine.

12. The peptide according to claim 1, characterised in that X.sub.9, X.sub.13, X.sub.14 and X.sub.16 are selected independently from each other from arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl arginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, 3-aminotyrosine, cysteine, selenocysteine, phenylalanine, tryptophan, phenylglycine, homophenylalanine, 4-tert-butylphenylalanine, methyltryptophan, naphtylalanine, diphenylalanine, methylphenylalanine, phenyl-phenylalanine, benzoylphenylalanine, histidine, N-methylhistidine, and -cyclohexylalanine.

13. The peptide according to claim 1, characterised in that the amino acid residue X.sub.10 is selected from arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methylarginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2-diaminopimelic acid, ornithine, sym-dimethyl arginine, asym-dimethyl arginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, 3-aminotyrosine, cysteine, selenocysteine, glutamine, citrulline, isoleucine, leucine, N-methylleucine, tertbutyl glycine, cyclohexylalanine, 1-aminocylcohexyl carboxylic acid, N-methylisoleucine, norleucine, norvaline, N-methylvaline, phenylalanine, tryptophan, phenylglycine, homophenylalanine, 4-tert-butylphenylalanine, methyltryptophan, naphtylalanine, diphenylalanine, methylphenylalanine, phenyl-phenylalanine, benzoylphenylalanine, histidine, N-methylhistidine, 3,5-dinitrotyrosine and tyrosine.

14. The peptide according to claim 1, characterised in that the amino acid residue X.sub.11 is selected from arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methylarginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2-diaminopimelic acid, ornithine, sym-dimethyl arginine, asym-dimethyl arginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, 3-aminotyrosine, cysteine, proline, -cyclohexylalanine, 3,4-cis-methanoproline, 3,4-dehydroproline, homoproline, mercaptoproline, thioproline, fluoroproline and hydroxyproline.

15. The peptide according to claim 1, characterised in that the amino acid residue X.sub.12 is selected from arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methylarginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2-diaminopimelic acid, ornithine, sym-dimethyl arginine, asym-dimethyl arginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid and 3-aminotyrosine.

16. The peptide according to claim 1, characterised in that the amino acid residue X.sub.15 is selected from histidine, N-methylhistidine, arginine, lysine, -hydroxylysine, homoarginine, -homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methylarginine, -N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexanoic acid, p-aminobenzoic acid, 3-aminotyrosine and cysteine.

17. The peptide according to claim 1, wherein the N-terminal amino acid and/or the C-terminal amino acid is modified.

18. The peptide according to claim 17, wherein the N-terminal amino acid modification is guanidination.

19. The peptide according to claim 1, in which the N-terminus and/or the C-terminus is connected directly or through a linker with at least one further peptide, protein, polymer and/or carrier.

20. The peptide according to claim 1, characterised in that at least one of the peptide bonds of the peptide backbone is chemically modified.

21. The peptide according to claim 1, wherein X.sub.2 is selected from tryptophan, arginine and cysteine.

22. The peptide according to claim 1, wherein X.sub.15 is a cysteine.

23. A pharmaceutical composition, characterised in that it comprises at least a peptide according to claim 1.

24. A host cell, which contains a peptide according to claim 1.

25. A peptide-multimer, comprising at least two peptides according to claim 1, wherein the at least two peptides are bound to each other through a linker peptide.

Description

(1) Below, the invention will be specified in greater detail in reference to the following embodiment examples and figures, without the invention being limited to these:

(2) FIG. 1 shows the results of the substitution analysis of apidaecin 1b (GNNRPVYIPQPRPPHPRLSEQ ID No. 2) with the amino acids indicated in the form of the one-letter code. Values of less than 1 indicate an enhancement of microbial activity. Values of greater than 1 indicate a deterioration in microbial activity. The left column shows the native sequence. The top line shows the substituents. The value of 0.55 directly below the C, for example, is a measure of the activity of the native apidaecin sequence that is substituted at position 1 with cysteine.

(3) FIG. 2 shows a specific embodiment of the peptides described herein. Each peptide marked with X is more effective than native apidaecin and is particularly advantageous in relation to gram-positive bacteria.

