New Insight into the Mechanism of Accumulation and Intraerythrocytic Compartmentation of Albitiazolium, a New Type of Antimalarial

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Sharon Wein, Christophe Tran Van Ba, Marjorie Maynadier, Yann Bordat, Julie Perez, Suzanne Peyrottes, Laurent Fraisse and Henri J. Vial Antimicrob. Agents Chemother. 2014, 58(9):5519. DOI: 10.1128/AAC.00040-14. Published Ahead of Print 7 July 2014.

New Insight into the Mechanism of Accumulation and Intraerythrocytic Compartmentation of Albitiazolium, a New Type of Antimalarial Sharon Wein,a Christophe Tran Van Ba,a Marjorie Maynadier,a Yann Bordat,a Julie Perez,a Suzanne Peyrottes,b Laurent Fraisse,c Henri J. Viala

Bis-thiazolium salts constitute a new class of antihematozoan drugs that inhibit parasite phosphatidylcholine biosynthesis. They specifically accumulate in Plasmodium- and Babesia-infected red blood cells (IRBC). Here, we provide new insight into the choline analogue albitiazolium, which is currently being clinically tested against severe malaria. Concentration-dependent accumulation in P. falciparum-infected erythrocytes reached steady state after 90 to 120 min and was massive throughout the blood cycle, with cellular accumulation ratios of up to 1,000. This could not occur through a lysosomotropic effect, and the extent did not depend on the food vacuole pH, which was the case for the weak base chloroquine. Analysis of albitiazolium accumulation in P. falciparum IRBC revealed a high-affinity component that was restricted to mature stages and suppressed by pepstatin A treatment, and thus likely related to drug accumulation in the parasite food vacuole. Albitiazolium also accumulated in a second high-capacity component present throughout the blood cycle that was likely not related to the food vacuole and also observed with Babesia divergens-infected erythrocytes. Accumulation was strictly glucose dependent, drastically inhibited by Hⴙ/Kⴙ and Naⴙ ionophores upon collapse of ionic gradients, and appeared to be energized by the proton-motive force across the erythrocyte plasma membrane, indicating the importance of transport steps for this permanently charged new type of antimalarial agent. This specific, massive, and irreversible accumulation allows albitiazolium to restrict its toxicity to hematozoa-infected erythrocytes. The intraparasitic compartmentation of albitiazolium corroborates a dual mechanism of action, which could make this new type of antimalarial agent resistant to parasite resistance.

M

alaria is still a major health problem, and the spread of resistance of Plasmodium falciparum, the most virulent human parasite, to current drugs, recently including artemisininbased combination therapy (1–3), is a major obstacle to the eradication of the disease. Few new approaches with a novel mechanism of action have been designed and thus are not found in the current international research pipeline (4). Plasmodium synthesizes considerable amounts of membranes for its growth and proliferation inside host cells. At its blood stage, phospholipids, which constitute the bulk of malarial lipids (5–9), mostly originate from the Plasmodium-encoded enzymatic machinery (7, 10). In this setting, the inhibition of phospholipid biosynthesis has been proposed as a novel therapeutic strategy (11– 13). The most advanced approach is based on the use of choline analogues (12, 14–17), whose primary interference has been associated with blocking the parasite choline carrier, thus preventing phosphatidylcholine biosynthesis (13, 18). Bis-thiazolium salts exert a rapid cytotoxic effect against all P. falciparum blood stages in vitro (17, 19) and cure in vivo malaria infections without recrudescence at very low doses for rodent malaria (⬍1 mg/kg of body weight delivered intraperitoneally) and primate malaria (17). The lead compound, albitiazolium (formerly named T3/SAR97276) (17) (Fig. 1), is currently being evaluated in phase 2 clinical trials by the parenteral route for the treatment of severe malaria. Bisthiazolium salts are also powerful in the low nanomolar range (20) against Babesia, another hematozoan parasite which also belongs to the Apicomplexa phylum. One prominent feature of choline analogues is their ability to irreversibly accumulate in P. falciparum-infected erythrocytes (17,

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21, 22) and in B. divergens-infected erythrocytes (20), with the major part of the accumulated compound being recovered in the intracellular parasite (21). This accumulation restricts compound toxicity to hematozoa-infected erythrocytes, being pharmacologically active at a low nanomolar concentration, while high micromolar concentrations are required against mammalian cells (17, 20). Accumulation can thus be considered a key feature of their specificity and potency against these two hematozoan parasites. Here, we thoroughly characterized processes that mediate the accumulation of the clinical candidate albitiazolium in mammalian P. falciparum-infected host cells. This process appears to be totally energy dependent and highly altered by any change in ionic contents. Remarkably, a comparative analysis of Plasmodium and Babesia indicated that albitiazolium accumulation in Plasmodium occurred in two distinct compartments, with one probably being related to the food vacuole. Hence, the albitiazolium accumula-

Received 15 January 2014 Returned for modification 24 February 2014 Accepted 2 July 2014 Published ahead of print 7 July 2014 Address correspondence to Henri J. Vial, [email protected], or Sharon Wein, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.00040-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.00040-14 The authors have paid a fee to allow immediate free access to this article.

