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Kinase Inhibitors and Nucleoside Analogues As Novel Therapies to Inhibit HIV-1 Or ZEBOV Replication

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Kinase Inhibitors and Nucleoside Analogues As Novel Therapies to Inhibit HIV-1 Or ZEBOV Replication Kinase Inhibitors and Nucleoside Analogues as Novel Therapies to Inhibit HIV-1 or ZEBOV Replication by Stephen D. S. McCarthy A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Stephen D. S. McCarthy (2017) Kinase Inhibitors and Nucleoside Analogues as Novel Therapies to Inhibit HIV-1 or ZEBOV Replication Stephen D.S. McCarthy Doctor of Philosophy Graduate Department of Laboratory Medicine and Pathobiology University of Toronto 2017 Abstract Without a vaccine for Human Immunodeficiency Virus type 1 (HIV-1), or approved therapy for treating Zaire Ebolavirus (ZEBOV) infection, new means to treat either virus during acute infection are under intense investigation. Repurposing tyrosine kinase inhibitors of known specificity may not only inhibit HIV-1 replication, but also treat associated inflammation or neurocognitive disorders caused by chronic HIV-1 infection. Moreover, tyrosine kinase inhibitors may effectively treat other infections, including ZEBOV. In addition, established nucleoside/nucleotide analogues that effectively inhibit HIV-1 infection, could also be repurposed to inhibit ZEBOV replication. In this work the role of two host cell kinases, cellular protoncogene SRC (c-SRC) and Protein Tyrosine Kinase 2 Beta (PTK2B), were found to have key roles during early HIV-1 replication in primary activated CD4+ T-cells ex vivo. siRNA knockdown of either kinase increased intracellular reverse transcripts and decreased nuclear proviral integration, suggesting they act at the level of pre-integration complex (PIC) formation or PIC nuclear translocation. c-SRC siRNA knockdown consistently reduced p24 levels of IIIB(X4) and Ba-L(R5) infection, or luciferase ii activity of HXB2(X4) or JR-FL(R5) recombinant viruses, prompting further drug inhibition studies of this kinase. Four c-SRC kinase inhibitors (dasatinib, saracatinib, KX2-391 and SRC Inhibitor-1) significantly reduced HXB2 and JR-FL infection in primary CD4+ T-cells. Thus, these potent c-SRC inhibitors should be further evaluated in humanized mouse models of HIV-1 infection. During 2014-16, the Ebola outbreak in West Africa prompted us to rapidly assess whether conventional nucleoside analogs could inhibit in vitro ZEBOV replication. Employing a new lifecycle model of ZEBOV infection in level 2 biocontainment, combinations of nucleoside analogues and interferons were found to synergistically inhibit ZEBOV replication. These included zidovudine, lamivudine and tenofovir, confirmed to show antiviral activity against fully infectious ZEBOV-GFP in level 4 biocontainment. Findings from this thesis provided the rationale for further preclinical development of nucleoside analogue combination treatments, and a phase II EVD trial evaluating recombinant interferon in Guinea. Pre-clinical results using c- SRC kinase inhibitors also suggest that this approach could also be effective in EVD. iii Acknowledgments First and foremost, I will thank my mentor Dr. Donald R. Branch, for his inspiration, invaluable advice and tremendous support throughout my time in his laboratory at Canadian Blood Services. I also extend thanks to my Ph D advisory committee, Dr. Rupert Kaul and Dr. Dwayne Barber, for their valuable advice, direction and input. I will next acknowledge the hard work of Dr. Thomas Hoenen, Dr. Danila Leontyev, Dr. Anton Neschadim, Dr. Daniel Jung, Dr. Trina Racine and Beata Majchrzak-Kita, who contributed immensely to the many joint projects related to this work. As well, I am very grateful to Darinka Sakac, Yulia Petrenko and Amanda Harrison Wong, who generously gave their time to train me, and Dr. Reed Siemieniuk for insightful conversations. I also thank Dr. Eleanor Fish and Dr. Gary Kobinger for their collaboration on a completely novel direction of this project, which has proven to be fruitful new avenue of research. I also thank my dear colleagues in the Branch laboratory, Evgenia Bloch, Carlyn Figueiredo, Cindy Tong, Megan Blacquiere, Bonnie Lewis, Minji Kim, Eric Lai and Beth Binnington, for the many opportunities to collaborate, abundant discussions, support of my ideas and work, and for creating a joyful and lively lab environment. I also want to thank undergraduate summer students Hannah Kozlowski, Shawn Goyal, Pauline Nicoletti, Ninon Guichard and Janette Spears, whom I closely mentored and had good fortune to collaborate with over the years. I also want to thank organizations that shaped my thinking around HIV and Ebola research. I am eternally grateful for the