Fig 1.
Screening the MMV ‘Pathogen Box’ for inhibitors of O2 consumption in T. gondii.
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.
Table 1.
Effects of the identified MMV Pathogen Box compounds on T. gondii proliferation.
Determination of the inhibitory properties of the identified compounds on the proliferation of (A) human foreskin fibroblast (HFF) cells or (B) T. gondii parasites, including wild type (WT) RH, WT ME49, atovaquone-resistant (ATVR) ME49, and ELQ-300-resistant (ELQR) RH and the corresponding ELQ sensitive (ELQS) parental strains. Data are reported as average EC50 (nM) ± SEM from three or more independent experiments. The selectivity index (SI) was calculated by dividing the EC50 against HFF cells by the EC50 against WT RH T. gondii parasites, with SI values >1 indicating increased selectivity towards the parasite. The fold change (FC) was calculated by dividing the EC50 against ATVR or ELQR parasites by the EC50 against WT ME49 or ELQS parasites respectively, with FC values >1 indicating increased resistance and FC values <1 indicating increased sensitivity to the tested compounds in the ATVR or ELQR strains. Paired t-tests were performed to compare the EC50 of the compounds in WT vs ATVR or ELQS vs ELQR parasites, and p-values are depicted as ns = not significant (p > 0.05), * p < 0.05, ** p < 0.01. ND = not determined.
Table 2.
Effects of the identified MMV Pathogen Box compounds on P. falciparum proliferation.
Determination of the inhibitory properties of the identified compounds on the proliferation of P. falciparum parasite strains, including WT 3D7, yeast dihydroorotate dehydrogenase (yDHODH)-expressing 3D7, and ATVR 3D7 and the equivalent ATV-sensitive parental WT 3D7 strain. As the yDHODH and ATVR strains were generated in different laboratories, proliferation of the WT 3D7 background strain of each was determined for comparisons. Data are reported as average EC50 (nM) ± SEM from three or more independent experiments. The fold change (FC) was calculated by dividing the EC50 against ATVR parasites by the EC50 against WT parasites, with FC values >1 indicating increased resistance to the tested compounds in the ATVR strain. Paired t-tests were performed to compare the EC50 of the compounds in WT vs ATVR parasites, and p-values are depicted as ns = not significant (p > 0.05), * p < 0.05, ** p < 0.01. ND = not determined.
Fig 2.
Identification of selective inhibitors of the ETC in P. falciparum.
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.
Table 3.
Inhibitory activities of MMV Pathogen Box compounds against O2 consumption rate in T. gondii and P. falciparum.
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.
Fig 3.
Identification of selective and off-target inhibitors of the ETC in T. gondii parasites.
(A) O2 consumption rate (OCR) versus extracellular acidification rate (ECAR) of T. gondii parasites treated with either no drug (black square), atovaquone (black triangle; 10 μM), azoxystrobin (light blue; 80 μM), MMV024397 (red; 20 μM), MMV688853 (dark blue; 20 μM), trifloxystrobin (pink; 10 μM), buparvaquone (burgundy; 20 μM) or auranofin (orange; 80 μM) assessed using a Seahorse XFe96 flux analyzer. Data represent the mean OCR and ECAR ± SEM of three independent experiments, and are derived from the top concentration of inhibitor tested in S3 Fig. Statistical analyses of these data are presented in S4 Fig. (B) Viability of extracellular T. gondii parasites treated with atovaquone (black triangles, 10 μM) or auranofin (orange circles, 1–100 μM) for 35–140 minutes. Viability was assessed by flow cytometry of propidium iodide-stained parasites and normalized to a DMSO-treated vehicle control, with the gating strategy outlined in S5 Fig. Data represent the mean ± SEM of three independent experiments; error bars that are not visible are smaller than the symbol.
Fig 4.
MMV688853 dually targets TgCDPK1 and the ETC in T. gondii parasites.
(A) Schematic depicting the small glycine gatekeeper residue of WT TgCDPK1 (white) which enables inhibition by 3-MB-PP1 and MMV688853. Mutation of this residue to a larger methionine residue (TgCDPK1G128M, red) blocks inhibitor access to the binding site and thereby confers resistance to these compounds. (B) Percent invasion of parasites expressing WT TgCDPK1 (white) or TgCDPK1G128M (red) into host cells in the absence of drug (DMSO vehicle control), or the presence of MMV688853 (5 μM), 3-MB-PP1 (5 μM) or atovaquone (1 μM), normalized relative to the no-drug control. At least 100 parasites were counted per experiment, with data representing the mean ± SEM of three independent experiments. ANOVA followed by Tukey’s multiple comparisons test was performed with relevant p-values shown. (C) Intracellular proliferation assays depicting the percent of vacuoles containing 1–8+ (gray tones) or abnormal (orange) parasites when parasites expressing WT TgCDPK1 (white) or TgCDPK1G128M (red) were cultured in the absence of drug (DMSO vehicle control), 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 (representative images in S6 Fig). At least 100 vacuoles were counted per condition, with data representing the mean ± SEM of three independent experiments. (D) Dose-response curves depicting the O2 consumption rate (OCR) of parasites expressing WT TgCDPK1 (black) or TgCDPK1G128M (red) incubated with increasing concentrations of MMV688853 as a percentage of a no-drug (DMSO vehicle) control. Data represent the mean ± SEM of three independent experiments. Inset bar graph depicts the EC50OCR ± SEM (nM) of three independent experiments. The p-value from a paired t-test is shown. (E) OCR of parasites expressing WT TgCDPK1 (white) or TgCDPK1G128M (red) incubated in the absence of drug (DMSO vehicle control), or in the presence of MMV688853 (5 μM), 3-MB-PP1 (5 μM) or atovaquone (1 μM), expressed as a percentage of the OCR prior to addition of compounds. Data represent the mean ± SEM of three independent experiments. ANOVA followed by Tukey’s multiple comparisons test was performed with relevant p-values shown. (F) Dose-response curves depicting the percentage proliferation of parasites expressing WT TgCDPK1 (black) or TgCDPK1G128M (red) in the presence of increasing concentrations of MMV688853 over 6 days. Values are expressed as a percent of the average fluorescence from the no-drug control at mid-log phase growth in the fluorescence proliferation assay, and represent the mean ± SEM of five independent experiments conducted in triplicate; error bars that are not visible are smaller than the symbol. Inset bar graph depicts the EC50 ± SEM (nM) of five independent experiments. The p-value from a paired t-test is shown.
