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Heme Binding Contributes to Antimalarial Activity of Bis-Quaternary Ammoniums

Abstract

Quaternary ammonium compounds have received recent attention due to their potent in vivo antimalarial activity based on their ability to inhibit de novo phosphatidylcholine synthesis. Here we show that in addition to this, heme binding significantly contributes to the antimalarial activity of these compounds. For the study, we used a recently synthesized bis-quaternary ammonium compound, T16 (1,12-dodecanemethylene bis[4-methyl-5-ethylthiazolium] diodide), which exhibits potent antimalarial activity (50% inhibitory concentration, ∼25 nM). Accumulation assays reveal that this compound is readily concentrated several hundredfold (cellular accumulation ratio, ∼500) into parasitized erythrocytes. Approximately 80% of the drug was shown to be distributed within the parasite, ∼50% of which was located in the parasite food vacuoles. T16 uptake was affected by anion substitution (permeation increasing in the order Cl− < Br− = NO3− < I− < SCN−) and was sensitive to furosemide—properties similar to substrates of the induced new permeability pathway in infected erythrocytes. Scatchard plot analysis of in situ T16 binding revealed high-affinity and low-affinity binding sites. The high-affinity binding site Kd was similar to that measured in vitro for T16 and ferriprotoporphyrin IX (FPIX) binding. Significantly, the capacity but not the Kd of the high-affinity binding site was decreased by reducing the concentration of parasite FPIX. Decreasing the parasite FPIX pool also caused a marked antagonism of T16 antimalarial activity. In addition, T16 was also observed to associate with parasite hemozoin. Binding of T16 to FPIX in the digestive food vacuole is shown to be critical for drug accumulation and antimalarial activity. These data provide additional new mechanisms of antimalarial activity for this promising new class of antimalarial compounds.

Worldwide resistance to traditional antimalarial drugs such as chloroquine and pyrimethamine sulfadoxine raises the urgency for the development of new drugs aimed at new drug targets (18). Inhibition of phosphatidylcholine synthesis—which leads to the inhibition of membrane biogenesis during the asexual intraerythrocytic stage of the human malaria parasite Plasmodium falciparum—has recently attracted attention as a potentially new chemotherapeutic target (18, 26). Over recent years, numerous (>600) compounds have been rationally synthesized to mimic choline structure. The resulting compounds from these structure-activity studies are quaternary and bis-quaternary ammoniums with lead compounds that exert potent (i.e., low nM) in vitro activity against P. falciparum and P. vivax (2, 5, 6, 26) as well as good in vivo activity against P. falciparum and P. cynomolgi in aotus and rhesus monkeys, respectively (26).

Biochemical investigations show that in P. knowlesi, the protein-mediated transport of choline into the infected erythrocyte is a rate-limiting step for phosphatidylcholine synthesis (1). In this malaria parasite, the in vitro and in vivo activity exerted by the quaternary and bis-quaternary ammonium compounds can be directly correlated to a reduction in the transport of choline and subsequent reduction of phospholipid metabolism (1, 2). However, questions concerning the mechanism of entry of these drugs into infected erythrocytes have not been addressed. Based on kinetic and pharmacological inhibitor profiles of isosmotic hemolysis and radiolabel flux experiments, several quaternary ammonium compounds have been demonstrated to permeate P. falciparum-infected erythrocytes via the parasite-induced new permeation pathway (NPP [11, 22]). Similarly, diamidine compounds, which—being highly charged and hydrophilic—are structurally comparable to the bis-quaternary ammonium compounds, have also been demonstrated to enter the host erythrocyte via the NPP (23). In this study, we have investigated the mechanism of entry and antimalarial activity of a newly synthesized bis-quaternary ammonium compound, T16 (1,12-dodecanemethylene bis[4-methyl-5-ethylthiazolium] diodide; Fig. 1), which exhibits potent in vitro activity (50% inhibitory concentration [IC50], ∼25 nM). Here we show that T16 is a substrate for the NPP, resulting in T16's entry into infected red blood cells. Subsequently, accumulation of T16 occurs in the parasite and in the digestive food vacuole. Binding of T16 to ferriprotoporphyrin IX (FPIX) (10) in the digestive food vacuole is shown to be critical for drug accumulation and antimalarial activity. These data provide new mechanisms of antimalarial activity for this promising new class of antimalarial compounds.