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Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR 5235, Université Montpellier 2, Montpellier, Francea; Institut des Biomolécules Max Mousseron, CNRS UMR 5247, Université Montpellier 2, Montpellier, Franceb; Sanofi, TSU-Infectious Diseases Unit, Toulouse, Francec

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FIG 1 Structure of the bis-thiazolium salt albitiazolium. Albitiazolium has potent activity against the in vitro growth of P. falciparum, with an IC50 (concentration of drug inhibiting parasite growth by 50%) of 2.3 nM (17). The IC50 for B. divergens was 65 nM (unpublished data).

MATERIALS AND METHODS Chemicals. Albitiazolium [albitiazolium bromide; 3,3=-dodecane-1,12diylbis[5-(2-hydroxyethyl)-4-methyl-1,3-thiazol-3-ium] dibromide] was synthesized in-house (17), and thiazole-2,2=-[14C]albitiazolium was provided by Sanofi. [3H]-chloroquine diphosphate salt was from Movarek Biochemicals and Radiochemicals. Carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and bafilomycin A1 were from Interchim. Solvable was from PerkinElmer. Other reagents were from SigmaAldrich. Ionophores and inhibitors were dissolved in dimethyl sulfixde (DMSO). The maximum DMSO concentration in the incubation medium was 0.125%. Biological materials. The P. falciparum 3D7 strain (MRA-102 from MR4) was cultured using standard methods (23) in human erythrocytes (Etablissement Français du Sang, Montpellier, France) in complete medium composed of RPMI 1640 (Gibco) supplemented with 25 mM HEPES (pH 7.4), 15 ␮g/ml hypoxanthine, and 0.5% AlbuMAX I (Gibco). Parasites were synchronized by using a 5% sorbitol treatment (24) or a VarioMACS magnetic cell separator (Miltenyi Biotech) (25). Babesia divergens (strain Rouen 1987, clone 4) was cultured in human erythrocytes at a 5% hematocrit in RPMI 1640 supplemented with 25 mM HEPES and 10% AB⫹ human serum in a 5% CO2 incubator. Standard assay for measuring drug uptake in uninfected and infected erythrocytes. Normal or infected erythrocytes with 5 to 10% parasitemia for P. falciparum and 30 to 40% for B. divergens were incubated at 1% or 5% hematocrit in medium containing [14C]albitiazolium or [3H]CQ. Unless otherwise specified, incubations were conducted in complete medium composed of RPMI 1640 supplemented with 25 mM HEPES and 0.5% AlbuMAX I (Plasmodium) or 10% human serum (Babesia), until the drug inside the infected erythrocyte was at steady state, i.e., typically 2 h for albitiazolium and 30 min for CQ. After incubation, suspensions were overlaid onto ice-cold dibutylphtalate in a microtube and centrifuged (10,000 ⫻ g, 1 min, 2°C). Radioactivity in the supernatant was counted by liquid scintillation and the supernatants were discarded. The vial walls were washed and the dibutylphtalate cushions were discarded. Cellular pellets were lysed with 100 ␮l H2O, transferred to scintillation vials, and decolorized using 200 ␮l of a cocktail of Solvable-acetic acid-H2O2 (30%) (5:2:2) before liquid scintillation counting. Cellular uptake of albitiazolium and CQ at 2°C in red blood cells (RBC) and infected red blood cells (IRBC) was nonsignificant. Albitiazolium or CQ uptake in 100% IRBC (uptakeIRBC) was expressed in pmol/107 IRBC and calculated as follows: uptakeIRBC ⫽ {uptake(RBC ⫹ IRBC, 37°C) – [(100% – P) ⫻ uptake(RBC ⫹ IRBC, 2°C)]}/P, where uptake(RBC ⫹ IRBC) is the total uptake measured in the mixed cell population and P is the level of parasitemia. The cellular accumulation ratio (CAR) is the ratio of the amount of drug within the cells to the amount of drug in the same volume of medium after incubation. CAR was calculated for IRBC on the basis of 75 fl (26), the mean volume of normal and infected human erythrocytes. For the intracellular parasite only, CAR was calculated on the basis of the assumption that 80% of the accumulated albitiazolium was located in the parasite (21) and that the parasite volume ranged from 1.7 to 41 fl accord-

