Artemisinin Activity-Based Probes Identify Multiple Molecular Targets Within the Asexual Stage of the Malaria Parasites Plasmodium Falciparum 3D7

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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. performed 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 relevant concentrations of the drug. Im- proteins with the biotin azide reporter followed by LC-MS/MS portantly, labeling was not shown using uninfected erythrocytes identification confidently reported 58 proteins using P1; how- or when infected cells were treated with the inactive analog, ever, four proteins were detected at very low abundance in two of deoxyether (12). Our strategy allows definitive identification of five replicates in the positive control treatment (CP1) (Fig. 2 and tagged proteins by comparison of active probe alkylation products Fig. S2A). Multiple t test analysis confirmed that the levels of these with those of their nonperoxidic equivalent controls (synthesized proteins tagged by the active probe were significantly higher than in as detailed in SI Text). Earlier work suggests that long peptide the control experiments (Fig. S2A). This result shows the impor- linkers can introduce cytotoxicity (13). To avoid this issue, pep- tance of having a positive control to avoid identification of proteins tide linkers of maximum three carbons were used for our alkyne that bind nonspecifically to the streptavidin column. When DMSO and azide probes. solvent was used as a negative control, nine proteins at very low, insignificant levels were detected in two of five replicates (Fig. 2 and Antimalarial Activity of ART-ABPPs. The antimalarial activity of the Fig. S2B). These observations emphasize the need to control for probes and controls was determined in vitro against P. falciparum nonspecific binding in these complex proteomic experiments. 3D7 parasites (SI Text). The azide/alkyne function did not sig- Studies performed with copper (I) salts and copper complexes nificantly affect antimalarial activity against 3D7 (Fig. 1A). The have shown the potential for ART activation and rearrangement, IC50 values for probe 1 (P1) and probe 2 (P2) were 43.86 (SD = similar to that generated by iron (16) under these conditions. ART 0.441 nM; n = 3) and 30.26 nM (SD = 0.314 nM; n = 3), re- activation based on copper was considered an unlikely problem spectively, compared with the IC50 of ART of 14 nM (SD = 0.41 during the click reactions used in this study because of the mul- nM; n = 3). Importantly, control probes CP1 and CP2 were in- tiple washing steps involved and the use of a reducing environ- active in agreement with expectations (12, 14). This result sup- ment [DTT and Tris(2-carboxyethyl)phosphine] during protein ports the essentiality of the endoperoxide bridge and validates extraction and click reactions, conditions known to block antima- our probe plus control pair strategy. larial action of the endoperoxides (17, 18). Nevertheless, to de- finitively rule out this possibility and validate the results more Parasite Protein Labeling and Identification. As a next step, we stringently, we have also introduced the bioorthogonal copper-free moved to the direct identification of ART molecular target(s) in click methodology by preparation of the azide analog of the alkyne situ as explained in Methods and Fig. 1B. The Mascot search probe (P2) (Fig. 1). After the azide probe is introduced into algorithm was used to identify proteins from the resultant pep- target biomolecules after peroxide activation, the azide can be tides identified by liquid chromatography (LC)–MS/MS, and the tagged with reporter using several selective reactions (19). Here, exponentially modified protein abundance index (emPAI) was we used strain-promoted [3 + 2] azide-alkyne cycloaddition to used to provide semiquantitation of protein abundance (15). Heat verify the results obtained from copper-catalyzed azide-alkyne map analysis (Fig. 2) shows the essentiality of the endoperoxide cycloaddition (CuAAC) with P1. The critical tagging reagent, a

