* Department of Pharmacology and Therapeutics, The University of Liverpool, Liverpool L69 3BX, United Kingdom; and Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
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Abstract |
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Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ-FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ-FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX.
Key words: Plasmodium falciparum; chloroquine; drug resistance; heme binding; Na+/H+ exchanger ![]() |
Introduction |
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CHLOROQUINE (CQ)1 has been one of the most successful antimalarial agents ever developed. Unfortunately, the emergence and spread of resistant
strains of Plasmodium falciparum has now rendered the
drug almost useless in most malaria endemic areas. The
immense clinical importance of falciparum malaria and
the universal success of CQ before the development of
resistance have provided the impetus for investigations
about the mode of action of CQ and the basis of resistance
at the cellular level. The activity of CQ depends on a high-level accumulation within the malarial parasite and drug
resistance stems from reduced drug accumulation. Unfortunately, a definitive mechanistic explanation for these observations has remained elusive (Fitch, 1970; Krogstad et
al., 1987
; Ginsburg and Stein, 1991
; Martiney et al., 1995
;
Bray et al., 1998
).
However, Wünsch and co-workers recently provided
compelling evidence for a novel mechanism of CQ resistance based on the differential stimulation of the parasite
Na+/H+ exchanger (NHE; Wünsch et al., 1998). Previous
work from this group linked altered saturation kinetics
of initial CQ uptake to the CQ resistance phenotype
(Sanchez et al., 1997
). They found that CQ uptake was inhibited competitively by specific inhibitors of NHE, providing evidence that the drug is actively transported through the parasite NHE. They also showed that CQ
stimulates the NHE of chloroquine-sensitive (CQS) parasites and suggested that CQ is taken up by the NHE of
these parasites in the ensuing rapid burst of sodium-proton exchange (Wünsch et al., 1998
). Conversely, it was
proposed that the NHE of chloroquine-resistant (CQR)
parasites did not transport CQ, since it was constitutively activated and insensitive to further stimulation by CQ
(Wünsch et al., 1998
). The reversal of CQ resistance by
verapamil was proposed to occur by modulating the activity of parasitic NHE via the calcium/calmodulin-dependent pathway (Sanchez et al., 1997
).
Since mammalian NHE is incapable of CQ transport
(Sanchez et al., 1997; Wünsch et al., 1998
), this unusual
mechanism of drug uptake should be responsible for the
specificity of CQ for malarial parasites. Therefore, this
model is incompatible with the notion that the specificity
of CQ's antimalarial action is caused by the formation of a
drug-ferriprotoporphyrin IX (FPIX) complex accumulating in the parasite upon exposure to CQ (Chou et al., 1980
;
Fitch, 1983
; Balsubramanian et al., 1984; Sullivan et al., 1996
; Ginsburg et al., 1998
). In a process unique to the malaria parasite, the FPIX released during proteolysis of hemoglobin is polymerized into an inert crystalline substance
called hemozoin (Francis et al., 1997b
). CQ inhibits this
polymerization process, causing a buildup of free FPIX
and/or CQ-FPIX complex that may ultimately kill the parasite (Slater, 1993
; Dorn et al., 1995
). Our own studies
suggest that the specificity, accumulation, and antimalarial
activity of CQ are all determined by the saturable equilibrium binding of CQ to FPIX (Bray et al., 1998
).
We found that CQR parasites have a reduced apparent
affinity of CQ-FPIX binding compared with CQS parasites. We propose that the resistance mechanism acts specifically at the site of FPIX generation to alter the affinity
of CQ-FPIX binding rather than changing the active
transport of CQ across the parasite plasma membrane
(Bray et al., 1998). Here we provide definitive evidence that CQ uptake is determined by the binding of CQ to
FPIX. In no part is the uptake of CQ controlled by the differential stimulation of the NHE as proposed (Wünsch et
al., 1998
). Furthermore, in CQR parasites reduced uptake
of CQ, reduced apparent affinity of CQ-FPIX binding,
and reversal of these parameters by verapamil are totally
independent of NHE activity. We also propose a mechanistic basis for the reversal of CQ resistance in which resistance reversers increase the pH of acid vesicles where
FPIX is generated. Therefore, this increases the affinity of
CQ-FPIX binding.
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Materials and Methods |
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Reagents
Leupeptin and trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E64) were obtained from Boehringer Mannheim. Ro 40-4388, Ro 61-7835, and Ro 61-9379 were provided by Dr. R.G. Ridley and Dr. R. Moon of Hoffmann LaRoche. Triethylamine (TEA), diethylamine (DEA), dibutylamine (DBA), 1-methyl-piperazine (1-MP) and dipropylamine (DPA) were purchased from Aldrich Chemical Co. Silicon oil was purchased from Dow Corning. 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein-acetoxymethylester (BCECF-AM) was purchased from Molecular Probes Inc. [3H]CQ (50.4 Ci per mmol) was purchased from DuPont NEN. [3H]Hypoxanthine was purchased from Amersham and [3H]amodiaquine (AQ; 106 mCi per mmol) was synthesized in house. All other reagents were purchased from Sigma Chemical Co.
Culture of P. falciparum and Drug Sensitivity Assays
Parasites were cultured and synchronized using standard techniques
(Trager and Jensen, 1976; Lambros and Vandenburg, 1979). Drug sensitivity in the presence or absence of potential resistance reversers and hemoglobinase inhibitors was determined according to the method of Desjardins et al. (1979). IC50 values were calculated for each assay using the
four parameter logistic method (Grafit program, Erithacus software; Kent
Laboratories). The effect of the combination of CQ and Ro 40-4388 on
parasite growth was tested by titration of the two drugs at fixed ratios proportional to their IC50 values. The fractional inhibitory concentrations of the resulting IC50 values were plotted as isobolograms (Berenbaum, 1978
).
Measurement of the Effect of Proteinase Inhibitors on Parasite Hemoglobin Digestion and Parasite Hemozoin Content
Erythrocytes infected with the K1 strain were synchronized and cultivated
to the trophozoite stage and suspended in growth medium at a parasitemia of 10% and a hematocrit of 5%. At time 0, a sample was taken and
the infected cells were separated, counted, and analyzed for hemoglobin
and hemozoin as described below. The remaining cell suspension was incubated for 3 h at 37°C in the absence or presence of 10 µM Ro 40-4388, or 20 µM N-acteyl-L-leucyl-L-leucyl-methional (ALLM), or 20 µM
N-acetyl-L-leucyl-L-leucyl-norleucinal (ALLN), or 20 µM leupeptin. After
incubation, each was split into two aliquots. The infected cells from one aliquot were immediately separated from the uninfected cells using a Percoll-alanine gradient and counted as described (Ginsburg et al., 1998).
