Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic alpha-helical structure isolated from the mast cells of hybrid striped bass. In our previous study, we showed that Pis-1[PG] with a substitution of Pro(8) for Gly(8) in Pis-1 had higher bacterial cell selectivity than Pis-1. We designed peptoid residue-substituted peptide, Pis-1[NkG], in which Gly(8) of Pis-1 was replaced with Nlys (Lys peptoid residue). Pis-1[NkG] had higher antibacterial activity and lower cytotoxicity against mammalian cells than Pis-1 and Pis-1[PG]. We determined the tertiary structure of Pis-1[PG] and Pis-1[NkG] in the presence of DPC micelles by NMR spectroscopy. Both peptides had a three-turn helix in the C-terminal region and a bent structure in the center. Pis-1[PG] has a rigid bent structure at Pro(8) whereas Pis-1[NkG] existed as a dynamic equilibrium of two conformers with a flexible hinge structure at Nlys(8). Depolarization of the membrane potential of Staphylococcus aureus and confocal laser-scanning microscopy study revealed that Pis-1[NkG] effectively penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas Pis-1[PG] did not penetrate the membrane but remained outside or on the cell surface. Introduction of a lysine peptoid at position 8 of Pis-1 provided conformational flexibility and increased the positive charge at the hinge region; both factors facilitated penetration of the bacterial cell membrane and conferred bacterial cell selectivity on Pis-1[NkG].

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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-NH(2) (X indicates Leu or Lys/Arg), was synthesized. K(6)L(2)W(3) displayed an approximately 40-fold increase in cell specificity, compared with the natural Trp-rich AMP indolicidin (IN). Lys-containing peptides (K(8)W(3), K(7)LW(3) and K(6)L(2)W(3)) showed approximately 2- to 4-fold higher cell specificities than did their counterparts, the Arg-containing peptides (R(8)W(3), R(7)LW(3) and R(6)L(2)W(3)), 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 (K(6)L(2)W(3)-D and R(6)L(2)W(3)-D) and Orn/Nle-containing peptides (O(6)L(2)W(3) and O(6)L(2)W(3)) to trypsin digestion compared with the rapid breakdown of the l-enantiomers (K(6)L(2)W(3) and R(6)L(2)W(3)), highlights the clinical potential of such peptides. K(6)L(2)W(3), R(6)L(2)W(3), K(6)L(2)W(3)-D and R(6)L(2)W(3)-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 K(6)L(2)W(3), K(5)L(3)W(3), R(6)L(2)W(3), O(6)L(2)W(3), O(6)L(2)W(3), and K(6)L(2)W(3)-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.

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