MMV006833 resistant parasites show mutations in PfSTART

With resistance alleles to frontline artemisinin based antimalarial drugs now appearing in most malaria endemic regions of the world it is imperative that new antimalarial drugs be developed to replace artemisinins in the future. Small molecule inhibitors that can reduce the capacity of Plasmodium falciparum and other Plasmodium species to invade human red blood cells (RBCs) where they grow and reproduce, could complement current antimalarials which target intraerythrocytic parasite forms. We previously reported on a screen of the Medicines for Malaria Pathogen Box for compounds that inhibit RBC invasion and identified an aryl acetamide compound, MMV006833, that appeared to block the ability of newly invaded merozoites to efficiently differentiate into amoeboid intraerythrocytic ring-stage parasite forms. Here we show that thorough selection for parasite resistance to MMV006833 followed by whole genome sequencing, two independent mutations arose in a START domain phospholipid transferase protein (PF3D7_0104200, PfSTART). Introduction of the PfSTART mutations into wildtype parasites reproduced resistance to both MMV006833 and highly potent analogue compounds, confirming PfSTART was their mechanism of resistance. Binding of these analogues to recombinant PfSTART with nanomolar affinity was demonstrated through isothermal titration calorimetry and organic solvent profiling experiments were utilised for the first time in Plasmodium to demonstrate engagement with PfSTART in the parasite. Live imaging of newly invaded merozoite stage parasites showed the inhibitors prevented the expansion of the encasing parasitophorous vacuole membrane (PVM) formed during invasion indicating START may transfer phospholipids from the parasite to the nascent PVM to allow the parasites to expand into larger intracellular parasite forms. With transmission blocking activity, we show PfSTART targets multiple stages of the parasite's lifecycle and presents a novel drug target.