The hemoglobin and hemozoin concentrations of these cells were measured as described below. The infected cells from the remaining aliquot
were washed three times in prewarmed complete medium and returned to
culture for an additional 3-h incubation period. After incubation, the infected cells were separated, counted, and the hemozoin concentration was determined.
Hemoglobin concentration was measured using Drabkins reagent as
described (Ginsburg et al., 1998). The hemozoin concentration in the
pellet was determined after suspension in 2% (wt/vol) SDS in 0.1 M
NaHCO3, pH 9.1, followed by centrifugation at 14,000 rpm for 5 min. The
supernatant was discarded and this step repeated three times to remove all
nonhemozoin heme. The remaining hemozoin pellet was dissolved in 1 ml of
0.1 M NaOH and the absorbance was read at 400 nm using a Beckman
DU640 spectrophotometer. The amount of hemozoin/1010 cells was calculated from a calibration curve of known amounts of FPIX dissolved in 0.1 M NaOH.
Measurement of the Uptake of [3H]CQ by Intact Parasitized Erythrocytes
Infected erythrocytes were suspended in the appropriate buffer containing [3H]CQ, at a parasitemia of 1-2% and a hematocrit of 0.5%. At the required times, 100-µl samples were removed and the reaction was terminated upon centrifugation of the cells (14,000 rpm for 2 min) through
silicon oil and processed for scintillation counting as described in Bray et al.
(1996). Parasite specific uptake was determined by subtracting counts because of an equal number of uninfected erythrocytes. Unless otherwise
stated, CQ accumulation is expressed as the cellular accumulation ratio
(CAR) which is the ratio of the amount of radiolabeled CQ in the parasites to the amount of radiolabeled CQ in a similar volume of buffer after incubation.
The effect of the proteinase inhibitors Ro 40-4388, Ro 61-7835, Ro 61-9379, ALLM, ALLN, leupeptin, and E64, on the steady-state uptake of CQ was measured for 1 h at 37°C, in complete medium containing 1 nM [3H]CQ. The reversibility of Ro 40-4388 (10 µM), ALLM (20 µM), and ALLN (20 µM) was determined as follows: [3H]CQ was added at a concentration of 4 nM to inhibitor-treated and control groups. After 1 h, an aliquot was taken and processed for scintillation counting as described above. The remaining aliquot was washed three times in prewarmed medium (37°C). [3H]CQ was re-introduced to the washed and control cells at a concentration of 4 nM. After 1 h, samples were removed and processed for counting. For the K1 isolate, parallel incubations were performed in the presence of 10 µM verapamil. In the case of ALLM and ALLN, inhibitors were added 45 min before the initial addition of CQ and a 10-min equilibration period was introduced after each wash.
CQ-FPIX binding parameters, in the absence or presence of 10 µM Ro
40-4388, were determined as described in Bray et al. (1998). For the K1
isolate, equilibrium binding parameters were obtained in sodium-free
buffer (122.5 mM choline chloride, 5 mM KCl, 1.2 mM CaCl2, 0.8 mM
MgCl2, 5.5 mM D-glucose, 1.0 mM K2HPO4, 10 mM Hepes, pH 7.4) in the
absence or presence of 2 mM NH4Cl, 10 µM verapamil, or 100 nM monensin. Saturable uptake of CQ was calculated by subtracting nonsaturable CQ uptake from the total CQ uptake as described previously (Bray et
al., 1998
). Binding data are presented as Scatchard plots of saturable CQ
uptake. The apparent Kd of binding is given by the reciprocal of the slope
and the amount of bound drug is given by the x-intercept.
The effect of resistance modulators was determined over 1 h in growth medium containing 1 nM [3H]CQ in the absence or presence of 10 µM verapamil, 100 nM nigericin, 100 nM monensin, 2 mM NH4Cl, 2 mM MTA, 2 mM TEA, 2 mM DEA, 2 mM DBA, 2 mM 1-MP, or 1 mM DPA. Care was taken to ensure the medium pH was 7.4 throughout. Optimum concentrations of these compounds were obtained in preliminary experiments by measuring the effect of serial 1:3 log dilutions of drugs on CQ uptake (data not shown).
To determine the effect of bicarbonate on the uptake of [3H]CQ, growth medium containing various concentrations of bicarbonate was shaken in atmospheric air at room temperature until the pH drift was complete. The pH was adjusted to 7.4 with 1 M HCl. Parasites were incubated in growth medium containing various concentrations of bicarbonate and 3 nM [3H]CQ for 1 h and terminated thereafter.
Measurement of the Uptake of [3H]CQ Into Isolated Parasites
Parasites were isolated from the host cell by selective lysis of the host cell
compartment (Elford, 1993). Ringer's buffer modified to simulate an intracellular milieu was used throughout (Wünsch et al., 1998
). In selected
experiments, sodium chloride in the buffer system was replaced with molar equivalents of either choline chloride or N-methyl-D-glucamine chloride. CQ uptake was determined by suspending the cell pellet containing
purified free parasites, in 200 µl of the appropriate buffer containing 50 nM [3H]CQ, prewarmed to 37°C. Drug uptake was determined in the absence or presence of 100 µM 5-N-ethyl-N-isopropyl amiloride (EIPA) or 10 µM
verapamil. At the appropriate time points, aliquots were removed and the
reaction was terminated by centrifugation (14,000 rpm, 1 min, at 4°C). The
sample was placed in an ice-bath, the supernatant was removed, and
the pellet was washed once in the appropriate ice-cold buffer without
[3H]CQ. After centrifugation (14,000 rpm, 1 min, at 4°C), the supernatant was carefully removed with a drawn out glass Pasteur pipette and the
pellet was processed and counted for infected erythrocytes.
CQ uptake into isolated parasites was ~30% of intact infected erythrocytes' uptake with over 30 min of CQ exposure. To investigate whether
this reduced CQ uptake occurs because of impaired NHE activity, we
compared the cytosolic pH of isolated parasites to that of parasites within
intact host erythrocytes. In agreement with previous reports (Wünsch et al.,
1998), we found no significant differences in cytosolic pH in these preparations. For example, the mean cytosolic pH for the K1 clone in its host
cell was 6.902 (SEM = 0.045, n = 12) compared with 6.992 (SEM = 0.038, n = 22) when the parasite is liberated from the host cell. In addition, free
parasites exhibited a marked cytosolic acidification in sodium-free buffer
(0.76 pH unit ± SEM 0.045, n = 6), in full agreement with previous reports (Bosia et al., 1993
). Thus, NHE is functional in isolated parasites.
