Title: Functional characterisation of essential enzymes in Plasmodium
Funder: The Francis Crick Institute

Abstract: Malaria is a devastating parasitic disease requiring the identification of novel drug targets. Our group combines chemical biology with Plasmodium genetics to identify novel enzymatic targets, determine their biological functions, and validate them as viable therapeutic targets. There are two major areas of research in my lab: the first studies the biological function of malarial proteases belonging to families proven to be druggable; the second aims to identify the targets of anti-parasitic compounds through chemical proteomic approaches. 1) Functional characterization and therapeutic validation of Plasmodium proteases. Our group uses the DiCre conditional knockout (cKO) approach to identify essential Plasmodium proteases. This method allows for the first time to study the phenotype associated with the loss of an essential genes and therefore, to prove or disprove previously published hypotheses. Dipeptidyl aminopeptidases (DPAPs) are the Plasmodium homologues of mammalian cathepsin C. Two DPAPs (DPAP1 and DPAP3) are expressed during the parasite blood cycle. Previous studies suggested that DPAP1 is important for parasite growth and that DPAP3 regulates parasite egress from infected RBCs. However, our cKO studies have disproved these hypotheses and shown that both DPAPs are only important for RBC invasion, where they play complementary rather than redundant functions. We are currently identifying the natural substrates of these proteases using quantitative proteomic methods. Using DiCre, we have also shown that one of the 3 Plasmodium homologues to DPP4, a current drug targets for type 2 diabetes, is important for intracellular parasite growth. Finally, we have also proven that the activity of DDI, a proteasome shuttler containing a HIV retropepsin-like domain, is essential for RBC invasion. We are also collaborating with GSK to chemically validate Plasmodium DPAPs, DPP4, and DDI as antimalarial targets by repurposing some of their protease inhibitor libraries. 2) Identification of novel antimalarial targets through chemical proteomics. Our group has developed a novel HTS FACS/microscopy-based phenotypic assay to monitor the parasite 48h blood cycle with hourly temporal resolution. We used this assay to screen 300 reactive compounds and identified 12 hits that block parasite development at different stages. We are now building the chemical proteomic tools to identify their target. Plasmodium metabolic serine hydrolases are very poorly annotated, little is known about their biological function, and they have very little homology to know enzymes from other organisms. This suggests that they might perform parasite-specific metabolic functions and have therefore the potential of being very good drug targets. We are currently using activity-based probes (ABPs) to profile and chemically annotate all metabolic serine hydrolases in the malaria parasite. ABPs are small molecules that covalently modify the catalytic residue of an enzyme. A tag (fluorophore or biotin) within the probe allows for visualisation and/or pull-down of the modified enzymes. Finally, by competing a library of 400 known antiparasitic compounds against a broad-spectrum serine hydrolase ABP, we have identified 3 compounds that inhibit 3 different serine hydrolases, which we are currently trying to identify using chemical proteomics. The targets identified through these diverse chemical biology approaches will then be genetically validated using the DiCre cKO approach.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;5.1 PHARMACEUTICALS
Health categories: INFECTION