Haemoglobin degradation underpins the sensitivity of early ring stage Plasmodium falciparum to artemisinins

Abstract

Current first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d). Quantitative modelling of the antagonism accounted for its complex dependence on the chemical features of the artemisinins and on the drug exposure time, and showed that almost all artemisinin activity in early rings (>80%) is due to haem-mediated activation. The surprising implication that haemoglobin uptake and digestion is active in early rings is supported by identification of active haemoglobinases (falcipains) at this stage. Genetic down-modulation of the expression of the two main cysteine protease haemoglobinases, falcipains 2 and 3, renders early ring stage parasites resistant to artemisinins. This confirms the important role of haemoglobin-degrading falcipains in artemisinin activation, and shows that changes in the rate of artemisinin activation could mediate high-level artemisinin resistance.

Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic

“…ART is highly active against trophozoites, in which Hb catabolism reaches its peak 7 . Unlike most other antimalarials, ART is also active against early ring-stage parasites 8 . Recent data suggest that parasite-mediated endocytosis and proteolysis of host Hb begin in very early ring stages (within several hours of RBC invasion), which could provide a potential source of activation for ART 7,8 .…”

Artemisinin Action and Resistance in Plasmodium falciparum

“…ARTs are less active against mid-ring stage parasites than against trophozoites [27], and are inactive against mature (stage V) gametocytes [28] and liver stages [29], consistent with decreased or absent hemoglobin digestion at these stages. However ARTs are active against very early asexual ring-stage parasites , just after erythrocyte invasion [27, 30]. This finding suggests that early rings can already import and digest host hemoglobin, even before formation of the digestive vacuole where most of the hemoglobin is degraded during the trophozoite stage.…”

“…This finding suggests that early rings can already import and digest host hemoglobin, even before formation of the digestive vacuole where most of the hemoglobin is degraded during the trophozoite stage. In support of this idea, ART action can be substantially mitigated in very early rings as well as trophozoites by inhibiting falcipains, which proteolytically cleave host hemoglobin and release heme-iron [30]. Recent evidence suggests that biosynthetic heme produced by the parasite might also contribute to ART activation in very early rings [31–33].…”

“…Additional iron may be released via hydrogen peroxide-mediated degradation of heme in the digestive vacuole [35] or by reaction with reduced glutathione in the parasite cytoplasm [36]. Iron chelators weakly antagonize ART activity [30, 37, 38]. However, this antagonism is much less potent than that observed with hemoglobinase inhibitors, providing further evidence that heme-iron rather than free iron is the main activator.…”

“…Moreover, in vitro drug-pressured lines have been reported that carry genetic changes other than at the K13 locus, notably copy number changes in pfmdr1 , which might associate with ART resistance [90]. Genetic down-modulation of the falcipain 2 and 3 cysteine protease hemoglobinases conferred a degree of ART resistance to early rings, although this is presumably because of the role of these hemoglobinases in producing heme as the ART activator as opposed to these being resistance modulators per se [30]. A central question underlying the role of secondary determinants is whether these are involved in reducing parasite susceptibility to ARTs during the trophozoite stage, when hemoglobin metabolism is at its peak.…”