Parasites treated with 3-MB-PP1 and, to a lesser extent, MMV688853 exhibit abnormal cellular morphology.

Representative images of (A) WT or (B) TgCDPK1G128M parasites expressing tdTomato observed during intracellular proliferation assays, with tdTomato fluorescence (top) and differential interference contrast (DIC; bottom) images depicted. WT or TgCDPK1G128M parasites were cultured in the absence of drug (no drug), or the presence of MMV688853 (5 μM), 3-MB-PP1 (5 μM) or atovaquone (1 μM) for 20 h. Abnormal morphology was defined as vacuoles that contained misshapen parasites as depicted in the images of MMV688853 and 3MB-PP1. Scale bars are 2 μm. (TIF)</p

Identified compounds inhibit O₂ consumption in <i>T</i>. <i>gondii</i>.

(A-B) Traces depicting the changes in O2 consumption rate (OCR) over time of intact T. gondii parasites incubated with no drug (pink) or with (A) atovaquone (ATV; two fold serial dilution from highest concentration—10 μM, colored dark green—to lowest concentration—0.01 μM, colored light green) or (B) auranofin (AUR; two fold serial dilution from highest concentration—80 μM, colored dark green—to lowest concentration—0.08 μM, colored light green)). FCCP (1 μM) was injected into the well to uncouple electron transport from the proton gradient and thus elicit the maximal OCR. A range of concentrations of the test compounds were then injected and the inhibition of OCR measured over time. A final injection of an inhibitory concentration of atovaquone (ATVi; 5 μM) maximally inhibited mitochondrial OCR. Values represent the mean ± SD of two technical replicates from a single experiment and are representative of three independent experiments. Similar OCR inhibition traces were obtained for each test compound. (C-I) Dose-response curves depicting the percent of T. gondii OCR in the presence of increasing concentrations of (C) atovaquone, (D) buparvaquone, (E) auranofin, (F) trifloxystrobin, (G) azoxystrobin, (H) MMV024397 or (I) MMV688853. Values represent the percent OCR relative to the no-drug (100% OCR) and inhibitory atovaquone-treated (0% OCR) controls, and depict the mean ± SEM of three independent experiments, each conducted in duplicate; error bars that are not visible are smaller than the symbol. (J) Comparison of the EC50 values determined for T. gondii OCR (OCR EC50; this figure and Table 3) and WT T. gondii proliferation (proliferation EC50; S1 Fig and Table 1). Coloring of compounds is as in Figs 1 and 3 (atovaquone, black; buparvaquone, burgundy; auranofin, orange; trifloxystrobin, pink; azoxystrobin, light blue; MMV024397, red; and MMV688853, dark blue). (TIF)</p

ELQ-300-resistant parasites exhibit cross-resistance to MMV688853 in O₂ consumption rate activity assays.

(A-C) Dose-response curves depicting the OCR of intact WT (black) or atovaquone-resistant (ATVR, red) T. gondii parasites in the presence of increasing concentrations of (A) atovaquone, (B) ELQ-300 or (C) MMV688853. (D-F) Dose-response curves depicting the OCR of intact parental ELQ-300 sensitive (ELQS; black) or ELQ-300-resistant (ELQR, blue) T. gondii parasites in the presence of increasing concentrations of (D) atovaquone, (E) ELQ-300 or (F) MMV688853. Values represent the percent OCR relative to the no-drug (100% OCR) and inhibitory atovaquone-treated (D-F) or antimycin A-treated (A-D; 0% OCR) controls, and depict the mean ± SEM of three independent experiments, each conducted in at least duplicate; error bars that are not visible are smaller than the symbol. Inset bar graphs depict the EC50OCR ± SEM (nM) of three independent experiments. Where relevant, paired t-tests were performed and p-values are shown.</p

Quantification of the ETC target determination assay.

Quantification of the change in the (A) malate- or (B) glycerol 3-phosphate-dependent O2 consumption rate (OCR) of plasma membrane-permeabilized T. gondii parasites after injection of inhibitors (atovaquone, 1.25 μM; buparvaquone, 5 μM; trifloxystrobin, 2.5 μM; azoxystrobin, 80 μM; MMV024397, 20 μM; MMV688853, 20 μM; auranofin, 10 μM). (C) Quantification of the rescue of OCR by TMPD after inhibition by the above compounds. Data were normalized relative to the baseline OCR level pre-substrate injection (0% OCR) and the malate/G3P-elicited OCR level pre-drug injection (100%). Data represent the mean ± SEM of three independent experiments each conducted in at least duplicate. Note that the TMPD graph combines data from both the malate and G3P experiments. Error bars that are not visible are smaller than the symbol. ANOVA followed by Dunnett’s multiple comparisons test were performed and p-values are shown. Representative traces from which these data were quantified are depicted in Fig 5. (TIF)</p

Inhibitory activities of MMV Pathogen Box compounds against O₂ consumption rate in <i>T</i>. <i>gondii</i> and <i>P</i>. <i>falciparum</i>.

