Physiology Directed Drug Discovery in Multidrug Resistant Plasmodium falciparum

Abstract

Resistance to front-line antimalarial therapies is a widespread issue, from early chloroquine failure to the more recent concerns of Artemisinin Combination Therapy (ACT) failure. Traditional methods of quantifying drug resistance focused on growth inhibitory, or IC50, concentrations of drug. In the clinical setting, however, parasites are exposed to much higher concentrations of drug that kill parasites within hours. Defined as cytocidal activity, these concentrations have only recently begun to be explored. Recent reports suggest that drug targets and molecular mechanisms of drug resistance differ at cytostatic versus cytocidal levels. To investigate these possible targets and mechanisms of resistance at cytocidal levels of drug, immunofluorescence assays were conducted to visualize PfAtg8 localization in chloroquine sensitive versus resistant parasites treated at cytocidal drug concentrations.

In addition to understanding chloroquine resistance, the recent emergence of a delayed clearance phenotype (DCP) and piperaquine resistance further warrant novel methods of probing molecular mechanisms of drug resistance. Mutations in the propeller domains of a Kelch domain-containing protein on chromosome 13 (K13) have been linked to DCP and traditional methods of quantifying cytostatic or cytocidal activities of drugs fail to distinguish K13-mutant parasites from wild-type parasites. In 2013, Witkowski et al. developed the ring-stage susceptibility assay (RSA) using dihydroartemisinin to correlate DCP with an in vitro measurement: increased parasite survival. This method was modified to investigate other endoperoxide drugs and of the seven compounds tested, OZ439 was only compound that circumvented the endoperoxide cross-resistance pattern.

A high-throughput screen identified several potent antimalarials, including phosphatidylinositol 3-kinase (PI3K) inhibitors. These PI3K inhibitors are not only extremely potent antimalarials, but they were also found to inhibit the formation of Atg8 puncta in parasites and were highly synergistic with ART derivatives. Based on recent studies suggesting the involvement of elements in the autophagy cascade in artemisinin resistance, immunofluorescence assays were done with K13 mutant and wild type parasites and results suggest a dysregulation of an autophagy-like process in the K13-mutant parasites. These results guided us towards conducting single-point Chou-Talalay combination analyses to investigate the efficacy of traditional ACTs in comparison with novel combinations involving an endoperoxide compound plus various PI3K inhibitors. Synergy was observed in OZ439-PI3K combinations in both DCP and wild-type strains.