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

Abstract

Pseudin-2 (Ps), isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity and cytotoxicity. To develop antimicrobial peptides with anti-inflammatory activity and low cytotoxicity, we designed Ps analogues with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. We further substituted Gly11 with Pro (Ps-P analogues) to increase bacterial cell selectivity. Ps analogues retained antimicrobial activity and exhibited reduced cytotoxicity, whereas Ps-P analogues exhibited lower cytotoxicity and antimicrobial activity. Tertiary structures revealed that Ps has a linear α-helix from Leu2 to Glu24, whereas Ps-P has a bend at Pro11 between two short α-helixes. Using various biophysical experiments, we found that Ps analogues produced much higher membrane depolarization than Ps-P analogues, whereas Ps-P analogues may penetrate bacterial cell membranes. Ps and its analogue Ps-K18 exhibited potent anti-inflammatory activity in LPS-stimulated RAW264.7 and mouse dendritic cells via a mechanism involving the Toll-like receptor 4 (TLR4) pathway. These activities may arise from their direct inhibition of the formation of TLR4-MD-2_LPS complex, implying that amphipathic α-helical structure with an optimum balance between enhanced cationicity and hydrophobicity may be essential for their anti-inflammatory activity. The bent structure provided by Pro substitution plays an important role in enhancing bacterial cell selectivity and cell penetration.

3,6‐Dihydroxyflavone: A Potent Inhibitor with Anti‐Inflammatory Activity Targeting Toll‐like Receptor 2

“…3,6-DHF was purchased from the Indofine Chemical Company (Hillsborough, NJ, USA) and purified using HPLC with an analytical Vydac C 18 column at Korea Basic Science Institute (KBSI, Ochang, Korea). 38 This assay includes a 30-s baseline step, a 60-s loading step, a 30-s baseline step, a 60-s association step followed by a 120-s dissociation step. HEK293 cells that are over-expressing and lacking in TLR2 (HEK293-hTLR2 and HEK293-null cells, respectively) were purchased from Invitrogen (San Diego, CA, USA).…”

Short Antimicrobial Peptides Exhibiting Antibacterial and Anti‐Inflammatory Activities Derived from the N‐Terminal Helix of Papiliocin

“…Bacterial‐membrane depolarization by AMPs was measured using diSC3‐5 dye (Sigma‐Aldrich, St. Louis, MO, USA). Peptide‐induced membrane depolarization was measured using intact Escherichia coli KCTC 1682 as described previously . Bacteria were grown to an OD 600 of 0.5, washed twice in a buffer containing 5 μM HEPES and 20 μM glucose (pH 7.4), and diluted and resuspended to an OD 600 of 0.05 using a buffer consisting of 5 μM HEPES, 20 μM glucose, and 0.1 M KCl (pH 7.4).…”

Structure-Activity Relationships of Peptide Antibiotics with Improved Bacterial Cell Selectivity of Pseudin

“…6 Hemolytic activity-Hemolytic activity of the peptides was tested against human red blood cells (hRBCs) as described previously. 7 Controls for no hemolysis (blank) and 100% hemolysis consisted of human red blood cells suspended in PBS and 0.1% Triton-X 100, respectively. The percent hemolysis was calculated using the following equation: NMR experiments and assignment-Perdeuterated DPC was purchased from Cambridge Isotope Laboratories (Andover, MA, USA).…”