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COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Director of Copyright Services sydney.edu.au/copyright IDENTIFICATION AND CHARACTERISATION OF NEW COMPOUNDS TO TREAT CHRONIC BACTERIAL LUNG INFECTIONS Samantha Ellis Supervisor: Associate Professor Jamie Triccas A thesis submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY SYDNEY MEDICAL SCHOOL THE UNIVERSITY OF SYDNEY January 2014 Department of Infectious Diseases and Immunology Faculty of Medicine University of Sydney Disclaimer This PhD thesis is based on work carried out at the University of Sydney, under the supervision of Dr. Jamie Triccas, between February 2010 and January 2014. This thesis incorporates original research that has not been previously submitted for a higher degree to any other university. It does not contain any material previously published or written by any other person except where reference has been made in the text. …………………………………… Samantha Ellis 2014 ii | P a g e Acknowledgments First and foremost, I would like to thank my supervisor, Associate Professor Jamie Triccas. Thank you for allowing me to complete this PhD and taking a chance on me. You always knew how to encourage me and I would not have gotten this far with anyone else. I hope that I have not disappointed. Secondly, I would like to thank my associate supervisor Professor Des Richardson and Danuta Kalinowski. Both of you were a great help, and of particular importance was the role you played in my results chapter and paper. Similarly, to Associate Professor Matt Todd and Associate Professor Peter Rutledge. Thank you for assisting me with my work, providing the compounds I needed to complete my study, and for helping me understand chemistry. To my lab. Rach and Lisa. You made coming into the lab so much more enjoyable and when I really needed someone to talk to you were both there to offer support and encouragement. I will be forever grateful for that. To Jono and Carlyn. Two of the best PhD buddies a person could ask for. You are both incredibly wonderful people and I’m glad that I can say that even after you both have moved on that we are still friends. I was truly blessed to be a part of such an amazing, fun lab. Thank you to the Myco lab for their help and guidance over the last 4 years. Without you I would not have been able to perform any of my TB experiments or mouse experiments. Your assistance and knowledge was greatly appreciated. Thanks to Rebecca and Catherine, who always had the answer and assisted me whenever I needed it. It can’t be an easy task looking after students but you both made it look easy. I appreciate all the help you gave. iii | P a g e To my significant PhD others. Jeremy, you were a great desk buddy and definitely made the day more enjoyable. Jenna, you were the best friend a person could ask for. You certainly made the day more interesting with our coffee runs, lunches and you screaming and hitting your computer, and I wouldn’t have had it any other way. And Claudio, you were definitely the best thing to come out of Italy. You have been an amazing friend and you are a wonderful person. It has been a privilege getting to you know. To Chris. Your love and support has gotten me through these last few years. I know you had to put up with a lot of whinging and complaining but I appreciate you listening and that you were just there for a cuddle if I needed it. You were definitely the best thing to come out of this degree. And finally a huge thank you to the most important people in my life. My family. To my brother, for being there and encouraging me when I was down, and celebrating with me when everything was going well. Thanks buddy. And to my mum. Words can’t express the help, love and support you have offered me over the years. I know the last 4 years have not been easy but without you, I would not have made it through. You are my inspiration and I am so proud to be your daughter. I dedicate this thesis to you. iv | P a g e Abbreviations oC Degrees Celsius µ Micro µg Microgram µL Microlitre µM Micromolar ADC Albumin-dextrose catalase AES Australian Epidemic Strain ANOVA Analysis of variance BCG Mycobacterium bovis (Bacille Calmette Guérin) BCG pSMT1 BCG