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1600459113 Ismail et al. Downloaded by guest on September 25, 2021 within minutes (19, 20). Both P1 and P2 exhibited similar labeling patterns as indicated by pulldown experiments, including 42 identical proteins (Fig. 3A). However, the pulldown exper- iment with P2 exhibited higher sensitivity compared with P1 (Fig. 2). The higher sensitivity could be attributed to the higher efficiency of the strain-promoted [3 + 2] azide-alkyne cycloaddition reaction compared with the CuAAC reaction in aqueous media (19, 20). More importantly, samples treated with the nonperoxidic equivalent of P2 (CP2) did not identify any proteins. Moreover, the comparison between samples treated in situ with P2 vs. cellular homogenates did not show any significant qualita- tive differences in the tagged protein profiles. Additionally, cellular homogenates exposed to P2 with the iron-chelating agent desfer- oxamine (DFO) (100 μM) showed that ART–protein alkylation was significantly reduced but not completely abolished by chelation (Fig. S3 and Table S1). These data may support a role for a nonferrous activation process. Alternatively, although 100 μM DFO was previously shown to inhibit the antimalarial activity of this class using live parasite (14), incomplete iron chelation remains a possibility under this experimental condition using processed parasite protein homogenate. Free sulfhydryl groups (thiols) of cysteine residues in the proteins of Plasmodium parasites are sensitive to oxidation and alkylation by ART-derived radicals, generating cysteine adducts (21, 22). Kehr et al. (23) showed that the free thiols in cysteine are reversibly glutathionylated, forming blended protein glutathione disulfides. This posttranslational modification is believed to function primarily as a redox-sensitive switch to facilitate re-

dox regulation and signal transduction (23). Metaanalysis on the BIOCHEMISTRY protein list identified by ART-ABPPs (P1 and P2) looking for a glutathione binding motif (Fig. 3B) indicated a high positive correlation (χ2 = 230.1 with a P value < 0.0001) as shown in Fig. 2. This positive correlation may be explained by the availability of free thiol groups in cysteine residuesactingastargetsforART–protein alkylation. In particular, thiol alkylation may directly contribute to the observed specific inhibition of cysteine proteases and aspartic proteases by the endoperoxides that results in decreased hemoglobin degradation in the parasite’s food vacuole (24).

Interaction of ART with the P. falciparum Hemoglobin Digestion Pathway. In this study, ART-ABPPs labeled a substantial number of proteases from the parasite’s digestive food vacuole (Fig. 2, Fig. S4, and Table S2). Plasmepsin-2, in particular, was identified in the high confidence list of the ART proteome. Together with plasmepsin-1, these two proteins belong to the aspartic proteases that coordinate with cysteine proteases (falcipain-2 and falcipain-3) in the process of hemoglobin degradation in the parasite’s food Fig. 2. Heat map of the entire proteomic dataset identified by the alkyne vacuole (25–27) and are considered good drug targets (28). In- and azide ART-ABPPs. Heat map analysis was carried out by plotting the hibition of vacuolar digestion could contribute to the pleiotropic average exponentially modified protein abundance index values for each action of the ART drug class. Neither falcipain-2 nor falcipain-3, protein. The data were acquired from multiple independent experiments for = = = = = considered to be the activators of plasmepsins (29), was targeted by each probe [P1, n 5; CP1, n 5; DMSO, n 5; P2, n 2; CP2, n 2; P2* the ART-ABPPs, which may reflect the steric effect of the ART (cellular homogenate)]. Each independent replicate was injected into the LC-MS/MS four times to improve the accuracy of protein identification. The structure in selective binding of aspartic proteases involved in heat map plot was clustered into four categories from high confidence to noise according to the hit frequency in all replicates. Protein hits found in seven to nine independent replicates were denoted as being of high confidence, hits seen in four to six replicates were denoted as medium confi- AB dence, three hits were denoted as low confidence, and hits seen in only one or two replicates were considered as noise. Proteins were manually searched for the presence (+) and absence (−) of a glutathione (GSH) binding motif according to data published by Kehr et al. (23). Complete datasets of ART proteomes are illustrated in Dataset S1. DMSO, dimethyl sulfoxide treatment; MW, protein molecular weight in kilodalton; ORF, open reading frame names.

substituted biotin cyclooctyne, possesses ring strain and elec- – tron-withdrawing substituents that together promote the [3 + 2] Fig. 3. Overview of ART-ABPP tagged proteins. (A) Venn diagram showing overlap between proteins identified with the alkyne (P1) and azide (P2) ART dipolar cycloaddition with azides installed into the P2 molecule probes. (B) Trend analysis between the confidence in protein identification as illustrated in Fig. 1B (19). The Cu-free click reaction follows as described in Fig. 2 and the proportion of those proteins carrying a glu- comparable kinetics to the CuAAC click reaction and proceeds tathione (GSH) binding motif (χ2 = 230.1 with a P value < 0.0001).

