The hydrophobicity (H), hydrophobic moment (|i) and the angle subtended by the positively charged helix face () of a set of model and magainin 2 amide peptides with conserved charge and helix propensity have been shown to be effective modulators of antibacterial and haemolytic activity. Except peptides of low hydrophobicity which are inactive, changing the parameters has little influence on the activity against Gramnegative bacteria, thus revealing the dominance of electrostatic interactions for the effect. However, the increase of H, |i and <5> substantially enhances haemolytic and Gram-positive antibacterial activity and is related to a reduction of peptide specificity for Gram-negative bacteria.

Pexiganan, a 22-amino-acid antimicrobial peptide, is an analog of the magainin peptides isolated from the skin of the African clawed frog. Pexiganan exhibited in vitro broad-spectrum antibacterial activity when it was tested against 3,109 clinical isolates of gram-positive and gram-negative, anaerobic and aerobic bacteria. The pexiganan MIC at which 90% of isolates are inhibited (MIC90) was 32 μg/ml or less forStaphylococcus spp., Streptococcus spp.,Enterococcus faecium, Corynebacterium spp.,Pseudomonas spp., Acinetobacter spp.,Stenotrophomonas spp., certain species of the familyEnterobacteriaceae, Bacteroides spp.,Peptostreptococcus spp., and Propionibacteriumspp. Comparison of the MICs and minimum bactericidal concentrations (MBCs) of pexiganan for 143 isolates representing 32 species demonstrated that for 92% of the isolates tested, MBCs were the same or within 1 twofold difference of the MICs, consistent with a bactericidal mechanism of action. Killing curve analysis showed that pexiganan killed Pseudomonas aeruginosa rapidly, with 106 organisms/ml eliminated within 20 min of treatment with 16 μg of pexiganan per ml. No evidence of cross-resistance to a number of other antibiotic classes was observed, as determined by the equivalence of the MIC50s and the MIC90s of pexiganan for strains resistant to oxacillin, cefazolin, cefoxitin, imipenem, ofloxacin, ciprofloxacin, gentamicin, and clindamicin versus those for strains susceptible to these antimicrobial agents. Attempts to generate resistance in several bacterial species through repeated passage with subinhibitory concentrations of pexiganan were unsuccessful. In conclusion, pexiganan exhibits properties in vitro which make it an attractive candidate for development as a topical antimicrobial agent.

The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an alpha-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic analogs for negatively charged membranes. Likewise, the peptide affinity to PC-rich membranes increases in the order of hydrophobicity. Correlation of peptide binding and membrane permeabilization of PC/PG (3:1) vesicles revealed that the observed differences in peptide activity on membranes of low negative surface charge are mainly caused by the different binding affinities. The antibacterial and hemolytic activity of the peptides increases with enhanced hydrophobicity. A strong correlation was found between the hemolytic effect and the bilayer-permeabilizing activity against PC-rich vesicles. Whereas the antibacterial specificity of the more hydrophobic analogs is retained for Escherichia coli, the specificity for Pseudomonas aeruginosa decreases with increasing hydrophobicity.

Human atopic asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of allergic inflammation and airway hyperresponsiveness. Recent studies demonstrate that the degree of airway responsiveness is strongly associated with interleukin (IL)-9 expression in murine lung. To investigate the contribution of IL-9 to airway hyperresponsiveness, and to explore directly its relationship to airway inflammation, we studied transgenic mice overexpressing IL-9. In this report we show that IL-9 transgenic mice (FVB/N-TG5), in comparison with FVB/NJ mice, display significantly enhanced eosinophilic airway inflammation, elevated serum total immunoglobulin E, and airway hyperresponsiveness following lung challenge with a natural antigen (Aspergillus fumigatus). These data support a central role for IL-9 in the complex pathogenesis of allergic inflammation.

To investigate the influence of the angle subtended by the positively charged helix face on membrane activity, six amphipathic alpha-helical peptides with angles between 80 degrees and 180 degrees, but with retained hydrophobicity, hydrophobic moment, and positive overall charge, were designed starting from the sequence of the antibacterial peptide magainin 2. CD investigations revealed that all analogs are in an alpha-helical conformation in vesicle suspension. The ability of the peptides to induce dye release from negatively charged phosphatidylglycerol (PG) vesicles decreased with increasing angle. However, peptides with a large angle of positively charged residues (140-180 degrees) exhibited a considerably higher permeabilizing activity at zwitterionic phosphatidylcholine (PC) and mixed PC/PG (3:1) vesicles than analogs with a small angle (80-120 degrees). In addition, analogs with large angles were more active in antibacterial and hemolytic assays. The antibacterial specificity of these analogs was decreased. Binding investigations showed that peptide binding is favored by a large angle and a high content of negatively charged phospholipid. In contrast, a small angle and a low negative membrane charge enhanced the membrane-permeabilizing efficiency of the bound peptide fraction. All analogs stabilized the bilayer phase of phosphatidylethanolamine over the inverted hexagonal phase. Therefore, a class L mechanism of permeabilization can be excluded. Furthermore, the analogs do not act by the induction of positive curvature strain or by a "carpet-like" mechanism. Our results are in accordance with a pore mechanism: The membrane-permeabilizing efficiency of analogs with enhanced angle of positively charged residues is reduced due to electrostatic repulsion between adjacent helices within the pore, thus resulting in a decreased pore-forming probability and/or pore destabilization.

Starting from the sequences of magainin 2 analogs, peptides with slightly increased hydrophobic moment (/x) but retained other structural parameters were designed. Circular dichroism investigations revealed that all peptides adopt an (Xhelical conformation when bound to phospholipid vesicles. Analogs with increased \i were considerably more active in permeabilizing vesicles mainly composed of zwitterionic lipid. In addition, the antibacterial and hemolytic activities of these analogs were enhanced. Correlation of permeabilization and binding indicated that the activity increase is predominantly caused by an increased membrane affinity of the peptides due to strengthened hydrophobic interactions.

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