Dihydroartemisinin–Ferriprotoporphyrin IX Adduct Abundance in Plasmodium falciparum Malarial Parasites and the Relationship to Emerging Artemisinin Resistance

Abstract

preceding paper in this issue), we quantified free ferriprotoporphyrin IX (FPIX) heme abundance for control versus delayed clearance phenotype (DCP) intraerythrocytic Plasmodium falciparum malarial parasites. Because artemisinin drugs are activated by free FPIX, these data predict that the abundance of long-hypothesized toxic artemisinin drug−FPIX covalent adducts might differ for control versus DCP parasites. If so, this would have important repercussions for understanding the mechanism of the DCP, also known as emerging artemisinin resistance. To test these predictions, we studied in vitro formation of FPIX−dihydroartemisinin (DHA) adducts and then for the first time quantified the abundance of FPIX− DHA adducts formed within live P. falciparum versus the stage of intraerythrocytic development. Using matched isogenic parasite strains, we quantified the adduct for DCP versus control parasite strains and found that mutant PfK13 mediates lower adduct abundance for DCP parasites. The results suggest improved models for the molecular pharmacology of artemisininbased antimalarial drugs and the molecular mechanism of the DCP.

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

“…Rab5 proteins have been implicated in hemoglobin import processes, suggesting that the differential associations might impact hemoglobin uptake [63][64][65][66]. These findings recall the recent report of reduced heme and heme-DHA adducts in K13 mutant parasites [29,30]. Birnbaum et al also recently showed reduced endocytosis of host cytoplasm, which consists mainly of hemoglobin, in ring-stage parasites expressing mutant K13 (compared to an isogenic WT control) or WT K13 parasites in which this protein was conditionally knocked sideways to cause loss of function [27].…”

“…Mechanistic studies have led to several proposals for how mutant K13 might counter ARTmediated cellular toxicity. These proposals include lowering the levels of the heme activator of ART including via reduced hemoglobin endocytosis in rings [27][28][29][30], upregulating endoplasmic reticulum (ER) stress-response pathways [31], reducing the levels of ubiquitinated proteins [32], promoting translational arrest via differential phosphorylation of the translation initiation factor eIF2α [33], or increasing levels of the phospholipid phosphatidylinositol-3phosphate (PI3P) [34,35]. To gain additional insight into the biology of this protein, we raised K13-specific monoclonal antibodies (mAbs) and used these to interrogate this protein's subcellular localization in DHA-exposed or vehicle-treated asexual blood-stage parasites.…”

Artemisinin-Based Antimalarial Drug Therapy: Molecular Pharmacology and Evolving Resistance

“…However, a number of details related to pro drug activation and subsequent target alkylation are yet to be elucidated. For example, the source of Fe 2+ for pro drug activation is not fully defined, although the principle source is likely to be a reduced (ferrous) ferriprotoporphyrin IX (FPIX) heme released during obligate parasite hemoglobin (Hb) catabolism [12].…”

Genetic screens reveal a central role for heme biosynthesis in artemisinin susceptibility

“…Artemisinins require activation within cells by scission of an endoperoxide bridge through the Fe 2+ center of heme 1,3 . Cleavage of this bond results in the production of ART radicals, which react with hundreds of proteins 4,5 , lipids 6 , and metabolites, 7 quickly leading to cell death.…”