Ismail et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 hemoglobin digestion. It also suggests that the targeting of proteins explanation of this phenomenon, observed in ART-resistant par- is not promiscuous. However, neither plasmepsin-2 nor M1-family asites, is that slowing down growth at ring stage by closing down alanyl aminopeptidase, which identified with high confidence by aspects of energy production is a protective mechanism, whereas ART-ABPPs, was significantly affected by the iron-chelating agent the parasite experiences ART-induced stress and possible protein (DFO), suggesting that the alkylation and inhibition of proteases damage. Normal growth resumes after up-regulation of the un- + with ART may not be solely dependent on free Fe 2 (Fig. S3). This folded protein response pathways that are capable of resolving observation requires additional investigation. The observation that protein damage caused by ART (38). Interestingly, in this study, of ART-ABPPs target multiple components of this degradation 11 enzymes participating in glycolysis, 7 enzymes were labeled and pathway is credible evidence of ART activation and may implicate detected with ART-ABPPs (Table S2). The disruption of the disturbance in the parasites feeding process as an important aspect microassembly of glycolysis in the parasite by ART should lead to of drug action (Fig. S4) as indicated by work using alternative ap- the parasite death. In support of this observation, studies in proaches (7, 30–32). Schistosoma japonicum-infected mice exposed to artemether also showed an effect of drug treatment on important glycolytic Interaction of ART with P. falciparum Antioxidant Defense Systems. enzymes in the parasite (39, 40). In general, these findings are Hemoglobin digestion in the malaria parasite is a highly dynamic consistent with the vital and essential role that glycolytic en- catabolic process. Hemoglobin digestion leaves the parasite under zymes play in supporting rapid growth and proliferation, similar intense oxidative stress by generating active redox byproducts, in- to that seen in other rapidly proliferating cells, such as cancer − cluding heme and a superoxide anion radical (O•2), with the latter cells (41, 42). The high glycolytic flux of intraerythrocytic de- being rapidly converted to H2O2 (33).Tocopewithsuchhighly velopmental cycle maintains rate-limiting glycolytic intermedi- cytotoxic processes, the parasite recruits an efficient enzymatic ates to support other pathways (e.g., nucleotide and lipid antioxidant defense system that includes glutathione and many biosynthesis) (Fig. S4) (41). thioredoxin-dependent proteins. Of particular interest in this study Overall, apart from glucose-6-phosphate isomerase, none of the is the labeling of ornithine aminotransferase (OAT), a thioredoxin- glycolytic proteomes identified by ART-ABPPs were significantly dependent protein identified with ART-ABPPs in high confidence affected by iron chelation using cellular homogenate (Fig. S3). andhighabundance(Fig.2andTable S2). PfOAT is essential in the coordination of ornithine homeostasis, polyamine synthesis, Interaction of ART with P. falciparum Nucleic Acid and Protein proline synthesis, and mitotic cell division (34). In malaria parasites, Biosynthesis Pathways. Plasmodium parasites cannot synthesize pu- PfTrx1 regulates PfOAT redox activity by interaction with two rine nucleotides de novo and instead, rely on the purine salvage highly conserved cysteine residues (Cys154 and Cys163), which do pathway that is essential for parasite growth and survival (43) and not exist in any other species and may interfere with substrate has been exploited as a drug target. ART-ABPPs identified five binding (34). PfOATlabelingwassignificantly reduced after iron enzymes (Table S2) involved in purine and pyrimidines synthesis chelation (Fig. S3), suggesting the supportiveroleoffreeironin (nucleic acid biosynthesis). Many of the enzymes involved in Plas- drug activation and leading to irreversible binding to PfOAT. modium nucleic acid biosynthesis differ from those of their human ART itself can generate ROS in the parasite through endoper- hosts. Accordingly, nucleic acid biosynthesis pathways have long + oxidebridgeactivation(8,35),andfreeFe 2 can enhance the been considered exploitable targets for novel antimalarial drug generation of ROS by the Fenton process (35). Therefore, it has design (43–46). For example, P. falciparum bifunctional dihy- been presumed that ART-activated ROS may lead to parasite death drofolate reductase-thymidylate synthase mediates the conversion by overwhelming the parasite’s antioxidant defense systems (36). of dihydrofolic acid (dihydrofolate; vitamin B9) to tetrahydrofolic Identification of stress-related proteins (Table S2) by ART-ABPPs acid by dihydrofolate (44). Tetrahydrofolate is needed to make both supports the hypothesis that ART is involved in the activation and purines and pyrimidines, the building blocks of DNA and RNA, and generation of ROS in vivo. Apart from Actin 1, none of these some of our most successful antimalarials, such as pyrimethamine proteins were significantly affected by iron chelation under the and proguanil, target this enzyme, resulting in parasite death (44, condition used here (Fig. S3). 