Inhibition and Enhancement of Respiratory Syncytial Virus Replication by Nucleoside Analogues and Bis(indole) Compounds by Lionel Dylan Jensen A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology Department of Medical Microbiology and Immunology University of Alberta © Lionel Dylan Jensen, 2018 Abstract Respiratory syncytial virus (RSV) is an Orthopneumovirus that infects the epithelium of the airways. Severe RSV infection of the lower respiratory tract in infants is a leading cause of pediatric hospitalizations. RSV also causes substantial morbidity in immunocompromised and elderly populations. Palivizumab, a humanized monoclonal antibody, is available for the prophylactic treatment of high-risk infants. However, this intervention is expensive and has a limited impact on annual hospitalization rates caused by RSV. No vaccine is available to prevent RSV infection and no efficacious antivirals are available to treat active infection. To address the burden of disease imposed by RSV, this project sought to develop and implement a screening assay to identify compounds with antiviral activity against RSV. Different screening protocols were examined as platforms for testing antiviral activity. The first protocol quantified changes to RSV replication complex morphology during antiviral treatment. Through confocal microscopy, changes to replication complex morphology were identified as early as six hours post infection. However, this assay was hindered by the low signal intensity produced by replication complexes. Therefore, alternative RSV-antiviral screening protocols were investigated. Subsequent protocols quantified initial monolayer infection, or quantified RSV progeny production, via colourimetric or immunofluorescence (IF) staining. Automated detection of IF-stained RSV-infected cells was conducted using a high content imaging system. This protocol (referred to as ‘the IF protocol’) offered the highest throughput screening capacity and most reliable detection of RSV infection versus the other methods that were tested. Using the IF protocol, the chemotherapeutic nucleoside analogue cytarabine was investigated and antiviral activity against RSV was observed. The IF protocol was then used to screen a series of bis(indole) compounds for antiviral activity against RSV. Bis(indole) compounds were hypothesized to have antiviral activity as they were derived from Isatisine A, a naturally occurring compound with modest antiviral activity. Bis(indole) ii compounds with antiviral activity against RSV were identified; the IF protocol was then used to guide the synthesis of novel bis(indole) compounds with improved cytotoxicity profiles. The Toll-like receptor 7 (TLR7) agonist loxoribine was investigated for antiviral activity against RSV, however, enhancement of RSV replication during loxoribine treatment was observed. This observation was unexpected as TLR7 is a pattern recognition receptor which contributes to the identification of pathogens by the innate immune system and TLR7 stimulation typically elicits an antiviral immune response. Furthermore, TLR7 agonists are undergoing clinical investigations to examine their potential as immunomodulatory treatments for airway diseases. As enhancing the severity of RSV infections in this population could be hazardous, it was considered essential to further characterize the relationship of loxoribine with RSV replication. Loxoribine-mediated enhancement of RSV replication in human airway epithelial cells was determined to be concentration-dependent and this effect was reproducible with the distinct TLR7 agonist CL097. Inhibition of TLR7 stimulation by the antagonist IRS-661, or by siRNA knock down of TLR7, prevented enhancement of RSV replication by loxoribine. Finally, TLR7-mediated enhancement of RSV replication was determined to be dependent on extracellular signal-regulated kinase activation. These results support the novel conclusion that exogenous stimulation of TLR7 benefits RSV replication. These results also suggest caution is warranted during the ongoing development of TLR7-based therapeutics, especially for therapeutics being developed for the treatment of airway diseases. iii Preface A portion of research conducted for this thesis forms part of a collaboration with Dr. Rachel Fearns at Boston University Medical Center, Boston, MA. The in vitro RSV transcription assay described in chapter 2 was designed by Dr. Rachel Fearns, and guidance with this protocol was provided by Dr. Sarah Noton. All subsequent experimentation and analyses of results using this protocol were completed by myself. A portion of research conducted for this thesis forms part of collaboration between Dr. Frederick West, Department of Chemistry at the University of Alberta, and Dr. David Marchant, Department of Medical Microbiology and Immunology at the University of Alberta. Resultantly, a portion of chapter four has been previously published as “Dual Catalytic Synthesis of Antiviral Compounds Based on Metallocarbene-Azide Cascade Chemistry” in The Journal of Organic Chemistry (available online April 17, 2018). With respect to this manuscript, B. Atienza was responsible for synthesis of bis(indole) compounds and composure of the manuscript relating to these chemical syntheses. I was responsible for data collection, analysis, and manuscript composition with respect to respiratory syncytial virus. F. West and D. Marchant were supervisory authors responsible for concept formation. Lastly, experimentation to produce figures 5.5c and 5.5e in chapter five of this thesis was completed by L. Bilawchuk, Department of Medical Microbiology and Immunology at the University of Alberta. iv Acknowledgements This work would not have been possibly without the endless encouragement and input of my project supervisor, Dr. David Marchant. I would also like to thank Cameron Griffiths. This project benefitted immensely from his wealth of knowledge, his undefeatable enthusiasm for science, and the endless thoughtful discussions we held - Cameron Griffiths, I am truly grateful. It is not hyperbole to say that every experiment I conducted benefitted from the input of our laboratory manager Leanne Bilawchuk. I must thank Leanne for her patience and expertise. I would like to thank my committee members Dr. Edan Foley and Dr. Michael Houghton. Throughout my project their guidance and critiques inspired me to attempt new techniques and greatly accelerated my research project. I greatly appreciate the help of the supporting administrative staff of Medical Microbiology and Immunology. They are the unsung heroes of the research conducted our department. In particular, I would like to thank Tabitha Vasquez and Debbie Doudiet. I have many fellow Medical Microbiology and Immunology students to thank for their input on techniques and experimentation. Namely, I would like to thank Farah Elawar, Ninad Mehta, David Fast, Brittany Fraser, Ben Kostiuk, Brittany Umer, Jason Wong, Amanda Scott, Adil Mohamed, Ken Gavina, and Kevin James. I would also like to thank all of those outside of academia who have supported me. This includes my close friends Alanna Poole, Sierra Poole, Laurence Pryer, Heather Nixdorff, Danielle Gregoire and David Strahl, and the good people of the Alpine Club of Canada, Edmonton section. Lastly, the most important contributors to any of my past, present, or future achievements are my parents, Randy and Nomi Jensen. v Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Acknowledgements ....................................................................................................................... v List of Tables ................................................................................................................................ ix List of Figures ................................................................................................................................ x List of Abbreviations ................................................................................................................... xi Chapter 1: Introduction ............................................................................................................... 1 1.1. RSV history ........................................................................................................................................................ 1 1.2. RSV burden of disease ....................................................................................................................................... 2 1.2.1. Burden of disease in Canada and worldwide ............................................................................................... 2 1.2.2. Risk factors and groups associated with severe RSV infection ..................................................................... 2 1.2.3. RSV clinical presentation ............................................................................................................................. 3 1.2.4. RSV infection and the development of asthma .............................................................................................. 4 1.3. RSV communicability .......................................................................................................................................