We also investigated the possibility that the reduced CQ uptake occurs because of a loss of viability. Isolated parasites were found to incorporate radiolabeled hypoxanthine and isoleucine at the same rate as intact infected erythrocytes for at least 6 h (data not shown). Since the parasites are viable and NHE is functioning normally, we assumed that the reduced accumulation of CQ happens because of cessation of hemoglobin trafficking from the host cell at time 0 that reduces the amount of FPIX available for CQ binding.
Determination of the Effect of Amiloride Analogues and Proteinase Inhibitors on FPIX Polymerization
Measurement of FPIX polymerization inhibition by amiloride analogues
and proteinase inhibitors employed a modification of the procedure described by Raynes et al. (1996). An aliquot (100 µl) of trophozoite lysate
and FPIX (100 µl of 3 mM in 0.1 M NaOH) was mixed with an aliquot of
1 M HCl (10 µl) and sodium acetate (500 mM, pH 5.2) to give a volume of
900 µl in each tube. A series of drug concentrations was prepared in ethanol and 100 µl of each was added to the appropriate samples. The effect of
ethanol on the polymerization process was assessed by adding 100 µl of
ethanol to the control samples. Samples were mixed and incubated for 12 h,
with occasional mixing. After incubation, samples were centrifuged (14,000 rpm, 15 min, at 21°C) and the hemozoin pellet repeatedly washed
with 2%(wt/vol) SDS in 0.1 M sodium bicarbonate, pH 9.0, with sonication (30 min, at 21°C; FS100 bath sonicator; Decon Ultrasonics Ltd.) until
the supernatant was clear (usually 3-4 times). After the final wash, the supernatant was removed and the pellet was resuspended in 1 ml of 0.1 M
NaOH, incubated for an additional 1 h at room temperature. Afterwards,
samples were mixed with a pipette. The hemozoin content was determined
by measuring the absorbance at 400 nm (Beckmann DU640 spectrophotometer) using a 1-cm quartz cuvette. The amount of hemozoin formed
during incubation was corrected for preformed hemozoin (the amount of
preformed hemozoin in the parasite extract was determined from a sample containing extract, but no substrate, which was incubated and repeatedly washed with 2% SDS as previously stated). The concentration of
drug required to produce 50% inhibition of polymerization (IC50) was determined graphically as described for the drug sensitivity assays.
Displacement of CQ Bound to FPIX-loaded Ghost Membranes and Parasite Debris
Erythrocyte ghost membranes were prepared as described previously
(Ginsburg et al., 1998). Membranes (0.27 mg protein) were loaded with
FPIX (2-5 µM) in 0.2 M Hepes buffer, pH 7.0 (HB7) at 37°C for 7 min followed by centrifugation (14,000 rpm, 10 min). The supernatant was discarded and the pellet was washed once in HB7. Samples of FPIX-loaded
membrane (0.01 mg protein) were suspended in 1 ml of HB7 containing
50 nM [3H]CQ and incubated in the absence or presence of various concentrations of the proteinase inhibitors, amiloride and the amiloride
analogues EIPA, 5-N-N-hexamethylene amiloride (HMA), and 5-N-isobutyl-N-methyl amiloride (IBMA) for 10 min at 37°C. The membrane suspension was centrifuged (14,000 rpm, 2 min), the supernatant removed,
and the pellet washed once in ice-cold HB7 without [3H]CQ. The remaining pellet was solubilized and processed for counting, as described above.
To measure CQ-FPIX binding affinity in a parasite-free system, the
membranes were loaded in HB7 with 2 µM FPIX as above and samples
(0.01 mg protein) were incubated in 1 ml HB7 containing 3 nM [3H]CQ
and 5-10 µM nonradioactive CQ for 10 min at 37°C. The reaction was terminated by centrifugation and samples were processed as above. Nonspecific CQ binding was estimated from parallel incubation of FPIX-free
membranes and subtracted from the total binding for each CQ concentration. Binding affinity was estimated by a computer fit of the data to the
Michaelis-Menten equation. The displacement of preaccumulated [3H]CQ
from parasite debris by EIPA was measured as described previously (Bray
et al., 1998). Before lysis, erythrocytes infected with the CQS (HB3 strain)
were loaded with 50 nM [3H]CQ in complete medium for 30 min at 37°C.
Cell-free Assay for the Generation of CQ Binding Sites from Hemoglobin
Pure isolated food vacuoles were prepared by modifying the methods of
Goldberg et al. (1990) and Saliba et al. (1998)
. Suspensions of synchronized trophozoites of the HB3 strain (~15% parasitemia) were washed
three times in PBS, pH 7.4. Each 5-ml sample of washed cell-pellet was
resuspended in PBS containing 0.15% saponin, incubated for 5 min, and
centrifuged (4,500 rpm for 5 min). The isolated trophozoites were washed
repeatedly in ice-cold PBS until the supernatant was clear. The trophozoite pellet was collected and resuspended in 10 vol of ice-cold trituration
buffer (0.25 M sucrose, 10 mM sodium phosphate, 0.5% streptomycin sulfate, pH 7.1) and triturated three times on ice, using a 27-gauge 3/4 in needle. The suspension was centrifuged (13,000 rpm, 2 min, at 4°C), supernatant discarded, and the pellet resuspended in 5 vol of buffer (2 mM magnesium sulfate, 100 mM potassium chloride, 10 mM sodium chloride, 25 mM Hepes, 25 mM sodium bicarbonate, 5 mM sodium phosphate, pH
7.1). To each 1 ml of the suspension 20 µl of 5 mg/ml DNaseI was added
and the suspension was incubated at 25°C for 5 min, followed by centrifugation (13,000 rpm, 2 min, at 4°C). The supernatant was discarded and the
pellet was resuspended in 5 vol of ice-cold trituration buffer and triturated
once. The suspension was layered on top of 7 ml of ice-cold 42% Percoll
solution containing 0.25 M sucrose, 1.5 mM magnesium chloride, pH 7.1, and centrifuged (12,000 rpm, 30 min, at 4°C). After centrifugation, the purified food vacuoles were harvested from the bottom of the gradient and washed three times in ice-cold buffer (0.25 M sucrose, 10 mM sodium phosphate, 1.5 mM magnesium chloride). Purity was checked by electron microscopy and by using assays of host cell and parasite cytosol marker
enzymes as described previously (Saliba et al., 1998
). Electron microscopy
revealed no contamination with other organelles or membranes and
~50% of the vacuoles had intact membranes (data not shown). Contamination with the parasite cytosolic enzyme lactate dehydrogenase was
<0.7%. Compared with isolated trophozoites and contamination with
host cell acetylcholine esterase was below the limits of detection in the assay.