47). Furthermore, P. falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase, an ART-ABPPs target identified with Interaction of ART with the P. falciparum Glycolysis Pathway. Pre- high confidence, is also central in purine salvage (46). Labeling of vious work by our group showed that artemether, the methyl the P. falciparum hypoxanthine-guanine-xanthine phosphoribosyl- derivative of ART, negatively alters some key glycolytic enzymes transferase was significantly reduced after iron chelation (Fig. S3), while increasing the expression of stress response proteins after suggesting a role of chelatable iron in ART activation leading to pharmacologically relevant drug exposure in the P. falciparum alkylation of the enzyme. ART-ABPPs also identified many ri- (37). Increased levels of stress-associated proteins coupled with a bosomal proteins (Table S2), suggesting that protein synthesis is decrease in glycolytic enzymes suggest a mechanism where the an additional target for ART. parasite designates to protect its machinery for generating its energy supply from disruption by endoperoxides. This hypothesis Interaction of ART with P. falciparum Chaperone Proteins and the is further supported by the recent study, where ART-resistant Cytoskeleton. ART-ABPPs identified several chaperone proteins parasites exhibited a slower growth rate through the early ring (Table S2). The majority of chaperonins were initially identified as stage of the intraerythrocytic developmental cycle (38). A credible heat shock proteins expressed in parasites in response to elevated

Fig. 4. Gene Ontology (GO) analysis for biological pathways identified from ART proteome. (A) Black bars represent the numbers of proteins identified within each biological process (pathway) using the STITCH 4 web tool (stitch.embl.de). (B) Multiple test- ing corrected P values for enriched GO categories. The confidence view of ART–protein and protein– protein interactions networks built up using the STITCH 4 web tool is illustrated in Fig. S4. A complete dataset of ART interactome is in Datasets S2 and S3.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1600459113 Ismail et al. Downloaded by guest on September 25, 2021 temperatures, a prominent symptom of the disease, and/or other interactions has functional relevance regarding parasite le- cellular stresses. In general, molecular chaperones are involved in thality and resistance. many cellular functions, including helping newly synthesized proteins to fold, protein trafficking, and signal transduction (48). Conclusion and Future Perspective. Using a chemical proteomic TCP-1 and cytoskeletal proteins α-tubulin, β-tubulin, and actin 1 approach, we have identified with a high level of confidence key were all labeled by ART-ABPPs. The fact that actin and tubulin proteins that are alkylated by ART; the biological functions of folding and dimerization are chaperoned by the TCP-1 chaperonin the tagged proteins are consistent with many of the presumed system gives rise to suggest a potential link between ART, the mechanisms proposed to explain ART drug action, in which parasite cell structure, protein trafficking systems, and signal the ART causes extensive damages to key proteins that have a transduction. wide spectrum of cellular activity. Collectively, copper-based and copper-free ART-ABPPs approaches present significant evi- Interaction of ART with Transport Proteins in P. falciparum. ART- dence in support of the argument that ART can target two im- + ABPPs identified the two subunits of the V-type H -ATPase portant sources of carbon/energy and amino acid supply (i.e., (subunits A and B) at various levels of confidence from high to glycolysis and hemoglobin digestion pathways) within the eryth- medium, respectively (Table S2). In the parasite, a V-type rocytic stage of infection. ART-ABPPs also provide additional ATPase generates a proton motive force across the parasite evidence that ART targets other essential parasite metabolic plasma membrane as well as across the digestive food vacuole pathways, including DNA synthesis, protein synthesis, and lipid (49, 50). It has previously been shown that ART results in rapid synthesis. The application of ART-ABPPs to monitor the activity depolarization of the parasite plasma membrane (51). Although of protein target(s) as well as their abundance in different par- the depolarization of the plasma membrane was previously asite lines could have significant implications for understanding shown to be partially attributed to lipid peroxidation, the ART- the problem of ART resistance, especially combined with ge- ABPPs data presented here could be interpreted as showing the nomic approaches. The data generated here are a significant step potential for a direct inhibition of the V-type ATPase activity by forward in identifying the complete array of protein targets that ART. Structural similarity between ARTs and thapsigargin, an are susceptible to alkylation with ART. The next key step will be inhibitor of SERCA (PfATP6), led to a hypothesis that PfATP6 to define formally which of these alkylation reactions is func- was a target for ART (5). This proposal received additional