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RESULTS

Albitiazolium entry into Plasmodium and Babesia IRBC. We first investigated the kinetics of albitiazolium entry at a therapeutic concentration, 125 nM. Albitiazolium uptake was not significant when normal RBC, P. falciparum IRBC (Fig. 2A), or B. divergens IRBC (data not shown) were incubated at 2°C. At 37°C (Fig. 2A), less than 0.08 pmol albitiazolium was recovered in 107 RBC after 5 h of incubation. This was less than twice the radioactive background measured at 2°C and corresponded to a cellular concentration of 107 nM and a CAR of ⬍1. On the other hand, P. falciparum IRBC took up albitiazolium to a high extent through a time-dependent and saturable process. At the ring stage, entry reached a plateau at about 1 pmol/107 IRBC after 60 min of incubation (Fig. 2A), which corresponded to an intracellular concentration of 1.33 ␮M and a CAR of around 10. At the trophozoite stage, albitiazolium uptake was much higher, reaching a plateau at 24 pmol/107 IRBC with an intracellular concentration of 32 ␮M after 90 min incubation. The CAR was already higher than 200 after 30 min of incubation, reaching a maximum of 405. Similarly, rapid albitiazolium uptake occurred in B. divergens IRBC, with maximal uptake of 2.5 ⫾ 0.2 pmol/107 IRBC (mean ⫾ standard error of the mean [SEM]) after 2 h of incubation. At the plateau, the CAR was 67, indicating high albitiazolium accumulation also in B. divergens IRBC (data not shown). For most of the following experiments, cells were incubated for 2 h, when the cellular albitiazolium concentration had reached steady state. At steady state, albitiazolium uptake in P. falciparum IRBC at the ring stage, as a function of external concentration, increased gradually up to 9 ␮M and reached 31.4 pmol/107 IRBC/2 h, corresponding to an intracellular concentration of 41.9 ␮M (Fig. 2B). At the trophozoite stage, as in the ring stage, albitiazolium uptake reached a plateau at 9 ␮M but with a much higher maximum uptake of 270 ⫾ 28 pmol/107 IRBC, indicating an intracellular concentration of 360 ␮M (Fig. 2B). Reversibility of albitiazolium uptake in Plasmodium IRBC. The maximal accumulation observed after 2 h of incubation may reflect saturation or a dynamic steady state for drug entry in infected erythrocytes that is compensated by equal exit from the cell.

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tion process differs completely from that of the well-known antimalarial chloroquine (CQ).

ing to its developmental stage (27). The volume of the Babesia divergens parasites was estimated to be 6.4 fl, based on data of Spencer et al. (28) and on the size of a human erythrocyte. Determination of the extent of albitiazolium accumulation at steady state. The amount of [14C]albitiazolium that accumulated at various concentrations, in both P. falciparum and B. divergens IRBC, was determined at steady state after 2 h of incubation. Saturation curves with nonlinear regression (GraphPad Prism) and Scatchard plots were drawn to estimate the number of maximum apparent binding sites (Bmax) and ligand affinities (dissociation constant, Kd). Nonspecific binding was determined from similar incubation mixtures that contained 10 mM unlabeled albitiazolium. Drug uptake in isolated P. falciparum parasites. Parasites were isolated from mature IRBC at 5% hematocrit by using 0.01% saponin for 1 min in cold phosphate-buffered saline (PBS; 116 mM NaCl, 8.3 mM Na2HPO4, 3.2 mM KH2PO4; pH 7.4). They were washed twice and counted in a Neubauer hemocytometer. They were then incubated at 4 ⫻ 106 to 7 ⫻ 106 parasites/ml for 30 min with 48 nM [14C]albitiazolium or 20 nM [3H]CQ in bicarbonate-free RPMI 1640 buffered with 25 mM HEPES (pH 7.4) (here called bicarbonate-free RPMI medium). The parasite suspensions were then overlaid onto a 5:4 dibutylphtalate-dioctylphtalate mixture instead of pure dibutylphtalate and treated as described above for IRBC.

Albitiazolium Partitioning in Infected RBC in Malaria

nM [14C]albitiazolium at ring () or trophozoite () stage was measured at 37°C (closed symbols) and 2°C (open symbols). Uptake into normal erythrocytes at 37°C (}) was also determined. (B) Concentration dependence of accumulation at ring () and trophozoite () stages after 2 h of incubation at 37°C. (C) Retention of accumulated albitiazolium in mature-stage P. falciparum. IRBC were preloaded for 90 min with 125 nM () or 100 ␮M [14C]albitiazolium (●) in complete medium and then washed to remove external albitiazolium and reincubated at 37°C in complete medium. At the indicated time intervals, aliquots of the suspensions (approximately 2.5 ⫻ 108 cells) were overlaid onto dibutylphtalate and then treated as described for the standard assay. Quantities of albitiazolium remaining within the cells were expressed as percentages of the amount measured after washing (time zero). In all cases, infected or normal erythrocytes were incubated at 5% hematocrit in complete medium. The results are expressed as means ⫾ SEM (n ⫽ 3) (small SEMs are hidden by the symbols) of one representative experiment.

