Biochemical and molecular characterisation of purine transporters of Trypanosoma brucei brucei

Abstract

This thesis describes the elucidation and comparison of substrate recognition relationships for the H2 nucleobase transporter of T. b. brucei and the main nucleobase transporter of human erythrocytes. Using standard transport kinetics, application of the Cheng-Prusoff equation and a derivation of the Nernst equation, it was possible to determine the Gibbs free energy (DG°) for the interactions of purine analogues with each transporter, which allowed predictions about the nature of the interactions that are essential for uptake. A range of unusual tricyclic and "fleximer" purine analogues was also assayed for ability to interact with the various purine transporters in T. b. burcei and human erythrocytes. This provided further insights into the extent that the substrate-binding pocket can accommodate unusual and large substrates. Some of the purine analogues used for the substrate-recognition study also displayed limited trypanocidal activity in vitro. More importantly, these results provide a foundation for the design and development of purine nucleobase analogues with anti-trypanosomal action that are efficiently and selectively accumulated by the parasite. One of the main aims of the project was to clone and characterise nucleobase transporters from T. b. brucei. The initial functional complementation strategy in nucleobase-transport deficient trypanosomes proved unsuccessful due to the presence of an additional, previously uncharacterised purine transporter in the trypanosome selection background and other technical obstacles. Homology searching of the T. b. brucei genome database led to the identification of a sequence with substantial similarity to the Adenosine Transporter 1 (TbAT1) gene previously shown to be responsible for the P2 nucleoside transport activity. The AT-like sequence was cloned, functionally expressed in heterologous systems (Saccharomyces cerevisiae and Xenopus oocytes), and characterised as the high-affinity Nucleobase Transporter 1 (TbNBT1). This also marked the first time a nucleobase-specific transporter had been cloned and characterised from any protozoan