(4) FIG. 3 shows a permeabilization assay, E. coli BL21AI with apidaecin derivatives. Api88 corresponds to the peptide having SEQ ID No. 92, Api137 corresponds to the peptide having SEQ ID No. 93; Api1341 corresponds to the peptide having SEQ ID No. 89.

EXAMPLES

Example 1: Substitution Analysis by Means of Peptide Array

(5) To optimize the antimicrobial activity of apidaecin 1b, a substitution analysis was carried out using this peptide (according to SEQ ID No. 2). The substitution library was synthesized by means of SPOT synthesis, and was analyzed for antibacterial activity against Pseudomonas aeruginosa by means of bioluminescence assay.

(6) The SPOT synthesis of the peptide libraries was carried out on Whatman 50 filter paper (Sigma-Aldrich, Germany) measuring 1929 cm by means of the Fmoc method and a SPOT synthesizer (Intavis, AG, Germany) (according to Reineke U et al. 2001). The luminescence screening method that was used is based on the publication by Hilpert and Hancock (Hilpert, 2007). The peptides synthesized on the membrane were split off and the peptide spots were punched out of the peptide membrane using a hole punch, and were transferred to a 96-well microtiter plate (Corning, USA), after which 200 L distilled water per well was added. The plate was sealed with aluminum foil (Biorad, Germany) and shaken lightly for 18 hours at RT. Each peptide of the array was thereby transferred to precisely one well in a microtiter plate, referred to as a master plate. The sealed master plates were stored at 20 C. The master plates were designed such that each row contains 10 peptides and two control samples (positive and negative).

(7) In the second step, the actual screening was performed. For this purpose, an overnight culture (37 C., 225 rpm, 18 b) of a strain of luminescent bacteria (P. aeruginosa) was used. The overnight culture was diluted 100 times and allowed to grow to an optical density of 0.35 at 600 nm [OD600] (approx. 2 hourslogarithmic phase cultureLog C). The incubation suspension (4 vol % Log C in 100 mM Tris-HCl buffer (pH 7.3) with 40 mM sterile filtered glucose) was then distributed to 96-well plates (VWR, Germany) that are suitable for luminescence, and incubated with a concentration series of the peptide library for 4 hours at 37 C. After incubation, luminescence measurement was performed using a luminometer (Thermo, Finland).

(8) From the results of the substitution analysis, those peptide sequences that showed the greatest activity in the assay were selected. These peptides were synthesized by conventional means on a polymeric carrier, and were analyzed in terms of their antibacterial activity against P. aeruginosa, E. coli, and S. aureus by means of MIC assay.

Example 2: Determining Minimum Inhibitory Concentrations and Growth Kinetics

(9) The minimum inhibitory concentrations (MIC) of the peptides were determined in a double assay of triplicates using s positive control sample (gentamycin) and a negative control sample (0.9% NaCl solution), according to a modified protocol from Wiegand et al. (Wiegand, 2008).

(10) For this purpose, the peptides were dissolved in water and were diluted in a double dilution series with MH (eight-fold diluted Mueller-Hinston-Medium2.6 g/L, Merck) in sterile 96-well, plates (Greiner Bio-One GmbH) in twelve dilution steps from 128 g/mL to 62.5 ng/mL. Overnight cultures were adjusted using MHB to approximately 1.510.sup.7 colony forming units per mL. Of these, 50 L of peptide solution per well was mixed with 50 L bacteria solution each to obtain an initial concentration of 410.sup.5 bacterial per well. After 20 hours of incubation at 37 C., the absorption was determined at 595 nm (microplate reader, Wallac Victor3, Perkin Elmer). The minimum inhibitory concentration was identified as the lowest peptide concentration at which no bacterial growth could be detected.

