Investigation of the export pathway in Plasmodium parasites utilising small molecule inhibitors of plasmepsin V

The human malaria parasite Plasmodium falciparum exports several hundred proteins into the host cell erythrocyte that are involved in cellular remodelling and severe virulence. The majority of proteins exported to the erythrocyte possess a conserved N-terminal export motif termed the Plasmodium export element (PEXEL). In order for proteins to be exported, the PEXEL motif (RxLxE/Q/D) must be processed by an ER-resident aspartic protease called plasmepsin V. Plasmepsin V is conserved across all Plasmodium species, including the most virulent human parasites P. falciparum and P. vivax, and is essential for blood-stage parasite survival. Therefore, plasmepsin V is considered a prime target for the development of new antimalarial therapies. Transition-state peptidomimetics of the natural PEXEL substrate are the most potent documented inhibitors of plasmepsin V to date. One such inhibitor, WEHI-916, showed that plasmepsin V plays a crucial role in protein export and that this process is essential for parasite survival, confirming that plasmepsin V is an important antimalarial target in the asexual blood stages. WEHI-916 has high affinity for plasmepsin V (IC50 20 nM), but only has moderate potency in blocking P. falciparum growth (EC50 2.5 μM). The large disconnect between biochemical and parasite growth inhibition can be attributed to the mimetic’s peptide-like character, which are known to possess poor membrane permeability and are susceptible to proteolytic degradation. Here, two strategies are described to overcome the liabilities associated with peptide-like molecules. These strategies were applied to transition-state mimetics that are potent inhibitors of plasmepsin V, with the aim of improving their membrane permeability and proteolytic stability, in order to enhance their activity against Plasmodium parasites. The first strategy utilised to improve these properties involved N-methylation of the backbone amide bonds. The second approach that was undertaken explored the structure-activity relationship within the S3 pocket of plasmepsin V to find a suitable isostere to replace the highly polar PEXEL P3 arginine, with the aim to improve membrane permeability. To further explore the biological role of plasmepsin V across multiple stages of the Plasmodium lifecycle, preliminary studies were also preformed towards the generation of a novel fluorescent probe that would fluoresce only once irreversibly bound to plasmepsin V. The synthesis of these analogues and the outcome of these strategies are discussed