Peptoid-Substituted Hybrid Antimicrobial Peptide Derived from Papiliocin and Magainin 2 with Enhanced Bacterial Selectivity and Anti-inflammatory Activity

Abstract

Antimicrobial peptides (AMPs) are important components of the host innate immune system. Papiliocin is a 37-residue AMP purified from larvae of the swallowtail butterfly Papilio xuthus. Magainin 2 is a 23-residue AMP purified from the skin of the African clawed frog Xenopus laevis. We designed an 18-residue hybrid peptide (PapMA) incorporating N-terminal residues 1-8 of papiliocin and N-terminal residues 4-12 of magainin 2, joined by a proline (Pro) hinge. PapMA showed high antimicrobial activity but was cytotoxic to mammalian cells. To decrease PapMA cytotoxicity, we designed a lysine (Lys) peptoid analogue, PapMA-k, which retained high antimicrobial activity but displayed cytotoxicity lower than that of PapMA. Fluorescent dye leakage experiments and confocal microscopy showed that PapMA targeted bacterial cell membranes whereas PapMA-k penetrated bacterial cell membranes. Nuclear magnetic resonance experiments revealed that PapMA contained an N-terminal α-helix from Lys(3) to Lys(7) and a C-terminal α-helix from Lys(10) to Lys(17), with a Pro(9) hinge between them. PapMA-k also had two α-helical structures in the same region connected with a flexible hinge residue at Nlys(9), which existed in a dynamic equilibrium of cis and trans conformers. Using lipopolysaccharide-stimulated RAW264.7 macrophages, the anti-inflammatory activity of PapMA and PapMA-k was confirmed by inhibition of nitric oxide and inflammatory cytokine production. In addition, treatment with PapMA and PapMA-k decreased the level of ultraviolet irradiation-induced expression of genes encoding matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in human keratinocyte HaCaT cells. Thus, PapMA and PapMA-k are potent peptide antibiotics with antimicrobial and anti-inflammatory activity, with PapMA-k displaying enhanced bacterial selectivity.

Investigation of cationicity and structure of pseudin-2 analogues for enhanced bacterial selectivity and anti-inflammatory activity

“…Phase-sensitive two-dimensional experiments, including nuclear NOESY, TOCSY and DQF-COSY, were performed on a Bruker 700 and 800 MHz spectrometer at the Korea Basic Science Institute (Ochang, Korea) as previously described 49, 50 . Structure calculations of the peptides were performed by following the standard protocol of the Cyana3.9 program in Linux 51 .…”

“…coli and S . aureus as described previously 50 . Outer membrane permeability was measured using NPN dye and E .…”

“…An ONPG hydrolysis assay was performed to measure inner membrane permeability of E . coli ML-35p cells as described previously 50 .…”

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides

“…Thus, it has become increasingly important for researchers to reliably determine whether AMPs are able to effectively translocate into bacterial cells (3). Many researchers have turned to confocal microscopy in order to assess cell entry (4)(5)(6)(7)(8)(9)(10)(11). However, bacterial cells are so small that effective imaging is limited by the resolution of conventional light microscopes.…”

Advances in Development of Antimicrobial Peptidomimetics as Potential Drugs

“…Likewise, replacement of Pro residues in Trp/Pro-rich AMPs appears to constitute a promising approach for development of cell-selective AMPs with intracellular mechanisms [ 196 ]. In most cases the modification with α-peptoid residues confers broad-spectrum antibacterial activity against common Gram-negative and Gram-positive pathogens [ 191 , 192 , 193 , 194 , 195 , 196 , 197 , 198 ]. With few exceptions preparation involves solid-phase peptide synthesis (SPPS) where Fmoc-protected peptoid building blocks are used at the appropriate positions.…”