ARF regulating proteins as novel drug targets in Trypanosoma brucei

Abstract

Human African Trypanosomiasis (HAT) and Animal African Trypanosomiasis (AAT) are neglected tropical diseases that pose a huge socioeconomic and health burden in poorer countries in sub-Saharan Africa. The causative agents of these diseases are the Trypanosoma brucei (T. brucei) spp., kinetoplastid parasites that is transmitted by tsetse flies (Diptera Glossina). Drugs against HAT and nagana are toxic, species and in nagana, host specific. With reports of emerging resistance to current drugs, it is essential to identify novel drug targets against T. brucei. This thesis focuses on identifying and characterising regulating proteins of ADP-ribosylation factors (ARFs) in T. brucei. ARFs are small GTPase binding proteins that have been shown to be essential in bloodstream form T. brucei. However the T. brucei ARFs share a high level of sequence identity with human ARFs, thus making them unsuitable as drug targets. Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) regulate the activity of ARFs and have been shown to have highly diverse protein sequences. A total of 3 putative ARF GEFs and 4 putative ARF GAPs were identified in bloodstream form T. brucei by bioinformatics RNA interference (RNAi) cell lines were successfully generated for two GEFs and two GAPs in Lister 427 bloodstream form T. brucei. Tetracycline-induced knockdown was used to demonstrate that TbGEF3 is essential for viability of bloodstream form T. brucei. A decrease in cell growth was observed from 24 hours post RNAi, correlating with a decrease in cell cycle progression and an increase in cell death. The ‘BigEye’ phenotype could also be seen from 24 hours post RNAi, and cells were identified to have an endocytosis defect. These results combined with low level of sequence similarities at the essential ARF binding regions of TbGEF3 demonstrated that TbGEF3 may be a potential drug target. The TbGEF3 RNAi cells were used to assess the sensitivity of suramin, a drug taken up via endocytosis, in the presence of a partial endocytosis defect. Cells incubated in tetracycline for 24 hours prior to drug treatment had a reduced sensitivity to suramin compared to cells grown in the absence of tetracycline. The Pathogen Box set of compounds was screened for molecules with activity against T. brucei parasites. Identified hit compounds were used in preliminary studies to initiate development of an assay to distinguish compounds that are taken up via endocytosis. In summary, the putative ARF-regulating protein TbGEF3 has been shown to be essential for viability in T. brucei and has promise as a potential drug target. Further work is required in order to validate the use of the TbGEF3 RNAi line as a drug screening tool.