Design of antimicrobial peptide arenicin analogs with improved therapeutic indices

Abstract

β-Hairpin antimicrobial peptides are among the most potent peptide antibiotics of animal origin. Arenicins, isolated earlier from marine polychaeta lugworm Arenicola marina, belong to a family of β-hairpin antimicrobial peptides and display a broad spectrum of biological activities. However, despite being potent antimicrobials, arenicins are partially unapplicable as therapeutics as a result of their relatively high cytotoxicity against mammalian cells. In this study, a template-based approach was used to create therapeutically valuable analogs of arenicin-1 and identify amino acid residues important for antibacterial and cytotoxic activities of the peptide. The plasmids encoding recombinant analogs were constructed by mutagenesis technique based on inverse PCR amplification of the whole arenicin-1 expression plasmid. The analogs were produced as a part of the fusion proteins in Escherichia coli. It was shown that an obvious reduction in hemolytic activity without lose of antimicrobial activity can be achieved by a single amino acid substitution in the non-polar face of the molecule with hydrophilic residues such as serine and arginine. As the result, the selective analog with 50-fold improved therapeutic index was developed. The circular dichroism spectra demonstrated that the secondary structure of the analog was similar to the natural arenicin-1 in water solution and sodium dodecyl sulfate micelles but significantly differed in the presence of dodecylphosphocholine micelles mimicking mammalian membranes. Similarly to arenicin-1, the designed analog killed bacteria via induction of the membrane damage, assessed using the fluorescent dye SYTOX Green uptake. Our results afford molecular insight into mechanism of antimicrobial action of the designed arenicin analogs and their possible clinical application.

Analysis of Synergistic Effects of Antimicrobial Peptide Arenicin-1 and Conventional Antibiotics

“…Recombinant arenicin-1 and tachyplezin-1 were obtained by heterologous expression in E. coli cells [9,10]. For antibiotic activity assessment, bacterial strains E. coli C600, S. aureus 209P and P. aeruginosa PAO1 were used as test cultures.…”

Dimerization of the antimicrobial peptide arenicin plays a key role in the cytotoxicity but not in the antibacterial activity

“…The recombinant arenicin-1 and its mutant analogs were prepared as described previously [9]. 15 were induced with 0.2 mM IPTG.…”

“…Vice versa, the analogs with low antimicrobial activity and pronounced hemolytic properties were found suggesting different mechanisms of the peptide action against pro-and eukaryotic cells. Interestingly, substitutions of the N-terminal β-strand aliphatic residues for the basic side chain amino acid arginine (Val4Arg, Ala6Arg, or Val8Arg) resulted in the most notable decrease in hemolytic activity [9]. The CD spectroscopy demonstrated that secondary structures of the analogs were quite similar in water and DPC micelles in contrast to the wildtype arenicin-1.…”

“…It was proposed that the peptide dimeric assembly by association of the N-terminal β-strands in membrane-mimicking environment of DPC micelles is retained in the bilayer membranes containing negatively charged lipids, and arenicin antimicrobial activity is mediated by the formation of toroidal pores assembled of several β-structural peptide dimers and lipid molecules [8]. Recently, we performed the structure-activity relationship study of arenicin-1 to reveal correlations between antibacterial and hemolytic properties of the peptide [9]. The analogs with single amino acid substitutions were obtained, and the ones with high antimicrobial activity and dramatically decreased hemolytic properties were revealed among them.…”

Improved strategy for recombinant production and purification of antimicrobial peptide tachyplesin I and its analogs with high cell selectivity

“…Initially, the expression and purification scheme, described in details in our previous work [5], was used to obtain recombinant THI. Briefly, induction of log-phase cells grown in LB media was performed by IPTG at 30…”