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IDENTIFICATION AND CHARACTERIZATION OF DRUG TARGETS IN THE PYRIMIDINE AND PURINE PATHWAYS OF TRYPANOSOMA BRUCEI APPROVED BY SUPERVISORY COMMITTEE Margaret Phillips, Ph.D. Richard Bruick, Ph.D. Benjamin Tu, Ph.D. Jennifer Kohler, Ph.D. Michael Reese, Ph.D. DEDICATION To my parents Guillermo and Lonnie, and grandparents Luis and Juana, for a lifetime of encouragement and support. My wife Alli, for all the love and support throughout this endeavor. IDENTIFICATION AND CHARACTERIZATION OF DRUG TARGETS IN THE PYRIMIDINE AND PURINE PATHWAYS OF TRYPANOSOMA BRUCEI by CHRISTOPHER LUIS LEIJA DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas December, 2016 Copyright by Christopher Luis Leija 2016 All Rights Reserved IDENTIFICATION AND CHARACTERIZATION OF DRUG TARGETS IN THE PYRIMIDINE AND PURINE PATHWAYS OF TRYPANOSOMA BRUCEI Christopher Luis Leija, Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2016 Supervising Professor: Margaret A. Phillips, Ph.D. The single-celled extracellular parasite Trypanosoma brucei causes Human African Trypanosomiasis (HAT), which is fatal if untreated. Current therapies result in severe side effects and require complex treatment regimens. In an effort to spur the development of effective, safe, and simple to administer drugs, my work sought to identify and characterize novel drug targets in the parasite pyrimidine and purine pathways. The pyrimidine de novo biosynthetic pathway has been well characterized, however little work had been done to evaluate the importance of pyrimidine salvage enzymes. Specifically, my research validates the essentiality two seemingly redundant enzymes: thymidine kinase (TK) and cytidine v deaminase (CDA). Using a combination of genetic and analytical techniques, a novel pathway linking cytosine and thymine nucleotides was discovered. This pathway is composed of the salvage enzymes TK and CDA in addition to a newly discovered 5’- nucleotidase. I demonstrate that the function of this pathway is to convert de novo synthesized cytosine deoxynucleotides into the deoxycytidine, which is ultimately converted to thymine deoxynucleotides. The vital role for TK in bridging pyrimidine nucleotide pools may represent a shared vulnerability unique to kinetoplastids, providing an opportunity to target multiple human pathogens. In contrast to the pyrimidine pathway, the parasite lacks the ability to generate purine nucleotides de novo. As a consequence, they are dependent on the salvage of purine nucleosides/bases from the host through a redundant and interconnected network of purine salvage and interconversion enzymes. In theory, any single precursor is capable of sustaining the formation of all purine nucleotides. We demonstrate that strategic inhibition of key metabolic routes circumvents the redundant nature of this pathway. The enzyme guanosine- 5’-monophosphate synthase (GMPS) catalyzes the formation of GMP from xanthosine-5’- monophosphate. The generation of a GMPS null cell line restricts the parasite to the salvage of guanine to maintain GMP nucleotide pools, which is only viable in supraphysiological concentrations of guanine. Using a similar approach, we also genetically validated the essentiality of adenylosuccinate lyase (ADSL), which catalyzes the formation of AMP and fumarate from adenylosuccinate. In this case, depletion of this enzyme is lethal in all conditions. These two novel drug targets offer a solution to bypassing the redundancy in the purine pathway for the development of anti-trypanosomal therapies. TABLE OF CONTENTS TITLE-FLY ............................................................................................................................. i DEDICATION ....................................................................................................................... ii TITLE PAGE ........................................................................................................................ iii COPYRIGHT ......................................................................................................................... iv ABSTRACT ........................................................................................................................... v TABLE OF CONTENTS ....................................................................................................... vii PRIOR PUBLICATIONS ....................................................................................................... ix LIST OF FIGURES AND TABLES....................................................................................... x LIST OF ABBREVIATIONS ............................................................................................... xiv CHAPTER 1 INTRODUCTION ................................................................................................................ 1 HUMAN AFRICAN TRYPANOSOMIASIS ................................................................. 2 CURRENT TREATMENTS ........................................................................................... 3 PYRIMIDINE PATHWAY ............................................................................................. 4 PURINE PATHWAY ...................................................................................................... 6 RESEARCH GOALS ...................................................................................................... 9 CHAPTER 2 PYRIMIDINE SALVAGE ENZYMES ARE ESSENTIAL FOR DE NOVO BIOSYNTHESIS OF DEOXYPYRIMIDINE NUCLEOTIDES ................................... 13 INTRODUCTION ......................................................................................................... 14 MATERIALS AND METHODS ................................................................................... 15 RESULTS ...................................................................................................................... 28 CONCLUSIONS ............................................................................................................ 40 CHAPTER 3 GMP SYNTHASE IS ESSENTIAL FOR VIABILITY AND INFECTIVITY OF T. BRUCEI DESPITE A REDUNDANT PURINE SALVAGE PATHWAY ................ 74 INTRODUCTION ......................................................................................................... 75 MATERIALS AND METHODS ................................................................................... 76 RESULTS ...................................................................................................................... 87 CONCLUSIONS ............................................................................................................ 95 CHAPTER 4 GENETIC VALIDATION OF ADENYLOSUCCINATE LYASE ESSENTIALITY AND KINETIC CHARACTERIZATION ..................................................................... 113 INTRODUCTION ....................................................................................................... 114 MATERIALS AND METHODS ................................................................................. 115 RESULTS .................................................................................................................... 119 CONCLUSIONS ........................................................................................................... 122 CHAPTER 5 PERSPECTIVES .............................................................................................................. 133 REFERENCES .................................................................................................................. 140 PRIOR PUBLICATIONS Leija C, Rijo-Ferreira F, Kinch LN, Grishin NV, Nischan N, et al. (2016) Pyrimidine Salvage Enzymes Are Essential for De Novo Biosynthesis of Deoxypyrimidine Nucleotides in Trypanosoma brucei. PLoS Pathog 12(11): e1006010. doi: 10.1371/journal.ppat.1006010 Li, Q.*, Leija, C.*, Rijo-Ferreira, F., Chen, J., Cestari, I., Stuart, K., Tu, B. P. and Phillips, M. A. (2015), GMP synthase is essential for viability and infectivity of Trypanosoma brucei despite a redundant purine salvage pathway. Molecular Microbiology, 97: 1006–1020. *Equal contribution Nguyen, S., Leija, C., Kinch, L., Regmi, S., Li, Q., Grishin, N.V., Phillips, M.A. (2015), Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins. Journal of Biological Chemistry, 290(32):199987-98 ix LIST OF FIGURES FIGURE 1.1 T. brucei pyrimidine pathway ......................................................................... 10 FIGURE 1.2 T. brucei purine pathway ............................................................................... 11 FIGURE 2.1 PCR analysis confirms replacement of tk alleles with selectable markers..... 46 FIGURE 2.2 TK is essential for in vitro growth and infectivity in mice ............................. 48 FIGURE 2.3 Infection of mice with TK c-null cells and wild-type SM cells...................... 48 FIGURE 2.4 Catalytically active TK is required to rescue the TK RNAi growth phenotype .. 49 FIGURE 2.5 Growth analysis of TK RNAi cells (±Tet) expressing Tet regulated HsvTK grown in parallel with the TK RNAi control (±Tet). ............................................................. 50 FIGURE

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