This was investigated by measuring the drug retention within IRBC preloaded with albitiazolium. After loading IRBC with 125 nM albitiazolium, washing, and reincubating the loaded IRBC, a limited rapid release (⬍13.6% ⫾ 4.0%) occurred within the first 15 min, while further incubation until 3 h led to a very limited 7% additional release, with 79.3% ⫾ 8.4% still retained within IRBC (Fig. 2C). Most of the albitiazolium accumulated within P. falciparum IRBC at a pharmacological concentration and thus appeared to have accumulated in an irreversible way. When preloaded with an extreme concentration of 100 ␮M albitiazolium, drug release was similar within the first 15 min (11.7% ⫾ 1.0%). Thereafter, albitiazolium was still significantly

retained, since 56.3% ⫾ 6.9% of albitiazolium was still recovered in IRBC after 3 h (Fig. 2C). Albitiazolium uptake during the Plasmodium erythrocyte cycle. We then determined the extent to which albitiazolium uptake and accumulation were dependent on the parasite developmental stage by measuring its uptake after 3 h of incubation at different points during the cell cycle in a tightly synchronized IRBC suspension (Fig. 3). At 1,500 nM albitiazolium, accumulation was significant at the very early ring stage and appeared to be constant during the first 12 h of the parasite cycle, at approximately 15 pmol/107 IRBC (i.e., 20 ␮M intracellular concentration and a CAR of 13). Accumulation peaked at 36 h (schizont stage), with a maximum uptake of

FIG 3 Albitiazolium accumulation throughout the P. falciparum erythrocytic cycle. Doubly synchronized P. falciparum IRBC at the ring stage were incubated

at 5% hematocrit in complete medium. At the indicated time, aliquots of 7.9 ⫻ 107 cells (5.3% parasitemia) were incubated in 300 ␮l of complete medium for 3 h in the presence of 250 nM [14C]albitiazolium. (A) Morphology of the parasites at the beginning of each incubation and distribution of the stages for mixed populations. (B) Extent of accumulation expressed per 3-h period. (C) CAR in total IRBC after 3 h of incubation or in the parasites. The parasite volumes were obtained from Hanssen et al. (27) The results are expressed as means ⫾ SEM (n ⫽ 3).

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FIG 2 Uptake (A), accumulation (B), and reversibility (C) of albitiazolium accumulation in P. falciparum-infected erythrocytes. (A) Kinetics of uptake of 125

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FIG 4 [14C]albitiazolium accumulation in P. falciparum- and B. divergensinfected erythrocytes at steady state. (A and B) P. falciparum IRBC at ring (A) and mature (B) stages were incubated for 2 h in complete medium at 37°C (5% hematocrit and 5 to 10% parasitemia). Cells were incubated in the absence (solid line) or the presence (dashed line) of 100 ␮M pepstatin A. (C) B. divergens IRBC were incubated for 2 h in complete medium at 37°C (5% hematocrit and 20 to 25% parasitemia).The results are shown as Scatchard plots and represent data from one of at least two experiments. Nonlinear analysis of data by using GraphPad Prism software indicated one component of accumulation in the experiments shown in panels A and C, while two components appeared highly significant in the experiment in panel B. The points used for the linear regression of the high-affinity process are shown as open symbols, and those used for the second process are shown as closed symbols. The shift of the low-affinity curve in the presence of 100 ␮M pepstatin A was due to the disappearance of the high-affinity accumulation process.

Effect of ammonium chloride on albitiazolium and CQ accumulation in Plasmodium IRBC. Although albitiazolium accumulation cannot be due to a lysosomotropic process as it is for CQ (32, 33) because of the permanent cationic charges of albitiazolium (Fig. 1), we alkalinized the malaria parasite food vacuole pH by incubating IRBC with 5 mM NH4Cl, an acidotropic agent (33, 34) to identify a possible relationship with the food vacuole pH. When incubated with 48 nM albitiazolium or 20 nM CQ (35) until steady state, accumulation in IRBC at mature stages was 36.2 ⫾ 1.1 and 39.2 ⫾ 0.8 pmol/107 IRBC, respectively (Fig. 5A),

