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 CQ, PPQ, or DHA. Wild-type (WT) K13 corresponds to the 3D7 genotype.

CQ and 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, grey, pink, blue, purple, and teal, respectively. Data from three independent replicates are indicated by different shading. Figure supplement 1. CQ, DHA,sity of different parasites.

CQ co-treatment confers artemisinin resistance in K13WT parasites.

(a) DHA dose response assays were performed on 0-3 hours post invasion (hpi) ring stage parasites. Two-fold 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 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 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. 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. Figure supplement 1. Early ring stage dose-response curves.

CQ and PPQ antagonize 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 h. (f) Dd2 PfCRTDd2 and Dd2 PfCRT3D7 trophozoites were mock treated or treated with 150 nM CQ or 10 μM CQ for 5.5 h. (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 Turkey’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 h. 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 Turkey’s test for multiple comparisons.****p < 0.0001; ***p < 0.001; **p < 0.01; ns = not significant. Figure supplement 1. Effect of pH on H-FluNox and Ac-H-FluNox activity. Figure supplement 2. Equimolar comparison of CQ, PPQ, and MFQ on Ac-H-FluNox fluorescence. Figure supplement 3. Representative images of Dd2 PfCRTDd2 and Dd2 PfCRT3D7 Ac-H-FluNox fluorescence. Figure supplement 4. Additional K48-ubiquitin western blots. Table supplement 1. Mean parasite intensity for Ac-H-FluNox experiments. Source data 1. Uncropped western blots used for ImageJ quantification.

Quinolines antagonize peroxides.

(a and b) Trophozoite-stage DHA dose-response assays were performed with Dd2 parasites in the presence or absence of 15 nM PPQ, 15 nM 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 Turkey’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 3 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 CQ, 150 nM ADQ, 500 nM LM, or 150 nM FQ for 5.5 h 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 3e and 4i 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. Figure supplement 1. Additional MFQ isobolograms. Figure supplement 2. ADQ, LM, and FQ IC50 values. Table supplement 1. Mean parasite intensity for Ac-H-FluNox experiments.