Figure 1.

Results of the initial screen of the Pathogen Box for effects on [Na⁺]_cyt, pH_cyt and cell volume in isolated asexual blood-stage 3D7 parasites. (a) Representative traces showing the effects of KAE609 (50 nM, green trace), 0.1% v/v DMSO (solvent control, red trace), the Na⁺ ionophore gramicidin (5 µM, grey trace) and two different Pathogen Box compounds (a black trace for the ‘non-hit’ MMV676269 and a blue trace for the ‘hit’ MMV020081, each at 1 µM) on [Na⁺]_cyt in isolated SBFI-loaded parasites. For the initial screen each of the four hundred compounds comprising the Pathogen Box was tested for its effect on [Na⁺]_cyt a single time (i.e. n = 1). (b) Representative traces showing the effects of concanamycin A (100 nM, grey trace), DMSO (0.1% v/v, vehicle control, red trace), and three Pathogen Box compounds (a black trace for the non-hit MMV676401, an orange trace for the hit MMV102872 and a purple trace for the hit MMV676477, each at 1 µM) on pH_cyt in isolated BCECF-loaded parasites. For (a) and (b) parasites were suspended in physiological saline at 1–2 × 10⁷ cells/mL, and compounds were added 5–30 s prior to the start of the measurement. Fluorescence was measured using the Tecan fluorimeter in 96-well plate format. (c) The number of Pathogen Box compounds (each tested at 5 µM) that caused a ≥3% change in the volume of isolated trophozoites following a 20 min exposure (n = 2). Pathogen Box compounds that caused an increase in [Na⁺]_cyt are shown in blue and KAE609 (10 nM) is shown in green. Pathogen Box compounds that caused a decrease in pH_cyt are shown in orange. Parasites were suspended in physiological saline at a density of 4 × 10⁵ parasites/mL.