support of my graduate department at the University of Toronto, Laboratory Medicine and Pathobiology. In particular, I deeply appreciate the wisdom and mentorship of Dr. Harry Elsholz, Dr. Maria Rozakis, Ferzeen Dharas-Sammy, Rama Ponda and Katie Babcock, who encouraged my participation in the Three Minute Thesis competition, to represent the department at the CIHR Canadian Student Health Research Forum in Winnipeg, and to participate in our vibrant student union CLAMPS. Additionally, I am thankful to Sergio Martinez, Duncan MacLachlan and Adam Busch, who at the AIDS Committee of Toronto taught me valuable perspectives on people living with HIV-1, access to STI testing, and health care inequality. I am also very thankful of Dr. Dana Devine and Don LaPierre at Canadian Blood iv Services, who gave me a platform to advocate for blood donor equality at a national level. And I am deeply thankful of Dr. Logan and Elizabeth Cohen, who through courageous actions, reported potential alternative therapies to treat Liberian Ebola patients in 2014. I will also take the opportunity to acknowledge the financial support of NSERC (CGS M scholarship), Canadian Blood Services (GFP scholarship and support from the Centre for Innovation to Dr. Branch), CIHR (Vanier CGS D scholarship, and support to Dr. Fish), the Ontario HIV Treatment Network (Dr. Branch) and Health Canada (Dr. Branch), which have financially supported me for the entirety of this project. Finally, I would like to thank my dear parents, brothers and close friends. Your love and unconditional support was the foundation of my success in graduate school. v Table of Contents Page Abstract ........................................................................................................................................... ii Acknowledgments.......................................................................................................................... iv Table of Contents ........................................................................................................................... vi List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Abbreviations .................................................................................................................... xii Chapter 1: Introduction ................................................................................................................... 1 1.1 HIV-1 and Ebola: Global Health Problems .......................................................................... 2 1.2 Human Immunodeficiency Virus (HIV) ............................................................................... 5 1.2.1 HIV-1 Epidemiology, Transmission and Replication Cycle .......................................... 5 1.2.2 HIV-1 Treatments, Potential Vaccines, and New Therapeutics ................................... 13 1.2.3 Host Kinases as Targets for HIV-1 Inhibition .............................................................. 17 1.2.4 The SRC Family of Non-Receptor Tyrosine Kinases in HIV-1 Infection ................... 22 1.2.5 Role of c-SRC in HIV-1 Infection ................................................................................ 37 1.2.6 Role of PTK2B in HIV-1 Infection .............................................................................. 42 1.3 Ebola Virus (EBOV) ........................................................................................................... 47 1.3.1 ZEBOV Epidemiology, Transmission and Replication Cycle ..................................... 47 1.3.2 ZEBOV Clinical Trials, Potential Vaccines and New Therapeutics ............................ 55 1.3.3 Repositioning Nucleoside Analogues for ZEBOV Inhibition ...................................... 59 1.3.4 Testing Other Potent Nucleoside/Nucleotide Analogues: Zidovudine, Lamivudine and Tenofovir ............................................................................................................................... 69 1.4 Statement of the Problem, Rationale, Hypotheses and Objectives of this Work ................ 87 1.4.1 Statement of the Problem ............................................................................................. 87 1.4.2 Rationale ....................................................................................................................... 87 1.4.3 Hypotheses.................................................................................................................... 88 1.4.4 Objectives of this Work ................................................................................................ 89 1.4.5 Organization of the Thesis ...........................................................................................
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