Validating protein kinases of Trypanosoma brucei as drug targets

Trypanosoma brucei spp. are protozoan parasites that cause Human African Sleeping Sickness and Nagana, a disease of cattle. The diseases have a very high mortality rate if untreated and the current drug treatments are inadequate due to toxicity and resistance. In order to develop new treatments, potential drug targets can be investigated in a candidate approach by genetic validation. In trypanosomes this can be performed by using RNA interference (RNAi) technology, which is well developed for this organism. One category of potential targets are protein kinases; members of this enzyme family have been shown to be suitable targets for relatively specific and selective, therapeutic inhibition- especially in the field of human oncology. T. brucei possesses 183 eukaryotic protein kinases, atypical protein kinase and pseudokinases that may be suitable for chemical inhibition. This study intended to genetically validated these targets and pursue interesting leads as potential drug targets. In order to perform RNAi studies on large numbers of candidate genes an existing stem-loop RNAi system was adapted to contain Gateway recombination sites. This created a highly efficient system, allowing the rapid generation of RNAi plasmids to target the entire complement of the predicted kinome of this organism. This collection of plasmids was used to generate a library of RNAi cell lines in bloodstream form parasites (BSF) which were screened for growth defects using an alamar blue cell viability assay. This identified 53 genes which were essential or important for proliferation of the BSF parasites. During this screen, a cell line was identified that contained an RNAi construct targeting two NEK kinases (NEK12.1: Tb927.8.7110, NEK12.2: Tb927.4.5310) which displayed a severe growth defect. This was replicated in a mouse model of infection. NEK 12.1 possesses an alanine gatekeeper residue and molecular modelling and virtual screening predicted this would allow the accommodation of bulky protein kinase inhibitors and therefore was investigated in more detail. Expression and purification of NEK12.1 in kinase active and dead forms allowed activity assays to be performed; these could be inhibited with the bulky inhibitor 3-MB-PP1, which also possessed activity against BSF parasites. Experiments to confirm the individual essentiality of NEK 12.1 (and its activity) remain to be performed, therefore NEK 12.1 has been partially genetically and chemically validated in this study. A unique “Orphan” kinase, containing a putative zinc-finger domain and a potential homologue of PDK1 were also investigated after the pTL RNAi screen. In vivo RNAi studies (using a mouse model) correlated well with the in culture RNAi data, demonstrating that these targets are essential in mammalian infections. During the pTL RNAi screen it was noted that the knockdown of one protein kinase, (DFK) led to a change in the morphology of the cells, yet no reduction in the alamar blue ratio was observed. Further investigation showed that after 72 h of RNAi induction 17% of cells expressed EP procyclin. This was associated with detectable changes in the transcriptome (ascertained by RNA -Seq), that were consistent with a BSF-PCF differentiation event. DFK was predicted to contain multiple transmembrane domains and features suggestive of a receptor kinase. Epitope tagging of DFK followed by cell fractionation and immunofluorescence microscopy demonstrated that the protein localised to the cell membrane. Transmission immuno-electron microscopy confirmed the cell membrane localisation and suggested that the protein kinase domain of DFK was intracellular. This study reveals several protein kinases as new drug targets, in turn identifying one as a key regulator of BSF-PCF differentiation. Due to the tractability of T. brucei it also provides a collection of plasmids and cell lines that should further the investigation of other key biological processes in this important pathogen