CRISPR-Cas9 based validation of potential antimalarial targets

With the ability of Plasmodium falciparum, the pathogen responsible for the most mortality from malaria, to evade the front-line drug treatment artemisinin and ongoing resistance to other antimalarial drugs. There is an urgent need for continued development of new therapeutics to treat malaria and to combat the resistant parasites. In this research project, the aim was to validate UMP-CMP kinase (UCK), pivotal in the exclusive pyrimidine biosynthesis for DNA and RNA in malaria parasites, as an essential enzyme and druggable target. Herein, the essentiality of UMP-CMP kinase (UCK) gene for parasite survival and reproduction during the blood stages was discovered by using CRISPR/Cas9-based gene replacement combined with dimerisable Cre-mediated recombination. Induced deletion of the sequence encoding a catalytic domain of UCK via the Di-Cre system resulted in defective asexual growth and parasite development failure. The phenotypic analysis showed truncated parasites were arrested in the trophozoite stage and failed to progress to schizonts. The presence of an antisense circular RNA of UCK (antisense circRNA-UCK) and its cellular functions were also investigated. We hypothesised that an antisense circRNA-UCK might regulate the parental gene (UCK) expression given the coincident expression of the UCK gene initiating during early nucleic acid synthesis stages and antisense circRNA-UCK expression near the end of this stage. Experimental detection of antisense circRNA-UCK was confirmed and the antisense circRNA-UCK knockout parasite generated using CRISPR-Cas9 recombination. An antisense circRNA-UCK knockout parasite line E4 showed a significant increase in the growth rate when compared to the control parasite with two other lines (B11 and D10) slightly increased in their growth rate. This finding suggests that antisense circRNA-UCK might play a role in blood stage parasite replication although further experimentation is needed to verify these observations. In addition, recombinant PfUCK was kinetically similar in substrate preference of CMP and UMP and was more active in reducing conditions as compared to human UCK. Screens of novel compounds from in silico prediction identified the first selective PfUCK inhibitors, active at single micromolar concentrations for future lead identification and optimisation. Altogether, these data suggest that PfUCK is a promising target for antimalarial drug development.