β-hairpin antimicrobial peptides: structure, function and mode of action

Abstract

A ‘state of emergency’ was declared by the World Health Organization three years ago to combat the increasing rate of resistance arising in bacteria to all currently available antibiotics on the market. Antimicrobial resistance is now a worldwide concern, and renewed efforts are needed in the search for new replacement drugs for obsolete antibiotics. Antimicrobial peptides (AMPs) have been discovered and studied over decades; importantly very limited bacterial resistance has been reported to date. The work here aims to better characterize and understand the structure-function relationships of select β-hairpin AMPs, leading to the design of novel, optimized and potentially therapeutically valuable peptides.Chapter 1 reviews the field of β-hairpin AMPs and provides a background for the specific AMPs studied in this thesis. Chapter 2 consists of an original data set that strengthens the current knowledge of β-hairpin AMPs by comparing their activity profile under similar conditions. This work analysed the contribution of amphipathicity and hydrophobicity to antimicrobial activity and cytotoxicity of β-hairpin peptides, concluding that a very fine balance between charge, hydrophobicity, amphipathicity, secondary and tertiary structure and mode of action is needed for a peptide to be therapeutically valuable. From this study, two distinct but linked areas of further investigation were identified (i) a structure activity and function relationship study and (ii) a determination of the mode of action of select β-hairpin AMPs.Chapter 2 identified that tachyplesin-1, a 17 amino acids β-hairpin AMP originating from the hemocyte debris of a horseshoe crab, Tachypleus tridentatus, was an ideal β-hairpin AMP candidate to study structure-activity and -toxicity relationships, as it possessed an 8-fold higher therapeutic index than other β-hairpin AMPs studied. Thus, chapter 3 contains an evaluation of the role of each amino acid of tachyplesin-1 through an alanine scan. New analog peptides were designed as a systematic approach to develop more therapeutically valuable peptides as replacements for antibiotics that have become ineffective. Measurements of activity and toxicity, combined with preliminary ADME data, led to the identification of two tachyplesin-1 analogs with great potential for future drug development.Chapters 4 and 5 consist of studies of the mode of action of arenicin-3, a 21 amino acid β-hairpin AMP originating from the coelomocytes of marine polychaeta lugworm, Arenicola marina, and its synthetic analog AA139. AA139 is a lead drug candidate of Adenium Biotech Ltd. currently in preclinical development for MDR urinary tract infection and MDR Gram-negative pneumonial diseases. Their 3D NMR solution structures revealed right handed twisted β-hairpin structures of 21 amino acids rigidified by two disulfide bridges (Cys3-Cys20 and Cys7-Cys16). The mode of action of arenicin peptides and their selectivity toward bacteria cells, and more specifically their interaction toward different lipid membrane systems, were examined. Chapter 4 focused on label free mode of action investigations, leading to a proposed model of arenicin mode of action involving three steps: (1) binding to the outer membrane through electrostatic interactions, (2) insertion into the hydrophobic core of the outer membrane creating partial permeabilization due to the ability of the peptide to modify its secondary structure allowing it to enter into the lipid bilayer without pore formation, (3) accessing and partially permeabilizing the cytoplasmic membrane. To provide further evidence of the arenicin peptides mode of action, chapter 5 contains a comprehensive structural investigation employing NMR experiments with 15N labelled AA139 in the presence of lipid bilayer model membranes, nanodiscs or vesicles, using solution and solid-state NMR, respectively. This work suggested that the C- and N-terminal of AA139 are the first point of interaction with lipid bilayer membranes. Following that first interaction, AA139 possibly undergoes a loss of its well-defined 3D solution structure, becoming dynamic and without a distinct fold whilst embedded in the membrane and causing the permeabilization of both outer and inner membranes. This study allowed us to gain a deeper understanding of AA139 mode of action, and of the differences between the successful lead drug candidate, AA139 and its therapeutically less valuable progenitor, arenicin-3. This study provides an example of potential approaches to employ when designing other improvements to antimicrobial peptides. More importantly, this study allowed us to confirm the great potential of nanodiscs, not only to study membrane proteins, but also to evaluate the mode of action of membrane-active peptides.