Pellets of pure food vacuoles were added to 20 vol of ice-cold 500 mM
sodium acetate, pH 5, and immediately subjected to five cycles of rapid
freezing in liquid nitrogen and thawing at room temperature. The suspension was triturated 10 times with a 27-gauge needle and centrifuged
(13,000 rpm, 3 min, 4°C). The proteinase-rich supernatant was collected
and used for the cell-free assay. Protein content was measured using the
bicinchoninic acid assay (Smith et al., 1985). Samples of the enzyme extract
(2-10 µl) were added to 200 µl of 500 mM sodium acetate, pH 5, containing [3H]CQ or [3H]AQ. 100 µl of a 50-µM solution of human hemoglobin
was added to the mixture. Purified erythrocyte ghost membranes (0.01 mg protein) were added to act as carriers for the CQ-FPIX complex. The samples were incubated for 1 h at 37°C in the absence or presence of 10 µM Ro
40-4388, or 20 µM Ro 61-7835, or 20 µM Ro 61-9379, or 20 µM leupeptin,
or 20 µM E64. After incubation, the samples were centrifuged (14,000 rpm,
1 min) and the supernatant was removed gently with a drawn out glass Pasteur pipette to avoid disturbing the pellet. The pellet was washed once in
ice-cold sodium acetate buffer without radiolabel and the remaining pellet
was solubilized and processed for counting as described above.
Measurement of Parasite Cytosolic pH
Parasite cytosolic pH was estimated using BCECF-AM as described by
Wünsch et al. (1998). The parasite suspension preloaded with BCECF was
incubated at 37°C for 15 min in a perfusion chamber and the cells were allowed to settle on a glass coverslip coated with poly-L-lysine. The experimental chamber was transferred to the stage of an inverted Diaphot microscope (Nikon). At appropriate time points, 10 µM Ro 40-4388, or 2 mM
NH4Cl, or 2 mM methylamine was added to the perfusion buffer and the
pH was monitored. NHE activity in sodium-free buffer was monitored by
measuring the ability of the cell to recover from an acid load: the cells
were perfused with Ringer's buffer containing 40 mM NH4Cl, after which the perfusion buffer was changed to Ringer's with one molar equivalent of
choline replacing sodium. When the fall in cytoplasmic pH stabilized, the
perfusion buffer was changed to sodium Ringer's to allow the cytosolic pH
to recover.
Digital imaging microfluorimetry was carried out with an image analysis system (Quanticell 700 series; Applied Imaging International Ltd.) incorporating an intensified camera (CCD; Photonic Science). Background
subtraction was performed independently for each excitation wavelength
used. The autofluorescence of unloaded parasites was negligible. The excitation wavelengths used were 440 and 490 nm with emission measured
above 510 nm. Calibration was performed for each cell using the nigericin/
high K+ method that uses two or three buffers of known pH (Wünsch et al., 1998). Since no ultrastructural detail could be observed under light microscopy, other than the food vacuole, the reported pH values are average
values for the entire parasite cytosol minus the food vacuole.
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Results |
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Saturable CQ Uptake in P. falciparum Is Driven by Binding to FPIX
We determined the effect of a range of proteinase inhibitors on CQ uptake. In addition to Ro 40-4388, two other
specific plasmepsin inhibitors, Ro 61-7835 and Ro 61-9379, inhibited CQ uptake at low micromolar concentrations
(Fig. 1 A). Similar inhibition was observed with the tripeptide aldehydes, ALLM and ALLN, which are known inhibitors of the processing of proplasmepsins I and II into
active mature enzymes (Francis et al., 1997a). The other cysteine proteinase inhibitors tested, E64 and leupeptin,
were much less effective. Inhibition of proplasmepsin processing by the tripeptide aldehydes and the direct inhibition of the plasmepsins with Ro 40-4388 are reversible
(Francis et al., 1997a
; Berry, C., personal communication).
We have established that the inhibition of CQ uptake by
both classes of compounds is also reversible, as predicted
by our hypothesis (Fig. 1, B and C). In CQS parasites, CQ
uptake is inhibited almost completely by the proteinase inhibitors (Fig. 1, A and B). In CQR parasites, the effect was
smaller but the inhibition of the resistance reversing effect
of verapamil was spectacular (Fig. 1 C). This observation
is in agreement with our hypothesis that verapamil increases CQ uptake by increasing the affinity of CQ-FPIX
binding (Bray et al., 1998
). Our data show that CQ-FPIX
binding determines the amount of drug that is taken up by
the parasite. The marked antagonism of CQ activity in the
presence of Ro 40-4388 (Fig. 1 D; Moon et al., 1997
) suggests that binding to FPIX also determines the antimalarial activity of CQ.
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Our model of the mechanism of CQ uptake is supported by the demonstration that proteinase inhibitors efficiently inhibit the degradation of hemoglobin and liberation of FPIX under the same conditions used to inhibit CQ uptake (Fig. 2). Hemoglobin digestion is almost completely inhibited by ALLM, ALLN, and Ro 40-4388. On the other hand, the effect of leupeptin was smaller. It is very difficult to accurately measure the concentration of free FPIX in parasites, so we have measured the concentration of hemozoin (polymerized FPIX). At the concentrations used, none of the compounds had any direct effect on the polymerization of FPIX in vitro (data not shown). However, all four compounds inhibited the production of hemozoin by intact parasites to some extent (Fig. 2). These data show that the concentration of FPIX is reduced when hemoglobin digestion is blocked. The inhibition was much more pronounced with ALLM, ALLN, and Ro 40-4388 than with leupeptin and was found to be reversible. Hemozoin production resumed at the normal rate when the proteinase inhibitors were removed by washing (Fig. 2). All these results are in agreement with the effect of the same inhibitors on CQ uptake (Fig. 1, A and B) and provide compelling evidence that CQ uptake results from binding of the drug to FPIX generated inside the infected erythrocyte.
