DihydroartemisininâFerriprotoporphyrin IX Adduct Abundance in

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Dihydroartemisinin - Ferriprotoporphyrin IX Adduct Abundance in Plasmodium falciparum Malarial Parasites and Relationship to Emerging Artemisinin Resistance Laura E Heller, Eibhlin Goggins, and Paul David Roepe Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00960 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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Biochemistry

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

Laura E. Heller, Eibhlin Goggins and Paul D. Roepe* Departments of Chemistry and of Biochemistry & Cellular & Molecular Biology Georgetown University 37th and O Streets NW Washington DC 20057

* Address

all correspondence to PDR: [email protected]

Keywords: Delayed Clearance Phenotype; Endoperoxide activation

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ABSTRACT Previously (Heller L. and Roepe, P.D.; preceeding paper, this issue) we quantified free ferriprotoporphyrin IX (FPIX) heme abundance for control vs delayed clearance phenotype (DCP) intraerythrocytic Plasmodium falciparum malarial parasites.

Since

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 vs 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 vs the stage of intraerythrocytic development. Using matched isogenic parasite strains we quantified adduct for DCP vs control parasite strains and find that mutant PfK13 mediates lower adduct abundance for DCP parasites. The results suggest improved models for the molecular pharmacology of artemisinin-based antimalarial drugs and the molecular mechanism of the DCP.

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INTRODUCTION Plasmodium falciparum malaria remains a threat to more than 1/2 the world's population and kills more than 1/2 million annually. Antimalarial drugs have been, and remain, the most effective treatment for malaria. Since resistance to quinoline - based and antifolate antimalarial drugs compromises their use in many areas of the globe, the currently recommended treatments for P. falciparum malaria are several available "artemisinin combination therapies" (ACTs). These combine the use of an artemisinin (ART) - based drug (e.g. the "parent" drug) with one of several possible "partner" drugs. The most commonly used ACT is CoArtem, which is comprised of artemether (ATM, the ether derivative of the natural product ART) and lumefantrine (LF). Other examples include dihydroartemisinin + piperaquine (DHA / PPQ) and artesunate + mefloquine (ATS/MQ). Although some data for ART are conflicting1 all ART - based drugs, save one (artemisone) presumably convert to DHA. Rates of conversion vary (e.g. ART vs ATM, ATS) but conversion is not necessarily metabolic, rather, at least for ATS and ATM it is known to be spontaneous in plasma and other aqueous media. Much more data (particularly for ART, which also decays to deoxy-ART under acidic conditions1) is needed to better understand relative spontaneous conversion rates.

In the clinic,

detailed study of the plasma ratios of ART/DHA, ATM/DHA or ATS/DHA at different times post ACT treatment has not been done, but available data suggest that, once administered to malaria patients, common ACTs reduce to predominantly DHA + partner drug within approximately 20 minutes.2

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Currently ACTs are the only universally effective treatments vs all malaria worldwide. Troublingly however, a harbinger of ART drug resistance has emerged in South East Asia,3,4 referred to as the "delayed clearance phenotype" (DCP). Shifts in either ART cytostatic potency (quantified as IC50) or cytocidal (parasiticidal) potency (quantified as LD50)5 have not generally been observed for parasite isolates cloned from malarial patients exhibiting a DCP, although one intriguing report does find LD50 shifts for ring stages of laboratory cultured strains of DCP P. falciparum.6

DCP is more

routinely defined by clinical criteria.4,7,8 In brief, patients infected with DCP malaria are typically still cured by ACTs, but the profound ~ 3 logarithmic drop in parasitemia that typically occurs in ~ 3 hrs upon ACT administration requires hours' longer treatment (e.g. 5-6 hrs to occur). In the laboratory DCP is quantified in one of several ways, most popularly via a ring stage assay (RSA) that uses very young ring stage parasites (0 - 3 hr post invasion) and that involves bolus dosing of cultured parasites with higher levels of ART - based drug (corresponding to those found in human plasma, not to laboratory IC50 levels; ≥ 700 nM is typically used), typically in the absence of relevant ACT partner drug.9 These assays clearly show that most ART - based drugs have reduced potency vs DCP parasites,10 but whether DCP parasites with reduced ring stage susceptibility to DHA are also resistant to common ACT partner drugs (e.g. LF, PPQ, MQR) is often not known.

