Substitution of the Arginine/Leucine Residues in Apidaecin Ib with Peptoid Residues: Effect on Antimicrobial Activity, Cellular Uptake, and Proteolytic Degradation

Abstract

Several aspects of the mechanism of action of Pro-rich antimicrobial peptides, together with their low toxicity in mammalian cells, make them good candidates for the development of new antibiotic agents. We investigated the effect induced in the insect antimicrobial peptide apidaecin Ib by the replacement of a single arginine/leucine residue with a N-substituted glycine. The resulting peptoid-peptide hybrids are more resistant to proteolysis and devoid of any significant cytotoxic activity, but moving the [NArg]residue from the N- to the C-terminal end of the molecule progressively reduces the antibacterial activity. Cell uptake experiments in E. coli cells suggest that the loss of antibacterial activity of [NArg(17)]apidaecin is a consequence of its inability to translocate into bacterial cells. Conversely, apidaecin and its peptoid-peptide hybrids are able to cross the plasma membrane in eukaryotic cells and to diffuse in the cytosol, although their translocating ability is far less effective than that of other known cell permeant peptides.

The Honeybee Antimicrobial Peptide Apidaecin Differentially Immunomodulates Human Macrophages, Monocytes and Dendritic Cells

“…In this study, we document for the first time that apidaecin associates with human macrophages, monocytes and dendritic cells with no need of specific membrane receptors, extending our previous observations that this bee AMP can cross the plasma membrane of epithelial eukaryotic cells, without determining cytotoxic effects [10] . A physiological temperature (37 ° C) is required for optimal cell binding, suggesting that either membrane fluidity or cooperation of protein carriers with low affinity and high capacity are necessary for optimal cell interaction.…”

“…Competition assays ( fig. 1 c) proved that this process does not require high-affinity receptors, similarly to what was observed in epithelial cells [10] . Cell association with a fluorescent, shorter peptide corresponding to the C-terminal sequence 13-18, conserved in different apidaecin isoforms, was significantly decreased at 37 ° C, but comparable to that of full-length apidaecin at 0 ° C, suggesting that the lacking N-terminal sequence is necessary for efficient cell capture of apidaecin at physiological temperature ( fig.…”

“…Full-length apidaecin Ib (GNNRPVYIPQPRPPHPRL), the peptide sequence corresponding to amino acids 13-18 of apidaecin and their fluoresceinated analogues were synthesized by solid-phase synthesis as previously reported [10] . Following the same protocol, an apidaecin derivative extended at the N-terminus with a cysteine residue (Cys-apidaecin) was prepared and used after HPLC purification in affinity chromatography.…”

“…Among these, the prorich 18-20 residues of apidaecins are potentially exploitable in medical and biotechnology applications as novel antibiotic drugs [9] , being poorly structured, devoid of S-S bridges, easily synthesizable and not toxic to human cells [10] . Apidaecin, like similar pro-rich peptides (drosocin, pyrrhocoricin and Bac7), acts via a three-step pro-cess involving: (i) bacterial surface binding, (ii) translocation into the cytoplasm and (iii) binding to DnaK, the Escherichia coli homologue of human HSP70 [2,[11][12] .…”

A practical method for the synthesis of peptoids containing both lysine-type and arginine-type monomers

“…18,19 We were interested in combining arginine-and lysine-type monomer residues within the same peptoid so that we could begin to probe their biological activities, and, in particular the relationship between antimicrobial properties and cytotoxicity. Herein, we describe the development of an efficient synthetic approach that can be utilised to synthesise novel peptoids (linear and cyclic) containing both lysine-type and arginine-type monomers within the same sequence.…”