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The final piece of experimental evidence that proves the
central role of FPIX binding in the uptake of CQ was obtained using a cell-free system. The binding characteristics
of intact CQS parasites can be reconstituted using an
erythrocyte ghost membrane preparation preloaded with
FPIX. The capacity (Bmax) of saturable CQ binding to
these membranes was dependent on the FPIX concentration, although at higher loading concentrations the apparent binding affinity was reduced due to aggregation of
FPIX (data not shown). We measured an affinity (Kd) for
this interaction of 25 nM (Fig. 3 A). This value is the same
as the apparent Kd of saturable CQ uptake in CQS P. falciparum, again indicating that FPIX binding is all that is required to account for the saturable uptake of CQ (Bray et al.,
1998 and see below). Other experiments indicate that CQ
binding sites can be generated from native hemoglobin by
an extract from purified food vacuoles (Fig. 3 B). There is little doubt that CQ binding is absent when either hemoglobin or vacuole extract is omitted, or when the extract
has been heat-treated (data not shown). The same proteinase inhibitors that block the uptake of CQ into intact cells
block CQ binding in the cell-free system (Fig. 3 B). In fact,
the percent inhibition of CQ binding in the cell-free system is almost perfectly correlated with the inhibition of
CQ uptake into intact cells by similar concentrations of
these compounds (R2 = 0.99, P < 0.001). AQ binding sites
can be generated in a similar fashion and have a similar inhibitor profile (Fig. 3 C). These data suggest that the
mechanism of AQ uptake is the same as that of CQ, i.e.,
the uptake of both drugs is driven by their binding to
FPIX.
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The initial rate of uptake of CQ into CQS parasites is
rapid, prompting some authors to suggest that CQ is actively transported (Warhurst, 1986; Ferrari and Cutler,
1991
; Sanchez et al., 1997
). However, our data suggest that
the initial rate of CQ uptake is determined by the intraparasitic binding of CQ to FPIX rather than active transport.
We found that after only a 5-min exposure of HB3 (CQS)
parasites to CQ, the number of CQ binding sites (Bmax) was reduced from 17.2 µmol/liter to 6.9 µmol/liter by the
specific hemoglobinase inhibitor Ro 40-4388 (Fig. 4).
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The Uptake of CQ Is Independent of the Activity of the NHE
In contrast to our explanation, others believe that CQ is
transported through the NHE of CQS parasites (Sanchez
et al., 1997; Wünsch et al., 1998
). The NHE of CQR isolates was activated constitutively, incapable of CQ transport, and responsible for an elevated cytoplasmic pH
(Wünsch et al., 1998
). We were not able to reproduce
these observations. In our hands, cytosolic pH is subject to
a greater degree of variation and is actually lower in the
resistant isolate (HB3, pH 7.183 ± SEM 0.04, n = 17; K1,
pH 6.902 ± SEM 0.046, n = 12). The reason for this discrepancy is unknown but there may be a methodological
basis: our pH measurements are average values for the
whole cytosol (minus the food vacuole), whereas those of
Wünsch et al. (1998)
refer to smaller regions of interest within the cytosol. Elevated cytoplasmic pH of CQR isolates is a fundamental tenet of the NHE hypothesis that
probably does not apply to the isolates used in this study.
Nonetheless, we have assessed the likely impact of small
variations in cytosolic pH on the binding of CQ to FPIX.
We found that the binding of CQ to FPIX in ghost membranes is sensitive to buffer pH, but this effect is probably
not significant across the range of reported cytosolic pHs
(Fig. 5 A). We have also tested the effect of hemoglobinase inhibition on cytosolic pH although such an effect
seems unlikely. We found that concentrations of Ro 40-4388, sufficient to block CQ uptake, had no effect on cytosolic pH, indicating that blocking hemoglobin degradation
does not affect the cytosolic pH and that this compound
does not block CQ uptake by interacting with the parasite
NHE (Fig. 5 B).
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It is proposed that CQ is driven through the NHE of
CQS parasites in a rapid burst of sodium-proton exchange
stimulated by CQ itself (Wünsch et al., 1998). We tested
this proposal directly by replacing the sodium ions in the
media with nonexchangeable cations and monitoring the
uptake of CQ into isolated parasites. This maneuver resulted in a marked inhibition of the NHE of isolated parasites, as evidenced by the inability to recover from an acid load (0.76 pH unit ± SEM 0.045, n = 6). It follows that sodium-free buffer should produce a very marked effect on
CQ uptake if the NHE is involved in this process. It was
observed that the initial rate of CQ uptake and steady-state CQ accumulation into isolated CQS parasites (HB3
strain) remained constant regardless of the concentration
of sodium ions in the buffer (Fig. 6, A and B). These data
demonstrate that CQ uptake is independent of the parasite's NHE activity status. The reported ability of CQ to
stimulate the NHE of CQS parasites (Wünsch et al., 1998
)
is interesting but it appears to have no involvement in the
mechanism of CQ uptake.
|
We also tested the effect of the sodium ion concentration on the uptake of CQ into isolated CQR parasites (K1
strain). The results, presented in Fig. 6 C, again show that
CQ uptake is unchanged when sodium ions are removed
from the buffer. In addition, the reversal of CQ resistance
by verapamil was independent of NHE activity, as it is retained in sodium-free medium (Fig. 6 C). CQ accumulation of CQR parasites is reduced by a similar ratio compared with CQS parasites regardless of the sodium ion
concentration. Moreover, it is independent of the presence
or absence of the host cell (Fig. 6, A and C; Bray et al.,
1998). Therefore, all of the phenotypic features of CQ uptake that distinguish CQR parasites from CQS parasites
are retained in sodium-free medium thereby providing
compelling evidence that the parasite NHE is not involved in CQ resistance.
Amiloride Derivatives Bind to FPIX and Displace CQ
EIPA significantly inhibits the uptake of CQ even though
there are no sodium ions in the buffer and NHE is inactive
(Fig. 6, A and B). These results are interesting because
they indicate that EIPA inhibits CQ uptake by a mechanism distinct from the inhibition of NHE. One possibility
is that EIPA inhibits the binding of CQ inside the parasite.
This assessment is supported by the data presented in Fig.