FIG. 1.

FIG. 1.

Chemical structure of T16 (1,12-dodecanemethylene bis[4-methyl-5-ethylthiazolium] diodide) and [3H]T16.

MATERIALS AND METHODS

Reagents.

T16 and [3H]T16 were synthesized in house. Ro 40-4388 was kindly provided by R. G. Ridley and R. Moon of Hoffman LaRoche. [3H]hypoxanthine was purchased from Amersham. Tissue solubilizer was purchased from BDH. All other reagents were purchased from Sigma-Aldrich Chemical Company.

Parasite, culture, and drug sensitivity assays.

P. falciparum strain TM6 (chloroquine resistant, IC50 ≥ 100 nM), was obtained from P. Tan-Ariya (Mahidol University, Bangkok, Thailand) and maintained in continuous culture. Cultures contained a 2% suspension of O+ erythrocytes in RPMI 1640 medium (R8758, glutamine, and NaHCO3) supplemented with 10% pooled human AB+ serum, 25 mM HEPES (pH 7.4), and 20 μM gentamicin sulfate (25). Radiolabel uptake experiments were performed on mature trophozoites synchronized by treatment with sorbitol (15). The sensitivity of P. falciparum-infected erythrocytes to T16 was determined by using the [3H]hypoxanthine incorporation method (8) with an inoculum size of 0.5% parasitemia (ring stage) and 1% hematocrit. IC50s were calculated by using the four-parameter logistic method (Grafit program; Erithacus Software, Surrey, United Kingdom). The effect of the combination of T16 and Ro 40-4388 on parasite growth was tested by titration of the two drugs at fixed ratios proportional to their IC50s. The fractional inhibitory concentrations of the resulting IC50s were plotted as isobolograms (3).

Uptake of [3H]T16 into uninfected and parasitized erythrocytes.

Uninfected erythrocytes or erythrocytes infected with synchronized cultures of P. falciparum were suspended (typically [3 to 5] × 108 cells ml−1) in HEPES (25 mM, pH 7.4)-buffered RPMI 1640 medium containing 50 nM [3H]T16 (specific activity, 69 Ci mmol−1). At specific time intervals, aliquots of the suspension were overlaid onto oil (400 μl of dibutyl phthalate) in a microcentrifuge tube and centrifuged at 10,000 × g for 20 s, thereby sedimenting the cells below the oil. Cell pellets were lysed by the addition of distilled water (100 μl) and then solubilized and decolorized by the addition of a cocktail (100 μl) containing five parts tissue solubilizer, two parts H2O2 (30%), and two parts glacial acetic acid. Samples were then counted by liquid scintillation counting. T16 binding affinity was measured after 2 h in 25 mM HEPES-buffered RPMI 1640 medium (pH 7.4) containing 50 nM [3H]T16 and various concentrations of unlabeled T16, in the presence or absence of Ro 40-4388.

Specific uptake of [3H]T16 into infected erythrocytes was calculated by subtracting the counts attributable to an equal number of uninfected erythrocytes. The cellular accumulation ratio (CAR) is defined here as the ratio of the amount of [3H]T16 in infected erythrocytes to the amount of [3H]T16 in the same volume of the suspending solution after incubation. The volumes of infected and uninfected erythrocytes were assumed to be equal (75 fl [20]). The intracellular drug concentration was calculated by multiplying the CAR by the suspending solution drug concentration after incubation.

