Phenotypic characterization of early-acting compound activity against the complete Plasmodium berghei liver stage

Malaria is a deadly disease caused by Plasmodium parasites, with more than 200 million cases in 2015. Plasmodium parasites only cause malaria while replicating in blood, but must first undergo development in the liver, making this asymptomatic stage a key intervention point for chemoprophylaxis. Drug resistance is arising against current antimalarials, proving impetus to identify and develop compounds with new mechanisms of action. The MMV Malaria Box, 400 compounds with validated activity against P. falciparum asexual blood stages, was screened against the complete liver stage of P. berghei, a rodent model parasite, at 5μM (Medina Vera, et al. In preparation). Seven compounds that killed the parasite early in liver stage development were selected for validation and further characterization. The goal of the work was to see if these 7 compounds, which appear to provoke similar terminal phenotypes, could be differentiated from each other and from two known early-killing compounds (Atovaquone and Torin2) by examining stage specificity of compound activity, and the resulting terminal parasite phenotypes. To accomplish this, we first validated the screening data in the P. berghei- HepG2 infection model, assessing both parasite size and abundance after continuous compound exposure, 2-48 hours post-infection (hpi). EC50s were then determined with a parasite-encoded luciferase activity readout, and compound activity was compared during 24-48, 48-66, and 2-8 hpi treatment windows using 3x, 5x, 10x EC50, and multiple linked readouts. Our results with the 6 validated compounds, having unknown mechanism of action, suggest we can use P. berghei liver stage activity and phenotypic profiling to identify compounds with novel or desirable properties.