Thus, although often also referred to as "ART resistance" (ARTR), DCP

actually refers to a spectrum of complex resistance phenomena that continue to evolve. Although the molecular mechanism of DCP is currently unknown, one class of genetic markers for DCP have recently been identified. These are single nucleotide polymorphisms (SNPs) in the pfk13 gene of P. falciparum, leading to single amino acid

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substitutions in the encoded PfK13 protein. The function of wild type PfK13 protein is unknown, although one hypothesis is that the protein may play a role in protein folding and / or ubiquitinylation.11 How the function of mutated PfK13 promotes DCP is unknown. One recent study suggested12 that PfVps34 (the sole P. falciparum class III phosphatidyl inositol 3' kinase,13) forms a complex with mutant PfK13 and that mutant PfK13 may indirectly affect phosphatidyl 3' phosphate (PI3'P) production by PfVps34. Via this model, the activity of ART drugs was proposed to be due to non covalent binding to PfK13, stabilized via hydrogen bond interactions involving an intact ART endoperoxide bridge. However, to our knowledge, all other studies indicate that the activity of ART based drugs is dependent upon cleavage of the drugs' endoperoxide bridge to generate highly reactive carbon centered radicals that are then capable of alkylating numerous targets within the parasite.14-16 Indeed, two recent reports identify many dozens of protein targets for endoperoxide - cleaved, or "activated", ART - based drugs,17,18 but there is only partial overlap between the two sets of proteins identified in these studies (123 protein targets are found in [18], 49 in [17], with 26 overlapping between the two sets). This suggests that alkylation of protein targets within the parasite may be somewhat random. Consistent with these observations, inhibition of PfVps34 by ART based drugs was recently shown to in fact also require activation of the drug endoperoxide and covalent binding of drug to PfVps34, with sites of covalent DHA PfVps34 interaction appearing to be random.19 In addition to the PfVps34 target hypothesis, due to the requirement for Fe2+ in catalyzing endoperoxide activation of DHA and other artemisinin - based drugs, it has

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also been hypothesized that FPIX heme released during parasite Hb catabolism could be an important target of ART - based drugs.16,18,20-23

That is, the most biologically

relevant means of activating DHA and other ART - based drug endoperoxide groups to create carbon centered radicals capable of alkylating ART targets is via Fe2+ catalyzed Fenton chemistry.14,24

In the malarial parasite, the by far most abundant potential

source of Fe2+ is ferriprotoporphyrin IX (FPIX) heme, released during obligate hemoglobin (Hb) catabolism by the intraerythrocytic parasite. FPIX Fe2+ is as potent as free Fe2+ in activating the ART drug endoperoxide bridge.19 To the best of our knowledge, free Fe2+ does not exist in eukaryotic cells, however, levels of "labile" iron are 2 - 5 µM.25,26 In contrast, concentrations of iron in the form of FPIX can exceed 40 mM within the malarial parasite DV.27 Taken together, these observations predict that FPIX is the primary activator of ART - based drugs within P. falciparum. Also, considered along with the known short lifetime and highly reactive nature of ART - based drug carbon centered radicals, this also predicts the formation of drug FPIX covalent adducts during ART - based drug activation by FPIX liberated within the DV of P. falciparum. Such FPIX mediated DHA activation and subsequent FPIX - DHA adduct formation is easily observed in vitro using pure drug, pure heme, and adequate reducing agent to form ferrous FPIX, but such adducts have not previously been isolated from live P. falciparum parasites. ART - heme adduct has however been observed in the blood of P. vinckei vinckei infected mice after ART treatment16 suggesting that adduct could possibly be formed within some rodent malarial parasites. ART drug alkylation of FPIX would inhibit detoxification of free FPIX to inert Hz, and would lead to the build up of toxic drug - FPIX adducts that would then have a

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variety of additional effects on the parasite. In this paper we test whether FPIX-DHA adducts are formed within P. falciparum parasites, test whether their abundance varies vs the stage of intraerythrocytic parasite development, and test whether differences in their abundance exist for control vs DCP parasites. The data suggest improved models for ART drug activity vs malarial parasites and for emerging artemisinin resistance.