7, showing EIPA displacing prebound CQ from parasite
debris in a concentration-dependent manner. This interaction is not due to displacement of CQ bound to the NHE protein as this could only account for ~5% of the measured CQ binding (calculated from binding data given in
Sanchez et al., 1997).
|
Amiloride and its derivatives were shown to competitively inhibit the saturable uptake of CQ (Wünsch et
al., 1998). Since we attribute saturable uptake of CQ to
CQ-FPIX binding, we have looked for an interaction of
these compounds with FPIX. Such an interaction is indicated by the data in Fig. 8, showing amilorides inhibiting
FPIX polymerization. Indeed, HMA is almost as efficient an inhibitor of FPIX polymerization as CQ itself. These
data suggest that amiloride derivatives might inhibit the
uptake of CQ into P. falciparum by preventing CQ-FPIX
binding. We tested this possibility directly and demonstrated that these compounds inhibit CQ-FPIX binding in
loaded ghost membranes in a dose-dependent manner
(Fig. 9). Interestingly, the 50% inhibitory concentration
for EIPA was within the range reported for 50% inhibition of CQ uptake into P. falciparum by EIPA (Sanchez et
al., 1997
). Furthermore, the rank order of inhibition of
CQ-FPIX binding by the amiloride analogues (HMA > EIPA > IBMA
amiloride) is the same as the rank order of their inhibition of CQ uptake into intact parasitized
erythrocytes (Wünsch et al., 1998
). These data indicate
that amiloride analogues do inhibit CQ uptake by blocking CQ-FPIX binding rather than inhibiting the parasite
NHE. We also hypothesized that verapamil reverses CQ
resistance by increasing the apparent affinity of CQ binding to FPIX (Bray et al., 1998
). If our working model is
correct and if EIPA binds to FPIX at the expense of CQ,
then EIPA should ablate the verapamil effect; this is
clearly demonstrated in Fig. 6 C.
|
|
Lysosomotropic Compounds Selectively Increase the Apparent Affinity of CQ-FPIX Binding in CQR Isolates
Although CQ is not actively transported through NHE
(Fig. 6, A-C) it is still possible that CQ resistance may
arise from changes in the cytosolic pH (Martiney et al.,
1995). Accordingly, we examined the effect of various
agents known to perturb intracellular pH on the uptake of
CQ by CQS and CQR parasites. We found that ammonium chloride, methylamine, a variety of alkylamines, and
the carboxylic ionophores, monensin and nigericin, selectively increase the CQ accumulation of CQR parasites.
The maximum effect was found at concentrations of 1-2 mM
for amines and 100 nM for ionophores. In contrast, CQ accumulation by CQS parasites was either unaffected or reduced (Fig. 10 A). In this respect, the amines and ionophores mimic the effect of verapamil (Krogstad et al.,
1987
). Ammonium chloride (Fig. 10 B) and methylamine
were also capable of reversing CQ resistance in the same
way as verapamil (although the effect was not as pronounced). The remaining weak bases could not be assessed for their resistance reversal potential because of
their inherent cytotoxicity.
|
Ammonium chloride, methylamine, and verapamil were selected to investigate the possibility that resistance reversers alter cytosolic pH of CQR parasites. We found that these compounds had no significant effect on cytosolic pH at the low concentrations required to reverse resistance (data not shown). Therefore, the ability of these compounds to increase CQ accumulation is unlikely caused by any interaction with cytosolic pH regulators like NHE. Indeed, any possible contribution of the NHE in the process of resistance reversal is negated by the data showing that ammonium chloride, monensin, and verapamil all increase the apparent affinity of CQ binding to FPIX in sodium-free medium, when the NHE is inactivated (Fig. 11, A-D).
|
We were also interested to see if any other cytosolic pH
regulators could influence CQ uptake. In common with
other eukaryotic cells, it is possible that P. falciparum parasites possess a chloride/bicarbonate exchange mechanism
(Bosia et al., 1993). If so, and if this protein can influence
CQ uptake as suggested (Martiney et al., 1995
), then CQ
uptake should be influenced by the bicarbonate concentration of the medium. It was found that CQ uptake into
CQS and CQR parasites was not effected by the concentration of bicarbonate in the medium (Fig. 12). Taken together, our data suggest that none of the major cytosolic
pH regulators play a major role in the uptake of CQ or in
the mechanism of CQ resistance.
|
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Discussion |
---|
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---|
The specificity of antimalarial activity of CQ stems from
the potential of malaria parasites to accumulate more CQ
than any other type of eukaryotic cell. CQR parasites of
some species, including P. falciparum, have evolved ways
to reduce the extent of CQ accumulation. A better understanding of the mechanism of CQ uptake in P. falciparum
would be a significant step forward since it should explain
the specificity of the drug and may also offer clues to the
basis of resistance. CQ undoubtedly penetrates the parasite by passive diffusion as observed in other eukaryotic cells (MacIntyre and Cutler, 1993). CQ is a diprotic weak
base and, as such, undoubtedly accumulates to some extent inside the acidic compartments of the parasite by a
proton trapping mechanism (Yayon et al., 1985). This
mechanism may account for a considerable concentration
of CQ inside cells (Geary et al., 1986
). However, it probably does not account for the full extent of CQ accumulation by P. falciparum, which is notably greater than CQ accumulation by other eukaryotic cells with large acidic
compartments (MacIntyre and Cutler, 1993). Pioneering
studies by Fitch in the 1970s demonstrated that CQ uptake
into P. falciparum is saturable, energy-dependent, and
possibly inhibited by various compounds (Fitch, 1970
;
Fitch et al., 1974
). These studies suggest that malaria parasites possess an additional CQ concentrating mechanism
acting in concert with passive diffusion and proton trapping. Two putative CQ concentrating mechanisms are currently under consideration: active CQ import (Warhurst,
1986
) mediated by the parasite NHE (Sanchez et al., 1997
;
Wünsch et al., 1998
); and intracellular binding of CQ to FPIX (Chou et al., 1980
; Bray et al., 1998
).
The NHE hypothesis initially appears plausible and
many of the supporting data are robust. However, a close
scrutiny of the available literature reveals some intriguing
inconsistencies that call into question the mechanistic explanation offered by Wünsch et al. (1998). Particularly
problematic is the uptake of AQ, a close structural analogue of CQ. Early studies showed that AQ competitively
inhibits the uptake of CQ in P. falciparum, suggesting that
the same mechanism drives the uptake of both drugs
(Fitch et al., 1974
). We reported that the uptake of AQ
into CQR parasites is equivalent to the uptake of CQ into
CQS parasites (Bray et al., 1996
). It is hard to see how the
extensive uptake of AQ into CQR parasites can be driven
by an NHE that apparently is activated constitutively and
incapable of drug transport (Wünsch et al., 1998
).
