Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7 Hanafy M. Ismaila,b, Victoria Bartonc, Matthew Phanchanaa, Sitthivut Charoensutthivarakulc, Michael H. L. Wongb, Janet Hemingwayb,1, Giancarlo A. Biaginia, Paul M. O’Neillc, and Stephen A. Warda,1 aResearch Centre for Drugs and Diagnostics, Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom; bVector Biology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom; and cDepartment of Chemistry, University of Liverpool, Liverpool L69 7ZD, United Kingdom Contributed by Janet Hemingway, January 12, 2016 (sent for review December 24, 2015; reviewed by Kelly Chibale, Gary Posner, and Donatella Taramelli) The artemisinin (ART)-based antimalarials have contributed signif- alkyne/azide-coupling partners through “click chemistry,” we icantly to reducing global malaria deaths over the past decade, but identified several cytochrome P450 enzymes that metabolized we still do not know how they kill parasites. To gain greater deltamethrin in rat liver microsomes (10). More recently, a insight into the potential mechanisms of ART drug action, we chemical proteomic approach was developed to identify parasite developed a suite of ART activity-based protein profiling probes proteins targeted by an albitiazolium antimalarial drug candidate to identify parasite protein drug targets in situ. Probes were in situ using a photoactivation cross-linking approach (11). designed to retain biological activity and alkylate the molecular However, this generic approach can introduce significant pro- target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins miscuity in the proteins tagged based on the intracompartmental tagged with the ART probe can then be isolated using click chemistry distribution of drug independent of actual mechanisms. before identification by liquid chromatography–MS/MS. Using these Here, we introduced the design and synthesis of click chem- probes, we define an ART proteome that shows alkylated targets in istry-compatible activity-based probes incorporating the endo- the glycolytic, hemoglobin degradation, antioxidant defense, and peroxide scaffold of ART as a warhead to alkylate and identified BIOCHEMISTRY protein synthesis pathways, processes essential for parasite survival. the ART molecular target(s) in asexual stages of the malaria This work reveals the pleiotropic nature of the biological functions parasite (Fig. 1). A major advantage of this strategy is that the targeted by this important class of antimalarial drugs. reporter tags are introduced under “click” reaction conditions performed after the drug has achieved its biological effects, en- artemisinin | antimalarial | bioactivation | chemical proteomics | abling purification, identification, and quantification of alkylated molecular targets 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 “con- alaria is a global health problem with 214 million new trol” nonperoxide partners to improve the specificity and biological cases of malaria and 438,000 deaths reported in 2015, M relevance of our resultant tagged protein list. 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 multi- Significance drug-resistant parasites (2) and lack of effective vaccines (3, 4). Given the effectiveness of the ART class, the question arises: The mechanism of action of the artemisinin (ART) class of an- how do these drugs kill parasites? A suggested mechanism of timalarial drugs, the most important antimalarial drug class in action involves the cleavage of the endoperoxide bridge by a use today, remains controversial, despite more than three de- + source of Fe2 or heme. This cleavage results in the formation of cades of intensive research. We have developed an unbiased oxyradicals that rearrange into primary or secondary carbon-cen- chemical proteomic approach using a suite of ART activity- tered radicals. These radicals have been proposed to alkylate based protein profiling probes to identify proteins within the parasite proteins that somehow result in the death of the parasite malaria parasite that are alkylated by ART, including proteins (5). However, this proposal remains a subject of intense debate involved in glycolysis, hemoglobin metabolism, and redox de- (6, 7), while these alkylated proteins are yet to be formally fense. The data point to a pleiotropic mechanism of drug action identified. So far, the proposed targets of ART action include a for this class and offer a strategy for investigating resistance PfATP6 enzyme, the Plasmodium falciparum ortholog of mam- mechanisms to ART-based drugs as well as mechanisms of ac- malian sarcoendoplasmic reticulum Ca21-ATPases (SERCAs) tion of other endoperoxide-based drugs. (5), translational controlled tumor protein, and heme (5). Addi- tionally, Haynes et al. (8) proposed that ART may act by impairing Author contributions: H.M.I., P.M.O., and S.A.W. designed research; H.M.I. and M.P. per- formed research; V.B., S.C., M.H.L.W., P.M.O., and S.A.W. contributed new reagents/ parasite redox homeostasis as a consequence of an interaction be- analytic tools; H.M.I., V.B., M.P., J.H., and G.A.B. analyzed data; H.M.I. and S.A.W. wrote tween the drug and flavin adenine dinucleotide (FADH) and/or the paper; H.M.I. contributed to the initial concept, carried out the initial biological studies, other parasite flavoenzymes in the parasite, leading to the genera- validated the methodology, and wrote the initial draft of the manuscript; V.B. and S.C. were responsible for the generation of probe molecules; M.P. carried out confirmatory tion of reactive oxygen species (ROS). New approaches are re- biological studies independent of H.M.I.; and P.M.O. and S.A.W. conceived of the original quired for definitive identification of ART molecular targets. This concept and were responsible for the biological materials and chemistry, respectively. insight into the drug activation-dependent mechanism of action will Reviewers: K.C., University of Cape Town; G.P., John Hopkins University; and D.T., Universita be invaluable in the target-led development of more potent drugs di Milano. with the potential to circumvent the emergence of resistance to The authors declare no conflict of interest. current first-line ART-based therapies. The goal of this study was to Freely available online through the PNAS open access option. identify ART-targeted proteins and their interacting partners in 1To whom correspondence may be addressed. Email: [email protected] or P. falciparum. We recently adopted a proteomic approach de- [email protected]. veloped by Speers and Cravatt (9) to synthesize a suite of pyre- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. throid activity-based protein profiling probes (ABPPs) (10). Using 1073/pnas.1600459113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1600459113 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Fig. 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. Results and Discussion in protein alkylation. In general, many essential proteins were Rationale of ART Activity-Based Protein Profiling Probes Design and identified as ART targets with P1 and P2 (Figs. 2 and 3). These Synthesis. We synthesized the click probes using techniques op- results are further supported by 1D gel fluorescence analysis timized and developed in house with the alkyne/azide introduced (Fig. S1) for proteomes treated with a trifunctional azido-biotin- by an optimized peptide coupling protocol (more details are in SI rhodamine tag (instead of the biotin azide tag) and subjected to Text). We varied click handles to expand the utility of ART-ABPPs protein purification followed by in-gel fluorescence visualization. and compare the efficiency of two distinctive tagging chemistries With this probe, significant protein labeling was apparent with (i.e., copper-catalyzed and bioorthogonal copper-free click chem- the active probe P1, whereas no labeling was evident with its istry). Previous work by Meshnick and coworkers (12) showed that alkyne deoxy-ether negative control analog. This result again rein- six uncharacterized malaria proteins bands were labeled with forces the importance of the endoperoxide function for bioacti- semisynthetic derivatives of ART (e.g., [3H]-dihydroartemisinin) vation (Fig. S1). Using on-bead trypsin digestion of captured at pharmacologically