Abstract
BackgroundKnowledge of the rate of action of compounds against cultured malaria parasites is required to determine the optimal time-points for drug mode of action studies, as well as to predict likely in vivo parasite clearance rates in order to select optimal hit compounds for further development. In this study, changes in parasite ATP levels and transgenic luciferase reporter activity were explored as means to detect drug-induced stress in cultured parasites.MethodsIn vitro cultures of Plasmodium falciparum 3D7 wild-type or firefly luciferase-expressing parasites were incubated with a panel of six anti-malarial compounds for 10 hours and parasite ATP levels or luciferase activity determined at two-hour intervals using luminescence-based reagents. For comparative purposes, parasite morphology changes were evaluated by light microscopy, as well as the extent to which parasites recover after 48 hours from a six-hour drug treatment using a parasite lactate dehydrogenase assay.ResultsChanges in parasite ATP levels displayed three phenotypes: mild or no change (chloroquine, DFMO); 2–4 fold increase (mefloquine, artemisinin); severe depletion (ritonavir, gramicidin). The respective phenotypes and the rate at which they manifested correlated closely with the extent to which parasites recovered from a six-hour drug treatment (with the exception of chloroquine) and the appearance and severity of morphological changes observed by light microscopy. Luciferase activity decreased profoundly in parasites treated with mefloquine, artemisinin and ritonavir (34-67% decrease in 2 hours), while chloroquine and DFMO produced only mild changes over 10 hours. Gramicidin yielded intermediate decreases in luciferase activity.ConclusionsATP levels and luciferase activity respond rapidly to incubation with anti-malarial drugs and provide quantitative read-outs to detect the appearance and magnitude of drug-induced stress in cultured parasites. The correlation between the observed changes and irreversible parasite toxicity is not yet sufficiently clear to predict clinical clearance rates, but may be useful for ranking compounds against each other and standard drugs vis-à-vis rate of action and for determining early time-points for drug mode of action studies.
Highlights
Knowledge of the rate of action of compounds against cultured malaria parasites is required to determine the optimal time-points for drug mode of action studies, as well as to predict likely in vivo parasite clearance rates in order to select optimal hit compounds for further development
Morphological evaluation and drug IC50 determination Plasmodium falciparum 3D7 cultures were maintained at 37°C in medium consisting of RPMI 1640 supplemented with 2mM L-glutamine, 25mM Hepes, 20mM glucose, 0.65 mM hypoxanthine, 60 μg/mL gentamycin, 2.5% (w/v) Albumax II and 3% (v/v) type O+ red blood cells in flasks suffused with a mixture of 5% CO2, 5% O2, 90% N2
In parasites exposed to chloroquine and difluoromethyl ornithine (DFMO) (Figures 1A and 1B, respectively), ATP levels matched those of untreated control parasites over the entire 10-hour incubation period (t-test P values >0.05 at all time-points)
Summary
Knowledge of the rate of action of compounds against cultured malaria parasites is required to determine the optimal time-points for drug mode of action studies, as well as to predict likely in vivo parasite clearance rates in order to select optimal hit compounds for further development. Large-scale screens of synthetic chemical libraries have been on specific biochemical or cell biological pathways, or by a more global approach, e.g. transcriptomic, proteomic and/or metabolomic profiling [3] These studies require knowledge of the rate of action of a compound and should ideally be performed at early time-points when the compound starts exerting its primary effect(s), as opposed to later time-points when the primary mode of action may conceivably be obscured by non-specific secondary responses in the parasite. A highly desirable property of anti-malarial compounds is that they should kill parasites rapidly, in order to reduce the required dosages in clinical use, minimize the likelihood of resistance development, and increase patient compliance This requires an accurate determination of the rate of action of promising compounds against malaria parasites, to enable researchers to rank compounds for further pre-clinical and clinical development. Making the distinction between parasites with normal vs aberrant drug-induced morphologies is challenging due to the heterogeneous morphology of individual parasites under routine culture conditions, the tendency of individual cells to display a spectrum of mild to severe morphological abnormalities, at early time-points, and the challenge of preparing uniform microscopy preparations on separate occasions
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