A further difficulty is encountered when the effect of
verapamil is considered. Many studies have highlighted
the ability of verapamil to selectively increase the uptake
of CQ into CQR parasites (Krogstad et al., 1987; Wellems
et al., 1990
; Bray et al., 1992
; Walter et al., 1993
; Bray et al.,
1996
). Furthermore, this property of verapamil was linked
perfectly to CQ resistance in the progeny of a genetic cross
of CQR and CQS clones (Wellems et al., 1990
). If NHE is
responsible for CQ uptake, then verapamil would somehow have to selectively stimulate NHE of resistant parasites that is constitutively activated and incapable of CQ
transport. However, this is an unlikely scenario, doubly so,
when one considers the large quantity of literature demonstrating verapamil functioning to inhibit rather than stimulate drug transporters and ion channels (Gottesman and
Pastan, 1993
; Bray and Ward, 1998
). The active transport
model is inconsistent with our own data showing a role for
CQ-FPIX binding in the uptake of CQ, in verapamil-sensitive CQ resistance and in the uptake of AQ (Bray et al.,
1998
). We rigorously tested these two hypotheses and
found that the FPIX binding model remains valid, whereas
the altered NHE model failed.
We have tested the assertion that a rapid burst of sodium-proton exchange is required to drive uptake of CQ
into CQS parasites. We used isolated parasites for these
experiments and assumed that NHE activity is not impaired under such conditions. We show here as others
have shown that parasitic NHE is fully functional after the
parasite has been isolated from its host cell (Bosia et
al., 1993). We found that replacing sodium ions in the
buffer with nonexchangeable cations, such as choline or
N-methyl-D-glucamine, inactivated NHE but had no effect
on the amount of CQ taken up at steady-state by isolated
CQS parasites (Fig. 6 B). Moreover, sodium-free buffer
had no effect on the initial velocity of CQ uptake into
CQS parasites (Fig. 6 A). This was measured over the first
5 min after the addition of CQ, which coincides with the reported period of maximal stimulated NHE activity
(Wünsch et al., 1998
). Since there can be no rapid burst of
sodium-proton exchange in sodium-free buffer and CQ
uptake is not altered by these conditions, our results
strongly suggest that any ability of CQ to stimulate the
NHE of CQS parasites (Wünsch et al., 1998
) is unrelated
to the mechanism of CQ uptake. In addition, we found that stimulation of CQ uptake into CQR parasites, produced by verapamil and other resistance reversers, was unrelated to the activity of the NHE because these effects
are retained in sodium-free buffer (Figs. 6 C and 11, A-D).
Consequently, the effect of verapamil on the uptake of CQ
cannot be attributed to modulation of NHE activity via
the calcium-calmodulin regulatory pathway, as postulated by Sanchez et al. (1997)
. Hence, the clone-specific phenotypic characteristics of CQ uptake into CQS and CQR
parasites are retained in conditions which completely inactivate the NHE (Figs. 6, A-C, and 11, A-D). Therefore, it
is difficult to see how these characteristics can be related
in any way to the activity of the parasite NHE.
We have demonstrated that amiloride analogue inhibition of CQ uptake occurs because of inhibition of CQ-FPIX binding by these compounds, rather than the inhibition of the parasite NHE. In a critical series of experiments, we were able to demonstrate that the inhibition of CQ uptake by EIPA was distinct from any activity of this drug against NHE. CQ uptake is undiminished in sodium- free buffer when NHE is inactive, yet it is effectively inhibited when EIPA is present in this buffer (Fig. 6, A-C). These data strongly suggest that blocking CQ uptake by EIPA is caused by inhibition of a process that does not require sodium-proton exchange for CQ uptake. This directly violates the fundamental requirement of the NHE hypothesis.
There is solid experimental support for the FPIX model
(Balsubramian et al., 1984; Sullivan et al., 1996; Bray et al.,
1998
). Therefore, the demonstration that EIPA displaces
prebound CQ from parasite debris (Fig. 7), binds to
FPIX (Fig. 8), and inhibits the binding of CQ to FPIX-loaded ghost membranes (Fig. 9) provides direct evidence
that EIPA inhibits CQ uptake into parasites by binding
to FPIX. Furthermore, the rank order of activity of
the amiloride analogues (HMA > EIPA > IBMA
amiloride) is the same for the inhibition of CQ-FPIX
binding (Figs. 8 and 9) as it is for the inhibition of CQ uptake (Wünsch et al., 1998
). Examination of the chemical
structure of amiloride reveals that either of the two terminal amino groups of the guanidine function has the potential to coordinate with the iron center of the porphyrin as
an axial ligand (Rocha Gonslaves et al., 1991). Hoe 370, a
specific NHE inhibitor that is structurally unrelated to the
amiloride analogues, also has been shown to inhibit CQ
uptake (Wünsch et al., 1998
). Note, this compound also
contains a guanidine function and may also bind to FPIX.
Inhibition of CQ uptake by these specific NHE inhibitors
provided the best evidence of active import of CQ through the NHE. However, in the light of the data reported here,
we believe that this property of NHE inhibitors must now
be considered to support the alternative theory that CQ
uptake is governed by its binding to FPIX.
The central theme of the studies presented here is the
definitive proof, after some thirty years of controversy,
that saturable CQ uptake is driven by its binding to FPIX.
We recently demonstrated that Ro 40-4388, a potent and
specific inhibitor of the parasite proteolytic enzyme plasmepsin I (Moon et al., 1997), produces a concentration-dependent reduction in the number of CQ binding sites of
intact parasitized erythrocytes (Bray et al., 1998
). Here we
have extended this observation blocking hemoglobin degradation by two distinct mechanisms. The plasmepsins are
thought to initiate hemoglobin degradation by cutting the
Phe33-Leu34 bond of the alpha chain. This unfolds the hemoglobin tetramer, allowing further proteolysis and the
release of FPIX (Goldberg et al., 1991
; Gluzman et al.,
1994
; Francis et al., 1997b
). Although the parasite contains other hemoglobinase enzymes, there is good evidence that
the inhibition of plasmepsin I alone is sufficient to stop the
digestion of hemoglobin and release of FPIX (Francis et al.,
1994
, 1997b
). We have used Ro 40-4388, Ro 61-7835, and
Ro 61-9379 to block hemoglobin cleavage. All three compounds are potent inhibitors of the parasite aspartic hemoglobinase enzymes, plasmepsin I and plasmepsin II
(Moon et al., 1997
; Bray et al., 1998
; Moon, R.P., personal
communication). In addition we have prevented proplasmepsin processing with ALLM and ALLN. We provide
evidence that the reversible inhibition of CQ binding produced by Ro 40-4388 and other proteinase inhibitors stems
from a reversible cessation of hemoglobin digestion and FPIX release in the parasite (Figs. 1, A-C, and 2).
