Identification and Characterization of Drug Targets in the Pyrimidine and Purine Pathways of Trypanosoma brucei

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 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.