Fig 5.
An assay to characterize the targets of the candidate ETC inhibitors identifies chemically diverse Complex III inhibitors.
(A) Schematic of the assay measuring the O2 consumption rate (OCR) of plasma membrane-permeabilized T. gondii parasites. Parasites were starved for 1 hour to deplete endogenous substrates then permeabilized with digitonin before the addition of the following substrates and inhibitors: Port A, the substrates malate (Mal) or glycerol 3-phosphate (G3P); Port B, the test compound; Port C, TMPD; Port D, sodium azide (NaN3). CoQ, coenzyme Q; III, Complex III; CytC, cytochrome c; IV, Complex IV; e-, electrons. (B-H) Traces depicting parasite OCR over time when supplying Mal (red squares) or G3P (black triangles) as a substrate. The candidate ETC inhibitors were (B) atovaquone (1.25 μM), (C) buparvaquone (5 μM), (D) auranofin (10 μM), (E) trifloxystrobin (2.5 μM), (F) azoxystrobin (80 μM), (G) MMV024397 (20 μM), or (H) MMV688853 (20 μM). Values represent the mean ± SD of three technical replicates and are representative of three independent experiments; error bars that are not visible are smaller than the symbol. Dotted lines represent the time points of each injection, and data points for each condition have been connected by lines to aid interpretation. Quantifications of the data in (B-H) are presented in S7 Fig. (I) T. gondii Complex III enzymatic activity was assessed in the presence of DMSO (no-drug), atovaquone (1.25 μM), buparvaquone (5 μM), trifloxystrobin (2.5 μM), azoxystrobin (80 μM), MMV024397 (20 μM) or MMV688853 (20 μM). Data 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.
Fig 6.
Most of the candidate ETC inhibitors target the ETC upstream of cytochrome c in P. falciparum parasites.
(A) Schematic of the assay measuring the O2 consumption rate (OCR) of permeabilized P. falciparum parasites supplied malate (Mal) as a substrate. The following addition of substrates and inhibitors were performed: Port A, the test compound; Port B, TMPD; Port C, sodium azide (NaN3). CoQ, coenzyme Q; III, Complex III; CytC, cytochrome c; IV, Complex IV; e-, electrons. (B-G) Traces depicting parasite OCR over time when supplying Mal as a substrate. The candidate ETC inhibitors tested (all at 10 μM) were (B) atovaquone, (C) auranofin, (D) trifloxystrobin, (E) azoxystrobin, (F) MMV024397, or (G) MMV688853. Values represent the mean ± SD of three technical replicates and are representative of three independent experiments; error bars that are not visible are smaller than the symbol. Dotted lines represent the time points of each injection, and data points for each condition have been connected by lines to aid interpretation.
Fig 7.
Assessing the activity of ETC inhibitors against atovaquone-resistant T. gondii 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.
Fig 8.
Assessing the activity of ETC inhibitors against atovaquone-resistant P. falciparum parasites.
(A-H) Dose-response curves depicting the percent proliferation of WT (black) or atovaquone-resistant (ATVR, red) P. falciparum parasites in the presence of increasing concentrations of (A) atovaquone, (B) chloroquine, (C) buparvaquone, (D) auranofin, (E) trifloxystrobin, (F) azoxystrobin, (G) MMV024397, or (H) MMV688853 after 96 h of culture, as measured using a SYBR Safe-based proliferation assay. Values are expressed as a percent of the average fluorescence from the no-drug control, 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 of three independent experiments. Paired t-tests were performed and p-values are shown.
Fig 9.
Assessing the activity of ETC inhibitors against ELQ-300-resistant T. gondii 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.
Table 4.
O2 consumption rate inhibitory activity of MMV688853 against drug-resistant T. gondii strains.
Determination of the O2 consumption rate (OCR) inhibitory properties of MMV688853 against T. gondii parasite strains resistant to atovaquone (ATVR ME49) or ELQ-300 (ELQR RH) and the corresponding ATV-sensitive (WT ME49) and ELQ-300-sensitive (ELQS) parental strains. Data are reported as average EC50OCR (μM) ± SEM from three independent experiments. EC50OCR values against atovaquone and ELQ-300 were determined as controls in each strain. FC, fold-change in EC50OCR values between the atovaquone-resistant strain and corresponding atovaquone-sensitive parental strain. Paired t-tests were performed to compare the EC50OCR of the compounds in WT vs ATVR parasites, and p-values are depicted as ns = not significant (p > 0.05), * p < 0.05.
Fig 10.
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.