Calcium dysregulation is the critical initiator in high dose chloroquine-induced programmed cell death of Plasmodium falciparum

Understanding the mechanism of action of novel antimalarial drugs is a key goal in eradicating malaria. Previously we have explored a possible alternative cell death mechanism that is elicited by micromolar amounts of the antimalarial chloroquine (CQ) against Plasmodium falciparum. The phenotypes assessed were calcium redistribution, mitochondrial depolarization and parasite digestive vacuole membrane permeabilization. Identifying the source or initiator of calcium release in the parasite will reveal a potential new therapeutic target. From timepoint studies we revealed the simultaneous loss of mitochondrial integrity along with intracellular calcium dysregulation, followed by downstream membrane rupture. Using a series of calcium efflux inhibitors and antioxidants, mitochondrial calcium (Ca2+mt) was implicated in initiating this cascade of parasite cell death phenotypes. Having established the mechanism with a high dose concentration of CQ, we expanded the search for similar phenotypic outcomes in a high-content screening of the Medicines for Malaria Venture (MMV) Pathogen Box. Three “hit” compounds were subjected to further characterization assays and the top performing compound (MMV085071) was identified. Ten analogues based on the top hit were assessed with dose-response assays and one lead analogue (MMV1545561) was selected as the top candidate based on its efficacy. Both parent and lead compound were investigated for their ability to redistribute Ca2+mt. While the effects were statistically significant, the lack of complete reversal of compound-induced calcium dysregulation and mitochondrial collapse by inhibitors suggested that Ca2+mt's role is more limited as compared to that of chloroquine. Furthermore, it is likely that the novel screening compounds are directed at multiple parasite targets and due to their highly efficacious nature, further follow-up work to ascertain their mechanism of action is warranted. Overall design: P. falciparum 3D7 strain parasites were synchronized with sorbitol and MACS sorting to enrich those in schizont stage. The enriched schizonts were seeded at 1.5-2% parasitemia at 1.25% haematocrit. Further synchronisation by sorbitol was performed 12 hours post seeding to ensure all parasites were synchronised at the ring stage. Drug treatments starts when parasites reach mid-to-late trophozoite stage. The infected RBCs were given fresh MCM and incubated for 4 hours, with 3uM MMV 085071, MMV 1545561, CQ or the same volume of DMSO resuspended in MCM medium.