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Malaria continues to be an important global health issue. Plasmodium falciparum, the most virulent human-infecting malarial parasite, causes over 200 million cases per year. New drugs and drug targets are desperately needed to combat emerging resistance to front- line therapies. Two thirds of P. falciparum genes have predicted functions but very few have been verified. Broad-spectrum activity-based probes (ABPs) profile the activity of enzyme families based on common catalytic mechanisms. Activity-based protein profiling (ABPP) can functionally annotate many enzymes at once, as well as report on their activity in the presence of inhibitors. In this thesis, we use ABPP to interrogate two large enzyme families in P. falciparum: serine hydrolases (SHs) and cysteine proteases (CPs). We describe a novel ABP, W-sCy5-VS, for the profiling of papain-like CPs (PLCPs), a family that has roles in haemoglobin degradation and red blood cell invasion in P. falciparum. SH activities were profiled using well-characterised fluorophosphonate (FPP) ABPs. Our first aim was to develop a medium-throughput, gel-based ABPP screen combining the two ABPs to profile both families simultaneously. The screen was performed in competition with the Malaria Box, a diverse set of anti-parasitic compounds. We identified five Malaria Box compounds that targeted SHs or PLCPs. The SH inhibitors were the most potent and we concentrated follow-up studies on this family. P. falciparum has forty-eight predicted metabolic SHs, forty-three of which share a common α/β- fold. These SHs may play important roles in lipid scavenging, signalling and metabolism in P. falciparum, but few has verified biological function. We used FPP probes to develop chemical proteomic techniques to profile this large, uncharacterised family. We used chemical proteomics to identify possible targets of the Malaria Box SH inhibitors. By extending the approach to different life stages, we achieved unprecedented depth coverage of SHs, verifying the catalytic activity for more than half of the family. Life-stage dependent activation of human SHs was also observed, representing an exciting new area of host-targeted drug discovery. Making use of new technologies for gene editing in P. falciparum, we designed a conditional mutation strategy to determine if the hydrolytic activity of four SHs were important for parasite growth and replication. Contrary to data from recent large-scale genetic screens, none of the targeted genes appeared to be essential. The life-stage-dependent activation of SHs will help identify the roles that these enzymes play during the life cycle. This work illustrates how integrating chemical and genetic approaches can both efficiently identify new drug targets and chemically profile multiple enzymes. These outcomes are vital in malaria research due to the lack of functional genome annotation and the pressing need for new antimalarial targets.

Malaria remains a major cause of morbidity and mortality around the world. The emergence and spread of parasites resistant to all frontline antimalarials means that the identification of novel targets is vital for the development of drugs with novel mechanisms of action. The protozoan parasites of the Plasmodium genus cause malaria through the continual infection of and egress from red blood cells (RBCs). Plasmodium spp. encode uncharacterised orthologues to approved protease drug targets, namely HIV-1 protease and DPP4, for the treatment of AIDS and diabetes, respectively. In this project, DiCre-mediated conditional truncation of four putative proteases was performed in the malaria parasite, P. falciparum. Truncation of two of four proteins resulted in growth defects in the asexual blood stages. One putative serine protease, named S9C, was found to be important, but not essential, for intraerythrocytic development. The delay in growth was paired with stunted early parasite morphology and abnormal structure of the parasitophorous vacuole, in which the parasite resides within the RBC. A putative aspartyl protease that is conserved in eukaryotes, DNA-damage inducible protein 1 (Ddi1), is essential in the asexual blood stages, and its loss results in an almost complete block of erythrocyte invasion. We found evidence that Ddi1 was implicated in cellular proteostasis, potentially interacting with proteins involved in protein trafficking, sorting and quality control. We also find a potentially conserved interaction between Ddi1 and a subset of ubiquitinated proteins. For both S9C and Ddi1, a conditional allelic replacement with wildtype and mutant alleles demonstrated the importance of the putative proteolytic activity. The importance of the Ddi1 catalytic activity makes it a novel druggable antimalarial target. Furthermore, studying these putative proteases for the first time has uncovered new aspects of parasite biology in P. falciparum.