Whole-cell parasite viability assays.

P. falciparum 3D7 parasites were grown in type O-positive red blood cells at 1% hematocrit cultured in RPMI (plus l-glutamine without NaHCO₃) under a 5% CO₂, 5% O₂, and 90% N₂ atmosphere at 37°C. The medium was supplemented with 0.5% AlbuMAX, 0.37 mM hypoxanthine, 27 mM NaHCO₃, 11 mM glucose, and 10 μg/ml gentamicin. Cultures were assayed in triplicate in a total volume of 100 μl, and the DMSO vehicle was used as a negative control. The assays were initiated at approximately 0.5% parasitemia (percent infected erythrocytes). Following 72 h of incubation, parasitemias were enumerated by flow cytometry of 30,000 erythrocytes stained with acridine orange (1.5 μg/ml in phosphate-buffered saline, 5 min, RT) with a FACSCanto II flow cytometer (BD Biosystems, Franklin Lakes, NJ).

Dose-response curves for compounds that elicited >50% growth inhibition at 10 μM were pursued in a 96-well plate format, with 50% effective concentrations (EC₅₀s) being determined from the results of incubation with a 2-fold dilution series of inhibitor starting at 40 μM. Percent inhibition was calculated by comparison to the growth of parasites grown with the DMSO controls from each plate. Chloroquine was included in each assay as a positive control starting at 1 μM. Averages of the duplicates were calculated and fit to the dose-response curves for the determination of IC₅₀s using GraphPad Prism (version 6.0) software (GraphPad Software, Inc., La Jolla, CA).

Additional whole-cell follow-on assays of inhibitors included tests against cultured bloodstream-form T. brucei and Leishmania amazonensis parasites, using previously described methods (11, 18). The findings from these assays lend insight into compound specificity, as both T. brucei and Leishmania have HKs with limited similarity to PfHK (<35%).