METHODS Materials All chemicals were reagent grade or better, purchased from commercial sources, and used without further purification. Red blood cells and matched human serum for parasite culture were from Valley Biomedical (Winchester, VA). Model Compound Studies To probe DHA alkylation of FPIX heme via UV - vis spectroscopy, FPIX as hemin-Cl (200 µM) was dissolved in acetate buffer (250 µM) / 40% DMSO. Glutathione (GSH) was added to a final concentration of 1 mM and absorption spectra were acquired immediately. The cuvette was capped to prevent the entry of additional oxygen. UV spectra (200-1100 nm) were acquired every 5 min until heme was fully reduced, i.e. when the clear Fe3+ heme peak had completely disappeared and was replaced by the Fe2+ heme spectrum (see Results). ATS or DHA in pure DMSO were then added (to a final 1:1 molar ratio of drug:heme), and absorbance was immediately measured. The cuvette was again capped and absorbance was measured every 5 min for the first 90 min, and then every 30 min for 12 hr more. In order to monitor production of the final adduct (FPIX-Fe(III)-C4-DHA; see Results), different reducing conditions were explored.

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In some experiments dithiothreitol (1 mM) was used in place of GSH and found to be more efficient. After 5 hr (and formation of species B-D; see Results), the cuvette was uncovered in order to allow the entry of oxygen and final measurements were again taken at regular intervals over several hr to reveal the final oxidized adduct (see Results).

Malarial Parasite Culture Live parasite cultures of P. falciparum strains CamWT, CamWT-C580Y, Cam3.11R539T and Cam3.11-R539T-Revertant28 were maintained essentially as described.27,29

Live Parasite Drug Treatment In aqueous solution the clinically more useful and more widely used ART derivatives artemether (ATM) and artesunate (ATS) quickly and spontaneously convert to dihydroartemisinin (DHA). In patients, DHA exhibits a relatively short half life of approximately 1 hr.2 To model clinical exposure of control vs DCP parasites to ART based drugs, at different points across the parasite life cycle beginning with 8 hours post invasion (mid ring stage), 30 mL of synchronized culture at 8 - 9 % parasitemia and 2% Ht was bolus dosed with plasma levels of DHA (5 µM, see Results)30,31 for 6 hr. Mass populations of infected RBC (iRBC) (2% Hct and 8.75% P) were then fractionated to isolate parasites. Each isolation time point included at least two independent experiments (two independent parasite culture growths) with at least two replicates (two independent adduct measurements) of each experiment, for ≥ 4 determinations in total.

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Biochemistry

Extraction and Purification of FPIX and Hz Extraction and purification of free FPIX heme in drug adduct form was as described in the previous paper, this issue27. In brief, after 5 µM DHA bolus dose, parasites were lysed with 0.1% saponin, washed 3x with ice cold PBS and once more with 150 µL PBS / 50 µL H2O / 50 µL 1M HEPES, pH 7.3 to yield purified parasites containing no residual Hb. Samples were incubated with shaking in 400 µL 4% SDS / 0.1 M NaHCO3, pH 9.1, centrifuged to pellet Hz, which was washed 3x with bicarbonate buffer and stored at 4oC. 5% guanidinium-Cl was added to the supernatant containing free FPIX and FPIXDHA adduct to precipitate SDS. The mixture was centrifuged, supernatant removed, and the pellet extracted with 1.5 mL of 2:1 CHCl3:H2O. The organic layer was saved, dried en vacuo, and extracted FPIX and FPIX-DHA adduct re - dissolved in LCMSgrade MeOH / 0.1 % formic acid for LCMS Analysis.

Mass Spectrometry Free FPIX heme samples extracted from parasites were prepared for LCMS as described.27 Mass spectrometry was also as described27 and standard curves for quantification of drug - FPIX adducts were generated in the following manner: standard solutions of FPIX-DHA adduct were made by incubating FPIX, GSH, and DHA in a 1:10:1 molar ratio overnight in 40% DMSO solution as described under "Model Compound Studies", above. The ratio of known adduct concentration and adduct MS peak areas (major peak m/z 881; see Results) were plotted as described for underivitized FPIX27.

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RESULTS Several studies have demonstrated formation of covalent ART drug - FPIX heme adducts in vitro and have provided chemically logical schemes for the overall reaction leading to adduct formation.32-34 In brief, activation of the ART endoperoxide bridge by reduced FPIX Fe2+ leads to generation of an oxy - centered ART radical that then rearranges to yield an ART carbon centered radical (most commonly the "C4" centered radical) that then attacks one of three preferred meso carbons on the tetrapyrrole ring of FPIX33 to form a covalent adduct.