tionally most relevant to antimalarial drug action and which may support from a study connecting ART resistance in P. falciparum be modified to confer drug resistance. field isolates from French Guiana with the S769N mutation in PfATP6 (52). The functional importance of PfATP6 in ART Methods BIOCHEMISTRY action and resistance remains controversial; however, PfATP6 Synthesis of Endoperoxide Probes and Respective Controls. Endoperoxide was detected by the alkyne version of ART-ABPPs (P1), albeit at probes and corresponding deoxy ether analogs (controls) were synthesized as low yield and low confidence. In addition to metabolic pathways, detailed in SI Text. ART-ABPPs identified nine proteins related to parasite–host cell invasion and the host immune responses (Table S2). The Parasite Cultures and Drug Sensitivity Assays. P. falciparum 3D7 parasites functional relevance of these as targets remains to be tested. were cultured following the method illustrated in SI Text. Drug activities of ART-ABPPs were measured using a standard fluorometric DNA binding ART and Resistance Mechanisms in P. falciparum. Several genes have method as detailed in SI Text. been associated with ART resistance mechanisms, including the chloroquine resistance gene (Pfcrt), the multidrug resistance In Situ Parasite Treatment for Chemical Proteomic Pulldown Assays. Ten flasks pump (Pfmdr1), and the K13 propeller domain of the kelch-like of synchronized trophozoite-stage parasite cultures at 10–12% parasitemia protein (2, 38, 53). This study was not designed to probe re- and 4% hematocrit (0.5 L culture) were treated with ART-ABPPs P1 and P2 and their respective controls (100 μL 5 mM stock) to give a final concen- sistance, and only a single parasite isolate has been investigated; μ however, the gene products of Pfcrt and Pfmdr1 (Table S3), and a tration of 1 M probe per treatment, 10-fold the IC90 (100 nM) at short protein from the proteasomal system, Cdc48 protein (but not pulse time to ensure full inhibition of parasite growth and maximal alkylation (53). All treatments were carried out with a minimum of two repli- K13), were labeled with ART-ABPP. These findings indicate the cates per treatment (noted elsewhere). Probe-treated parasites were potential for the use of ART-ABPPs in investigating resistance maintainedincultureat37°Cinanatmosphereof3%CO,4%O,and and tolerance mechanisms using appropriate parasite isolates 2 2 93% N2 for 6 h, an incubation period that has already been shown to cause with well-defined resistance phenotypes. irreversible parasite lethality (53). An equivalent culture without drug probe (five replicates) was treated with 100 μL DMSO as a control treatment ART Interactome. We used a bioinformatics interaction network maintained under identical culture conditions. Parasite proteins were se- analysis approach using STICH 4 software (54) to build up and quentially extracted using a modification of the protocol described by predict functional ART–protein association networks and identify Molloy et al. (56) (SI Text). major pathways targeted by the drug based on compiled available experimental evidence, database information, and the literature. ART-ABPPs Labeling and the Effect of Iron Chelation. Parasite protein extracts Analysis of the data generated in silico connectivity for ART, (cellular homogenates) were prepared from 1 L untreated in vitro cultured with 67 proteins collectively identified with P1 and P2 termed parasite as described earlier. Protein extracts were adjusted to 2 mg/mL in the ART interactome shown in Fig. S5. As predicted, the pro- Eppendorf tubes and treated with P2 at 10 μM in the presence or absence of teome experimentally identified with ART-ABPPs revealed a the iron-chelating agent DFO at 100 μM for 1 h at 37 °C. Subsequent protein strong network association of functionally important proteins processing was as described in SI Text. (Fig. S5). The connectivity analysis confirmed that the glycolytic pathway is a primary interacting target for ART with a P value Identification of ART-ABPPs Alkylated Proteins. Alkylated proteins from all − of 1 × 10 9 (Fig. 4). Additional pathways were identified as experiments were subjected to click chemistry reaction with relevant biotin being targets for ART interaction, including the biosynthesis of reporter as detailed in SI Text. As the next step, biotin-tagged proteins was secondary metabolites, metabolic pathways, ribosomes, phag- affinity-purified using streptavidin agarose beads, and the captured proteins osomes, etc. (Fig. 4). Additional analysis to identify the cellular was identified using LC-MS/MS after bead trypsin digestion for the purified proteins as described in SI Text. components targeted by the ART interactome using Cytoscape software supports a role for ART acting akin to the “cluster ” ACKNOWLEDGMENTS. This work was funded by the Wellcome Trust bomb hypothesis (55), with multiple cellular targets mainly but through its Strategic Support Fund award, Medical Research Council Confi- not exclusively in the food vacuole and cytosol (Figs. S4 and S6). dence in Concept funding, and through Mahidol-Liverpool Chamlong The important next step will be to establish which of these Harinasuta PhD Scholarship.

