title: 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.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997, Moscow, Russia.
authors.forename: Pavel V
authors.initials: PV
authors.lastname: Panteleev
authors.forename: Ilia A
authors.initials: IA
authors.lastname: Bolosov
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Balandin
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Ovchinnikova
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004926:Escherichia coli / Q000187:drug effects*; D015801:Helminth Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D008826:Microbial Sensitivity Tests; D011550:Pseudomonas aeruginosa / Q000187:drug effects*; D013211:Staphylococcus aureus / Q000187:drug effects*; D013329:Structure-Activity Relationship
chemical_list: D023181:Antimicrobial Cationic Peptides; D015801:Helminth Proteins; C496888:arenicin-1, Arenicola marina
keywords: antimicrobial peptide; arenicin; heterological expression; site-directed mutagenesis; therapeutic index; β-hairpin