Mechanism of action of artemisinin antimalarials and implications for drug resistance in Plasmodium falciparum

© 2015 Dr. Stanley Cheng XieThe most deadly cases of malaria in humans are caused by Plasmodium falciparum. Artemisinin-based combination therapy is the current first-line treatment against this disease. However, artemisinin resistance has recently emerged in Southeast Asia, manifesting as delayed parasite clearance times in patients. Efforts to monitor and contain artemisinin resistance were initially frustrated by a lack of correlation between in vitro parasite sensitivity to artemisinins and in vivo drug efficacy in patients. Even though artemisinins have short in vivo half-lives, standard in vitro assays have traditionally employed extended drug exposure formats to assess parasite sensitivity to the drugs. We hypothesized that short drug pulses would better mimic in vivo conditions and be more clinically relevant for artemisinins. Using novel pulsed drug exposure assays, we demonstrate that laboratory and field (Pailin, Cambodia) parasite strains exhibit stage- and strain-dependent differences in drug sensitivities. Three stages with distinct drug sensitivities are identified in laboratory strains, namely hypersensitive early rings, insensitive mid-rings and sensitive trophozoites. Moreover, using this assay format, we are able to clearly distinguish the in vitro response of sensitive and resistant Pailin strains. We find that resistant field strains exhibit the highest levels of resistance at the very early ring stage and the late schizont stage. Our detailed analysis of field strains reveals that exposure to short pulses of artemisinins induces growth retardation in both sensitive and resistant parasites. Following this growth retardation, resistant strains survive, while sensitive parasites succumb. The data suggest that artemisinins are activated, and cause cellular damage, in both strains, but resistant parasites are better able to withstand the damage. As arrest in growth is often part of a cellular stress response, we postulated that artemisinin resistance is caused by an up-regulated parasite cellular defense mechanism. Consistent with this, we demonstrate that proteasome inhibitors effectively synergize artemisinin activity against both sensitive and resistance strains, with particularly strong synergism evident during the most resistant stage of the resistant strains. This suggests that the parasite proteasome system could be targeted to enhance drug action, offering a way to overcome artemisinin-resistant malaria. The mechanism of drug action is also investigated. Trophozoites take up and digest large amounts of host hemoglobin, and previous reports showed that the products of hemoglobin digestion can act as artemisinin activators in the trophozoite stage. However, the relevant activator or mechanism of action in rings is not clear. We show that chelating the labile iron pool has little effect on artemisinin activity against early rings. By contrast, hemoglobinases inhibitors strongly antagonize artemisinin action. We show for the first time that the hemoglobinases, falcipain-2 and -3, are expressed in rings. Moreover knockout of falcipain-2 and knockdown of falcipain-3, render the very early ring stage insensitive to artemisinins. These data lead to the surprising conclusion that hemoglobin digestion is active at the early ring stage and is involved in artemisinin activation