Ismail et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 1. WHO (2014) World Malaria Report 2014 (WHO, Geneva). 32. Meshnick SR, Thomas A, Ranz A, Xu CM, Pan HZ (1991) Artemisinin (qinghaosu): The 2. Winzeler EA, Manary MJ (2014) Drug resistance genomics of the antimalarial drug role of intracellular hemin in its mechanism of antimalarial action. Mol Biochem artemisinin. Genome Biol 15(11):544. Parasitol 49(2):181–189. 3. RTS,S Clinical Trials Partnership (2015) Efficacy and safety of RTS,S/AS01 malaria vac- 33. Becker K, et al. (2004) Oxidative stress in malaria parasite-infected erythrocytes: host- cine with or without a booster dose in infants and children in Africa: Final results of a parasite interactions. Int J Parasitol 34(2):163–189. phase 3, individually randomised, controlled trial. Lancet 386(9988):31–45. 34. Jortzik E, et al. (2010) Redox regulation of Plasmodium falciparum ornithine δ-ami- 4. Rts SCTP; RTS,S Clinical Trials Partnership (2014) Efficacy and safety of the RTS,S/AS01 notransferase. J Mol Biol 402(2):445–459. malaria vaccine during 18 months after vaccination: A phase 3 randomized, con- 35. Krishna S, Uhlemann AC, Haynes RK (2004) Artemisinins: Mechanisms of action and trolled trial in children and young infants at 11 African sites. PLoS Med 11(7): potential for resistance. Drug Resist Updat 7(4-5):233–244. e1001685. 36. Hunt NH, Stocker R (1990) Oxidative stress and the redox status of malaria-infected 5. Eckstein-Ludwig U, et al. (2003) Artemisinins target the SERCA of Plasmodium falci- erythrocytes. Blood Cells 16(2-3):499–526. parum. Nature 424(6951):957–961. 37. Makanga M, Bray PG, Horrocks P, Ward SA (2005) Towards a proteomic definition of 6. O’Neill PM, Barton VE, Ward SA (2010) The molecular mechanism of action of arte- CoArtem action in Plasmodium falciparum malaria. Proteomics 5(7):1849–1858. misinin–the debate continues. Molecules 15(3):1705–1721. 38. Mok S, et al. (2015) Drug resistance. Population transcriptomics of human malaria 7. Mercer AE, Copple IM, Maggs JL, O’Neill PM, Park BK (2011) The role of heme and the parasites reveals the mechanism of artemisinin resistance. Science 347(6220):431–435. mitochondrion in the chemical and molecular mechanisms of mammalian cell death 39. Xiao S, et al. (1998) Effect of artemether on hexokinase, glucose phosphate isomerase induced by the artemisinin antimalarials. J Biol Chem 286(2):987–996. and phosphofructokinase of Schistosoma japonicum harbored in mice. Zhongguo Ji 8. Haynes RK, et al. (2010) Facile oxidation of leucomethylene blue and dihydroflavins Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 16(1):25–28. by artemisinins: Relationship with flavoenzyme function and antimalarial mechanism 40. Xiao SH, et al. (1998) Effect of artemether on glyceraldehyde-3-phosphate de- of action. ChemMedChem 5(8):1282–1299. hydrogenase, phosphoglycerate kinase, and pyruvate kinase of Schistosoma japoni- 9. Speers AE, Cravatt BF (2004) Profiling enzyme activities in vivo using click chemistry cum harbored in mice. Zhongguo Yao Li Xue Bao 19(3):279–281. methods. Chem Biol 11(4):535–546. 41. Salcedo-Sora JE, Caamano-Gutierrez E, Ward SA, Biagini GA (2014) The proliferating 10. Ismail HM, et al. (2013) Pyrethroid activity-based probes for profiling cytochrome cell hypothesis: A metabolic framework for Plasmodium growth and development. P450 activities associated with insecticide interactions. Proc Natl Acad Sci USA 110(49): Trends Parasitol 30(4):170–175. 19766–19771. 42. Olszewski KL, Llinás M (2011) Central carbon metabolism of Plasmodium parasites. 