Medical Biology - Theses
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ItemNo Preview AvailableDiscovery of antimalarials with novel mechanisms of actionBailey, Brodie ( 2022)Today, despite an annual investment of $3 billion from governments, NGOs and pharmaceutical companies, malaria remains one of the most devastating parasitic diseases in human history. Of great concern is the emergence of resistance to all currently available antimalarial treatments, particularly the front-line artemisinin combination therapies. Therefore, an urgent need has arisen towards the development of antimalarials with novel mechanism of action to combat the rise of resistance. To discover new antimalarial chemotypes a high-throughput screen of the Janssen Jumpstarter library against asexual stage P. falciparum was undertaken and uncovered the novel 2-(N-phenyl carboxamide) triazolopyrimidine scaffold. This thesis describes the optimisation and mechanistic characterisation of the 2-(N-phenyl carboxamide) triazolopyrimidine antimalarial class. In Chapter 2, the optimisation and phenotypic characterisation of the triazolopyrimidine series was conducted. In the optimisation process, the structure activity relationship was defined and delivered analogues with EC50 values below 100 nM against the asexual stage parasite. Phenotypic characterisation determined this class exhibited a slow to moderate rate of kill and arrested asexual stage development at the trophozoite stage. Equipotent activity against P. falciparum multi-drug resistant field strains and moderately reduced activity in P. knowlesi indicated a distinct mechanism of action to clinically relevant antimalarials and a conserved target, albeit with potential species differentiation. In Chapter 3, the mechanism of action of triazolopyrimidine series was explored using chemoproteomic and chemogenomic techniques. Chemical probes were synthesised for use in affinity pulldown and fluorescent imaging. However, these probes were unable to clearly identify the molecular target of the chemical series. Resistance selection was undertaken and whole genome sequencing of resistant clones identified amplifications and non-synonymous point mutations in a putative mitochondrial carrier protein (PF3D7_0407500) of unknown function. Metabolomic analysis of drug treated parasites indicated disruptions in pyrimidine metabolism and depletion of pantothenate metabolites. The unique metabolic signature reveals a potential multifaceted and indirect effect on the function of the electron transport chain and CoA pathways of the mitochondria. Finally, Chapter 4 describes research towards the validation of the putative mitochondrial carrier protein (PF3D7_0407500) as the molecular target of the triazolopyrimidine series. Genetic validation of the target is described in which we attempt to introduce the resistance conferring mutations into wild-type parasites. Another component of the validation focuses on the characterisation of the indirect effect of the triazolopyrimidine series on the function of the mitochondria. Pantothenate uptake experiments show that a frontrunner triazolopyrimidine analogue blocks the flux of this metabolite into the parasite. To definitively confirm the function of the carrier protein, recombinant expression systems were trialed unsuccessfully to enable biochemical substrate screening and structural studies. Together this research describes the exploration of a novel antimalarial class with a distinct mechanism of action. Future research will aim to genetically validate the carrier protein and further mechanistically characterise its involvement in mitochondria function and development of the malaria parasite. Using these compounds as tools, we have uncovered a novel carrier protein that has a unique function that could represent a novel druggable target for future antimalarial development.