Cell death in the human malaria parasite: exploring autophagy as a drug target and early cellular events following drug perturbation

Abstract

The emergence and spread of resistance to antimalarial drugs has highlighted the demand to search for new drugs that target novel pathways in the human malaria parasite Plasmodium falciparum. This thesis explores the potential for targeting of the PfAtg8-PfAtg3 protein-protein interaction (PPI) that is required for lipidation of phosphatidylethanolamine (PE) within parasite autophagy pathway. Whilst P. falciparum lacks a complete cascade of the classical autophagy pathway, the homologues for key proteins such as PfAtg8 exist and have been shown to be involved in cellular processes such as vesicle trafficking and apicoplast biogenesis. Here, the antiplasmodial activity of a library of 131 compounds designed in silico to act as inhibitors of hLC3 (Atg8 homologue)-Atg3 interaction were evaluated. Two hits, SK1.47 and SK1.49, have been identified and shown to have moderate antiplasmodial activity (EC50 of 1-2 µM) against intraerythrocytic parasites, produce a rapid cytocidal activity against trophozoite stage parasites and have selectivity for the parasites over HepG2 cells. As a first proof of concept, both compounds inhibit the formation of PfAtg8-labelled vesicles, potential autophagosomes, induced by nutrient starvation. Computational modelling of SK1.47 and SK1.49 docking to PfAtg8 suggests that the naphthalene group binds to the W-pocket and the substituted phenyl binds to the L-pocket of the PfAtg3 interacting region of PfAtg8. This docking study also highlights aspects of the core structure of both molecules that should be further explored in terms of their antiplasmodial activity.

Ultrastructural changes and induction of biochemical markers of apoptotic cell death appear to suggest that SK1.47 and SK1.49 treated parasites do not undergo apoptotic cell death. This study was extended to exploit a bioluminescence assay of parasite viability following exposure to a range of benchmark antimalarial drugs, which allows samples to be prepared that match a defined and titrated kill effect applied using drugs of different chemical classes. These established the conditions for a comparative study of apoptotic markers of early cell death using these different chemical classes, and also highlighted the central role of mitochondrial membrane potential collapse for the majority of drugs that explored and Ca2+ redistribution from the digestive vacuole following treatment with 4-aminoquinolines.

This study also highlights the opportunity of autophagy related proteins (Atg) of P. falciparum parasite as a novel target for drug development. Although SK1.47 and SK1.49 compounds are not considered as lead compounds for drug development due to lack the required potency with unfavourable physicochemical properties (high LogP), they are available now as chemical probes to explore the contested role of autophagy in malaria parasite homeostasis and response to drugs.