Hapuarachchi SV , Cobbold SA , Shafik SH , Dennis AS , McConville MJ , Martin RE , Kirk K , Lehane AM .

	Fig 1. Representative traces illustrating the strategy employed to screen the Malaria Box for compounds that perturb the cytosolic pH (pHcyt) of the parasite.Malaria Box compounds (1 μM; from the May 2012 Malaria Box batch; identified here by their positions on the plates) were added successively to BCECF-loaded isolated 3D7 P. falciparum trophozoites (suspended at 37°C in pH 7.10 Experimental Saline Solution). A maximum of nine inactive compounds were tested on a single batch of parasites before the V-type H+-ATPase inhibitor concanamycin A (conA; 100 nM) was added as a positive control to ensure that a pH change was still detectable (A). A batch of parasites was also replaced with a new batch after a hit was detected (B) or a compound caused an optical effect (seen as an instantaneous perturbation of the fluorescence signal; C). In the latter case concanamycin A (100 nM) was added before changing to another batch of parasites. The purpose of adding the concanamycin A was to confirm (or otherwise) that any such optical effect did not obscure the pH-responsiveness of the fluorescence signal.
	Fig 2. Fluorescence traces showing the effects of DMSO (solvent control), MMV007839 and MMV000972 on the cytosolic pH (pHcyt) of 3D7 P. falciparum trophozoites under varying conditions.Each trace is representative of those obtained in at least three independent experiments. (A-C) The effects of DMSO (0.1% v/v; solvent control; A), MMV007839 (1 μM; B) and MMV000972 (1 μM; C) on the cytosolic pH of parasites suspended in (glucose-containing) Experimental Saline Solution (pH 7.10) at 37°C. (D-F) The effects of DMSO (0.25% v/v; solvent control; D), MMV007839 (2.5 μM; E), and MMV000972 (2.5 μM; F) on the cytosolic pH of parasites suspended (for 10 min prior to commencing recording, and throughout the initial recording period) in glucose-free Experimental Saline Solution (pH 7.10, 37°C), and the effects of the subsequent addition of glucose (22 mM) to the parasite suspension. (G-I) The effects of adding DMSO (0.25% v/v; solvent control; G), MMV007839 (2.5 μM; H), and MMV000972 (2.5 μM; I), followed by sodium L-lactate (10 mM), on the cytosolic pH of parasites suspended at 4°C in Experimental Saline Solution (pH 7.10).
	Fig 3. L-[14C]lactate influx into PfFNTGly107Ser mutant parasites and Dd2 parental parasites.(A) The uptake of L-[14C]lactate by isolated trophozoite-stage PfFNTGly107Ser mutant parasites (white bars) and Dd2 parental parasites (black bars) in the absence of compound (0.4% v/v DMSO; solvent control) and in the presence of the non-specific anion transport inhibitor NPPB (100 μM). The mean distribution ratio (i.e., [intracellular L-[14C]lactate]/[extracellular L-[14C]lactate]) and SEM from nine (parent) or seven (mutant) independent experiments for the control, and from three independent experiments for NPPB, are shown. The results of unpaired t-tests are shown; ***P < 0.001. (B-C) The effects of MMV007839 (B) and MMV000972 (C) on L-[14C]lactate uptake into isolated trophozoite-stage PfFNTGly107Ser mutant parasites (white symbols) and Dd2 parental parasites (black symbols). L-[14C]lactate uptake in the presence of each concentration of MMV compound is expressed as a percentage of that observed when there was no inhibition of transport. The data shown are the average ± SEM from n independent experiments performed on different days, where n was seven for the MMV007839 data with parental parasites, three for the MMV007839 data with PfFNTGly107Ser mutant parasites, three for the MMV000972 data with parental parasites, and four for the MMV000972 data with PfFNTGly107Ser mutant parasites (with the exception of the lowest concentration, for which there was one less experiment in all cases). In all cases (panels A-C), the experiments were performed at 4°C in pH 6.1 Experimental Saline Solution, and parasites were preincubated with the compound (at the concentration indicated) or DMSO (0.4% v/v; solvent control) for 1 min prior to the addition of L-[14C]lactate (1.3 μM). The concentrations of compound and DMSO after the addition of L-[14C]lactate were the same as those present during the preincubation. The uptake of L-[14C]lactate was measured over 20 s.
