Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei subspecies, is one of the most neglected diseases: available treatments are old, toxic, and difficult to administer; they are not efficacious against all parasite species or disease stages and drug resistance is an increasing problem.
Protein kinases are well validated drug targets for a variety of human diseases with many inhibitors under development or in the clinic. The T. brucei kinome has been annotated and there is evidence of essentiality of some of the members of this family. This thesis aims at evaluating the essentiality of Glycogen Synthase Kinase 3 (TbGSK3 short; Tb927.10.13780) and chemically validating it as a potential drug target in T. brucei.
TbGSK3 recombinant protein was biochemically characterised and screened against a focussed kinase library using the KinaseGlo assay method. Further repurchase and synthesis of novel compounds yielded 10 validated chemical series against TbGSK3 short. In particular two series showed anti-proliferative activity against the parasite. GSK3 07 series was further investigated by the Drug Discovery Unit with a phenotypic approach for its off-target effects, and GSK3 09 series was further validated to act “on target”. The latter series showed a good correlation between biochemical potency and cellular efficacy. Using a combination of chemical and genetic approaches TbGSK3 short was demonstrated to be specifically targeted by a GSK3 09 tool molecule in T. brucei lysates. Furthermore, the in vitro efficacy in trypanosomes could be reverted by target over-expression. Further validation of its activity “on target” was given by its ability to modulate the cell toxicity caused by TbGSK3 short over-expression.
The genetic validation of TbGSK3 short by generation of conditional null mutants was not possible due to the tight regulation of the protein levels and the cell toxicity associated with protein over-expression.
The validated TbGSK3 short chemical tool could be used to elucidate the functions of TbGSK3 short in T. brucei, identify its substrates and increase the chance to solve the crystal structure of this enzyme for the design of novel inhibitors with different mechanism of inhibition and/or increased selectivity.
|Date of Award||2014|
|Supervisor||Alan Fairlamb (Supervisor) & Julie A. Frearson (Supervisor)|