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211.7 ⫾ 8.8 pmol/107 IRBC (282 ␮M) and with the CAR increasing to 202 (data not shown). When we used a more therapeutic concentration of 250 nM, albitiazolium accumulation exhibited a similar profile, with an already substantial intracellular concentration of 5.2 ␮M at the early ring stage which increased to 50.7 ␮M when the parasites matured (Fig. 3B). The CAR ranged from 21 to 404, from the ring to schizont stages (Fig. 3C). Considering that approximately 80% of the drug appeared to be localized within the intracellular parasite (21), it is crucial to take into account that as the parasite grows, the volume of the parasite increases from 1.7 to around 41 femtoliters (27). In light of this consideration, CAR within the parasite appeared to be very high throughout the parasite cell cycle, with values ranging from 200 to 610 (Fig. 3C). For B. divergens, based on the same restriction of albitiazolium accumulation to 80% of the total accumulated drug within the intraerythrocytic parasite, the CAR appeared to be 250. Intracellular binding of albitiazolium in Plasmodium and Babesia IRBC. We investigated albitiazolium uptake at steady state when IRBC were incubated with increasing concentrations of the drug. In P. falciparum IRBC at the ring stage, analysis of the saturation binding curve by nonlinear regression and of the Scatchard plot indicated one class of binding sites with a Kd of 3.64 ⫾ 0.52 ␮M and a Bmax of 19.64 ⫾ 8.02 pmol/107 IRBC (Fig. 4A; see also Fig. S1B in the supplemental material). At mature stages, it is striking that two classes of binding sites were present. The highaffinity process showed a Kd of 0.82 ⫾ 0.08 ␮M and a Bmax of 67.47 ⫾ 22.67 pmol/107 IRBC. The lower-affinity process had a substantially lower Kd that was in the same range as that at the ring stage (11.73 ⫾ 3.07 ␮M) but a higher Bmax, 478 ⫾ 148.2 pmol/107 IRBC (Fig. 4B; see also Fig. S1C). Based on the Kd values, the low-affinity class of the accumulation process could thus be present throughout the P. falciparum life cycle in the blood. In the phylogenetically related apicomplexan B. divergens, only one class of binding sites was detected, with a Kd of 0.93 ⫾ 0.21 ␮M and a Bmax of 15.51 ⫾ 1.37 pmol/107 IRBC (Fig. 4C; see also Fig. S1A). The food vacuole is present in Plasmodium at its mature stages but not in Babesia spp. parasites (29, 30), and it represents a major difference between the two hematozoan parasites. We therefore investigated the possible involvement of the food vacuole in the accumulation process by adding to P. falciparum IRBC suspensions the aspartic protease inhibitor pepstatin A, which blocks plasmepsin-mediated hemoglobin degradation in the food vacuole and prevents ferriprotoporphyrin IX (FPIX) release (31). Addition of 100 ␮M pepstatin A did not significantly affect accumulation at the ring stage or the low-affinity component at the mature stages (P ⬎ 0.3). In contrast, the high-affinity accumulation of albitiazolium at P. falciparum mature stages was completely inhibited (Fig. 4B; see also Fig. S1B and C in the supplemental material). Preventing FPIX generation by pepstatin A reduced the albitiazolium high-affinity binding capacity by diminishing the number of available binding sites until completely suppressing the high-affinity component. Since part of the bis-thiazolium salt accumulation has been shown to be due to its binding to FPIX inside the food vacuole (reference 21 and unpublished data), pepstatin A-induced suppression of the high-affinity process suggests a link with the malaria parasite food vacuole.

Albitiazolium Partitioning in Infected RBC in Malaria

corresponding to a CAR of 1,031 ⫾ 44 and 3,826 ⫾ 42 (data not shown). The addition of 5 mM NaCl did not affect any accumulation (Fig. 5A), while the addition of 5 mM NH4Cl decreased albitiazolium accumulation surprisingly by 50%, to 18.0 ⫾ 0.8 pmol/107 IRBC, and CQ accumulation as expected by 63.5%, to 14.3 ⫾ 0.1 pmol/107 IRBC (Fig. 5A). We hypothesized that this effect was not due to the change in food vacuole pH but to the presence of NH4⫹ and/or NH3 in the medium. At physiological pH, most of the ammonium is in the NH4⫹ form and thus requires transport proteins for membrane passage. As for other inorganic or organic cations (choline, tetramethylammonium, or tetrapropylammonium) (18, 36, 37) and for albitiazolium, (18), NH4⫹ ion transport should be mediated by the new permeability pathways (NPP) and thus at least partly inhibits albitiazolium entry. Alter-

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FIG 5 Comparative effects of NH4Cl on accumulation of albitiazolium and CQ in P. falciparum IRBC and free parasites. IRBC were incubated at ⬃7.5 ⫻ 107 cells/ml and ⬃5% parasitemia in the presence of 48 nM [14C]albitiazolium (Albi) or 20 nM [3H]CQ for 2 h or 30 min, respectively. Free parasites were incubated at 4 ⫻ 106 to 7 ⫻ 106 parasites/ml under similar conditions for 30 min. (A) Uptake in IRBC incubated in bicarbonate-free RPMI medium (bfmedium), supplemented or not with 5 mM NaCl or 5 mM NH4Cl (pH 7.4). (B) Uptake in isolated parasites incubated in bf-medium supplemented or not with 5 mM KCl or 5 mM NH4Cl (pH 7.4). (C) Reversibility of accumulation in IRBC in the presence of NH4Cl. IRBC at mature stages were loaded in bfmedium for 2 h with 48 nM [14C]albitiazolium or 30 min with 20 nM [3H]CQ, to achieve maximum drug accumulation. The loaded IRBC were then washed and reincubated (time zero) for 30 min in medium supplemented or not with 5 mM NaCl or 5 mM NH4Cl. Quantities of radioactive drug remaining in IRBC are expressed as a percentage of the amount of drug measured at time zero. Data are from one of two experiments, each performed in triplicate. The results are expressed as means ⫾ SEM (n ⫽ 3). Significant differences with respect to controls in bicarbonate-free RPMI medium are shown, as follows: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

