What Goes around Comes around-A Comparative Study of the Influence of Chemical Modifications on the Antimicrobial Properties of Small Cyclic Peptides

Abstract

Tryptophan and arginine-rich cyclic hexapeptides of the type cyclo-RRRWFW combine high antibacterial activity with rapid cell killing kinetics, but show low toxicity in human cell lines. The peptides fulfil the structural requirements for membrane interaction such as high amphipathicity and cationic charge, but membrane permeabilisation, which is the most common mode of action of antimicrobial peptides (AMPs), could not be observed. Our current studies focus on elucidating a putative membrane translocation mechanism whereupon the peptides might interfere with intracellular processes. These investigations require particular analytical tools: fluorescent analogues and peptides bearing appropriate reactive groups were synthesized and characterized in order to be used in confocal laser scanning microscopy and HPLC analysis. We found that minimal changes in both the cationic and hydrophobic domain of the peptides in most cases led to significant reduction of antimicrobial activity and/or changes in the mode of action. However, we were able to identify two modified peptides which exhibited properties similar to those of the cyclic parent hexapeptide and are suitable for subsequent studies on membrane translocation and uptake into bacterial cells.

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

“…Although the tridecapeptides tritrpticin and indolicidin permeabilize lipid bilayers (43), hexapeptides such as Combi-1 and Combi-2 showed very poor permeabilization (44). It is important to note that even peptides with very small structural differences can have distinct mechanisms of interacting with membranes, resulting in different permeabilization capabilities and modes of action, as shown recently for very small cyclic RW-rich peptides (45). Thus, the observations noted in this study may not necessarily apply to all antimicrobial peptides.…”

Evidence for a Novel Mechanism of Antimicrobial Action of a Cyclic R-,W-Rich Hexapeptide

“…The 3-N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-2,3-diaminopropionic acid (Dap-NBD)-bearing analogue, c(RRRW[Dap-NBD]W) was developed for laser scanning microscopy investigations, and the introduction of lysine (K) in c(KRKW[Dap-NBD]W) was required for HPLC-based uptake studies. Both cWFW analogues conserved the non-membrane-permeabilising mode of action of the parent sequence [ 20 ]. The N-terminally carboxyfluorescein-labelled Buforin II (Fluos-TRSSRAGLQFPVGRVHRLLRK-NH 2 ) served as control.…”

“…subtilis (L-170) were used to quantify peptide uptake into the cytoplasm [ 20 , 27 ]. The approach, originally developed for the investigation of peptide uptake into eukaryotic cells, is based on chemical modification of the amino group of lysine side chains with o-nitroaniline and fluorescence-based HPLC detection of non-modified and modified sequences [ 20 , 28 ]. Fluorescence labelling was necessary for quantification of cyclic hexapeptide uptake.…”

“…HPLC analysis was performed as described before using a Jasco LC-2000 Plus (U.S.) with a ProntoSil 300-5-C18-H column (250 × 4.6 mm, 5 μm) (Bischoff Chromatography, Germany) and a precolumn with PolyenCap A300, 10 μm [ 20 ]. Peptide amounts were quantified via calibration curves for carboxyfluorescein (ex 445 nm, em 520 nm, gain 1000) and NBD fluorescence (ex 470 nm, em 520 nm, gain 1000), respectively.…”

“…In this study we investigated alternative mechanisms of action of cWFW with the aid of specifically designed peptide derivatives [ 20 ]. First, confocal laser scanning microscopy (CLSM) was used to visualise peptide translocation into the cytoplasm.…”