Β-Hairpin Antimicrobial Peptides
Total Page:16
File Type:pdf, Size:1020Kb
β-hairpin antimicrobial peptides: structure, function and mode of action Ingrid Alexia Edwards MSc Chemistry and chemical engineering A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Institute for Molecular Bioscience Abstract 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 I Abstract 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. II Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. III Publications included in this thesis Edwards, IA, Elliott, AG, Kavanagh, AM, Zuegg, J, Blaskovich, MAT, Cooper, MA, (2016), Contribution of amphipathicity and hydrophobicity to the antimicrobial activity and cytotoxicity of β-hairpin peptides, ACS Inf. dis. DOI: 10.1021/acsinfecdis.6b00045 Edwards, IA, Elliott, AG, Kavanagh, AM, Blaskovich, MAT, Cooper, MA, (2017), Structure-activity and -toxicity relationships of the antimicrobial peptide tachyplesin-1, ACS Inf. dis. DOI:10.1021/acsinfecdis.7b00123 Submitted manuscripts included in this thesis No manuscripts submitted for publication. Other publications during candidature Conference abstracts Edwards, IA, Mobli, M, Zuegg, J, Cooper, MA. Arenicin-3 peptides: β-hairpin AMPs structures and mode of action. 11th Australian peptides conferences, Kingscliff, Australia, October 2015 Edwards, IA, Elliott, AG, Blaskovich, MAT and Cooper, MA. Contribution of physicochemical parameters to the antimicrobial activity / toxicity index of β-hairpin peptides. 34th European peptide conference, Leipzig, Germany, September 2016. Edwards, IA, Elliott, AG, Blaskovich, MAT and Cooper, MA. Contribution of physicochemical parameters to the antimicrobial activity / toxicity index of β-hairpin peptides. Queensland Annual Chemistry Symposium, Brisbane, Australia, November 2016. Edwards, IA, Elliott, AG, Blaskovich, MAT and Cooper, MA. Contribution of physicochemical parameters to the antimicrobial activity / toxicity index of β-hairpin peptides. Solution for drug resistant infections, Brisbane, Australia, April 2017. Edwards, IA, Elliott, AG, Blaskovich, MAT, Mobli, M, Solution NMR characterisation of β-hairpin antimicrobial peptides in lipid bilayer nanodiscs, 12th Australian peptides conferences, Noosa, Australia, October 2017 IV Edwards, IA, Elliott, AG, Blaskovich, MAT, Mobli, M, Solution NMR characterisation of β-hairpin antimicrobial peptides in lipid bilayer nanodiscs, ANZMAG (Australian New Zealand Magnetic Resonance) conference, Kingscliff, Australia, December 2017 Contributions by others to the thesis Zhenling Cui designed the plasmid vector mentioned in chapter 5, page 5-110. Alan Zhang performed the expression and purification of cNW9 mentioned in chapter 5, page 5-113. Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis. Research Involving Human or Animal Subjects The work in this thesis was performed under the ethics approval number 2014000031 granted by NHMRC/ARC agent for the duration period: 17 March 2015 to 31 December 2019, to perform hemolytic assay of red blood cells for compounds with antimicrobial activity. V Acknowledgements Many people have contributed to make my journey through my PhD as smooth as possible