Spectroscopic evidence for predicted reaction

intermediates is rare however. To validate and further illuminate the proposed reaction scheme (Fig. 1)32,33 we monitored reaction of DHA with FPIX heme by UV - vis spectroscopy under conditions that both stabilize FPIX heme in monomeric form and that would slow conversion between putative intermediates (so that UV - vis spectra could be better resolved).

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Biochemistry

C

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Fig. 1 Intermediates in the production of FPIX-DHA covalent adduct. The first step in the formation of FPIX-DHA adduct is the reduction of Fe(III)FPIX (A) to Fe(II)FPIX (B) by glutathione, followed by the addition of DHA (2) which instantly forms an iron-oxo intermediate (C). Intramolecular rearrangement (3) gives the covalent complex (D) and final oxidation (4) yields (E).

Reduction of ferric FPIX with 5 x molar excess GSH is easily monitored by UV vis spectroscopy (Fig. 2) with a dramatic sharpening and redshift of the heme Soret band (present at 380 nm when FPIX is dissolved in aqueous DMSO solution) to 419 nm. Upon addition of equimolar DHA, heme absorbance immediately broadens and blueshifts to 387 nm as the ferric FPIX-DHA oxo radical dative complex ("C" Fig. 2) is formed. This intermediate is relatively short lived and rearranges to form the ferrous FPIX - C4- DHA covalent complex ("D" Fig. 2) which absorbs maximally near 421 nm. NMR data clearly show that drug deacetylation occurs during rearrangement of the oxo 11


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radical to the C4 radical and formation of the FPIX - C4- DHA complex (Fig. 2C vs 2D; LH, PDR, and Angel de Dios, unpublished; see also Meunier et al.16,33,35.) Finally, slow re oxidation of FPIX Fe2+ in complex D results in the final oxidized FPIX - DHA adduct ("E" Fig. 2) with reduced absorbance near 408 nm. Importantly, the first and third steps of the reaction (Fig. 1) that generate B and D from A and C, respectively, are both highly pH dependent with rates slower at more alkaline pH (data not shown) due to H+ dissociation associated with GSH/GSSG cycling and DHA carbon - FPIX meso carbon single bond formation, respectively.

A more detailed analysis of these pH

dependencies will be presented elsewhere.

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Biochemistry

Figure 2. Absorbance spectra of FPIX heme in the absence vs presence of GSH (A,B) and after the addition of DHA (C-E; see text). Structures corresponding to the observed spectra are shown to the right.

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Production of the ultimate FPIX-DHA adduct "E" is easily monitored by LCMS as well. After reacting fully reduced ferrous FPIX with equimolar DHA to completion, and extraction of the solution using the same protocol developed for extraction of heme from parasites27 a peak characteristic of adduct at 881 m/z (Fig. 3A), is clearly visible.

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Fig 3A. MS data for FPIX-DHA adduct synthesized in vitro using FPIX, GSH, DHA and excess phosphatidyl choline (PC). Adduct was extracted from solution using 2% SDS and 5% guanidinium-Cl to mimic the FPIX extraction process used for live parasites (see Methods).

Our perfected FPIX heme extraction protocol27 entails treatment with excess sodium dodecyl sulfate followed by precipitation of detergent with guanidinium hydrochloride. In our hands, these treatments result in loss of H+ and addition of one Na+ (coordinated to tetrapyrrole proprionate) to yield the observed m/z of 881 (Fig. 3B).

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N

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FPIX (Fe 2+, "B")

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+Na -H

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Fig 3B. MS ionization of FPIX-DHA adduct synthesized in vitro to yield m/z = 881, see text.

When FPIX is reacted in vitro with artesunate (ATS) instead of DHA (mass of 384 Da for ATS vs 284 Da for DHA) the 881 peak shifts to 981 m/z (data not shown) consistent with this interpretation. When FPIX is not reduced with GSH prior to addition of DHA, no adduct is formed (Fig. 3C). 100

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Fig 3C. MS spectrum of the product of FPIX + DHA without addition of GSH (see text). No adduct 881 m/z is observed (inset=adduct region).