	Fig 4. The effects of MMV007839 and MMV000972 on PfFNT-mediated L-[14C]lactate uptake by Xenopus oocytes expressing native PfFNT or PfFNTGly107Ser.The PfFNT-mediated uptake of L-[14C]lactate by oocytes expressing native PfFNT (black symbols) or oocytes expressing PfFNTGly107Ser (white symbols) was measured in the presence of a range of different concentrations of MMV007839 (A) and MMV000972 (B). The component of transport attributable to PfFNT (or to PfFNTGly107Ser) was determined by subtracting the uptake of L-[14C]lactate into non-injected oocytes (averaged from the values obtained across the full range of inhibitor concentrations tested, noting that the uptake of L-[14C]lactate into non-injected oocytes was unaffected by either inhibitor; S4 Fig) from the uptake of L-[14C]lactate measured (at each inhibitor concentration) into oocytes expressing either native PfFNT or PfFNTGly107Ser. In all panels, the PfFNT-mediated uptake of L-[14C]lactate is expressed as a percentage of that determined for native PfFNT in the absence of MMV compound (0.4% v/v DMSO; solvent control). To enable a visual comparison between the native PfFNT and PfFNTGly107Ser datasets, only part of the concentration range used for the latter is shown in the main panels. The insets show the data obtained for PfFNTGly107Ser over the full concentration range tested. In all cases, the data are averaged from four independent experiments (using oocytes from different frogs) and are shown ± SEM. The uptake was measured at 27.5°C over 10 min at pH 6.4 using 2.7 μM L-[14C]lactate and 600 μM unlabelled L-lactate.
	Fig 5. The effect of MMV007839 on the metabolite profile of parasitised erythrocytes.(A) Relative amounts of intracellular metabolites in MMV007839-treated parasitised erythrocytes (infected with the 3D7 strain) and untreated parasitised erythrocytes. The data shown are the mean log2 ratios of the level of each metabolite in the MMV007839-treated samples relative to its level in the untreated samples (+ MMV007839/- MMV007839), determined 1 h, 3 h and 6 h after the addition of MMV007839, obtained from three independent experiments. The concentration of MMV007839 used (6 μM) corresponds to approximately 20× the IC50 value for growth inhibition of 3D7 parasites. (B) The log2 ratios of metabolite levels in MMV007839-treated samples relative to those in untreated samples for all metabolites of known identity for which a statistically significant difference in abundance was observed between MMV007839-treated and untreated infected erythrocytes (see S1 Data for statistical analysis). The data shown are the mean + SEM from three independent experiments. GL3P, sn-glycerol-3-phosphate; Pyr, pyruvate; Lac, lactate; R5P, ribose-5-phosphate; AcCoA, acetyl coenzyme A; Gln, glutamine; VD, Val-Asp; PE, Pro-Glu; PVQ, Pro-Val-Gln; PDAV, Pro-Asp-Ala-Val; TNVK, Thr-Asn-Val-Lys; Oro, orotate; O5P, orotidine-5-phosphate; DC, deoxycytidine; GGPP, geranylgeranyl-pyrophosphate. (C) Relative amounts of metabolites in the extracellular medium in suspensions containing MMV007839-treated and untreated parasitised erythrocytes. The data shown are the mean log2 ratios (+ MMV007839/- MMV007839) + SEM from three independent experiments. There was a statistically significant difference in the concentrations of 3-phosphoglycerate and phosphoenolpyruvate in the extracellular medium between MMV007839-treated and untreated cell suspensions (see S1 Data). DHAP, glycerone phosphate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; Cit, citrate; αKG, 2-ketoglutarate; Mal, malate; Pip, pipecolate.
	Fig 6. The effect of MMV007839 on the volume of parasites and parasitised erythrocytes.MMV007839 (1 μM) and DMSO (0.1% v/v; solvent control) were tested for their effects on the volume of (A) saponin-isolated 3D7 P. falciparum trophozoites suspended at 37°C in pH 7.10 Experimental Saline Solution and (B) intact erythrocytes infected with 3D7 trophozoites suspended at 37°C in a pH 7.40 bicarbonate-free RPMI medium (containing supplements as outlined in the Materials and Methods). The data are expressed as a percentage volume increase relative to the starting volume, which for isolated parasites (A) ranged from 35 fL to 46 fL in the individual experiments, and for intact infected erythrocytes (B) ranged from 73 fL to 79 fL. In both cases the data shown are the mean ± SEM from three independent experiments performed on different days. For each condition, the (pre-normalised) volume data for each time point greater than 0 was tested for statistical significance compared to the time zero data for that condition; **P < 0.01, ***P < 0.001.

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