Design of novel indolicidin-derived antimicrobial peptides with enhanced cell specificity and potent anti-inflammatory activity.

Indolicidin (IN) is a 13-residue Trp-rich antimicrobial peptide isolated from bovine neutrophils. To develop novel IN-derived antimicrobial peptides with enhanced cell specificity (therapeutic index) and potent anti-inflammatory activity, several IN analogs were synthesized by Pro-->Lys substitution. All IN analogs displayed an increase in therapeutic index by 3- to 15-fold relative to parental IN. IN and its analogs induced a significant membrane depolarization against intact Staphylococcus aureus in a dose-dependent manner and depolarized membrane potential at 5 microg/ml (MIC for S. aureus) almost completely. However, these peptides caused less than 40% calcein leakage from negatively charged EYPG/EYPE liposomes mimicking bacterial membranes at 10 microg/ml. Based on these results, we hypothesize that IN and its analogs kill microorganisms via the formation of small ion channels that permit transit of ions or protons, but not molecules as large as calcein. Furthermore, IN and its analogs induced a remarkable suppression in nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression in LPS-stimulated mouse macrophage RAW264.7 cells. All IN analogs showed LPS-binding activity comparable to that of IN. Taken together, their potent antimicrobial, anti-inflammatory and LPS-neutralizing activities similar to those of IN, coupled with their no cytotoxicity, our designed IN analogs make excellent candidates for novel antimicrobial and anti-sepsis agents.