A number of compounds were tested for their ability to inhibit [3H]T16 uptake. These included choline, Ro 40-4388, furosemide, phloredzin, and niflumate. All compounds were preincubated with infected and noninfected erythrocytes for 5 min prior to incubation (1 h) with [3H]T16. Control samples were treated with dimethyl sulfoxide when appropriate. To determine the effect of anion substitution on [3H]T16 uptake, cells were suspended in the appropriate solutions (i.e., the same buffers as those used by Kirk and Horner [14]) for 10 min, and uptake was measured as described above.

Uptake of [3H]T16 into infected erythrocytes and its distribution into trophozoites and food vacuoles.

Uninfected and P. falciparum-infected (4 to 10% parasitemia) erythrocytes (typically [5 to 9] × 108 cells ml−1) suspended in HEPES-buffered RPMI 1640 medium (25 mM, pH 7.4) were incubated with 25 nM [3H]T16 (specific activity, 69 Ci mmol−1) for 1 h. Uninfected erythrocytes and an aliquot of the infected erythrocytes were overlaid onto oil (400 μl of dibutyl phthalate) and centrifuged at 10,000 × g for 20 s. The cell pellets were then processed, and the radioactivity was determined as described above. Preparation of free parasites, from an aliquot of infected erythrocytes, was performed by centrifugation (3,000 × g, 5 min) and resuspension of the pellet in 5 vol of 0.15% (wt/vol) saponin in phosphate-buffered saline (PBS) for 1 min, followed by three washes by centrifugation and resuspension in HEPES-buffered RPMI 1640 medium. An aliquot of this preparation containing the free parasites was overlaid onto oil (a 5:4 mixture of dibutyl phthalate-dioctyl phthalate) and centrifuged (10,000 × g, 20 s). The pellet of free parasites was then processed, and the radioactivity was measured as described above. Preparation of food vacuoles from an aliquot of free parasites was performed based on the method of Saliba et al. (19). Free parasites were hypotonically lysed by the addition of 10 vol of H2O in the presence of DNase 1 (10 μg ml−1) and triturated four times through a 27-gauge 1.2-cm needle. The food vacuoles were then washed by centrifugation in HEPES-buffered RPMI 1640 medium and overlaid onto a cushion (1 ml) of 0.85 M sucrose and centrifuged at 10,000 × g for 10 min. The pellet was then processed, and the radioactivity was measured as described above.

UV and visible spectral scans.

FPIX or protoporphyrin IX (PIX) solutions (3 mM) in 0.1 M NaOH were prepared on the day of experimentation. Before scanning, samples were diluted (3 μM) in 0.2 M HEPES (pH 7.0) in the presence and absence of 3 μM T16. Samples were scanned against the appropriate blank control in a Hewlett-Packard 8452A diode array spectrophotometer (Palo Alto, Calif.).

Determination of binding affinity (Kd) of T16 for FPIX-loaded ghost membranes.

The binding affinity of [3H]T16 to FPIX-loaded ghost erythrocytic membrane (12) was measured as described previously for [3H]chloroquine (4).

Binding and incorporation of [3H]T16 to FPIX and hemozoin crystal.

In order to determine whether T16 could bind or be incorporated into hemozoin crystal (β-hematin), T16 was incubated with FPIX-loaded ghost erythrocyte membrane in conditions that permitted hemozoin formation (4). For this experiment, erythrocyte ghost membrane (100 μl [12]) and FPIX (100 μl of a 3 mM solution in 0.1 M NaOH) were mixed with an aliquot (10 μl) of 1 M HCl and made up to a final volume of 0.9 ml with 0.5 mM Na acetate (pH 5.2). [3H]T16 (in distilled water) was then added (100 μl) to this assay mixture, giving a final [3H]T16 concentration of 50 nM. Controls were made without FPIX and/or without erythrocyte ghost membrane. Samples were well mixed and incubated at 37°C for 48 h. Following incubation, the samples were pelleted by centrifugation (14,000 × g, 10 min) and resuspended in 1 ml of 0.2 M sucrose. These suspensions were then applied to a discontinuous sucrose gradient composed of five cushions (1 ml each) of 0.2, 0.8, 1.2, 1.6, and 2.0 M sucrose. After centrifugation (100,000 × g for 1 h), the phases were collected separately and processed for radioactive counting as described above. Previous experiments had established that hemozoin crystals but not FPIX sediments to the 2.0 M fraction.

