Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs.Malaria is a global health problem with 214 million new cases of malaria and 438,000 deaths reported in 2015, mostly in sub-Saharan Africa (1). The endoperoxide class of antimalarial drugs, such as artemisinin (ART), is the first line of defense against malaria infection against a backdrop of multidrug-resistant parasites (2) and lack of effective vaccines (3, 4). Given the effectiveness of the ART class, the question arises: how do these drugs kill parasites? A suggested mechanism of action involves the cleavage of the endoperoxide bridge by a source of Fe²⁺ or heme. This cleavage results in the formation of oxyradicals that rearrange into primary or secondary carbon-centered radicals. These radicals have been proposed to alkylate parasite proteins that somehow result in the death of the parasite (5). However, this proposal remains a subject of intense debate (6, 7), while these alkylated proteins are yet to be formally identified. So far, the proposed targets of ART action include a PfATP6 enzyme, the Plasmodium falciparum ortholog of mammalian sarcoendoplasmic reticulum Ca²¹-ATPases (SERCAs) (5), translational controlled tumor protein, and heme (5). Additionally, Haynes et al. (8) proposed that ART may act by impairing parasite redox homeostasis as a consequence of an interaction between the drug and flavin adenine dinucleotide (FADH) and/or other parasite flavoenzymes in the parasite, leading to the generation of reactive oxygen species (ROS). New approaches are required for definitive identification of ART molecular targets. This insight into the drug activation-dependent mechanism of action will be invaluable in the target-led development of more potent drugs with the potential to circumvent the emergence of resistance to current first-line ART-based therapies. The goal of this study was to identify ART-targeted proteins and their interacting partners in P. falciparum. We recently adopted a proteomic approach developed by Speers and Cravatt (9) to synthesize a suite of pyrethroid activity-based protein profiling probes (ABPPs) (10). Using alkyne/azide-coupling partners through “click chemistry,” we identified several cytochrome P450 enzymes that metabolized deltamethrin in rat liver microsomes (10). More recently, a chemical proteomic approach was developed to identify parasite proteins targeted by an albitiazolium antimalarial drug candidate in situ using a photoactivation cross-linking approach (11). However, this generic approach can introduce significant promiscuity in the proteins tagged based on the intracompartmental distribution of drug independent of actual mechanisms.Here, we introduced the design and synthesis of click chemistry-compatible activity-based probes incorporating the endoperoxide scaffold of ART as a warhead to alkylate and identified the ART molecular target(s) in asexual stages of the malaria parasite (Fig. 1). A major advantage of this strategy is that the reporter tags are introduced under “click” reaction conditions performed after the drug has achieved its biological effects, enabling purification, identification, and quantification of alkylated parasite’s proteins and their interacting partners as shown in Fig. 1B. To avoid nonspecific probe-dependent tagging, a common limitation of these approaches, we generated the respective “control” nonperoxide partners to improve the specificity and biological relevance of our resultant tagged protein list.Open in a separate windowFig. 1.Rational design of the ART-ABPPs. (A) Conversion of ART to ART-ABPPs involves the addition of a clickable handle (i.e., an alkyne or azide to the ART drug pharmacophore by the peptide-coupling method illustrated in SI Text). The structures of the alkyne (P1) and azide (P2) probes and respective inactive deoxy controls CP1 and CP2 with in vitro IC₅₀ values are presented. (B) General workflow of copper-catalyzed and copper-free click chemistry approaches used in the identification of alkylated proteins after in situ treatment of P. falciparum parasite with alkyne and azide ART-ABPPs. The azide- and alkyne-modified proteins are tagged with biotin azide and biotin dibenzocyclooctyne (Biotin-DIBO), respectively, via click reactions followed by affinity purification tandem with LC-MS/MS for protein identification.