Perhaps the most convincing support for our hypothesis comes from the demonstration that a purified food vacuole extract can generate CQ binding sites in a cell-free system using human hemoglobin as a substrate (Fig. 3 B). The correlation of the inhibitor specificity of this process with that of CQ uptake into intact cells is compelling. Nevertheless, it is important that any proposed mechanism accounts quantitatively as well as qualitatively for CQ uptake. Further analysis of the data presented in Fig. 3 B reveals that this is the case. The extract from 106 vacuoles generates 123.72 fmol of CQ binding sites. At the external CQ concentration used (0.61 nM) this equates to a CAR of 2,399 (assuming one food vacuole per infected erythrocyte). This figure compares with a CAR of ~2,600 for intact parasites under similar conditions (Fig. 1 A). Much proteolytic activity has undoubtedly been lost in our assay because of poor enzyme extraction efficiency and leakage of enzymes from vacuoles during purification. Therefore, intact parasites clearly possess more than enough proteolytic activity to account for the uptake of CQ.
FPIX-CQ binding is the principal driving force for drug
uptake including the initial CQ uptake kinetics, measured
after 5 min exposure to CQ and previously attributed to a
carrier-mediated import mechanism (Fig. 4). Published estimates of the rate of hemoglobin digestion adequately defend this argument. It is estimated that each parasite degrades 0.06 fmol of hemoglobin per hour (Goldberg et al.,
1990). This would liberate 50 µmol FPIX per liter of parasites in 5 min, i.e., more than enough to account for initial
rate of bound CQ (17.2 µmol per liter in 5 min, Fig. 4)
even at a stoichiometry of 2 FPIX:1 CQ. If so, this indicates that CQ uptake may limit itself by stopping hemoglobin digestion since the steady-state Bmax is only 30-40
µmol per liter (Bray et al., 1998
). CQ uptake might be limited by CQ-FPIX inhibition of the hemoglobinase enzymes. It is possible that sufficient CQ-FPIX complex
remains within the vacuole to inhibit the proteolytic
enzymes (Vander Jagt et al., 1987; Gluzman et al., 1994
).
To quote Chou et al. (1980), "Unequivocal identification of an isolated substance as a drug receptor requires
(a) that affinities and specificities of binding of the drug
match those of the receptor, (b) that the drug is ineffective
when the putative receptor is absent from the organism,
and (c) that drug effectiveness returns when the receptor
is reintroduced into the organism." The specificity of hemoglobinase inhibitors that inhibit cellular CQ uptake is identical to their specificity in the cell-free enzymatic CQ
binding assay (Figs. 1 A and 3 B). These drugs are specific
and reversible inhibitors of FPIX generation (Fig. 2). This
biochemical knockout has permitted us to show that FPIX
can be reversibly removed, causing a reversible inhibition
of drug uptake (Figs. 1 B and 2). In addition to governing
the uptake of the drug, CQ-FPIX binding determines antimalarial activity since the combination of CQ and hemoglobinase inhibitors is markedly antagonistic (Fig. 3 C;
Moon et al., 1997
). Furthermore, we were able to match
the affinity of binding of CQ to CQS parasites to the affinity of CQ-FPIX binding in a parasite-free system (Fig. 3
A). Thus, all of the above criteria have been satisfied and
identify FPIX as the CQ receptor in P. falciparum.
Our data indicate that NHE has no involvement in the
mechanism of CQ resistance (Figs. 6 and 11). Indeed, any
involvement of cytosolic pH regulation seems unlikely. Instead, our data suggest that CQ resistance stems from an
alteration in the local environment of FPIX generation in
acid vesicles. We found that a wide range of lysosomotropic compounds mimics the effects of verapamil (Fig. 10
A). This could indicate the inhibition of a drug transporter similar to P-glycoprotein. However, since many of these
compounds are not known to interact with P-glycoprotein,
we suggest an alternative mode of resistance reversal. The
concentrations required to reverse resistance produced no
alkalinization of the parasite cytosol but might be expected to produce a significant alkalinization of lysosomes
and endosomes (Millot et al., 1998). There is evidence in
the literature that hemoglobin digestion begins in hemoglobin delivery vesicles, before they fuse with the food
vacuole (Slomianny and Prensier, 1990
). To protect the
parasite, the resistance mechanism must be operational
throughout the endocytic pathway. The intracellular localization of CQ resistance gene CG2 throughout the endocytic pathway is certainly consistent with this hypothesis
(Su et al., 1997
). It is our belief that CQ resistance results
from a selective change in vesicular function within relevant hemoglobin processing acidic compartments. This
reduces the affinity of CQ-FPIX binding that can be
reversed by lysosomotropic agents. CG2 and related proteins could potentially alter the binding of CQ to FPIX by directly binding to FPIX or by altering vesicular pH or
buffering capacity. All of these mechanisms could be modulated by vesicle alkalinization and are currently under investigation in our laboratory.
![]() |
Footnotes |
---|
Address correspondence to S.A. Ward, Department of Pharmacology and Therapeutics, Liverpool University, Liverpool L69 3BX, United Kingdom. Tel: 44-151-794-8219. Fax.: 44-151-794-8217. E-mail: saward{at}liv.ac.uk
Received for publication 22 July 1998 and in revised form 8 January 1999.
The authors would like to thank Dr. R.G. Ridley and Dr. R.P. Moon for their generosity in providing the proteinase inhibitors Ro 40-4388, Ro 61-7835, and Ro 61-9379.
This work was supported by a Research Program grant from the Wellcome Trust. O. Janneh is supported by the Overseas Research Students Award Scheme.
![]() |
Abbreviations used in this paper |
---|
AQ, amodiaquine; CQ, chloroquine; CQR, chloroquine-resistant; CQS, chloroquine-sensitive; FPIX, ferriprotoporphyrin IX; HB7, Hepes buffer at pH 7; NHE, Na+/H+ exchanger.
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