In situ binding of [3H]T16 to hemozoin.

Synchronized P. falciparum-infected erythrocytes at the trophozoite stage were incubated for 2 h at 37°C in the presence of 50 nM of [3H]T16. After incubation, cells were washed once in PBS and were saponin treated by incubation at a hematocrit of 10% for 5 min at 4°C in PBS containing 0.06% (wt/vol) saponin. After centrifugation at 4,000 × g for 10 min, the pellet was washed twice in PBS and the free parasites were resuspended in 1 ml of 0.2 M sucrose and sonicated in a probe type sonicator (Vibra Cell, Bioblock Scientific) for 30 s at 4°C. The suspension was then overlaid onto discontinuous sucrose gradients, ultracentrifuged, and treated as described above for radioactive counting.

RESULTS

T16 is a NPP substrate that selectively accumulates in the malaria parasite and is distributed in the digestive food vacuole.

The uptake of [3H]T16 into uninfected and infected erythrocytes suspended in HEPES-buffered RPMI 1640 solution (pH 7.4) was monitored over several hours. Uninfected erythrocytes were observed to accumulate relatively low levels of T16 (CAR < 2; Fig. 2); in contrast, P. falciparum-infected erythrocytes showed high levels of accumulation. At equilibrium, the level of T16 in infected erythrocytes was approximately 500 times that of the surrounding suspension (Fig. 2). Measurement of [3H]T16 distribution into parasitized erythrocytes after a 1-h incubation period revealed that the fraction accumulated by the intracellular parasite accounted for 77% ± 10% (n = 3) of the total uptake. Further analysis revealed that 40% ± 3% (n = 3) of the total uptake (∼50% of the parasite-specific uptake) was localized in the parasite's food vacuoles.

FIG. 2.

FIG. 2.

Time course of 50 nM [3H]T16 uptake into uninfected (•) and P. falciparum-infected (○) erythrocytes suspended in HEPES-buffered RPMI 1640 medium (pH 7.4). Data are the means ± standard error of three independent experiments.

The uptake of [3H]T16 into infected erythrocytes was observed to be inhibited by furosemide, phloredzin, and niflumate (Table 1) —known inhibitors of the parasite-induced NPP (13). Uptake of [3H]T16 into infected erythrocytes also showed a marked dependence on the nature of the counterion present in solution, increasing in the order Cl− < Br− = NO3− < I− < SCN− (Fig. 3), a physiological property characteristic of the transport of cations through the NPP (13, 14).

TABLE 1.

Effect of inhibitors on [3H]T16 uptake in P. falciparum-infected erythrocytes

Subtrate or inhibitor Concn Inhibition (%)a
Ro 40-4388 10 μM 76
Choline 1 mM 63
Furosemide 100 μM 69
Phloredzin 100 μM 22
Niflumate 100 μM 34

FIG. 3.

FIG. 3.

Effect of anion substitution from the suspending solution on the uptake (increase relative to chloride [n-fold]) of 50 nM [3H]T16 into P. falciparum-infected erythrocytes. Data represent the means ± standard error of three independent experiments.

T16 binds to FPIX.

The total binding of [3H]T16 to infected erythrocytes plotted on a Scatchard plot is shown in Fig. 4. This biphasic curve is indicative of at least two binding sites—one of high affinity and one of low affinity. Nonlinear-regression analysis of the high-affinity binding site reveals a Kd for T16 of 0.73 ± 0.26 μM (standard error [SE]; n = 5).

FIG. 4.

FIG. 4.