natively, presence of NH4⫹ ions in the host cytosol could change pump activities and thus ionic gradients, altering the transports dependent on these gradients and leading to inhibition of albitiazolium entry into IRBC. To circumvent the NH4Cl effect on the host cell, we investigated its effect on isolated P. falciparum parasites. Preliminary experiments indicated that bicarbonate-free RPMI medium was the most appropriate medium for incubation of isolated parasites (see Fig. S2 in the supplemental material). The addition of 5 mM NH4Cl to isolated parasites decreased CQ uptake by 30%, from 4.2 ⫾ 0.2 to 3.0 ⫾ 0.01 pmol/107 parasites, but it did not affect albitiazolium uptake, while the same KCl concentration had no effect on the corresponding uptake (Fig. 5B). This indicated that NH4Cl does affect albitiazolium entry into IRBC but not into the intracellular parasite. An additional strategy to identify a possible link between the pH and drug accumulation involved preloading IRBC with antimalarial drugs until steady state and then monitoring the effect of NH4Cl on drug retention and release. As expected, the presence of NH4Cl drastically decreased CQ retention by 38%, from 6.5 ⫾ 0.1 to 4.0 ⫾ 0.01 pmol/107 IRBC (Fig. 5C), with concomitant release into the external medium (increased by 51% [data not shown]). Conversely, NH4Cl did not modify albitiazolium retention in preloaded IRBC (P ⬎ 0.05) or induce its release (data not shown). Energy dependence of drug uptake into Plasmodium IRBC. Albitiazolium accumulation increased over time and reached 27.2 ⫾ 0.6 pmol/107 IRBC after 90 min of incubation (Fig. 6A). Deprivation of glucose, which considerably reduces the amount of energy available (38), almost completely abolished albitiazolium accumulation, which was less than 1 pmol/107 IRBC (Fig. 6A). In comparison, maximal accumulation of CQ was obtained after 20 to 30 min of incubation, and glucose deprivation only partly decreased (P ⬍ 0.05) its accumulation (e.g., by only 15% within the first 30 min) (Fig. 6B). To further test whether accumulation of albitiazolium is an energy-dependent process, accumulation was measured in the presence of fructose (which can be used alternatively in the glycolytic pathway of P. falciparum [39]) or of the P-ATPase inhibitor orthovanadate. Both the removal of glucose and the addition of vanadate drastically inhibited albitiazolium accumulation. Interestingly, addition of fructose restored albitiazolium accumulation (Fig. 6C), attesting to its accumulation being an energy-requiring process that is dependent on the generation of ATP. Effect of perturbation of cellular ionic contents and gradients. Drug accumulation may be the result of active and concentrative transport. As these processes are often energized by ionic gradients between cellular compartments, we analyzed the effects of compounds that are known to affect ionic pumps and transport or ion exchange between compartments. Accumulation of both albitiazolium and CQ appeared to be the most highly affected by the K⫹ ionophore nigericin, followed by the Na⫹-specific ionophore monensin A. The potent inhibition exerted by bafilomycin A1, an inhibitor of V-type H⫹-ATPase (40, 41), and FCCP suggests that albitiazolium uptake was even more energized than CQ by the proton-motive force across the erythrocyte and/or parasite plasma membrane. The calcium ionophore A23187 induced substantial inhibition of albitiazolium uptake, a pattern that was also observed for CQ (Fig. 7). Even though effectors were used at lower-than-usual concentrations, all of them markedly decreased albitiazolium uptake in a concentration-de-

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or 20 nM [3H]CQ (B) into IRBC at late stages was measured at 1% hematocrit and 5 to 10% parasitemia in bicarbonate-free RPMI 1640 without glucose buffered with 25 mM HEPES and supplemented (continuous line) or not (dashed line) with 11 mM D-glucose. The results are expressed as means ⫾ SEM (n ⫽ 3). All data obtained in the absence of glucose were significantly different from those obtained in the presence of glucose for both albitiazolium (P ⱕ 0.01) and CQ (P ⱕ 0.05). (C) Uptake of 48 nM [14C]albitiazolium into IRBC at late stages after 20 or 60 min of incubation in bicarbonate-free RPMI 1640 without glucose buffered with 25 mM HEPES. Supplementation with 11 mM D-glucose, 40 mM fructose, or 11 mM D-glucose in combination with 200 ␮M orthovanadate were tested as indicated. The results means ⫾ SEM (n ⫽ 3). The indicated significant differences are with respect to incubations with 11 mM D-glucose: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

pendent manner and led to complete inhibition of albitiazolium uptake (Fig. 7). DISCUSSION

