Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.26 page(s

In vivo synergy between DHA and Carfilzomib in a <i>P</i>. <i>berghei</i> mouse model.

<p>Balb/c mice were infected with 10⁶ parasites (<i>P</i>. <i>berghei</i>) and treatment was initiated at ~1% parasitaemia (i.e., day 3), indicated with an arrow. Data represent averages of data from three independent experiments. (A) Carfilzomib alone has no effect on parasite growth at doses of 0.5 and 1 mg/kg, but has a slight antimalarial effect at 1.5 mg/kg. (B) Treatment with DHA (5 mg/kg) + Carfilzomib (0.5 mg/kg) reduces parasite growth, while DHA (5 mg/kg) + Carfilzomib (1 mg/kg) prevents parasite growth. (C) DHA (10 mg/kg) + Carfilzomib (0.5 or 1 mg/kg) also reduces parasitemia by 2- to 3-fold. Error bars represent SEM and * and *** represent, respectively, <i>p</i> <0.05 and <i>p</i> <0.01, after Bonferroni correction for multiple testing.</p

Model of killing and survival-promoting events following treatment with ART.

<p>ART is activated by an Fe(II) source (e.g., heme released from hemoglobin degradation or from the cellular labile iron pool) to produce activated ART (ART*), which is reactive, leading to cellular damage and ultimately to parasite death (shown in black). The parasite mounts a stress response, that manifests as growth retardation and engagement of the proteasome-ubiquitin pathway (shown in red). The stress response in K13 mutants is enhanced (shown in blue). In contrast, epoxomicin inhibits the stress response (shown in green), thus promoting parasite death.</p

Synergy of DHA and a proteasome inhibitor against <i>P</i>. <i>falciparum</i>.

<p>The interaction of DHA and epoxomicin (Epo) was examined using (A) 3D7 and (B) PL1 parasites at the ages indicated. Left panels in (A and B) show the dose-responses to DHA (blue symbols) and the influence of sublethal concentrations (indicated) of epoxomicin. Right panels in (A and B) show isobolograms for the epoxomicin-DHA pair at the 50% <i>LD</i>_<i>50</i><i>(3h)</i> level. The dashed line is plotted between the <i>LD</i>_<i>50</i><i>(3h)</i> of each drug when used alone, emphasizing the concave nature of many of the isobolograms. The absence of a dashed line indicates the <i>LD</i>_<i>50</i><i>(3h)</i> value of one of the drugs was outside the range of concentrations examined. Error bars, where present, correspond to the range of duplicates.</p

DHA induces growth retardation prior to killing.

<p>(A, C) DHA-induced decrease in the SYTO-61 signal of viable parasites. Tightly synchronized 3D7 (A) and PL2 and PL7 (C) parasites were subjected to 4-h (A) or 3-h (C) DHA pulses at the ring-stage ages indicated in the figure. The median SYTO-61 signals of viable parasites were determined in the next cycle (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002132#sec013" target="_blank">Materials and Methods</a>). The concentration dependence of the SYTO-61 signal (growth effects; green symbols) is compared with that of parasite viability (grey symbols). Dashed green and grey lines represent the <i>IC</i>_<i>50</i> and <i>LD</i>_<i>50</i> values associated with each exposure time. Error bars correspond to the range of duplicates. (B,D) Changes in the SYTO-61 staining profile of viable parasites after drug exposure. 3D7 parasite (6 and 18 h p.i., B) were subjected to 4-h DHA pulses at 63 and 16 nM, respectively. Alternatively PL2 and PL7 late rings (18 h p.i.) were subjected to 1.5, 3, and 6-h DHA pulses (16 nM; D). The SYTO-61 signals were measured by flow cytometry in the next cycle. The SYTO-61 distributions of the viable population (dark green) are compared with those of untreated parasites (light green). Asterisks highlight an absolute increase in the number of younger parasites, indicating growth retardation.</p

Delayed parasite clearance of resistant strains reflects both reduced killing and delayed removal of unviable parasites.

<p>(A) Viable parasite load during DHA treatment regimen. The number of total (circulating and sequestered; orange curves) and circulating (grey curves) viable parasites during DHA therapy in hypothetical patients presenting with ring-stage PL7- and PL2-like infections were simulated using CED model parameters and an in vivo DHA pharmacokinetic profile (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002132#pbio.1002132.s001" target="_blank">S1 Appendix</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002132#pbio.1002132.s014" target="_blank">S3 Table</a>). Arrows indicate the times of drug administration. (B) Influence of unviable parasite removal on parasite clearance profiles. The circulating unviable parasites in (A) in the PL2 and PL7-like infections (red and blue curves, respectively) were modelled to disappear from circulation with a half-life of 5 h (dashed curves) or exhibit more complex removal (solid curves) involving strain-dependent (1- and 3.5-h half-lives for PL2 and PL7), in addition to a strain-independent (3-h half-life) component. Symbols correspond to median circulating parasite loads from patients in Pailin (blue) and Wang Pha (red) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002132#pbio.1002132.ref041" target="_blank">41</a>]. (C) Contribution of unviable parasites to parasite clearance curves. The solid curves represent the simulated parasite clearance curves shown in Fig 7B incorporating the complex clearance of unviable parasites. The dashed curves correspond to the total number of unviable parasites at each time point and the dotted curves to the unviable parasites produced during the course of the first treatment (i.e., formed during the first 24 h).</p

