Structural and biochemical studies of trypanosomatid drug target proteins

The work presented in this dissertation focuses on the potential drug target proteins from the parasites in the order of Trypanosomatidae. The diseases of main interest includes African sleeping sickness, American trypanosomiasis and various types of Leishmaniasis. The proteins studied are Trypanosoma brucei phosphoglycerate kinase (TbPGK), Leishmania mexicana glycerol-3-phosphate dehydrogenase ( LmGPDH) and T. brucei PEX14 (TbPEX14). Because of these parasites' heavy dependence on glycolysis for ATP production, the glycolytic enzymes such as TbPGK and LmGPDH are considered good drug targets. The structure of TbPGK in complex with an adenosine analogue revealed an unexpected binding mode and alternative conformations of the inhibitor, which were reproduced by a docking program. To solve the unambiguity of the inhibitor bound to LmGPDH, the anomalous signals of the halogen atoms from data collected at 1.77 Å was used to reveal the multiple binding modes of the inhibitor. The anomalous signal also helped to identify inhibitor molecules bound to a secondary binding site created by crystal packing. The ternary complex structure of LmGPDH with DHAP and NAD+, determined to 1.9 Å resolution, showed conformational changes including a domain motion, rearrangements of sidechains and ordering of a loop when compared to the apo and binary complex structures. DHAP and NAD+ appeared to form a covalent bond, producing an adduct in the active site of LmGPDH. From the results of site-directed mutagenesis, molecular modeling and comparison with related dehydrogenases, a catalytic mechanism of LmGPDH is proposed. TbPEX14, a peroxin involved in the import process of proteins into the glycosome, is also considered a promising drug target because of its essential role in the compartmentation of glycolytic enzymes and biogenesis of glycosome. The study about the interaction of Tb PEX14 with TbPEX5 using fluorescence assay showed the critical residues of TbPEX14 for the interaction and determined the affinities of various WXXXF/Y motif peptides from TbPEX5 with TbPEX14. The assay developed provides a basis for the search for inhibitors of PEX 14-PEX5 interaction, which is vital for the proper function of the glycosomes.