Trypanosomatid parasites cause a wide range of so-called neglected diseases which affect over 27 million people every year. Current treatments are toxic and prone to resistance; therefore, it is imperative to identify novel protein targets and to develop more efficient treatments. Phosphofructokinase (PFK) is the third enzyme in glycolysis, and its reciprocal enzyme in gluconeogenesis is fructose-1,6-bisphosphatase (FBPase); in trypanosomatid parasites (Trypanosoma brucei [Tb], Trypanosoma cruzi [Tc] and Leishmania [Lm] species), both enzymes are recognised drug targets. This thesis describes biochemical and structural studies on these two allosteric enzymes that have been studied with two main purposes: 1) To understand their intrinsic behaviour. The allosteric mechanism of T. brucei PFK is described with the help of two novel crystal structures: TbPFK with the allosteric activator AMP, and mutant A288D located in the effector site. These studies have provided a better understanding of the effect of evolution on the allostery of PFK; and have introduced the first reproducible crystallisation of TbPFK via its A288D mutant. 2) To find novel inhibitors using in silico and high-throughput methods, and to investigate how the intrinsic behaviour relates to the mechanism of inhibition. Nanomolar selective inhibitors against TbPFK and TcPFK have been obtained and optimised to a novel family with low micromolar inhibitory activity against cultured parasites. Crystal structures with three of these inhibitors on TbPFK have helped us understand the structure-activity relationship. Moreover, novel crystal structures of TcPFK and LmFBPase, as well as reproducible crystallisation conditions for the latter enzyme and a mutant of TbPFK (A288D) will undoubtedly facilitate future drug discovery on these targets. Our long-term aim of finding novel drugs against sleeping sickness has been supported by the Wellcome Trust which has recently granted a Seeding Drug Discovery Award with the name “Optimisation of a trypanosome phosphofructokinase lead series to give candidates for treatment of the trypanosomatid based neglected disease Human African Trypanosomiasis”.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:700015 |
| Date | January 2014 |
| Creators | Vasquez Valdivieso, Montserrat Guadalupe |
| Contributors | Walkinshaw, Malcolm ; Gilmore, Linda |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17980 |
Page generated in 0.0019 seconds