We report here new insights into a key property of the clinical candidate albitiazolium, which is accumulated specifically within hematozoa-infected erythrocytes. This choline analogue is thus able to exert a potent toxic effect against Plasmodium- and Babesia-infected erythrocytes while having weak toxicity against mammalian cells (17, 20, 21). At 37°C, albitiazolium did not significantly interact with normal erythrocytes but massively accumulated in P. falciparum IRBC (Fig. 2A). Its uptake occurred through a concentration-dependent and saturable process (Fig. 2B), reaching steady state after 60 to 90 min of contact with the drug (Fig. 2A). The outcome was that, at a 250 nM external concentration and assuming an equal distribution in all infected erythrocyte compartments, the intracellular concentrations of albitiazolium in IRBC were at least 5.2 ␮M and 50.7 ␮M at the ring and schizont stages, respectively, with CAR ranging from 21 to 404 (Fig. 3C). Previous studies showed that the structurally very close bis-thiazolium T16 is mainly localized (77%) within the intracellular parasite (21). Here again, at least 50% of the albitiazolium that accumulated within IRBC (14.3 pmol/107 IRBC/30 min) (Fig. 2A) was recovered in the intracellular parasite (7.2 pmol/107 IRBC/30 min) (Fig. 5C). Considering the volumes of intraerythrocytic P. falciparum parasites determined by cryo-X-ray tomography (27), concentrations of the drug within the intraerythrocytic parasite ranged from 42 to 193 ␮M. Meanwhile, CAR remained in the same range throughout the parasite cycle, with values between 200 and 600 (Fig. 3C), revealing that albitiazolium accumulated to a large extent throughout the P. falciparum infection cycle. Albitiazolium accumulation in the intracellular parasite ap-

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peared to be essentially irreversible (Fig. 2C). It probably follows serious physiological consequences, because the parasite massively accumulates the drug but cannot get rid of the toxic compound, and the parasite is thus sentenced to death. The rapid but limited release of the accumulated albitiazolium (11 to 14%) (Fig. 2C) within the first 15 min of incubation in drug-free medium likely corresponded to the release of the minor portion of drug contained in the erythrocyte cytosol of IRBC, which might have been released or reexported into the external medium via parasite-induced NPP in the erythrocyte membrane (37). We recently showed that albitiazolium enters IRBC mainly through these NPP (18), which can transport molecules bidirectionally (42, 43). While the recently described albitiazolium entry via NPP (18) likely explains its specific tropism for IRBC, these pore-like systems do not mediate concentrative processes (44). Albitiazolium accumulation requires intracellular binding sites or active transport in parasite-specific compartments, or a combination of both. Analysis of saturation curves of the accumulated bis-thiazolium in P. falciparum IRBC at steady state revealed only one component of accumulation at the ring stage (Fig. 4A) but two components at mature stages (Fig. 4B). This suggests that at mature stages accumulation in P. falciparum IRBC occurs through two distinct processes that differ in both affinity and extent and that the highaffinity process is not present at the ring stage. For the closely related T16, approximately 50% of the accumulated drug has been reported in the food vacuole, where it interacts with hemoglobindegradation products (20, 21). For albitiazolium, upon treatment with the aspartic protease inhibitor pepstatin A, which is widely reported to affect acidic P. falciparum food vacuole processes, the high-affinity component was selectively suppressed whereas the

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FIG 6 Comparative effects of glucose deprivation on albitiazolium and CQ uptake into P. falciparum IRBC. (A and B) Uptake of 48 nM [14C]albitiazolium (A)

Albitiazolium Partitioning in Infected RBC in Malaria

during 30 min in mature-stage IRBC was measured at 1% hematocrit and 5 to 10% parasitemia in bicarbonate-free RPMI medium in the presence of bafilomycin A1, FCCP, monensin A, nigericin, or A23187 at the indicated concentrations. The results are expressed as means ⫾ SEM (n ⫽ 3).

