4-Aminoquinolines block heme iron reactivity and interfere with artemisinin action
- Division of Infectious Diseases, Washington University School of Medicine, United States
- Department of Molecular Microbiology, Washington University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Chloroquine (CQ) and dihydroartemisinin (DHA) are superantagonistic in CQ-resistant parasites.
Shown are trophozoite-stage isobolograms for (a–e) CQ-DHA, (f–k) PPQ-DHA, and (l–q) MFQ DHA. The dotted line on each graph represents perfect additivity. 3D7, Dd2, Dd2 PfCRT3D7, Dd2 PfCRTDd2, Dd2 K13R539T, and Dd2 PfCRTM343L parasites are indicated in orange, gray, pink, blue, purple, and teal, respectively. Data from three independent replicates are indicated by different shading.
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Figure 1—source data 1
Isobologram analysis.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig1-data1-v1.zip
Figure 1—figure supplement 1
Chloroquine (CQ), dihydroartemisinin (DHA), piperaquine (PPQ), and mefloquine (MFQ) sensitivity of different parasites.
Shown are mean (a) CQ, (b) DHA, (c) PPQ, and (d) MFQ trophozoite-stage IC50 values ± SEM obtained as part of at least three independent isobologram replicates (see Figure 1). For (b) DHA, IC50 values from CQ-DHA and PPQ-DHA isobolograms are shown. Statistical significance between Dd2 versus all other parasites was determined using a one-way ANOVA with a Dunnett’s test for multiple comparisons. ****p<0.0001; **p<0.01; *p<0.05; ns = not significant.
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Figure 1—figure supplement 1—source data 1
Chloroquine (CQ), dihydroartemisinin (DHA), piperaquine (PPQ), and mefloquine (MFQ) IC50 values.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig1-figsupp1-data1-v1.zip
Figure 2 with 1 supplement
Chloroquine (CQ) co-treatment confers artemisinin resistance in K13WT parasites.
(a) Dihydroartemisinin (DHA) dose-response assays were performed on 0–3 hr post invasion (hpi) ring-stage parasites. Twofold serial dilutions of DHA were prepared in 96-well plates (DHA concentration represented by different shades of purple) starting at 1.4 μM so that both IC50 values and ring-stage survival assay (RSA) survival values at 700 nM DHA could be determined. To evaluate quinoline-DHA interactions in early ring stages, DHA titrations were prepared with fixed concentrations of 10 μM CQ or 200 nM piperaquine (PPQ). (b–d) Mean RSA survival values ± SEM (top panel) and mean DHA IC50 values ± SEM (bottom panel) are shown for (b) Dd2 PfCRT3D7, (c) Dd2, and (d) Dd2 K13R539T parasites. For the PPQ-resistant parasites (e) Dd2 PfCRTM343L and (f) MRA-1284, additional fixed PPQ concentrations of 500 nM and 10 μM were also tested. At least three independent replicates were performed for each experiment. For b bottom panel, a Student’s t-test was used to assess statistical differences between DHA alone versus DHA + PPQ. For all other graphs, statistical differences between DHA alone (-) and DHA + quinolines was assessed using a one-way ANOVA with a Dunnett’s test for multiple comparisons. ****p<0.0001; ***p<0.001; *p<0.05; ns = not significant.
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Figure 2—source data 1
Ring-stage survival assay (RSA) dose-response assays.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig2-data1-v1.zip
Figure 2—figure supplement 1
Early ring-stage dose-response curves.
Dihydroartemisinin (DHA) dose-response assays on 0–3 hr post invasion (hpi) ring-stage (a) Dd2 PfCRT3D7, (b) Dd2, and (c) Dd2 K13R539T parasites were performed with DHA alone, or DHA with the addition of 10 μM chloroquine (CQ), or 200 nM piperaquine (PPQ). For the PPQ-resistant parasites (d) Dd2 PfCRTM343L and (e) MRA-1284, additional PPQ concentrations of 500 nM and 10 μM were also tested. Shown are dose-response curves with mean % inhibition ± SEM from at least three independent replicates.
Figure 3 with 3 supplements
Chloroquine (CQ) and piperaquine (PPQ) antagonize dihydroartemisinin (DHA) activation by ‘inactivating’ heme.
