Copper-binding tripeptide motif increases potency of the antimicrobial peptide Anoplin via Reactive Oxygen Species generation

[Display omitted] •An ATCUN motif potentiates the antimicrobial activity of the antimicrobial peptide Anoplin.•The addition of the ATCUN motif results in a decrease of Anoplin’s α helical content.•The presence of the ATCUN motif promoted oxidative damage of the bacterial membrane. Antimicrobial peptides (AMPs) are broad spectrum antimicrobial agents that act through diverse mechanisms, this characteristic makes them suitable starting points for development of novel classes of antibiotics. We have previously reported the increase in activity of AMPs upon addition of the Amino Terminal Copper and Nickel (ATCUN) Binding Unit. Herein we synthesized the membrane active peptide, Anoplin and two ATCUN-Anoplin derivatives and show that the increase in activity is indeed due to the ROS formation by the CuII-ATCUN complex. We found that the ATCUN-Anoplin peptides were up to four times more potent compared to Anoplin alone against standard test bacteria. We studied membrane disruption, and cellular localization and found that addition of the ATCUN motif did not lead to a difference in these properties. When helical content was calculated, we observed that ATCUN-Anoplin had a lower helical composition. We found that ATCUN-Anoplin are able to oxidatively damage lipids in the bacterial membrane and that their activity trails the rate at which ROS is formed by the CuII-ATCUN complexes alone. This study shows that addition of a metal binding tripeptide motif is a simple strategy to increase potency of AMPs by conferring a secondary action.