Cell specificity,anti-inflammatory activity,and plausible bactericidal mechanism of designed Trp-rich model antimicrobial peptides

To develop novel short Trp-rich antimicrobial peptides (AMPs) with potent cell specificity (targeting bacteria but not eukaryotic cells) and anti-inflammatory activity, a series of 11-meric Trp-rich model peptides with different ratios of Leu and Lys/Arg residues, XXWXXWXXWXX-NH₂ (X indicates Leu or Lys/Arg), was synthesized. K₆L₂W₃ displayed an approximately 40-fold increase in cell specificity, compared with the natural Trp-rich AMP indolicidin (IN). Lys-containing peptides (K₈W₃, K₇LW₃ and K₆L₂W₃) showed approximately 2- to 4-fold higher cell specificities than did their counterparts, the Arg-containing peptides (R₈W₃, R₇LW₃ and R₆L₂W₃), indicating that multiple Lys residues are more important than multiple Arg residues in the design of AMPs with good cell specificity. The excellent resistance of d-enantiomers (K₆L₂W₃-D and R₆L₂W₃-D) and Orn/Nle-containing peptides (O₆L₂W₃ and O₆L₂W₃) to trypsin digestion compared with the rapid breakdown of the l-enantiomers (K₆L₂W₃ and R₆L₂W₃), highlights the clinical potential of such peptides. K₆L₂W₃, R₆L₂W₃, K₆L₂W₃-D and R₆L₂W₃-D caused weak dye leakage from bacterial membrane-mimicking negatively charged EYPG/EYPE (7:3, v/v) liposomes. Confocal microscopy showed that these peptides penetrated the cell membrane of Escherichia coli and accumulated in the cytoplasm, as observed for buforin-2. Gel retardation studies revealed that the peptides bound more strongly to DNA than did IN. These results suggested that one possible peptide bactericidal mechanism may relate to the inhibition of intracellular functions via interference with DNA/RNA synthesis. Furthermore, some model peptides, containing K₆L₂W₃, K₅L₃W₃, R₆L₂W₃, O₆L₂W₃, O₆L₂W₃, and K₆L₂W₃-D inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA expression, the release of nitric oxide (NO) following LPS stimulation in RAW264.7 cells and had powerful LPS binding activities at bactericidal concentrations. Collectively, our results indicated that these peptides have potential for future development as novel antimicrobial and anti-inflammatory agents.