11. Penarete-Vargas DM, et al. (2014) A chemical proteomics approach for the search of Mol Biochem Parasitol 175(2):95–103. pharmacological targets of the antimalarial clinical candidate albitiazolium in Plas- 43. Ting LM, et al. (2005) Targeting a novel Plasmodium falciparum purine recycling modium falciparum using photocrosslinking and click chemistry. PLoS One 9(12): pathway with specific immucillins. J Biol Chem 280(10):9547–9554. e113918. 44. Yuthavong Y, et al. (2012) Malarial dihydrofolate reductase as a paradigm for drug 12. Asawamahasakda W, Ittarat I, Pu YM, Ziffer H, Meshnick SR (1994) Reaction of development against a resistance-compromised target. Proc Natl Acad Sci USA antimalarial endoperoxides with specific parasite proteins. Antimicrob Agents 109(42):16823–16828. Chemother 38(8):1854–1858. 45. Cassera MB, Zhang Y, Hazleton KZ, Schramm VL (2011) Purine and pyrimidine path- 13. Liu Y, Wong VK, Ko BC, Wong MK, Che CM (2005) Synthesis and cytotoxicity studies of ways as targets in Plasmodium falciparum. Curr Top Med Chem 11(16):2103–2115. artemisinin derivatives containing lipophilic alkyl carbon chains. Org Lett 7(8): 46. Sarkar D, Ghosh I, Datta S (2004) Biochemical characterization of Plasmodium falci- 1561–1564. parum hypoxanthine-guanine-xanthine phosphorybosyltransferase: Role of histidine 14. Stocks PA, et al. (2007) Evidence for a common non-heme chelatable-iron-dependent residue in substrate selectivity. Mol Biochem Parasitol 137(2):267–276. activation mechanism for semisynthetic and synthetic endoperoxide antimalarial 47. Yuthavong Y, et al. (2005) Malarial (Plasmodium falciparum) dihydrofolate reductase- drugs. Angew Chem Int Ed Engl 46(33):6278–6283. thymidylate synthase: Structural basis for antifolate resistance and development of 15. Ishihama Y, et al. (2005) Exponentially modified protein abundance index (emPAI) for effective inhibitors. Parasitology 130(Pt 3):249–259. estimation of absolute protein amount in proteomics by the number of sequenced 48. Hartl FU, Bracher A, Hayer-Hartl M (2011) Molecular chaperones in protein folding peptides per protein. Mol Cell Proteomics 4(9):1265–1272. and proteostasis. Nature 475(7356):324–332. 16. Bousejra-El Garah F, Pitié M, Vendier L, Meunier B, Robert A (2009) Alkylating ability 49. Hayashi M, et al. (2000) Vacuolar H(+)-ATPase localized in plasma membranes of of artemisinin after Cu(I)-induced activation. J Biol Inorg Chem 14(4):601–610. malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of 17. Krungkrai SR, Yuthavong Y (1987) The antimalarial action on Plasmodium falciparum parasitized erythrocytes. J Biol Chem 275(44):34353–34358. of qinghaosu and artesunate in combination with agents which modulate oxidant 50. Saliba KJ, Kirk K (1999) pH regulation in the intracellular malaria parasite, Plasmo- stress. Trans R Soc Trop Med Hyg 81(5):710–714. dium falciparum. H(+) extrusion via a V-type H(+)-ATPase. J Biol Chem 274(47): 18. Meshnick SR, et al. (1989) Activated oxygen mediates the antimalarial activity of 33213–33219. qinghaosu. Prog Clin Biol Res 313:95–104. 51. Antoine T, et al. (2014) Rapid kill of malaria parasites by artemisinin and semi-syn- 19. Sletten EM, Bertozzi CR (2009) Bioorthogonal chemistry: Fishing for selectivity in a sea thetic endoperoxides involves ROS-dependent depolarization of the membrane po- of functionality. Angew Chem Int Ed Engl 48(38):6974–6998. tential. J Antimicrob Chemother 69(4):1005–1016. 20. Baskin JM, et al. (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc 52. Jambou R, et al. (2005) Resistance of Plasmodium falciparum field isolates to in-vitro Natl Acad Sci USA 104(43):16793–16797. artemether and point mutations of the SERCA-type PfATPase6. Lancet 366(9501): 21. Wu WM, Chen YL, Zhai Z, Xiao SH, Wu YL (2003) Study on the mechanism of action of 1960–1963. artemether against schistosomes: The identification of cysteine adducts of both car- 53. Ariey F, et al. (2014) A molecular marker of artemisinin-resistant Plasmodium falci- bon-centred free radicals derived from artemether. Bioorg Med Chem Lett 13(10): parum malaria. Nature 505(7481):50–55. 