Determination of the O2 consumption rate (OCR) inhibitory properties of the identified compounds on (A) WT RH strain T. gondii parasites, and on (B) WT 3D7 strain P. falciparum parasites, using a Seahorse XFe96 flux analyzer. T. gondii experiments were conducted on intact parasites, and P. falciparum experiments measured malate-dependent OCR in digitonin-permeabilized parasites. Data are reported as average EC50 value against OCR (EC50OCR) (μM) ± SEM from three or more independent experiments. ND = not determined.</p

Assessing the activity of ETC inhibitors against atovaquone-resistant <i>T</i>. <i>gondii</i> parasites.

(A-G) Dose-response curves depicting the percent proliferation of WT (black) or atovaquone-resistant (ATVR, red) T. gondii parasites in the presence of increasing concentrations of (A) atovaquone, (B) buparvaquone, (C) auranofin, (D) trifloxystrobin, (E) azoxystrobin, (F) MMV024397, or (G) MMV688853. Values are expressed as a percent of the average fluorescence from a no-drug control at mid-log phase growth in the fluorescence proliferation assay, and represent the mean ± SEM of three (or four for (E)) independent experiments performed in triplicate; error bars that are not visible are smaller than the symbol. Inset bar graphs depict the EC50 ± SEM (nM) of three (or four for (E)) independent experiments. Paired t-tests were performed and p-values are shown.</p

Identification of selective inhibitors of the ETC in <i>P</i>. <i>falciparum</i>.

Dose-response curves depicting the proliferation of WT (black) or yeast dihydroorotate dehydrogenase (yDHODH)-expressing (red) P. falciparum parasites in the presence of increasing concentrations of (A) the known ETC inhibitor atovaquone, (B) chloroquine, a compound that does not inhibit the ETC, (C) buparvaquone, (D) auranofin, (E) trifloxystrobin, (F) azoxystrobin, (G) MMV024397, or (H) MMV688853 after 96 h of culture. Values are expressed as a percentage of the average proliferation of the drug-free control, and represent the mean ± SEM of three independent experiments performed in triplicate; error bars that are not visible are smaller than the symbol.</p

All tested compounds except auranofin inhibit O₂ consumption rate but not extracellular acidification rate.

(A) O2 consumption rate (OCR) and (B) extracellular acidification rate (ECAR) of T. gondii parasites treated with either no drug (white), atovaquone (gray; 10 μM), trifloxystrobin (pink; 10 μM), azoxystrobin (light blue; 80 μM), MMV024397 (red; 20 μM), buparvaquone (burgundy; 20 μM), auranofin (orange; 80 μM) or MMV688853 (dark blue; 20 μM), assessed using a Seahorse XFe96 flux analyzer. Bars represent the mean ± SEM of three independent experiments each conducted in duplicate. ANOVA followed by Dunnett’s multiple comparisons test were performed and p-values are shown. A graphical output of these data comparing OCR to ECAR is depicted in Fig 3A. (TIF)</p

Assessing the activity of ETC inhibitors against ELQ-300-resistant <i>T</i>. <i>gondii</i> parasites.

(A-I) Dose-response curves depicting the percent proliferation of ELQ-300-resistant (ELQR, blue) T. gondii parasites, or the corresponding ELQ-300-sensitive parental strain (ELQS, black), in the presence of increasing concentrations of (A) ELQ-300, (B) antimycin A, (C) atovaquone, (D) buparvaquone, (E) auranofin, (F) trifloxystrobin, (G) azoxystrobin, (H) MMV024397, or (I) MMV688853. Values are expressed as a percent of the average fluorescence from a no-drug control at mid-log phase growth in the fluorescence proliferation assay, and represent the mean ± SEM of three independent experiments performed in triplicate; error bars that are not visible are smaller than the symbol. Inset bar graphs depict the EC50 ± SEM (nM) of three independent experiments. Paired t-tests were performed and p-values are shown.</p

Screening the MMV ‘Pathogen Box’ for inhibitors of O₂ consumption in <i>T</i>. <i>gondii</i>.

The O2 consumption rate (OCR) of extracellular T. gondii parasites was measured in a 96-well plate using a Seahorse XFe96 extracellular flux analyzer. Compounds from the MMV ‘Pathogen Box’ were added to wells at a final concentration of 1 μM, and the change in OCR was monitored in real-time after each addition. Percent inhibition of OCR by each of the 400 compounds was calculated relative to complete inhibition observed after addition of the known OCR inhibitors atovaquone (1 μM) and antimycin A (10 μM), with each compound represented by a dot. A >30% inhibition cut off was applied (dotted line), with seven compounds inhibiting OCR by >30% at 1 μM (coloring of dots corresponds to coloring of labels of the chemical structures shown below). These hits included MMV689480/buparvaquone (burgundy), the endochin-like quinolone (ELQ) MMV671636 (green), MMV688754/trifloxystrobin (pink), MMV688978/auranofin (orange), MMV024397 (red), the aminopyrazole carboxamide MMV688853 (dark blue), and MMV021057/azoxystrobin (light blue). Data are from a single experiment, with the plate layouts and data points summarized in S1 Table.</p