Following a similar approach as in the previous paper27 for FPIX quantification, quantification of FPIX - DHA LCMS data is shown in Fig. 3D, and yields a linear relationship similar to that found previously for underivitized FPIX heme. 2500

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Fig. 3D LCMS / DHA - FPIX adduct calibration curve, R2 = 0.90.

We next tested for the presence of FPIX - DHA adducts in live malarial parasites treated with bolus dose DHA for 6 hr (see Methods).

As shown in Fig. 4A, using

essentially the same extraction procedure as that used for FPIX,27 adduct is easily found for trophozoite stage CamWT parasites (Fig. 4A; note clear peak at 881 m/z characteristic of adduct extracted under these conditions (Fig. 4A inset; compare to Fig. 3A).

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881.3

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m/z Figure 4A. Adduct isolated from the parasite after bolus dose of 5 µM DHA for 6 hr (see Methods). Heme species are seen at m/z 616.3 (M+), 557.2 (M-C2H3O2), and 659.4 (M-2H +2Na).33,58 Phospholipid species is seen at m/z 760.6, and FPIX-DHA adduct is seen at m/z 881.3 (compare to Fig. 3A). Inset adjusts LCMS range to the narrower window of m/z 800-920 corresponding to the adduct region. Parameters for inset data collection were adjusted for visual representation to optimize the signal at m/z 881, with capillary voltage increased to 147.0 V and RF loading decreased to 105%.

Importantly, the efficiency of CHCl3 extraction of FPIX-DHA adduct is similar to the efficiency of extraction of free FPIX27 and is near 100% (Fig. 4B).

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1150

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Fig 4B. Calibration curves for known amounts of FPIX heme (squares) and FPIX-DHA (triangles) after extraction using SDS, guanidinium-hydrochloride, CHCl3 as described (see text), followed by drying and re dissolving in MeOH. The data show that the extraction procedure used in this work extracts known amounts of FPIX and FPIX-DHA adduct to near completion and with equal efficiency.

FPIX-DHA adduct is also easily extracted for ring and schizont stage CamWT parasites (Fig. 5A,E, left hand side) treated with DHA in the same manner, however absolute amounts are lower presumably due to the lower abundance of free FPIX at these stages.27 Due to the altered abundance of free FPIX found for DCP parasites relative to control,27 we synchronized DCP parasites and extracted FPIX - DHA adduct using identical methods. As expected, since FPIX abundance is significantly reduced at the trophozoite stage for Cam580Y (C580Y) relative to CamWT27, significantly less FPIX - DHA adduct is found at the trophozoite stage for Cam580Y treated identically to CamWT (Fig. 5D vs 5C; and Fig. 6) However, when adduct is extracted from identically

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treated ring or schizont stage parasites, surprisingly, less adduct is again found for CamC580Y DCP parasites relative to CamWT control (Fig. 5B vs 5A; Fig. 5F vs Fig. 5E; and Fig. 6) even though the DCP parasites show slightly elevated levels of free FPIX at these stages.27

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Ca mWT-C580Y

Figure 5. Heme-DHA Adducts measured by LCMS for Mid-Ring (A,B), Trophozite (C,D), and Schizont (E,F) stages of control CamWT (left) and DCP CamWT-C580Y (right) parasites. Asterisks indicates position of m/z 881 FPIXDHA adduct peak. 20


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To test the association between altered FPIX - DHA adduct levels and the DCP, a second DCP strain, Cam3.11 R539T as well as its isogenic control strain that does not harbor PfK13 mutations (strain Cam 3.11 R539Trev)28 were also analyzed. As shown in Fig. 6, although absolute abundance differs somewhat relative to the control

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[Adduct in Parasite] (µM)

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20

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10

5

0

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Cam3.11-Revertant CamWT-C580Y

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Figure 6. Quantification of FPIX-DHA adduct for control CamWT and Cam3.11R539T-Revertant strains vs DCP CamWT-C580Y and Cam.311-R539T parasites at mid-ring (black bars), trophozoite (blue bars) and schizont (green bars) stages. Data (+/- S.E.M.) represent at least two independent experiments (two independent parasite culture growths) with at least two replicates (two 21


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independent adduct measurements) of each (≥ 4 determinations in total). All stage - dependent differences between either DCP parasite and its matched isogenic control are statistically significant (p < 0.05; student's t test).

