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.