Scatchard plot of the total binding of [3H]T16 onto P. falciparum-infected erythrocytes. Shown is a single representative plot from five similar experiments. The curved line suggests the presence of at least two components—a high-affinity T16 binding site and a low-affinity T16 binding site.

FPIX, which is continuously generated by the intraerythrocytic malaria parasite during hemoglobin digestion, is known to be a receptor to several cationic aromatic antimalarial drugs, such as chloroquine (4) and the diamidines (23). We therefore investigated whether FPIX was the high-affinity receptor for T16 binding. T16 was shown in vitro to bind to a molar equivalent of FPIX, thus producing a marked increase in the Soret peak of FPIX (Fig. 5A). In addition, a small blueshift in the spectral profile of PIX in the presence of equimolar T16 (Fig. 5B) was also observed.

FIG. 5.

FIG. 5.

UV and visible spectral scans of (A) 3 μM FPIX (λmax, 395 nm in aqueous HEPES [pH 7.0]) and (B) 3 μM PIX (λmax, 410 nm in aqueous HEPES [pH 7.0]) in the presence (dashed line) or absence (solid line) of 3 μM T16. Data are representative of three independent experiments.

Binding of [3H]T16 to FPIX was also measured in FPIX-loaded erythrocyte ghost membranes. Binding isotherms revealed that the T16 radioligand bound to FPIX with an affinity (Kd) value of 2 μM (Fig. 6). This value is similar to that obtained for the high-affinity in situ binding (Kd, 0.7 μM), suggesting that FPIX is an intracellular high-affinity receptor for T16.

FIG. 6.

FIG. 6.

Nonlinear-regression analysis of [3H]T16 binding to FPIX-loaded ghost erythrocyte membrane. Data points are from two independent experiments performed in triplicate.

Further confirmation was provided with the use of the plasmepsin I inhibitor Ro 40-4388 (16). This inhibitor prevents hemoglobin digestion by the parasite, thereby reducing the amount of free FPIX (4). We were able to show that [3H]T16 uptake is inhibited by 10 μM Ro 40-4388 (Table 1). Treatment of infected erythrocytes with Ro 40-4388 would have had the consequence of reducing the number of binding sites available to T16 (by reducing the amount of free FPIX [4]) without affecting the affinity of the T16-FPIX binding. Scatchard plots of the high-affinity binding in the presence of Ro 40-4388 do indeed show this (Fig. 7), thereby confirming that FPIX is critical for driving T16 accumulation.

FIG. 7.

FIG. 7.

Scatchard plot of the high-affinity binding of [3H]T16 to P. falciparum-infected erythrocytes in the presence (•) and absence (○) of 10 μM Ro 40-4388. Data points are means of duplicate observations from three separate experiments.

Binding of T16 to FPIX is critical for antimalarial activity.

Reducing the parasite FPIX pool also had an effect on the antimalarial activity of T16. This is indicated in the isobole analysis experiments shown in Fig. 8, in which the reduction of the FPIX pool by use of the protease inhibitor Ro 40-4388 resulted in a marked antagonism of T16 activity.

FIG. 8.

FIG. 8.

Isobole plot of T16 in vitro antimalarial activity in combination with Ro 40-4388.

In vitro and in situ interaction of [3H]T16 with hemozoin.