1645–1647. 54. Kuhn M, et al. (2014) STITCH 4: Integration of protein-chemical interactions with user 22. Wu Y, Yue ZY, Wu YL (1999) Interaction of qinghaosu (artemisinin) with cysteine data. Nucleic Acids Res 42(Database issue):D401–D407. sulfhydryl mediated by traces of non-heme iron. Angew Chem Int Ed Engl 38(17): 55. Ridley RG (2003) Malaria: To kill a parasite. Nature 424(6951):887–889. 2580–2582. 56. Molloy MP, et al. (1998) Extraction of membrane proteins by differential solubilization 23. Kehr S, et al. (2011) Protein S-glutathionylation in malaria parasites. Antioxid Redox for separation using two-dimensional gel electrophoresis. Electrophoresis 19(5):837–844. Signal 15(11):2855–2865. 57. Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 24. Pandey AV, Tekwani BL, Singh RL, Chauhan VS (1999) Artemisinin, an endoperoxide 193(4254):673–675. antimalarial, disrupts the hemoglobin catabolism and heme detoxification systems in 58. Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum eryth- malarial parasite. J Biol Chem 274(27):19383–19388. rocytic stages in culture. J Parasitol 65(3):418–420. 25. Banerjee R, et al. (2002) Four plasmepsins are active in the Plasmodium falciparum 59. Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M (2004) Simple and in- food vacuole, including a protease with an active-site histidine. Proc Natl Acad Sci expensive fluorescence-based technique for high-throughput antimalarial drug USA 99(2):990–995. screening. Antimicrob Agents Chemother 48(5):1803–1806. 26. Sijwali PS, Rosenthal PJ (2004) Gene disruption confirms a critical role for the cysteine 60. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum. Proc Natl quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. Acad Sci USA 101(13):4384–4389. 61. Speers AE, Cravatt BF (2009) Activity-based protein profiling (ABPP) and click chem- 27. Klemba M, Gluzman I, Goldberg DE (2004) A Plasmodium falciparum dipeptidyl istry (CC)-ABPP by MudPIT mass spectrometry. Curr Protoc Chem Biol 1:29–41. aminopeptidase I participates in vacuolar hemoglobin degradation. J Biol Chem 62. Voronin D, et al. (2014) Wolbachia lipoproteins: Abundance, localisation and se- 279(41):43000–43007. rology of Wolbachia peptidoglycan associated lipoprotein and the Type IV Secre- 28. Rosenthal PJ, McKerrow JH, Aikawa M, Nagasawa H, Leech JH (1988) A malarial tion System component, VirB6 from Brugia malayi and Aedes albopictus. Parasit cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falci- Vectors 7:462. parum. J Clin Invest 82(5):1560–1566. 63. Gardner MJ, et al. (2002) Genome sequence of the human malaria parasite Plasmo- 29. Drew ME, et al. (2008) Plasmodium food vacuole plasmepsins are activated by falci- dium falciparum. Nature 419(6906):498–511. pains. J Biol Chem 283(19):12870–12876. 64. Saito R, et al. (2012) A travel guide to Cytoscape plugins. Nat Methods 9(11): 30. Klonis N, et al. (2011) Artemisinin activity against Plasmodium falciparum requires 1069–1076. hemoglobin uptake and digestion. Proc Natl Acad Sci USA 108(28):11405–11410. 65. Bindea G, et al. (2009) ClueGO: A Cytoscape plug-in to decipher functionally 31. Zhang S, Gerhard GS (2008) Heme activates artemisinin more efficiently than hemin, grouped gene ontology and pathway annotation networks. Bioinformatics 25(8): inorganic iron, or hemoglobin. Bioorg Med Chem 16(16):7853–7861. 1091–1093.

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