(a) Shown are mean ΣFIC50 values calculated from the trophozoite-stage isobolograms in Figure 1. Values close to 1 (pink) indicate additivity. Values ranging from 1.25 to 2 (light blue) indicate classical antagonism. Values >2.25 (dark turquoise) indicate superantagonism. (b top panel) Heme (Fe2+-FPIX) cleaves the N-O bond of H-FluNox, mediating probe fluorescence. (b bottom panel) Heme cleaves the peroxide bridge of DHA, which generates a carbon-centered radical that can alkylate proximal biomolecules. (c) H-FluNox was incubated with increasing concentrations of heme in the presence or absence of 10 μM CQ, 150 nM PPQ, 10 μM PPQ, 150 nM MFQ, or 10 μM MFQ. Shown are mean relative fluorescence units (RFU) ± SD from at least three independent replicates performed in technical triplicate. (d and e) Dd2 trophozoites were treated with 10 μM CQ, 150 nM PPQ, 150 nM MFQ, or mock-treated for 5.5 hr. (f) Dd2 PfCRTDd2 and Dd2 PfCRT3D7 trophozoites were mock-treated or treated with 150 nM CQ or 10 μM CQ for 5.5 hr. (d–f) Parasites were then incubated with 10 μM Ac-H-FluNox (the cell-permeable analog of H-FluNox) to visualize and quantify ‘active’ heme. Shown are (d) representative images and (e and f) mean fluorescence intensity ± SD of at least 70 parasites from at least three independent drug treatments. Statistical significance was assessed using a one-way ANOVA with a Tukey's test for multiple comparisons. (g and h) Dd2 trophozoites were treated with 10 μM CQ, 25 nM DHA, 10 μM CQ + 25 nM DHA for 6 hr. Parasite lysates were subjected to western blot and probed with anti-K48-linked ubiquitin (K48-Ub) antibodies or anti-plasmepsin V (PMV) antibodies. Shown is a (g) representative blot and (h) quantification of relative K48-Ub intensity ± SEM from three independent replicates. Statistical significance between mock-treated and antimalarial-treated parasites was determined using a one-way ANOVA with a Tukey's test for multiple comparisons. ****p<0.0001; ***p<0.001; **p<0.01; ns = not significant. Scale bar = 5 μm.
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Figure 3—source data 1
Mean sum FIC50 values used to generate the heat map.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig3-data1-v1.zip
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Figure 3—source data 2
H-FluNox relative fluorescence units (RFU) with chloroquine (CQ), piperaquine (PPQ), and mefloquine (MFQ).
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig3-data2-v1.zip
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Figure 3—source data 3
Ac-H-FluNox parasite fluorescence with chloroquine (CQ), piperaquine (PPQ), and mefloquine (MFQ).
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig3-data3-v1.zip
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Figure 3—source data 4
Ac-H-FluNox fluorescence of Dd2 PfCRTDd2 and Dd2 PfCRT3D7 parasites.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig3-data4-v1.zip
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Figure 3—source data 5
Uncropped, labeled western blots.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig3-data5-v1.zip
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Figure 3—source data 6
Western blot raw images.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig3-data6-v1.zip
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Figure 3—source data 7
Western blot quantifications.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig3-data7-v1.zip
Figure 3—figure supplement 1
Effect of pH on H-FluNox and Ac-H-FluNox activity.
(a) H-FluNox was incubated with increasing concentrations of heme at pH 7.5, 5.4, and 4. Shown are mean relative fluorescence units (RFU)± SD from two independent replicates performed in technical duplicate. (b) Dd2 trophozoites were treated with 10 μM chloroquine (CQ) or mock-treated for 5.5 hr. Parasites were then co-incubated with Ac-H-FluNox and 10 mM ammonium chloride to visualize free heme and alkalinize the digestive vacuole, respectively. Shown are two representative images per treatment condition. Note that exposure times were optimized for different treatment conditions to better visualize the digestive vacuole. Scale bar = 5 μm.
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Figure 3—figure supplement 1—source data 1
H-FluNox pH titration relative fluorescence units (RFU).
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
Equimolar comparison of chloroquine (CQ), piperaquine (PPQ), and mefloquine (MFQ) on Ac-HFluNox fluorescence.
Dd2 trophozoites were treated with 10 μM CQ, 10 μM PPQ, 10 μM MFQ, or mock-treated for 1 hr. Parasites were then incubated with Ac-H-FluNox to visualize and quantify ‘active’ heme. Shown are (a) representative images and (b) mean fluorescence intensity ± SD of at least 75 parasites from three independent drug treatments. Statistical significance was assessed using a one-way ANOVA with a Tukey’s test for multiple comparisons. Scale bar = 5 μm.
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Figure 3—figure supplement 2—source data 1
Ac-H-FluNox fluorescence for 10 μM chloroquine (CQ), 10 μM piperaquine (PPQ), 10 μM mefloquine (MFQ) treatment.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig3-figsupp2-data1-v1.zip
Figure 3—figure supplement 3
Representative images of Dd2 PfCRTDd2 and Dd2 PfCRT3D7 Ac-H-FluNox fluorescence.
Dd2 PfCRTDd2 and Dd2 PfCRT3D7 trophozoites were mock-treated or treated with 150 nM chloroquine (CQ) or 10 μM CQ for 5.5 hr. Parasites were then incubated with 10 μM Ac-H-FluNox (the cell-permeable analog of H-FluNox) to visualize and quantify ‘active’ heme. Shown are representative images relating to Figure 3f. Scale bar = 5 μm.
