Chemical Genetics of P. falciparum Plasmepsin V, Plasmepsin X, and Cytochrome b

Abstract

© 2025 Wenyin SuMalaria is caused by the Plasmodium parasite and results in about 600,000 deaths worldwide annually. The emergence of resistance against almost all antimalarial drugs has now become an obstacle to eliminating the disease. To address the issue, there is an urgent need for the discovery and development of new antimalarials with novel mechanisms of action. In this thesis, complementary forward and reverse genetics, and chemo-proteomic techniques were applied to characterise the mechanism of action (MoA) and explore resistance factors of three antimalarial classes. Plasmepsin V (PMV) is an aspartyl protease that is essential for processing the N-terminal PEXEL motif of proteins licensing them for export to the host red blood cell during the asexual segment of malaria parasite’s life cycle. Small molecule peptidomimetics mimicking the PEXEL motif have been previously developed as potent inhibitors of PMV. The peptidomimetics have been shown to block protein export and kill the malaria parasite, although confirmation of their on-target activity was required. In Chapter 2, resistance selection and genome sequencing revealed a mutation in PMV. Reverse genetics was then used to reverse engineer parasites to confirm the mutation in PMV. Biased and unbiased chemo-proteomics were then applied to demonstrate on-target engagement of PMV in P. falciparum parasites, definitively showing that the peptidomimetics kill the parasite by targeting PMV. Plasmepsin X (PMX) is an aspartyl protease that is vital for the maturation of proteins that enable the malaria parasite to enter and exit from the host red blood cell. Small molecules with an imino pyrimidinone chemotype have been developed as inhibitors of PMX that potently block red blood cell invasion and egress preventing asexual stage parasite development. Resistance selection using imino pyrimidinone inhibitors was previously performed to understand the risk of resistance in the field. In Chapter 3, parasites were genetically reversed engineered to confirm and investigate the mutations and amplifications in PMX responsible for the resistance observed. To uncover new starting points for antimalarial development, a phenotypic screen of the Janssen Jumpstarter library against the asexual stage parasite uncovered the cyclopropyl carboxamide hit class. In Chapter 4, forward genetics was used to identify the MoA of the hit class. Genome sequencing of cyclopropyl carboxamide-resistant parasites revealed mutations in the Qo site of cytochrome b, which is an essential component of the mitochondrial electron transport chain. Cytochrome b was confirmed as the molecular target by evaluating cyclopropyl carboxamide analogs against cytochrome b resistant parasite lines, and in a mitochondrial functional assay supporting this antimalarial class targeting cytochrome b. The investigation of the mechanisms of action of the three antimalarial classes undertaken in this thesis will assist in better understanding the role of the target proteins in parasite survival and resistance to facilitate the future development of these antimalarial classes