CamWT and DCP strain Cam580Y strains, a similar trend of reduced adduct abundance at all intraerythrocytic developmental stages is found for DCP strain Cam 3.11R539T relative to its genetically matched control, strain Cam3.11R539TRevertant. Upon quantifying these data vs those in the previous paper27 we find that, depending on the strain, 0.3 - 0.5 % and 0.6 - 1.2 % of total free FPIX present at the trophozoite and ring stages, respectively, are converted to FPIX - heme adduct. Finally, to test whether FPIX-DHA adduct is incorporated into or associated with Hz crystals, Hz from DHA treated parasites was isolated and solubilized as described27. LCMS data reveal only FPIX monomer and reciprocal dimer for these samples (Fig. 7) without measureable FPIX-DHA adduct or FPIX dimer - DHA adduct (note lack of peak at 881 m/z, Fig. 7).

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700 616.5

600

Intensity (kCounts)

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500

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659.5

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1231.9 1254.5

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0 400

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m/z Figure 7. Mass spectrum of FPIX from Hz isolated from CamWT parasites treated with DHA as described, after solubilizing Hz using NaOH.29 Both FPIX monomer (616.4) and reciprocal dimer peaks at 1254.5 and 1231.9 [2*FPIX – 2H, + Na] and [2*FPIX -H], respectively29 are observed, but no FPIX-DHA adduct is observed (compare to Fig. 4A).

DISCUSSION

The results of this study may be summarized as follows: 1) A covalent FPIX-DHA adduct, similar to what is easily produced in vitro using mixtures of pure DHA and pure FPIX in the presence of appropriate reducing agent, is

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also produced within live intraerythrocytic P. falciparum malarial parasites treated with plasma levels of DHA. 2) The relative abundance of FPIX-DHA adduct varies vs the stage of intraerythrocytic parasite development, with the largest abundance found at the trophozoite stage, corresponding to the stage that harbors the largest concentration of free FPIX heme within the parasite DV. 3) The relative abundance of FPIX-DHA adduct is lower for DCP parasites vs matched isogenic control strains, at all stages of parasite development. 4) Although FPIX-DHA adduct is formed within live parasites, no appreciable DHA or FPIX-DHA adduct is found incorporated in Hz isolated from DHA - treated parasites. It has been debated for some time whether FPIX heme is the primary activator of artemisinin prodrugs to produce artemisinin drug radicals, and whether covalent artemisinin drug - FPIX heme adducts are formed within P. falciparum malaria. This study provides important data on both points, and is the first to our knowledge to directly measure FPIX-DHA adduct for live P. falciparum. One previous study reported the presence of heme - ART adduct in the blood stream of mice infected with P. vinckei vinckei malaria and subsequently treated with ART16 but the origin of heme within the adduct, and the site of adduct formation, were not determined. Here we show that adduct FPIX is clearly formed within the P. falciparum parasite and that adduct is likely formed within the parasite DV. FPIX-DHA adduct is formed within live parasites treated with clinically relevant plasma levels of DHA (these initial experiments are done at 5.0 µM DHA for 6 hrs;

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Biochemistry

plasma DHA ranges from 0.5 µM - 5 µM and perhaps even higher30,31).

This

observation has very important implications for the mechanism of action of ART drugs. FPIX-DHA adducts are likely quite toxic36, with toxicity similar to FPIX alone, and perhaps even more toxic since the DHA moiety attached to FPIX would be predicted to improve membrane permeability properties allowing wider organellar access for FPIXDHA relative to underivitized FPIX. We find that FPIX-DHA adduct abundance is lower for two DCP parasites expressing two different DCP associated mutant PfK13 proteins vs their matched isogenic control parent strains, at all stages of parasite development. The observation of lower adduct for DCP trophozoites vs control trophozoites is not entirely surprising since DCP trophozoites harbor reduced levels of free FPIX relative to control trophozoites.27 However, the observation of reduced adduct for DCP rings and schizonts is curious, since DCP parasites harbor slightly increased levels of free FPIX relative to control parasites at these stages.27 This point is discussed further below. We propose that the lower abundance of ring stage FPIX-DHA adduct for DCP parasites is a key feature of the molecular mechanism of evolving artemisinin resistance. Importantly, lower abundance is found for two different DCP strains harboring two different DCP - associated PfK13 mutations, suggesting that lower adduct abundance is a general feature of PfK13 - mediated DCP. However, for lower FPIX-DHA adduct to be correlated with DCP, two riddles must be explained. First, toxic FPIX-DHA adduct is more abundant for trophozoites, yet several studies have found that ART drugs are more potent vs rings relative to trophozoites, and second, most studies have found that the reduced potency of ART

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drugs seen for DCP parasites is specific to the ring stage.37,38 If FPIX-DHA adduct is more abundant for trophozoites, how is ring stage ART drug selectivity explained ? Obviously much more work remains to be done related to this point but we propose that FPIX-DHA adduct targets metabolic processes specific to the ring more than metabolic processes specific to the trophozoite.

