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Methods to Identify and Develop Drugs for Cryptosporidiosis Rajiv Satish Jumani University of Vermont

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Methods to Identify and Develop Drugs for Cryptosporidiosis Rajiv Satish Jumani University of Vermont University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2018 Methods To Identify And Develop Drugs For Cryptosporidiosis Rajiv Satish Jumani University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Medical Sciences Commons, Microbiology Commons, and the Parasitology Commons Recommended Citation Jumani, Rajiv Satish, "Methods To Identify And Develop Drugs For Cryptosporidiosis" (2018). Graduate College Dissertations and Theses. 892. https://scholarworks.uvm.edu/graddis/892 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. METHODS TO IDENTIFY AND DEVELOP DRUGS FOR CRYPTOSPORIDIOSIS A Dissertation Presented by Rajiv Satish Jumani to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Cellular, Molecular and Biomedical Sciences May, 2018 Defense Date: February 27, 2018 Dissertation Examination Committee: Christopher D. Huston, M.D., Advisor Johnathan E. Boyson, Ph.D., Chairperson Wolfgang Dostmann, Ph.D. Markus Thali, Ph.D. Gary E. Ward, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT Cryptosporidiosis is a common diarrheal disease caused by intestinal infection with the apicomplexan parasite Cryptosporidium, in humans usually either with C. hominis or C. parvum. Unfortunately, given a large burden of disease in children and immunocompromised people like AIDS patients, the only currently approved treatment, nitazoxanide, is unreliable for these patient populations. To address the urgent need for new drugs for the most vulnerable populations, large phenotypic screening efforts have been established to identify anti-Cryptosporidium growth inhibitors in vitro (hits). However, in the absence of a gold standard drug, the in vitro and in vivo characteristics that should be used to prioritize screening hits are not known. This thesis is focused on identifying promising anti-Cryptosporidium hits and drug leads, and using them to establish validated methods to guide hit-to-lead studies for anti-Cryptosporidium drug development. A re-analysis of our phenotypic screen of the Medicines for Malaria Venture Open Access Malaria Box identified a promising C. parvum growth inhibitor, MMV665917. It had similar in vitro activity against C. hominis, C. parvum Iowa, and C. parvum field strains, and it was amenable to preliminary structural activity relationship studies using commercially available variants, with one variant demonstrating nanomolar potency. Furthermore, MMV665917 was effective in vivo in an acute interferon-γ mouse model of cryptosporidiosis; and it appeared to cure an established infection in the chronic NOD SCID gamma (NSG) mouse model, unlike nitazoxanide, paromomycin, and clofazimine. We hypothesized that anti- Cryptosporidium activity in the highly immunocompromised chronic NSG mouse model might relate to compounds being capable of killing and eliminating parasites (cidal), rather than only preventing growth (static). To test this, we developed a novel in vitro parasite persistence assay that showed that MMV665917 was potentially cidal, whereas nitazoxanide, paromomycin and clofazimine appeared static. This pharmacodynamic assay also provided the concentration of compound required to maximize rate of parasite elimination, which could help design in vivo experiments. To further characterize compounds based on mechanism of action, we developed a range of in vitro medium-throughput life-stage assays. To validate and gain value from the assays, a “learner set” of compounds from our in-house screens and collaborations were tested in all of the in vitro assays and in the in vivo NSG mouse model. Using these assays, it was possible to group molecules based on chemical class/mechanism of action. Because compounds from distinct groups showed activity in the NSG mouse model, these methods could be used to obtain a diverse set of early- stage Cryptosporidium inhibitors for prioritization. Furthermore, compounds that appeared static in the in vitro parasite persistence assay did not have activity in the NSG mouse model. In summary, we report the identification and development of a highly promising initial lead, MMV665917, and report a range of in vitro assays that can be used to prioritize anti-Cryptosporidium hits and leads. CITATIONS Material from this dissertation has been published in the following form: Jumani R.S., Bessoff K., Love M.S., Miller P., Stebbins E.E., Teixeira J.E., Campbell M.A., Meyers M.J., Zambriski J.A., Nunez V., Woods A.K., McNamara C.W., Huston C.D.. (2018). A Novel Piperazine-Based Drug Lead for Cryptosporidiosis from the Medicine for Malaria Venture Open Access Malaria Box. Antimicrobial Agents and Chemotherapy, 62, e01505-17. ii ACKNOWLEDGEMENTS First and foremost I would like to thank my advisor, Dr. Christopher D. Huston. No words can justify my gratitude for him. A long time ago, when I was rotating in his lab he mentioned to me that motivating students is not his strength; but his constant motivation all these years is the one thing that has kept me going all along, including the herculean task of writing this dissertation. Even at times when I lost confidence in my abilities, Chris has been extremely patient with me, finding innovative ways to help me. He has not only been a personal and professional guide and mentor, but I owe most of my personal and professional success to him. It is common saying that graduate students subconsciously take after their mentors; I would honestly consider myself very fortunate to have inherited his virtues. His values and teachings have not only made me a good scientist but a better human being as well. I am extremely thankful to my thesis committee for guiding me all through these years. Their critical insight have taught me so much that I always looked forward to learning from them at the committee meetings, while at the same time dreading that I am not smart enough. Dr. Jon Boyson has always provided very thorough and meaningful insights that have been extremely vital to the success of my training and thesis. Dr. Wolfgang Dostmann has not only taught me all the organic and medicinal chemistry knowledge I know, but also been like a beacon, guiding me with invaluable personal and professional advice at every critical juncture. Dr. Markus Thali was the first faculty that I got in touch with during my applications, iii and I have had some extremely memorable conversations with him. He is one of the main reasons why I love Switzerland and Swiss chocolates – thank you again for introducing me to Lindor! Dr. Gary Ward’s love for science and rigor has been contagious. I have always looked up to his professionalism, teaching and presentation skills. I would like to thank my labmates: Adam, Alvee, Connor, Erin, Kovi, Liam, Peter and Ze, who have been like a family to me. This thesis would have not happened without all of you. Kovi is an amazing personality and taught me all the Crypto; Adam’s been the prodigy scientific big brother; Ze is a scientific genius; Peter is extremely hard working and provides the most critical insights; Liam is extremely kind and smart; Connor is always lively, and a programming genius; Alvee is one of the most intelligent people I have known; and Erin is one of the smartest and kindest person I have met. The CMB, NGP and medicine department folks have always been there and made things happen; thank you Erin Montgomery, Carrie, Kristin, Jessica, Hayley, Ashley and Pam. Dozens of people inside and outside UVM have taught me immensely, and although too many to name here, I am extremely grateful to them. I am extremely fortunate to have such a loving and supportive wife, family and friends, who have been my pillar of support. This thesis is dedicated to my mother, Mrs. Meena S. Jumani, who is the real reason and inspiration behind my endeavors. iv TABLE OF CONTENTS Page CITATIONS .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................ iii LIST OF TABLES ........................................................................................................... x LIST OF FIGURES ........................................................................................................ xi CHAPTER 1: COMPREHENSIVE LITERATURE REVIEW ...................................... 1 1.1. Introduction........................................................................................................... 1 1.2. Historical Perspective ........................................................................................... 1 1.3. Transmission ......................................................................................................... 3 1.4. Epidemiology ........................................................................................................ 4 1.4.1. Children ........................................................................................................... 4 1.4.2. Immunocompetent and Immunocompromised Adults .................................... 5 1.5. Life Cycle ............................................................................................................
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