Kinase-dependent control of DNA replication and repair as a drug target in Trypanosoma brucei

The life of any living organism is dictated by the information contained in its genome, the genetic material that is contained within all cells and viruses. The propagation of an organism requires that the genome is copied, thereby generating offspring. This copying process is referred to as replication, and is a reaction that is carried out by specific proteins at a specific time during growth. The genome must also be protected from damage, which comes in many forms that can compromise the chemical composition of the genetic material. Protection is frequently due to reactions that repair damage that has already been inflicted, and these reactions are carried out, like replication, at specific times during growth. In fact, the two reactions can influence each other. The co-ordination of each reaction with growth is determined by a widely characterised set of enzymes, called protein kinases, which modify components of the cellular machineries that drive replication and repair. Trypanosomes are parasites that blight the health and economy of huge parts of the world and appear to have unconventional aspects of genome replication and repair. We wish to ask if we can identify the kinase enzymes that control these reactions, and thus understand if and how these reactions might be different from humans and other animals that are infected by the parasites. If we can identify these parasite protein kinases, we intend to try and find chemicals that will stop their activity and might then stop the growth of the parasites. If this is successful, we will have uncovered new strategies to treat the diseases caused by the parasites. These strategies will be robust because they target one of the most important aspects of the biology of any organism, and may be applicable to other parasites and infective organisms.

DNA replication and repair are central processes in the transmission of all genomes. The activity of each process is regulated to occur at specific cell cycle stages, allowing the cell to monitor their success. In kinetoplastid organisms, including Trypanosoma species, no work to date has analysed the protein kinase-dependent checkpoints that link replication and repair to cell cycle progression. Unconventional aspects of cell division, the cell cycle, replication and repair have been detailed in kinetoplastid parasites, potentially making the protein kinases (PKs) that co-ordinate these central cell reactions viable drug targets. Identifying these PKs is a key step in understanding and validating this. To do this comprehensively, we propose to use a recently developed RNAi library that targets the complete repertoire of 183 PKs in T. brucei. PKs involved in DNA replication and repair will be identified by comparing, before and after RNAi induction, nucleotide incorporation rates, subcellular localisation of components of the replication machinery and the level of chromatin-association of replication components. PKs that act in repair will be identified by using cell imaging to evaluate when RNAi alters a conserved response to induced DNA breaks, the formation of subnuclear foci, and using next generation sequencing to identify those PKs whose RNAi targeting results in cell killing specifically in the presence of DNA damage. This work will reveal the network of PKs that regulate genome transmission in these diverged eukaryotes. To complement the above RNAi approach, and to move rapidly to the identification of compounds that target PKs that act in replication and repair, we will employ high-content cell imaging and assays developed above to screen available libraries of chemicals that have been designed to target PKs. Any compounds identified will be tested thoroughly for their mode and strength of anti-parasite activity and for the PKs that they target.

The immediate beneficiaries (stakeholders) of this work will be academic researchers in the fields of parasitology, DNA replication and repair, and in cell cycle control. This will result from the fundamental findings that emerge from the screens that will be performed to identify protein kinases (PKs) that control DNA replication and repair in the parasite T. brucei, which will have relevance for such processes in other parasites, microbes and in all organisms. We anticipate, furthermore, that the novel approach we propose, linking medium-throughput genomic and compound screens with high content imaging, will be taken up by other researchers in the field, and that we will provide tools and expertise.

Integral to this approach is screens for compounds that inhibit T. brucei PKs, which may themselves be developed as anti-parasite therapies or provide the foundation for such therapies. This aspect of the project will have a wider impact, whose output will be realised at the end, and we will then consult on how this might be taken forward, either through the enrolment of a commercial pharmaceutical company, a not-for-profit organisation (e.g. the Drugs for Neglected Diseases Initiative) or a charity (e.g. the Wellcome Trust) as stakeholders in this development. This process could, then, contribute to local, national and international economies, as well as the health and wellbeing of the countries affected by these parasites.

The work on this project falls within the broad areas of genetics and microbial biology, which have a wide impact on the health of the population of the UK and beyond, and are frequently the subject of media discussion (e.g. through television programmes, such as Horizon or 'Monsters Inside Me', and in public science exhibits, such as at the Glasgow Science Centre). We will contribute to this discussion, throughout the course of the project, through public lectures and exhibits, school outreach programs and articles in local and national media.