Possibilities include dramatically upregulated

transcription and translation that occurs in the ring stage, early ring stage Hb catabolism that may differ from trophozoite state catabolism37, ring stage membrane biogenesis, and/or organellar development. If adduct affects these processes, then even small differences in the level of ring stage adduct abundance could conceivably confer DCP. It is well known that DCP parasites do not show shifts in drug IC50 similar to how chloroquine resistant (CQR) parasites show 10 - fold shifts in chloroquine (CQ) IC50 relative to control.

This predicts that the decrease in adduct abundance for DCP

trophozoites is not significant enough to have an equally profound effect on growth inhibition. With regard to this point, we note that adduct abundance corresponds to approximately 0.3 - 0.5 % of the total amount of free FPIX present at the trophozoite stage (depending on the strain of parasite), however, adduct abundance corresponds to 0.6 - 1.2 % of the total amount of free FPIX present at the ring stage. We suggest that the greater percent abundance for rings is sufficient to promote ring stage quiescence as previously observed,40,41 but not to shift drug IC50. With regard to the second riddle, adduct is found to be between 1-25 µM depending on stage and strain; for CamWT trophozoites, it is ~ 20 µM. We propose that an adduct toxicity threshold is achieved for control strain rings and that this threshold is well overachieved for trophozoites. Via this reasoning, the decrease in

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Biochemistry

trophozoite adduct abundance for DCP parasites then, although significant, still leaves > 5 µM levels of adduct present for DCP trophozoites, presumably still above this threshold, leading to only small effects on ART drug toxicity for trophozoites.

In

contrast, ~ 5 µM adduct is found for control strain ring stages, and ~ 2.5 µM for DCP ring stages. We propose that the decrease in DCP ring adduct moves the level of FPIXDHA adduct below the threshold relevant for toxic effects leading to quiescence, whereas copious remaining adduct in the DCP trophozoite or schizont is still sufficient to kill parasites at these stages. This idea predicts that evolution of heightened DCP correlated with further decreases in adduct abundance at these stages, would promote formal ARTR. Interestingly, we find a lower abundance of FPIX-DHA adduct for DCP rings and schizonts relative to control even though DCP rings and schizonts show increased levels of free FPIX at these stages relative to control. With regard to this apparent paradox we note that the level of free FPIX is not necessarily the relevant moiety for activating ART based drugs, rather it is the level of ferrous (Fe2+)FPIX that is relevant. Ferrous FPIX is either provided upon immediate release of FPIX (as Fe2+FPIX) from catabolized Hb, or upon reduction of the more abundant pool of FPIX within the parasite DV (likely ferric (Fe3+)FPIX). The principle reductant that would control Fe2+ / Fe3+ ratios for any eukaryotic cell is GSH. Reduced GSH is capable of donating e- to Fe3+ (Fig. 2), and in the process oxidizes to GSSG (the GSH dimer). GSH/GSSG ratios are typically 20 or higher for eukaryotic cytosol (the cytosol is highly reducing) whereas they are typically closer to 1 for lysosomes or vacuoles.42 This predicts that the ratio of ferrous to ferric FPIX within the DV is low, yet, ferrous FPIX is not zero. We estimate that the

