The aim of this thesis was to expand upon the knowledge of the mechanism of action and resistance of antifolate drugs in African trypanosomes with a specific focus on transport mechanisms. A media deficient in folate and thymidine was established which enabled the assessment of their modulation on antifolate in vitro potencies and also screen a small set of antifolate compounds. The phenomenon of ‘thyminelessdeath’ was found to account for methotrexate toxicity, as well as the primary mechanism of raltitrexed toxicity. This was confirmed by cell cycle studies demonstrating cell cycle arrest in S phase which could be rescued with thymidine. Transport kinetics of folate and methotrexate were characterised and found to be competitive substrates for uptake in T. brucei. Transport of these substrates was inhibited by classical antifolates, but not by non-classical antifolates. Genome-wide RNAi library screens with methotrexate and raltitrexed identified the putative folate transporter genes to be involved in drug resistance. RNAi knockdown of the folate transport genes resulted in a substantial reduction in folate transport was seen. RNAi knockdown also led to cross-resistance to classical antifolates, whereas these parasites became hypersensitive to non-classical antifolates. Methotrexate-resistant trypanosomes were generated in which transport of methotrexate and folate was substantially reduced. Amino acid changes were evident in the putative folate transporter genes but no change in transcription or copy number was evident. Cross resistance to classical antifolates was demonstrated in these resistant parasites and cells become hypersensitivity to non-classical antifolates (a similar phenotype to folate transporter RNAi knockdown). Proteomic studies were performed in drugresistant trypanosomes; however, no conclusive findings were evident due to limitations of these experiments. In conclusion, these studies demonstrate good evidence of both transport-mediated drug action and drug resistance.
|Date of Award||2017|
|Supervisor||Alan Fairlamb (Supervisor) & David Horn (Supervisor)|