Hemozoin was grown in vitro in the presence of [3H]T16 and thereafter separated by a discontinuous sucrose gradient. Analysis of the sucrose fractions revealed that 55% of [3H]T16 was recovered from the 0.2 M fraction (Fig. 9A). Control experiments (without FPIX and/or erythrocyte ghost membrane—see Materials and Methods) revealed that this fraction contains free [3H]T16 and (solubilized) FPIX-associated [3H]T16. The remaining (45%) [3H]T16 was recovered from the 2.0 M sucrose fraction containing the hemozoin-associated [3H]T16 (Fig. 9A). These data indicated that T16 can readily interact in vitro with the growing hemozoin crystals. In order to determine whether [3H]T16 interacts with hemozoin crystals in situ, an enriched (saponin freed) trophozoite suspension was incubated with [3H]T16 (2 h) and subsequently sonicated and separated by discontinuous sucrose gradients. Analysis of the sucrose fractions indicated that 35% of the recovered [3H]T16 was associated with hemozoin (localized in the 2.0 M fraction, Fig. 9B). A further 35% [3H]T16 was recovered in the 0.2 M fraction, signifying free T16 and FPIX-associated T16. In addition, 20% of the recovered [3H]T16 was localized in the 0.8 M fraction, which may reflect protein-associated T16. Together, these data show that in addition to binding to FPIX, T16 is also able to interact with hemozoin and/or with the hemozoin formation process.

FIG. 9.

FIG. 9.

(A) Binding of [3H]T16 to FPIX and hemozoin in vitro and subsequent separation by discontinuous sucrose gradient. (B) Binding of [3H]T16 to freed (saponin treated) Plasmodium falciparum trophozoites and subsequent separation by discontinuous sucrose gradient. Control experiments indicate that free [3H]T16 and [3H]T16-FPIX complexes were restricted to the 0.2 M fraction and that [3H]T16-hemozoin complexes were restricted to the 2 M fraction.

DISCUSSION

In this study we have described the transport, accumulation, and antimalarial activity of a novel bis-quaternary ammonium compound, T16. We have shown that this compound does not accumulate excessively in uninfected erythrocytes (CAR, ∼2; Fig. 2), whereas accumulation in P. falciparum-infected erythrocytes is very high (CAR, ∼500; Fig. 2)—probably accounting for the observed potent (low nM) antimalarial activity.

Transport of T16 through the host's erythrocyte plasma membrane is facilitated by passage via the parasite-induced NPP. This argument is supported by the partial inhibition of [3H]T16 uptake by the NPP inhibitors—furosemide, phloredzin and niflumate (Table 1)—as well as the observed stimulation of uptake by substitution of the counteranion in the bathing solution (Fig. 3). The NPP has been shown to favor the passage of low-molecular-weight anions (13) and has been characterized by patch clamp analysis as an inwardly rectified anion channel (7). Nevertheless, it is not unprecedented that a positively charged drug such as T16 could be transported through the NPP. Small cations (e.g., K+, Na+, Rb+, choline+, and carnithine+ [13]) as well as larger cations (e.g., pentamidine [23] and tetrapentyl ammonium [22]) have previously been shown to act as NPP substrates, albeit with permeation rates several orders of magnitude lower than that of Cl−.

Once inside the host cell, T16 was shown to accumulate largely (∼80% of total uptake) inside the intracellular parasite. The mechanism by which T16 crosses the parasite's plasma membrane is as yet unclear; however, the observation that choline exerted an inhibitory effect on [3H]T16 uptake (Table 1) suggests that choline and T16 may share a common pathway into the parasite. Alternatively, a similar competition of these substrates for the NPP may have resulted in the inhibition of [3H]T16 uptake.

The uptake of T16 was shown to be concentrative, suggesting either active transport or intracellular binding. As ∼50% of the total [3H]T16 uptake in the parasite was localized in the food vacuole, we investigated whether T16 could bind to FPIX, a well-known food vacuole-residing drug-binding target (e.g., chloroquine [4]). T16 was shown to be able to bind in vitro to FPIX, effecting a large increase in the Soret peak of FPIX (Fig. 5A). T16 was also shown to bind to FPIX when loaded into erythrocyte ghost membranes (Fig. 6), with an affinity (Kd, 2 μM) that is comparable to that measured in situ (Kd, 0.7 μM; Fig. 4).

