Year: 2009
Type: dissertation
Abstract: Trypanosoma brucei, the causative agent of the disease African sleeping sickness in humans and nagana in animals, is a scourge of sub-Saharan Africa. There is a desperate need for more efficacious therapies for the disease; here we describe research validating T. brucei hexokinase 1 (TbHK1) as a drug therapeutic target for T. brucei infection and the identification and characterization of novel inhibitors of the enzyme by both low throughput and high throughput means. Additionally this thesis introduces efforts at characterizing a second T. brucei hexokinase, focusing mostly on cell biology and the determination of localization. The small molecule quercetin (QCN) was tested and found to inhibit recombinant TbHK1 and spectroscopic study revealed that quercetin binds to TbHK1 proximal to the active site. Genetic experiments revealed that QCN may be cytotoxic to parasites as a result of TbHK inhibition. QCN localized to punctate bodies and the flagellum of T. brucei (consistent with the localization of TbHKs) and over expression of TbHK1 was partially protective. In order to identify additional TbHK1 inhibitors, high through-put screening (HTS) techniques were implemented. HTS of >220,000 compounds yielded 239 primary inhibitors of TbHK1. Of the 239 hits, 15 were confirmed in validation assays as TbHK1 inhibitors; these compounds had a spectrum of activity against trypanosomes, suggesting possible therapeutic leads. In order to initiate characterization of the unknown function of TbHK2, localization studies of TbHK2 were performed. Immunofluorescence assays were employed and TbHK2 was found to localize to the parasite's flagellum, a novel finding that suggests that glycolysis in the parasite may not be limited to glycosomes, organelles related to peroxisomes that heretofore were considered the cellular compartments of glycolysis. Immunofluorescence signal was not exclusive to the flagellum but was observed in the cytosol and glycosomes. To understand the mode of localization, the TbHK sequences were aligned with known and predicted flagellum proteins and a conserved flagellum localization peptide signal was identified in the C-termini of TbHK1 and TbHK2. The peptide signal (RAVLAK) was further explored as a flagellum targeting sequence using fusions to a reporter. These experiments, along with expression and localization of HA-tagged TbHK2 revealed that TbHK2 associates with the flagellum and basal body. This unusual localization suggested that TbHK2 may act as an environmental glucose sensor or perhaps in energy production for the active flagellum. The appendix is comprised of three chapters. The first appendix section is a published paper that focuses on AMP dependent protein kinase from T. brucei. I contributed microscopic expertise (and intellectual input) and am an author on this work. The second appendix section is a summary of research performed to characterize a library of lonidamine analogs against TbHK1 and trypanosomes. And lastly, the third appendix section is the developed assay for testing the viability of T. brucei cells and adaptation for high throughput screening.
Source: TigerPrints (Clemson University)
Author: Todd A. Lyda
Language: English
Subfield: Epidemiology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W181490087