Mode of Action of Daptomycin, a Lipopeptide Antibiotic

Mode of Action of Daptomycin, a Lipopeptide Antibiotic by Jawad Kadhum Muraih A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Chemistry (Biochemistry) Waterloo, Ontario, Canada, 2012 © Jawad Kadhum Muraih 2012 Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. Jawad K. Muraih iii Abstract Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally at- tached fatty acyl residue. The antibiotic kills Gram-positive bacteria by membrane depolariza- tion. It has long been assumed that the mode of action of daptomycin involves the formation of oligomers on the bacterial cell membrane; however, at the outset of my studies, this had not been experimentally demonstrated. In the work described in this thesis, I have used fluorescence energy transfer (FRET) between native daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the antibiotic indeed forms oligomers on bacterial cell membranes. In a liposome model, oligomer formation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly and is stable for a length of time longer than required for the bactericidal effect. Through variation of the ratio of FRET donor (native daptomycin) and acceptor (NBD-daptomycin), I have determined that the oligomer consists of approximately 6–7 molecules, or, depending on the structure of the oligomer, possibly up to twice that number. Oligomer formation on liposomes and on bacterial membranes was confirmed using excimer fluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was also used to demonstrate a stoichiometric interaction between daptomycin and PG. It has previously been shown that the bactericidal activity of daptomycin requires calcium and correlates with the concentration of PG in the bacterial cell membrane; these requirements mirror those observed here for oligomer formation. Furthermore, membrane permeabiliza- tion is selective, and electron microscopy of bacterial membranes exposed to daptomycin has revealed no discontinuities or accretions of electron density. Both of these findings suggest formation of a small membrane lesion, which is compatible with the small size of the oligomer that was determined here. In conjunction with these previous findings, the experiments con- tained in my thesis strongly suggest that the oligomer is the bactericidal form of daptomycin. v Acknowledgements Even though there are many people who have helped me to reach this point of my life, the one to whom I owe my deepest thanks and gratitude is Michael Palmer. I am not exaggerating when I say that he is one of the best supervisors and advisors in the Department of Chemistry at the University of Waterloo. I am highly indebted to Professor Palmer for his valuable and fruitful guidance, suggestions and discussions; without his knowledge, perceptiveness, enthusiasm, and encouragement, this thesis would never have seen the light. In particular, I thank Professor Palmer for his incredible patience for always keeping the door to his office open for questions, and for his continual support. Thank you for everything Professor Palmer. I would like to extend my thanks to my advisory committee members, Professors John Honek, Elizabeth Meiering and Shawn Wettig for their useful suggestions and comments. I would like to thank my lab members Eric Brefo-Mensah, Oscar Zhang, Muhamad Salah, and former members Mohammad Khan, Dr. Waseem el Huneidi, Dr. Lisa Pokrajac for their help and useful discussions; all the undergraduate students Ana, Alex, Jesse, Kathy, Minyoung, and Celine. All of you contribute to make our lab is a nice place to work. In addition, I would like to thank my friend Kamal Mroue for the moments we shared together and all of my friends. In particular, I want to thank my parents, my brothers and my sisters for their support and encouragement. Finally, I would like to express my gratitude to my wonderful wife Najah, for her patience and support, also I would like to thank my son Mohammad for his help and patience and my children Rand, Rahaf, Fatemah, Ali and Sarah whom have provided full joy and pleasure to our joint life. vii Dedication To my parents, brothers and sisters To my wife and children ix Contents Author’s Declaration iii Abstract v Acknowledgements vii Dedication ix List of Figures xv List of Tables xvii 1 Introduction 1 1.1 Antibiotics ..................................... 1 1.1.1 History .................................. 2 1.1.2 Natural and synthetic antibiotics ..................... 5 1.1.3 Mechanisms of action of specific antibiotics ............... 9 1.1.4 Resistance to antibiotics ......................... 10 1.1.5 Peptide antibiotics ............................ 