low-affinity component and the accumulation process at ring stage were not affected (Fig. 4A and B). Hence, the high-affinity accumulation process is likely localized within the parasite food vacuole, where albitiazolium can interact with FPIX (unpublished data). Accumulation of albitiazolium in P. falciparum IRBC involved a second process with a 10-fold-lower affinity but 7-fold-higher capacity that was apparently present throughout the cell cycle. Interestingly, albitiazolium also appeared to be highly accumulated in B. divergens IRBC (CAR, ⱖ60), but with only one process. Since B. divergens does not possess a food vacuole, these two hematozoan parasites might share this second accumulation compartment, which is not related to the food vacuole. Because bisthiazolium salts specifically inhibit both the P. falciparum and babesial biosynthesis of phosphatidylcholine (20), this compartment could be the parasite endoplasmic reticulum, where the machinery for phospholipid biosynthesis operates. The lipid-like albitiazolium molecule (a long central hydrophobic chain and two polar heads) (Fig. 1) might be inserted into some biological mem-

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branes, as is the case for cationic type I amphiphiles, such as dodecyltrimethylammonium bromide (45). Indeed, the long flexible alkyl chain could fold in a hairpin manner so that the two heads are side by side and become inserted into membranes. The high accumulation of albitiazolium in the parasite is reminiscent of CQ accumulation in the P. falciparum parasite, which operates through concentrative entry of weak bases into the acidic parasite food vacuole with subsequent binding to hemozoin, i.e., a degradative heme catabolite. Because albitiazolium is a permanently charged bis-cationic compound (Fig. 1), its entry into the food vacuole cannot occur through lysosomotropic properties, which are restricted to weak bases. The extent of accumulation thus likely does not depend on the food vacuole pH, as is the case for the weak base CQ. Alkalinization of the food vacuole by the addition of 5 mM NH4Cl (from pH 5.2 to ⬃6) decreased CQ accumulation in free parasites by 30% (33, 34, 46), whereas albitiazolium accumulation was not affected (Fig. 5B). Furthermore, this alkalinization induced the release of preloaded CQ but not of preloaded albitiazolium (Fig. 5C). Contrary to findings with CQ,

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FIG 7 Effects of ion effectors on albitiazolium and CQ uptake in P. falciparum IRBC. Accumulation of 48 nM [14C]albitiazolium during 2 h or of 20 nM [3H]CQ

Wein et al.

3. 4. 5. 6. 7. 8. 9. 10.

11. 12.

13.

14.

15.

ACKNOWLEDGMENTS We are grateful to Pascale Bette-Bobillo for her assistance in the detailed analysis of accumulation data. The research leading to these results benefitted from funding from the European Community Framework Programme, Antimal Integrated Project (LSHP-CT-2005-018834) and EviMalar Network of Excellence (FP7/ 2007-2013, number 242095), Sanofi and Innomad program of the Eurobiomed competitiveness cluster (Région Languedoc-Roussillon/FEDER/ OSEO). The P. falciparum 3D7 strain was obtained through the Malaria Research and Reference Reagent Resource Center (MR4; http://www.mr 4.org/). S.W. planned and designed the study, acquired and interpreted the data, performed the statistical analyses, and wrote the manuscript; M.M., C.T.V.B, Y.B., and J.P. acquired, analyzed, and interpreted the data; S.P. synthesized albitiazolium; L.F. designed the study; H.V. planned and designed the study and wrote the manuscript.

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albitiazolium accumulation in P. falciparum parasites therefore does not depend on the food vacuole pH. Finally, we showed that both albitiazolium accumulation components were dependent upon the presence of glucose or fructose, which are both metabolized via glycolysis. The inhibitory effect of orthovanadate emphasized that accumulation processes are energy dependent (Fig. 6). Albitiazolium accumulation was also drastically affected by effectors of cellular ionic contents and gradients (Fig. 7), even when these effectors were used at low concentrations. The reductions were so drastic that they did not allow us to identify the involvement of one particular ion. This suggests that perturbation of IRBC ionic contents and gradients may alter albitiazolium transport and access to the intracellular compartment. In conclusion, the massive accumulation of albitiazolium in P. falciparum IRBC resulted from at least two distinct components: a high-affinity component, likely due to accumulation in the food vacuole with binding to FPIX, and a lower-affinity process but with a higher capacity, which remains to be clearly identified. Specific targeting of albitiazolium to hematozoa-infected erythrocytes is likely a key feature of the specificity and potency of this new type of antiparasitic drug. Our CAR findings, which entailed ratios as high as 1,000, suggested that there were very high concentrations in the accumulation compartments. This massive, irreversible, and energy-dependent accumulation allowed albitiazolium to exert an antiplasmodial and antibabesial activity at a low nanomolar concentration, resulting in a high micromolar intraparasitic concentration, while being weakly toxic to mammalian cells (17). Considering that applying drug pressure of a closely related choline analogue for 9 months to a P. falciparum blood culture (47) did not lead to the appearance of drug-resistant parasites (unpublished data), it is therefore likely that this intraparasitic compartmentation underlies a dual mechanism of action (18, 20, 21) that thus makes this new type of antimalarial agent resistant to parasite resistance.

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