containing plasmid encoding luciferase gene BH Benzoyl hydrazide BSA Bovine-serum albumin CF Cystic Fibrosis CFU Colony forming units CO2 Carbon dioxide DETANO Diethylenetriamine-nitric oxide DFO Desferrioxamine dH2O Distilled water v | P a g e DNA Deoxyribonucleic acid DOTS Directly observed treatment short-course strategy ESAT-6 Early secreted antigen, 6kDa FDA Food and Drug Administration g Grams Hsp60 Heat shock protein 60 (used in plasmids as promoter) INH Isoniazid kb Kilobase kDa Kilodalton L Litre LB Luria-Bertani LPS Lipopolysaccharide m Milli M Molar MDR Multi-drug resistant MDRTB Multi-drug resistance Tuberculosis MIC Minimum Inhibitory Concentration min Minute mM Millimolar vi | P a g e MRSA Methicillin-resistant Staphylococcus aureus ng Nanogram nM Nanomolar OADC Oleic acid-albumin-dextrose-catalase (medium supplement) PBS Phosphate buffered saline PMA Physical medium attachment RO Reverse osmosis SD Standard deviation SEM Standard error of the mean TDW Triple distilled water v/v Volume per volume w/v Weight per volume W.H.O World Health Organisation XDR Extensively Drug resistant XDRTB Extensively Drug resistant Tuberculosis vii | P a g e List of Figures Figure 1.1: Global incidence of susceptible and multiple-drug resistant Tuberculosis. Figure 1.2: Incidence of Tuberculosis cases worldwide. Figure 1.3: The colonisation profiles of the most common pathogens in CF patients. Figure 1.4: Percentage of MDR-TB cases in newly diagnosed patients. Figure 1.5: Treatment success for new cases of tuberculosis treated under DOTS between 1994 and 2006. Figure 1.6: New drug pipeline for tuberculosis treatment. Figure 2.1. Map of the pSMT1 plasmid. Figure 2.2. Alamar blue assay reaction. Figure 3.1. Concentration-dependent inhibition of mycobacterial growth by PCIH. Figure 3.2. Establishing drug activity through testing of components against mycobacteria. Figure 3.3. Concentration-dependent inhibition of mycobacterial growth by PCIH. Figure 3.4. Effect of PCIH on intracellular bacteria and host cell viability. Figure 3.5. Inhibition of mycobacterial growth by modified class of chelators. Figure 3.6. Bacterial growth inhibition by high iron affinity chelators. Figure 3.7. In vivo efficacy against mycobacterial infection. Figure 3.8. Efficacy of PCIH against M. tuberculosis. Figure 3.9. Schematic of the possible mechanisms involved in the anti-mycobacterial viii | P a g e activity of PCIH. Figure 4.1. Screening of compounds. Figure 4.2. Screening of compounds against mycobacteria. Figure 4.3. Cell viability in human cell line. Figure 4.4. Effect of compounds against high bacterial load. Figure 4.5. Efficacy of compounds in macrophages. Figure 4.6a. Exposure of BCG to compounds in combination with INH. Figure 4.6b. Exposure of BCG to compounds in combination with Rifampicin. Figure 4.7a. Exposure of M. avium to compounds in combination with INH. Figure 4.7b. Exposure of M. avium to compounds in combination with Rifampicin. Figure 4.7c. Exposure of M. avium to compound 53 in combination with Rifampicin. Figure 4.8. Optimisation of dosage and incubation time for a non-replicating model of infection. Figure 4.9. Testing compound 48 in a non-replicating model. Figure 4.10. Effect of compound 48 against non-replicating bacteria. Figure 4.11. Structure of lead MCyCs inhibitors. Figure 5.1. Optimising conditions for use in mycobacterial High-Throughput screen. Figure 5.2a. Screening of compounds against P. aeruginosa CJ2009. Figure 5.2b. Screening of compounds against P. aeruginosa Bb2009. Figure 5.3. Screening of compounds against M. avium. ix | P a g e Figure 5.4. MIC90 of selected compounds against P. aeruginosa strains. Figure 5.5. MIC90 of selected compounds against M.avium. Figure 5.6. Effect of compounds against patient sputum samples. Figure 5.7. Toxicity of compounds effective against P. aeruginosa. Figure 5.8. Toxicity of compounds effective against M.avium. List of Tables Table 3.1.Structure and activity of PIH derivatives against mycobacteria. Table 4.1. MIC90 of selected Metal-Cyclam complexes. Table 4.2. Comparison of MIC90 values between different strains of M. tuberculosis. Table 5.1. MIC90 of control antibiotics against Pseudomonas to determine the most resistant strain.