In the present study, the 26-residue amphipathic ␣-helical antimicrobial peptide V13K L (Y. Chen et al., J. Biol. Chem. 2005, 280:12316-12329, 2005) was used as the framework to study the effects of peptide hydrophobicity on the mechanism of action of antimicrobial peptides. Hydrophobicity was systematically decreased or increased by replacing leucine residues with less hydrophobic alanine residues or replacing alanine residues with more hydrophobic leucine residues on the nonpolar face of the helix, respectively. Hydrophobicity of the nonpolar face of the amphipathic helix was demonstrated to correlate with peptide helicity (measured by circular dichroism spectroscopy) and self-associating ability (measured by reversedphase high-performance liquid chromatography temperature profiling) in aqueous environments. Higher hydrophobicity was correlated with stronger hemolytic activity. In contrast, there was an optimum hydrophobicity window in which high antimicrobial activity could be obtained. Decreased or increased hydrophobicity beyond this window dramatically decreased antimicrobial activity. The decreased antimicrobial activity at high peptide hydrophobicity can be explained by the strong peptide self-association which prevents the peptide from passing through the cell wall in prokaryotic cells, whereas increased peptide self-association had no effect on peptide access to eukaryotic membranes.Antibiotic resistance, due to the extensive clinical use of classical antibiotics (22, 32), has become a great concern in recent years, prompting an urgent need for a new class of antibiotics. Antimicrobial peptides have been proposed as potent candidates of a new class of antibiotics, with characteristics including an ability to kill target cells rapidly, an unusually broad spectrum of activity, activity against some of the more serious antibiotic-resistant pathogens in clinics, and the relative difficulty in selecting resistant mutants in vitro (13,35). Although the exact mode of action of antimicrobial peptides has not been established, it is generally accepted that the cytoplasmic membrane is the main target of antimicrobial peptides, whereby peptide accumulation in the membrane causes increased permeability and a loss of barrier function, resulting in the leakage of cytoplasmic components and cell death (13,28).Factors believed to be important for antimicrobial activity have been identified, including peptide hydrophobicity, the presence of positively charged residues, an amphipathic nature that segregates basic and hydrophobic residues, and secondary structure. Recently, Hodges and coworkers increased this list to include (i) the importance of a lack of structure in benign medium (nondenaturing conditions; see Materials and Methods) but an inducible structure in the presence of the hydrophobic environment of the membrane, (ii) the presence of a positively charged residue in the center of the nonpolar face of amphipathic cyclic ␤-sheet and ␣-helical peptides as a determinant for locating the peptides at the interface r...

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V 681 ) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80°C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V 681 was improved 90-and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V 681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.

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