17 1.1.6 Cyclic lipopeptide antibiotics ...................... 18 1.2 Daptomycin .................................... 22 1.2.1 Physicochemical properties ....................... 23 1.2.2 Biosynthesis ............................... 23 1.2.3 Mode of action .............................. 24 1.2.4 Spectrum of Activity and Clinical Use .................. 29 1.2.5 Structural Variation of Daptomycin ................... 31 xi Contents 1.3 Research Objectives ................................ 34 2 Oligomerization of daptomycin on membranes 35 2.1 Introduction .................................... 35 2.2 Materials and Methods .............................. 37 2.2.1 Preparation of NBD-daptomycin ..................... 37 2.2.2 Preparation of Alexa Fluor 350-daptomycin ............... 38 2.2.3 Preparation of large unilamellar vesicles (LUV) and of bacterial membrane vesicles ............................... 38 2.2.4 Antibacterial activity tests for daptomycin and NBD-daptomycin .... 39 2.2.5 Fluorescence studies ........................... 40 2.2.6 Numerical fitting ............................. 41 2.3 Results ....................................... 41 2.3.1 Characterization of NBD-daptomycin .................. 41 2.3.2 Calcium-dependent membrane interaction of daptomycin and NBD- daptomycin ................................ 43 2.3.3 Detection of daptomycin oligomerization on membranes by FRET ... 44 2.3.4 Detection of oligomerization by NBD self-quenching; oligomerization on bacterial membranes ......................... 47 2.3.5 Daptomycin does not form oligomers in solution at antimicrobially ac- tive concentrations ............................ 48 2.3.6 Effect of EDTA on membrane-bound daptomycin oligomers ...... 50 2.4 Discussion ..................................... 52 3 Characterization of daptomycin oligomerization with excimer fluorescence 55 3.1 Introduction .................................... 55 3.2 Methods and Materials .............................. 57 3.2.1 Synthesis of perylene-daptomycin .................... 57 xii Contents 3.2.2 Synthesis of pyrene-daptomycin ..................... 58 3.2.3 Antibacterial activity test for pyrene- and perylene-daptomycin ..... 59 3.2.4 Lipids and liposomes ........................... 59 3.2.5 Fluorescence measurements ....................... 59 3.2.6 Deconvolution of perylene excimer and monomer spectra ........ 60 3.2.7 Titration of perylene excimer fluorescence with phosphatidylglycerol . 61 3.3 Results ....................................... 61 3.3.1 Antibacterial activity of pyrene- and perylene-daptomycin ....... 61 3.3.2 Fluorescence of pyrene-daptomycin ................... 61 3.3.3 Observation of perylene-daptomycin excimer fluorescence ....... 63 3.3.4 Quantitative evaluation of excimer fluorescence ............. 65 3.3.5 Titration of excimer fluorescence with phosphatidylglycerol ...... 67 3.4 Discussion ..................................... 68 4 The subunit stoichiometry of the daptomycin oligomer 71 4.1 Introduction .................................... 71 4.2 Materials and Methods .............................. 72 4.2.1 Preparation of liposomes and daptomycin derivatives .......... 72 4.2.2 Fluorescence measurements ....................... 73 4.2.3 Determination of the corrected relative kynurenine fluorescence of daptomycin in mixed oligomer samples ................. 73 4.2.4 Numerical simulation of hetero-oligomer formation ........... 74 4.3 Results ....................................... 74 4.3.1 Rationale of the overall experimental approach ............. 74 4.3.2 Kinetics and linear range of the fluorescence signal ........... 76 4.3.3 Kynurenine fluorescence of daptomycin in the presence of NBD-daptomycin ............................. 76 4.3.4 Kynurenine fluorescence in hybrid oligomers formed on membranes .. 79 xiii Contents 4.4 Discussion ..................................... 80 5 Summary and future research work 87 5.1 Summary ..................................... 87 5.2 Further Research Work .............................. 90 5.2.1 Causative role of oligomerization in bactericidal action ......... 90 5.2.2 Contribution of the negatively charged phospholipids other than PG to daptomycin binding, oligomerization, and bactericidal action ...... 90 5.2.3 Functional characterization of the membrane defect ........... 91 5.2.4 Analysis of oligomer structure on membranes by NMR ......... 91 Permissions 93 Bibliography 113 xiv List of Figures 1.1 Structures of sulfamidochrysoidine, sulfanilamide and p-aminobenzoate. 4 1.2 Structures of pencillin G and benzyl-penicilloic

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