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concentration of total FPIX is near 4 mM for control trophozoite DV.27 If even a modest 1 % of total FPIX is reduced by available GSH, this predicts 40 µM ferrous FPIX heme available for activating DHA. In vitro, we and others19,43 find that DHA is activated to near completion in the presence of ferrous FPIX. Therefore, µM levels of ferrous FPIX within the DV are more than adequate for generating the levels of adduct that we observe. Lower levels of adduct for DCP rings, in spite of higher total free FPIX relative to control, suggests a change in either release of ferrous FPIX from catabolized Hb, or in GSH / GSSG traffic near the site of free FPIX (presumably, the nascent ring DV). Indeed, other work has found altered GSH / GSSG cycling linked to ART - based drug resistance phenomena.44,45 We suggest that once the DHA endoperoxide is activated (by FPIX Fe2+), it's proximity to FPIX, stabilized somewhat by a Fe-oxo radical bridge (Fig. 2B), confers "preferred target" status to free FPIX, leading to efficient adduct formation within P. falciparum as observed. However, as shown earlier17,18 other targets for DHA adduct formation are clearly possible within the parasite, but it is currently not known what percent of these targets are effectively alkylated, or how they would exert toxic effects sufficient to quickly kill the parasite. Much work clearly remains to be done to define the rank order of DHA "preferred targets". Here, FPIX-DHA adduct is found to be an abundant intracellular poison for DHA treated P. falciparum whose level is correlated with DHA sensitivity in DCP vs matched isogenic control strains. To the best of our ability to ascertain, all known DCP parasites evolve within a chloroquine resistant (CQR) background. That is, although ARTR can be selected in the laboratory via other pathways, to our knowledge, all naturally occurring DCP

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parasites that have been studied to date are also CQR. Association between CQR and DCP status could simply be a consequence of the fact that most parasites found in southeast Asia (S.E.A.), where DCP largely originates, are CQR, lowering the statistical likelihood of finding CQS/DCP parasites. However, the CQR phenotype is known to be characterized by the presence of one of many different possible CQR - associated mutant PfCRT proteins,46 as well altered digestive vacuolar (DV) physiology, including changes in DV volume and pH.47-50 Whether CQR - associated alterations in DV physiology are obligate, permissive or merely coincidental for development of DCP parasites is not known, however, interestingly, some studies have suggested that some PfCRT mutations may in some way facilitate PfK13 - mediated DCP.47 Along these lines, CQR parasites are measured to have lower DV pH and other changes in DV physiology,48-53 which are predicted to alter the availability of soluble FPIX. Changes in DV physiology measured for the CQR parasite DV might contribute to DCP via affects on FPIX-DHA solution chemistry. In addition, studies have suggested that PfCRT and a PfCRT plant ortholog may transport GSH.54,55 If so, perhaps some mutant isoforms of PfCRT associated with S.E.A. CQR phenotypes facilitate important alterations in GSH/ GSSG traffic that help to bias ferrous / ferric FPIX ratios. Lower ratios would act to reduce adduct abundance by lowering [FPIX(Fe2+)] necessary for ART - based drug activation, and thereby facilitate emergence of DCP. We note that a previous study has suggested higher levels of GSH in some CQR parasites but no direct measurements of GSH were made in this work and subcellular localization of any increased GSH was not determined56.

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Finally, we note that, formally, there are two possible sources for reduced FPIX: either DV localized, or DV in origin but cytosolically localized after diffusion of DV FPIX. The cytosol is a highly reducing environment that would strongly bias FPIX towards the ferrous form. Formally then, to explain lower adduct abundance, DCP parasites might harbor reduced concentrations of DV GSH or show reduced transport of free FPIX from the DV to the cytosol. Relevant to this point, a recent study finds ~ 1.5 µM heme within the cytosol that does not change across the parasite life cycle,57 suggesting that the large differences in free FPIX and FPIX-DHA adduct that we observe across life cycle stages are clearly due to DV localized FPIX.

Acknowledgements

This work was supported by the NIH (grant AI111962 to PDR) and the Dept. of Chemistry, Georgetown University. We thank Ms. Esther Wisdom for experimental help and our colleagues A. de Dios, M. Hassett, D. Sullivan, and G. Posner for many helpful discussions.

Dedication

This paper is dedicated to the memory of Professor Gary Posner, a generous and thoughtful colleague, pioneer in organocopper chemistry, and leader in the study of artemisinin chemistry.

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S.

Cobbett

(2010)

falciparum chloroquine-resistance

Plant

homologs

transporter, PfCRT,

of are

the Plasmodium required

for

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artemisinin-type and Fe(III)-heme by electrospray mass spectrometry and collisional activation tandem mass spectrometry. J. Am. Soc. Mass. Spec. 15, 1181-1190.

FOR TABLE OF CONTENTS USE ONLY

N

N

H

Fe N

N

HO HO H

O

O OH

OH

O

ONa

38


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