(11) In the experiment, the antibacterial activity of the peptides according to the invention in relation to the following bacterial strains was analyzed: Pseudomonas aeruginosa PAOI (wt strain), Pseudomonas aeruginosa DSM 9644, Staphylococcus aureus DSM 1104/ATCC 25923, Staphylococcus aureus ATCC 6247, Escherichia coli UB1005 (F, nalA37, metB1) and Escherichia coli ATCC25922. The following Table 4 shows the results of the test:

(12) TABLE-US-00013 TABLE4 Minimuminhibitoryconcentrationin1/8MHBing/mL ImprovementoverNative MIC[g/mL] Sequence(SEQNo.2) SEQ P. S.aureus P. S.aureus ID aeruginoas E.coli ATCC aeruginosa E.coli ATCC No. Peptide PAO1wt UB1005 25923 PAO1wt UB1005 25923 2* GNNRPVYIPQPRPPHPRL-OH 500 5 >125 1 1 1 30* GNNRPVYIPQPRPPHPRL-NH.sub.2 250 1.25 >125 2 4 54 GWNRPVYIPQPRPPHPRL-NH.sub.2 64 1.25 63 8 4 4 55 GRNRPVYIPQPRPPHPRL-NH.sub.2 64-128 0.625 32 4-8 8 8 56 GNNRCVYIPQPRPPHPRL-NH.sub.2 125 10 31 4 1 8 57 GNNRRVYIPQPRPPHPRL-NH.sub.2 64 5 32 8 1 8 58 GNNRPVYRPQPRPPHPRL-NH.sub.2 64 0.313 63 8 16 4 59 GNNRPVYIPQPRRPHPRL-NH.sub.2 125 10 31 4 1 8 60 GNNRPVYIPQPRPCHPRL-NH.sub.2 250 20 16 2 0 16 61 GNNRPVYIPQPRPPHCRL-NH.sub.2 125 20 32 4 0 8 62 GNNRPVYIPQPRPPHPRR-NH.sub.2 125 1.25-2.5 125 4 2-4 2 63 GWNRPVYIPRPRPPHPRL-NH.sub.2 16-32 0.63 16 16-32 8 16 52 GWNRPVYIPQPRRPHPRL-NH.sub.2 64 8 4-8 8 1 31-64 64 GNNRPVYIPRPRRPHFRL-NH.sub.2 64 2.5 4 8 2 64 50 GWNRPVYIPRFRRPHPRL-NH.sub.2 32 2.5 2 16 2 128 91* GNNDPVYIPQPRPPHPRL-NH.sub.2 121.0 >19.4 >60.5 4 <0.25 *Comparative examples

(13) The results show that the modifications (particularly N2W and/or P15R) significantly increase antimicrobial activity, particularly against P. aeruginosa and S. aureus.

(14) The antibacterial activity of the peptides according to the invention against the following pathogenic bacteria strains of the gram-positive bacterium S. aureus and of P. aeruginosa was also analyzed:

(15) TABLE-US-00014 TABLE5 AntimicrobialactivityagainstvariouspathogenicstrainsofS.aureus,andP.aeruginosa strains.MICvaluesweredeterminedintriplicatein1/8MHBing/mL. S.aureus DSM S.aureus 1104/ E.coli P.aeruginosa SEQID DSM ATCC ATCC PAODSM P.aeruginosa No. 6247 25923 25922 9644 PAO1wt 2* GNNRPVYIPQPRPPHPRL-OH 256 >125 2 >256 500 90* GNNRPVYIPQPRPPHPRL-NH.sub.2 64 >125 2 n.d. 250 52 GWNRPVYIPQPRPPHPRL-NH.sub.2 2 4-8 16 64 64 64 GWNRPVYIPRPRRPHPRL-NH.sub.2 2 4 8 32 64 50 GWNRPVYIPRPRRPHPRL-NH.sub.2 2 2 8 32 32 *Comparative examples, n.d.: Not determined

(16) In another experiment, the antibacterial activity of the peptides according to the invention having a guanidinated N-terminus against the following pathogenic bacteria strains of the gram-positive bacterium S. aureus and of P. aeruginosa was analyzed:

(17) TABLE-US-00015 TABLE6 AntimicrobialactivityagainstvariouspathogenicstrainsofS.aureus,andP.aeruginosa strains.MICvaluesweredeterminedintriplicatein1/8MHBing/mL. SEQID S.aureusDSM P.aeruginosa E.coli No. 6247 PAODSM9644 DSM1103 84 gu-ONNRPVYIPRPRPPHPRR-OH 8 64 4 85 gu-OWNRPVYIPRPRPPHPRL-OH 16 16 4 86 gu-ONNRPVYIPRPRRPHPRL-OH 32 32 8 87 gu-OWNRPVYIPRPRRPHPRL-OH 16 8 8 88 gu-ONNRPVYIPRPRRPHPRL-NH.sub.2 16 16 8 89 gu-OWNRPVYIPRPRRPHPRL-NH.sub.2 8-16 4 8 Gu: N-Terminus contains a tetramethylguanidino group (N-guanido-ornithine)

(18) Additional measurements produced the following results.

