Structure-based design of an indolicidin peptide analogue with increased protease stability.

Indolicidin is an antimicrobial cationic peptide with broad-spectrum activity isolated from bovine neutrophils. An indolicidin analogue CP-11, ILKKWPWWPWRRK-NH(2), with improved activity against Gram-negative bacteria had increased positive charge and amphipathicity while maintaining the short length of the parent molecule. The structure of CP-11 in the presence of dodecylphosphocholine (DPC) micelles was determined using nuclear magnetic resonance spectroscopy. CP-11 was found to be an amphipathic molecule with a U-shaped backbone bringing the N- and C-termini in close proximity. On the basis of this close proximity, a cyclic disulfide-bonded peptide cycloCP-11, ICLKKWPWWPWRRCK-NH(2), was designed to stabilize the lipid-bound structure and to increase protease resistance. The three-dimensional structure of cycloCP-11 was determined under the same conditions as for the linear peptide and was found to be similar to CP-11. Both CP-11 and cycloCP-11 associated with phospholipid membranes in a similar manner as indicated by circular dichroism and fluorescence spectra. The minimal inhibitory concentrations of CP-11 and cycloCP-11 for a range of bacteria differed by no more than 2-fold, and they were nonhemolytic at concentrations up to 256 microg/mL. Cyclization was found to greatly increase protease stability. The half-life of cycloCP-11 in the presence of trypsin was increased by 4.5-fold from 4 to 18 min. More importantly, the antibacterial activity of cycloCP-11, but not that of CP-11, in the presence of trypsin was completely retained up to 90 min since the major degradation product was equally active. A structural comparison of CP-11 and cycloCP-11 revealed that the higher trypsin resistance of cycloCP-11 may be due to the more compact packing of lysine and tryptophan side chains. These findings suggest that cyclization may serve as an important strategy in the rational design of antimicrobial peptides.

Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli.

Cationic bactericidal peptides are components of natural host defenses against infections. While the mode of antibacterial action of cationic peptides remains controversial, several targets, including the cytoplasmic membrane and macromolecular synthesis, have been identified for peptides acting at high concentrations. The present study identified peptide effects at lower, near-lethal inhibitory concentrations. An amidated hybrid of the flounder pleurocidin and the frog dermaseptin (P-Der), two other pleurocidin derivatives, and pleurocidin itself were studied. At 2 microg/ml, the MIC, P-Der inhibited Escherichia coli growth in a broth dilution assay but did not cause bacterial death within 30 min, as estimated by viable count analysis. Consistent with this, P-Der demonstrated a weak ability to permeabilize membranes but was able to translocate across the lipid bilayer of unilamellar liposomes. Doses of 20 microg/ml or more reduced bacterial viable counts by about 2 log orders of magnitude within 5 min after peptide treatment. Abrupt loss of cell membrane potential, observed with a fluorescent dye, dipropylthiacarbocyanine, paralleled bacterial death but did not occur at the sublethal, inhibitory concentrations. Both lethal and sublethal concentrations of P-Der affected macromolecular synthesis within 5 min, as demonstrated by incorporation of [3H]thymidine, [3H]uridine, and [3H]histidine, but the effects were qualitatively distinct at the two concentrations. Variations of the inhibition pattern described above were observed for pleurocidin and two other derivatives. Our results indicate that peptides at their lowest inhibitory concentrations may be less capable of damaging cell membranes, while they maintain their ability to inhibit macromolecular synthesis. Better understanding of the effects of peptides acting at their MICs will contribute to the design of new peptides effective at lower, less toxic concentrations.