Unpacking boxes: identification of novel inhibitors of malaria parasite invasion

With resistance to current frontline antimalarial treatments rapidly emerging in malaria endemic regions, there is an urgent need to identify new antimalarial compounds with novel mechanisms of action. Currently, no clinically used antimalarials target erythrocyte invasion, the process by which the short-lived, extracellular merozoite form of Plasmodium falciparum parasites enters a host erythrocyte. Following invasion, the parasite grows and then divides to form a schizont, containing merozoites that exit the host cell and invade new erythrocytes. To identify novel egress and invasion inhibitors, we screened the Medicines for Malaria Venture COVID Box (160 compounds) and Global Health Priority Box (240 compounds). Parasites were treated with the compounds at 1 μM during the egress and invasion period and across both libraries, 20 invasion inhibitors and two partial egress inhibitors were identified. A comparison of the growth IC₅₀ and invasion IC₅₀ of each hit compound revealed that invasion inhibition is likely central to the parasiticidal mechanism of six of the compounds, as their IC₅₀s for growth and invasion were similar. Of these six inhibitors, two (MMV006931 and MMV024850) were found to directly block the invasion of mechanically purified merozoites. We sought to identify the protein targets of these compounds through resistance selection and subsequent whole genome sequencing. We were able to select parasites with robust resistance to MMV006931 but not MMV024850. Genome sequencing of clonal MMV006931-resistant parasites identified mutations in the P. falciparum sterol exporter PfNCR1, which helps eliminate excess cholesterol from the parasite's plasma membrane. By demonstrating that MMV006931 sensitises parasites to lysis by a sterol-dependent detergent, we confirmed that PfNCR1 is the likely target of MMV006931.