(19) TABLE-US-00016 TABLE7 Antimicrobialactivityagainstvariouspathogenicstrains.MICvaluesweredeterminedin triplicatein1/8MHBing/mL. Dilutedmedium E.coli E.coli K.pneumoniae P.auruginosa S.aureus BL21A1 ATCC25922 Salmonellae. DMS681 DMS9644 DSM6247 SEQIDNo. PeptideSequence TSB TSB TSB TSB TSB MHB 92 gu-ONNRPVYIPRPRPPHPRL-NH.sub.2 1 2 2 8 8 89 gu-OWNRPVYIPRPRRPPRL-NH.sub.2 8 18 >64 8 2 93 gu-ONNRPVYIPRPRPPHPRL-OH 1 4 2 16 16-32 85 gu-OWNRPVYIPRPRPPHPRL-OH 2 4 2 8-16 4 87 gu-OWNRPVYIPRPRRPHPRL-OH 8 8 4-8 8 4 Normalmedium E.coli E.coli K.pneumoniae P.auruginosa S.aureus BL21A1 ATCC25922 Salmonellae. DMS681 DMS9644 DSM6247 SEQIDNo. PeptideSequence TSB TSB TSB TSB TSB MHB 92 gu-ONNRPVYIPRPRPPHPRL-NH.sub.2 89 gu-OWNRPVYIPRPRRPPRL-NH.sub.2 93 gu-ONNRPVYIPRPRPPHPRL-OH 85 gu-OWNRPVYIPRPRPPHPRL-OH 87 gu-OWNRPVYIPRPRRPHPRL-OH

(20) The serum stability of selected apidaecin derivatives was also measured. The results are shown in Table 8.

(21) TABLE-US-00017 TABLE8 SerumstabilityofapidaecinWRderivativesin25%and100%mouseserum. t [min] SEQID 25% t.sub.1/2[min] No. Sequence.sup.a serum 100%serum DegradationProducts 92 gu-ONNRPVYIPRPRPPHPRL-NH.sub.2 15 1 gu-O1-R17 89 gu-OWNRPVYIPRPRRPHPRL-NH.sub.2 16 1 gu-O1-R17,gu-O1-R12 93 gu-ONNRPVYIPRPRPPHPRL-OH 360 34 gu-O1-R17 85 gu-OWNRPVYIPRPRPPHPRL-OH 237 16 gu-O1-R17,gu-O1-R16 87 gu-OWNRPVYIPRPRRPHPRL-OH 0%after2hours gu-O1-R12(Apl341analogue)

(22) The following abbreviations are used in the description of the invention:

(23) BOC tert-butyloxy carbonyl

(24) .sup.tBu tert-butylether

(25) DCM dichloromethane

(26) DMF dimethylformamide

(27) eq. equivalents per mol, mol equivalents

(28) Fmoc fluorenylmethoxycarbonyl

(29) Guan guanidino group (at the N-terminus)

(30) Hyp trans-4-hydroxyproline

(31) HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate

(32) HOBt 1-hydroxybenzotriazole

(33) M mol/l

(34) MALDI-TOF matrix assisted laser desorption/ionization with time of flight analysis

(35) MIC minimum inhibitory concentration

(36) MS mass spectrometry

(37) Mtt 4-methyltrityl

(38) NHS N-hydroxysuccinimide

(39) NMM N-methylmorpholine

(40) O ornithine

(41) O.sup.tBu tert-butylester

(42) PBS phospho-buffered saline

(43) RP-HPLC reversed phase high performance liquid chromatography

(44) RT room temperature

(45) TCA trichloroacetic acid

(46) TFA trifluoroacetic acid

(47) Tris tris(hydroxymethyl)-aminomethane

(48) TSB tryptic soy broth

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