Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum

Abstract

The mechanism of action of artemisinin and its derivatives, the most potent of the anti-malarial drugs, is not completely understood. Here we present an unbiased chemical proteomics analysis to directly explore this mechanism in Plasmodium falciparum. We use an alkyne-tagged artemisinin analogue coupled with biotin to identify 124 artemisinin covalent binding protein targets, many of which are involved in the essential biological processes of the parasite. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using alkyne-tagged artemisinin coupled with a fluorescent dye to monitor protein binding, we show that haem, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The haem derives primarily from the parasite's haem biosynthesis pathway at the early ring stage and from haemoglobin digestion at the latter stages. Our results support a unifying model to explain the action and specificity of artemisinin in parasite killing.

A Click Chemistry‐Based Proteomic Approach Reveals that 1,2,4‐Trioxolane and Artemisinin Antimalarials Share a Common Protein Alkylation Profile

“…In the present study many cytoskeletal proteins including α‐tubulin, β‐tubulin, and actin 1 were labeled with trioxolanes and ART probes suggesting a potential link between these endoperoxide drugs and parasite cell structure, protein trafficking systems and signal transduction 9. Moreover, the global analysis of protein alkylation generated through P2 ( 7 a ), for both classes, that is, ART and trioxolanes, is consistent with the “cluster bomb” hypothesis,7, 9, 15 whereby Fe 2+ /heme‐activated drug alkylates multiple redox‐susceptible protein targets functioning in multiple cellular pathways (Figure S4) including the food vacuole, a site considered important for iron dependent activation, and also in the cytosol (Figure S5).…”

“…We proposed that carbon centered radicals generated from the reductive scission of the endoperoxide bridge from either P2 ( 7 a ) or P3 ( 11 a ) alkylate proteins by C‐radical attack at the disulfide bridges of the glutathionylated proteins identified in this study;11, 13 further work is on‐going to confirm this mode of reactivity. The major proteins in the mass range 30 to 75 KDa identified for P2 ( 7 a ) are, Pf LDH, Pf OAT and Pf HGPRT, similar to that seen earlier with the equivalent ART probes,7, 9 Collectively the data suggest that 1,2,4‐trixolanes efficiently target plasmodial energy supply, the antioxidant defence system and DNA synthesis. In addition, ART has been shown to modulate a variety of signaling pathways in cancer cells 14.…”

“…125 in Ref. 7) with an azide probe, demonstrating the improved specificity of our approach using optimized active and control probe based methodology.…”

“…In a recent study, Wang et al 7. used a non‐optimised ART‐alkyne activity‐based protein‐profiling probe that via click‐chemistry reactions was associated with some 124 P. falciparum proteins 7.…”

“…used a non‐optimised ART‐alkyne activity‐based protein‐profiling probe that via click‐chemistry reactions was associated with some 124 P. falciparum proteins 7. In this study, a further 125 proteins are reported as being identified in single replicate experiments only, raising concerns of the specificity of the approach 7. Concurrently, using both ART‐alkyne and azide optimized probes we were able to identify 59 P. falciparum proteins with high confidence that were specifically alkylated by ART pointing towards a pleiotropic mechanism of drug action 9.…”