The plasmepsin inhibitor Ro 40-4388 has previously been shown to reduce the intracellular pool of FPIX (4). Upon treatment with Ro 40-4388, the in situ binding of T16 was shown to be reduced without an effect on the binding affinity (Fig. 7). This result indicates that FPIX is likely to be the target high-affinity binding site of T16. Isobole analysis revealed that Ro 40-4388 antagonized T16 activity, indicating that binding of T16 to FPIX is essential for antimalarial activity (Fig. 8). Similar antagonism with Ro 40-4388 has been demonstrated for other FPIX-binding drugs such as chloroquine and pentamidine (4, 23). We should state here, however, that bis-quaternary compounds have not shown any cross-resistance with chloroquine (26) and that resistance modulators such as verapamil (10 μM) have no effect on the activity of T16 against chloroquine-resistant isolates (data not shown). Therefore, although heme binding contributes to T16 antimalarial activity, chloroquine resistance mechanisms—which essentially affect access to heme by modulating the transmembrane transport of chloroquine—do not affect T16 activity. This last point is further illustrated in the P. falciparum (chloroquine resistant) TM6 strain, in which differences in IC50s between chloroquine (128 nM) and T16 (25 nM) are not reflected by differences in heme affinity (Kd, 139 nM for chloroquine and 700 nM for T16).

Understanding what happens to the parasite once T16 is bound to FPIX and concentrated is not entirely clear. FPIX-binding drugs such as chloroquine are believed to exert their antimalarial activity by preventing the formation and growth of the hemozoin (β-hematin) crystal, thereby allowing FPIX to build up to toxic levels (9, 17, 21). In the case of chloroquine, hemozoin production is prevented by chloroquine-FPIX complexes incorporating in the growing polymer (hemozoin), resulting in the termination of chain extension (24). Our results showing [3H]T16 association with hemozoin (Figs. 9A and 9B) suggest that a similar mechanism may operate for T16; however, confirmation of this phenomenon would require a more detailed chemical analysis of the T16-hemozoin complex—which is outside the scope of this study. The structure of T16 also points to a second possible mode of action of antimalarial activity. As shown in Fig. 1, T16 has two positively charged heads linked by a hydrophobic hydrocarbon chain, properties resembling those of a cationic detergent. From our data, we can estimate that during a typical in vitro antimalarial activity assay, upon exposure to an IC50 of T16 (∼25 nM), the concentration of T16 found in the food vacuole (assuming the food vacuole volume is ∼3% that of the infected erythrocyte [27]) at equilibrium (∼500 CAR) would reach approximately 170 μM. At this relatively high concentration, it is possible that T16 disrupts the food vacuole membrane in a detergentlike manner, thereby nonspecifically affecting any number of membrane-bound proteins.

Finally, it should be noted that ∼50% of the T16 residing in the parasite was not found in the food vacuole. Previous experiments with bis-quaternary ammonium compounds in P. knowlesi have correlated the in vitro and in vivo antimalarial activity to a reduction in phospholipid metabolism (1, 2). The remaining non-heme-binding fraction of intracellular T16 may therefore be potentially bound to a target site affecting phospholipid metabolism.

Bis-quaternary ammonium compounds are emerging as potentially exciting drug alternatives to conventional antimalarial chemotherapy. The antimalarial activity of these compounds has been attributed to the inhibition of the parasite's phosphatidylcholine metabolism. Here we show that in addition to this mechanism, the potent antimalarial activity of bis-quaternary ammonium compounds, e.g., T16, can be attributed to the drug's ability to be transported via the parasite-induced NPP, its compartmentalization into the parasite's food vacuole, and finally, its binding to FPIX and to the growing malaria pigment (hemozoin). Binding of T16 to FPIX has been shown to be important for the high accumulation level of the drug and for the antimalarial activity.

Acknowledgments

This work was supported by the European Union (QLK2-CT-2000-01166), CNRS/DGA (GDR 1077), and MENRT (PRFMMIP and VIHPAL). E. Richier is a recipient of a PAL+/MRT fellowship. P.G.B. and S.A.W. receive support from the Wellcome Trust.

We thank Jean Louis Morgat for assistance in the synthesis of the radioactive molecule.

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