Figure 4 with 2 supplements
Quinolines antagonize peroxides.
(a and b) Trophozoite-stage dihydroartemisinin (DHA) dose-response assays were performed with Dd2 parasites in the presence or absence of 15 nM piperaquine (PPQ), 15 nM mefloquine (MFQ), 15 nM PPQ+15 nM MFQ, or 2 nM DHA. Shown are (a) dose-response curves with mean % inhibition ± SEM and (b) mean IC50 values ± SEM from at least three independent replicates. Statistical significance was determined using a one-way ANOVA with a Tukey’s test for multiple comparisons. Trophozoite-stage isobolograms were performed on Dd2 parasites to determine drug-drug interactions of (c) PPQ-MFQ, (d) ADQ-DHA, (e) LM-DHA, (f) PPQ-OZ439, and (g) FQ OZ439 using the following fixed ratios: 1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1. Shown are fractional IC50 values from at least three independent replicates. Independent replicates are indicated in different shading and the diagonal dotted line on each plot indicates perfect additivity. (h and i) Dd2 parasites were mock-treated or treated with 10 μM chloroquine (CQ), 150 nM ADQ, 500 nM lumefantrine (LM), or 150 nM ferroquine (FQ) for 5.5 hr and free heme was then labeled in live parasites with Ac-H-FluNox. Shown are (h) representative images and (i) mean fluorescence intensity ± SD of at least 90 parasites from three independent drug treatments. Statistical significance was assessed using a one-way ANOVA with a Dunnett’s test for multiple comparisons. ****p<0.0001; ns = not significant. (j) Mean relative fluorescence of Ac-H-FluNox from drug treatments in Figure 3 and (i) was plotted against Dd2 mean ΣFIC50 values for the indicated combinations. Combinations with DHA are indicated in black. Combinations with OZ439 are indicated in blue. Scale bar = 5 μm.
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Figure 4—source data 1
Dose response analyses for TACTs.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig4-data1-v1.zip
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Figure 4—source data 2
Isobologram analyses.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig4-data2-v1.zip
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Figure 4—source data 3
Ac-H-FluNox fluorescence for ADQ, FQ, and LM.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig4-data3-v1.zip
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Figure 4—source data 4
Values for FIC50 and Ac-H-FluNox correlation.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig4-data4-v1.zip
Figure 4—figure supplement 1
Additional mefloquine (MFQ) isobolograms.
Trophozoite-stage isobolograms were performed on (a) Dd2 PfCRTM343L and (b) Dd2 parasites to determine drug-drug interactions of using the following fixed ratios: 1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1. Shown are fractional IC50 values from three independent replicates.
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Figure 4—figure supplement 1—source data 1
Isobologram analysis.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig4-figsupp1-data1-v1.zip
Figure 4—figure supplement 2
Amodiaquine (ADQ), lumefantrine (LM), and ferroquine (FQ) IC50 values.
Shown are mean Dd2 trophozoite-stage IC50 values ± SEM obtained as part of three independent isobologram replicates (see Figure 4d, e and g).
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Figure 4—figure supplement 2—source data 1
Amodiaquine (ADQ), lumefantrine (LM), ferroquine (FQ), IC50 values.
- https://cdn.elifesciences.org/articles/108976/elife-108976-fig4-figsupp2-data1-v1.zip
Tables
Table 1
Parasite strains used in this study.
Strains are annotated with their origin (isolate or edited), chloroquine resistance transporter (PfCRT) genotype, Kelch 13 (K13) genotype, and whether they are resistant to chloroquine (CQ), piperaquine (PPQ), or dihydroartemisinin (DHA). Wild-type (WT) K13 corresponds to the 3D7 genotype.
| Parasite name | Origin | PfCRT genotype | K13 genotype | Resistance | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 74 | 75 | 76 | 97 | 220 | 271 | 326 | 343 | 356 | 371 | ||||
| 3D7 | Africa (1981) | M | N | K | H | A | Q | N | M | I | R | WT | – |
| Dd2 | Indochina (1980) | I | E | T | H | S | E | S | M | T | I | WT | CQ |
| Dd2 PfCRTDd2 | Edited (introns removed) | I | E | T | H | S | E | S | M | T | I | WT | CQ |
| Dd2 PfCRT3D7 | Edited | M | N | K | H | A | Q | N | M | I | R | WT | – |
| Dd2 K13R539T | Edited | I | E | T | H | S | E | S | M | T | I | R539T | CQ, DHA |
| Dd2 PfCRTM343L | Edited | I | E | T | H | S | E | S | L | T | I | WT | CQ, PPQ |
| MRA-1284 (IPC_6261) | Cambodia (2012) | I | E | T | Y | S | E | S | M | T | I | C580Y | DHA, PPQ |
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4-Aminoquinolines block heme iron reactivity and interfere with artemisinin action
eLife 14:RP108976.
https://doi.org/10.7554/eLife.108976.3