Ubiquitination of <i>P</i>. <i>falciparum</i> proteins following ART treatment.

<p>Uninfected RBCs or trophozoite-infected RBCs (24–44 h p.i.) (3% hematocrit) of 3D7, PL2, and PL7 strains were incubated with 1 μM QHS or 20 nM WR99210 for 90 min at 37°C. Cell extracts were subjected to SDS-PAGE and western blotting and probed with anti-ubiquitin IgG with ECL detection, then stripped and re-probed with anti-<i>Pf</i>GAPDH. (A) Representative western blots. (B) Densitometric analysis of the anti-ubiquitin signal for at least nine QHS and three WR99210 experiments. Significance was determined using a Student’s <i>t</i> test. * <i>p</i> <0.05; *** <i>p</i> <0.005.</p

Stage-dependent in vitro DHA responses of Pailin strains.

<p>(A,B) Tightly synchronized PL2 and PL7 (A, red and blue, respectively) and PL1 and PL5 (B, green and blue, respectively) cultures were assayed using 3-h DHA pulses across the blood cycle. The <i>a</i> panels show the variation of <i>LD</i>_<i>50</i><i>(3h)</i> and <i>V</i>_<i>min</i><i>(3h)</i> during the first 32 h p.i. Time points corresponding to 50% trophozoites (T; filled cytoplasm in Giemsa stained smear) are indicated. The <i>b</i> panels show the variation of <i>LD</i>_<i>50</i><i>(3h)</i> and <i>V</i>_<i>min</i><i>(3h)</i> across the schizont-to-ring transition. Post-invasion ages are normalized to those in the next cycle to account for the different lifecycle durations of the different strains (49, 52, 59, and 57 h for PL1, 2, 5, and 7, respectively). Negative ages represent pre-invasion ages and correspond to schizonts that are the indicated time from forming new rings. The midpoint of each box corresponds to the average parasite age at the start of each assay, normalized to a value of zero when 50% of the parasites had formed rings. Colored bar widths reflect the relative parasite populations at the beginning of each assay: schizonts (dark blue for PL7 and PL5, dark red for PL2, and dark green for PL1), rings (light blue for PL7 and PL5, light red for PL2, and light green for PL1), and schizonts forming rings during the course of the assay (grey for all strains). (C) Tightly synchronized PL2 (red) and PL7 (blue) cultures were subjected to DHA pulses of different durations at 2, 18, and 34 h p.i. Curves represent best fits obtained with the cumulative effective dose (CED) model, with the parameters shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002132#pbio.1002132.t001" target="_blank">Table 1</a>. Dose response profiles for each exposure time are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002132#pbio.1002132.s003" target="_blank">S1 Fig</a>.</p

CED model parameters for PL2 and PL7 strains.

<p>ND: parameters could not be reliably determined</p><p>CED model parameters for PL2 and PL7 strains.</p

Quantitation of growth retardation and transition to pyknotic forms following DHA treatment.

<p>(A) Early ring-stage PL7 parasites (1.5 h p.i.) were treated with a 3.5 h DHA pulse (1 μM) and the morphology of parasites (Giemsa smears) examined at 31 and 35 h after the drug pulse. Representative images of untreated and treated parasites are shown. The proportion of the parasite population corresponding to trophozoites (hemozoin-containing parasites) and non-trophozoites (rings or pyknotic forms) are indicated in the figure. (B) The size distribution of viable parasites (hemozoin-containing) in the drug-treated culture was quantitated and compared with untreated cultures at the indicated times after the drug pulse (<i>n</i> >50 per sample). Late rings are larger than early trophozoites and were counted as a separate category (filled grey bars). (C) PL2 (red) and PL7 (blue) parasites (1.5 h p.i.) were rendered unviable by treatment with a DHA pulse (1 μM, 3.5 h) and the fraction of unviable parasites exhibiting pyknotic morphology was quantitated (filled symbols). Open symbols denote untreated parasites. (D) Representative images of untreated PL2 parasites and treated unviable parasites showing ring-like and pyknotic morphologies (fraction of each morphology indicated).</p