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.

Reports of emerging resistance to first-line artemisinin antimalarials make it critical to define resistance mechanisms and identify in vitro correlates of resistance. Here we combine unique in vitro experimental and analytical approaches to mimic in vivo drug exposure in an effort to provide insight into mechanisms of drug resistance. Tightly synchronized parasites exposed to short drug pulses exhibit large stage-dependent differences in their drug response that correlate with hemoglobin digestion throughout most of the asexual cycle. As a result, ring-stage parasites can exhibit >100-fold lower sensitivity to short drug pulses than trophozoites, although we identify a subpopulation of rings (2-4 h postinvasion) that exhibits hypersensitivity. We find that laboratory strains that show little differences in drug sensitivity in standard in vitro assays exhibit substantial (>95-fold) difference in sensitivity when exposed to short drug pulses. These stage-and strain-dependent differences in drug sensitivity reflect differential response lag times with rings exhibiting lag times of up to 4 h. A simple model that assumes that the parasite experiences a saturable effective drug dose describes the complex dependence of parasite viability on both drug concentration and exposure time and is used to demonstrate that small changes in the parasite's drug response profile can dramatically alter the sensitivity to artemisinins. This work demonstrates that effective resistance can arise from the interplay between the short in vivo half-life of the drug and the stage-specific lag time and provides the framework for understanding the mechanisms of drug action and parasite resistance.endoperoxide | Plasmodium | qinghaosu | effective dose model