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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1426-1437
Antimalarial Activity of 77 Phospholipid Polar Head Analogs: Close
Correlation Between Inhibition of Phospholipid Metabolism and In
Vitro Plasmodium Falciparum Growth
By
Marie L. Ancelin,
Michèle Calas,
Jacques Bompart,
Gérard Cordina,
Dominique Martin,
Mohammed Ben Bari,
Taïb Jei,
Pierre Druilhe, and
Henri J. Vial
From CNRS UMR 5539, Department of Biologie-Santé, Montpellier,
France; the Laboratoire des Aminoacides, Peptides et protéines,
CNRS UMR, 5810, Montpellier, France; the Laboratoire de Chimie
Organique Pharmaceutique, Faculté de Pharmacie, Montpellier,
France; the Département de Chimie, Faculté des Sciences Ben
M'Sik, Casablanca, Morocco; and the Laboratoire de Parasitologie
Bio-Médicale, Institut Pasteur, Paris, France.
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ABSTRACT |
Seventy-seven potential analogs of phospholipid polar heads, choline
and ethanolamine, were evaluated in vitro as inhibitors of
Plasmodium falciparum growth. Their IC50 ranged
from 10 3 to 107 mol/L. Ten compounds
showed similar antimalarial activity when tested against three
different parasite strains (2 chloroquine-sensitive strains and 1 chloroquine-resistant strain). Compounds showing marked antimalarial
activity were assayed for their effects on phospholipid metabolism. The
most active compounds (IC50 of 1 to 0.03 µmol/L) were
inhibitors of de novo phosphatidylcholine (PC) biosynthesis from
choline. For a series of 50 compounds, there was a close correlation
between impairment of phospholipid biosynthesis and inhibition of in
vitro malaria parasite growth. High choline concentrations caused a
marked specific shift in the curves for PC biosynthesis inhibition.
Concentrations inhibiting 50% PC metabolism from choline were in close
agreement with the Ki of these compounds for the choline transporter in
Plasmodium knowlesi-infected erythrocytes. By contrast,
measurement of the effects of 12 of these compounds on rapidly dividing
lymphoblastoid cells showed a total absence of correlation between
parasite growth inhibition and human lymphoblastoid cell growth
inhibition. Specific antimalarial effects of choline or ethanolamine
analogs are thus likely mediated by their alteration of phospholipid
metabolism. This indicates that de novo PC biosynthesis from choline is
a very realistic target for new malaria chemotherapy, even against pharmacoresistant strains.
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INTRODUCTION |
THE INCREASING polypharmacoresistance of
Plasmodium falciparum to conventional antimalarials, combined
with the resistance of mosquitoes to various pesticides, has
contributed to a dramatic resurgence of malaria.1 New
therapeutic approaches to this endemic disease are now being actively
investigated. One approach consists of interfering with parasite
metabolic pathways to develop a new range of antimalarial drugs with
original structures and modes of action.
We previously characterized phospholipid (PL) metabolism as an ideal
target for new chemotherapy due to its vital importance to the
parasite. PL metabolism is absent from normal mature human erythrocytes,2 but the erythrocyte PL content increases by as much as 500% after infection.3-5 Phosphatidylcholine
(PC) and phosphatidylethanolamine (PE) are the major PL of the infected erythrocyte, representing about 85% of total PL. De novo pathways for
PC and PE biosynthesis from choline and ethanolamine, respectively, have been thoroughly characterized in Plasmodium-infected
erythrocytes.6 For de novo PC biosynthesis,
cholinephosphate cytidylyltransferase is a regulatory step in the
pathway, but choline transport (which regulates the supply of
precursor) is also a limiting step.7 For de novo PE
biosynthesis, ethanolaminephosphate cytidylyltransferase is probably
the rate-limiting step (personal observation), whereas ethanolamine entry occurs by mere passive diffusion.8 We
have previously shown that impairment of PL biosynthesis with polar head analogs, which interfere with natural polar head incorporation either by substitution or competition9-11 or with unnatural
fatty acids,12 is lethal to the intraerythrocytic stage of
P falciparum in vitro.
We have just reported the effects of 77 compounds that are analogs of
ethanolamine or, for most of them, of choline on in vitro P
falciparum growth.13 In the present study, systematic screening of compounds with antimalarial effects on PL metabolism, using a method that we developed,14 enabled us to show a
clear correlation between their antimalarial activity and PL metabolism impairment. Some pharmacologic target characteristics that will be
useful for designing very active specific antimalarial compounds could
thus now be defined.
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MATERIALS AND METHODS |
Chemicals.
Thirty products were chemically synthesized as described
previously.13 The compounds that were synthesized for this
study are noted in Table 1.
F14 and F19 were provided by Dr J. Berthe (Sanofi, Montpellier,
France). The other commercial products, as well as choline,
ethanolamine, and dibutyl phtalate, came from Sigma Chemical Co (St
Louis, MO).
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Table 1.
General Structures of Phospholipid Polar Head Group
Analogs and Their IC50 and PL50 Against P
falciparum In Vitro (Nigerian Strain)
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(Methyl-3H)choline, (1-3H)ethan-1-ol-2-amine,
and (G-3H)hypoxanthine were purchased from Amersham Corp
(Les Ulis, France) and (3H)thymidine was purchased from CEA
(Saclay, France). RPMI 1640 medium15 and special RPMI 1640 without choline, methionine, or serine were obtained from GIBCO
Laboratories (Eragny, France). Modified RPMI 1640 consisted of special
RPMI 1640 complemented with 25 mmol/L HEPES, 20 µmol/L methionine,
and 50 µmol/L serine. All reagents were of analytical grade.
Biologic materials.
Human blood or AB human serum came from the local blood bank. Three
P falciparum strains were routinely used: 2 chloroquine-sensitive strains, the Nigerian strain16 and
the NF54 strain,17 and a chloroquine-resistant strain from
Thailand (T23).18 P falciparum strains were
maintained by serial passages in human erythrocytes according to the
petri dish candle-jar method.19 P knowlesi-infected erythrocytes (Washington strain, variant 1) were collected from splenectomized Macaca fascicularis monkeys (Sanofi,
Montpellier, France) infected with cryopreserved parasites, as
previously described.9 The lymphoblastoid cell line (SAR
strain) was a gift from Dr B. Klein (Institut de Génétique
Moléculaire, Montpellier, France).
Antimalarial activity.
Effects of drugs on in vitro P falciparum growth were measured
in microtiter plates using (3H)hypoxanthine incorporation
into nucleic acids of an infected erythrocyte suspension (final
hematocrit level, 1%; parasitemia, 0.3% to 0.8%) according to the
method of Desjardins et al,20 with the drug placed in
contact with infected erythrocytes for one full parasite cycle (48 hours). Parasite viability was assayed by the capacity to incorporate
(3H)hypoxanthine (0.7 µCi/well) in parasite nucleic acids
for 15 hours. At the end of incubation, cells were collected on glass fiber filters (Whatman GF/C; Whatman, Maidstone, UK) with
a cell harvester (Micro 96; Skatron, Lier, Norway) and filters were
then counted for radioactivity in 2 mL of scintillation cocktail
(Packard no. 299) (Packard Instrument, Meriden, CT) in a Beckman LS
5000 spectrophotometer (Beckman, Fullerton, CA). The results are
expressed as the concentration resulting in 50% inhibition
(IC50) of hypoxanthine incorporation. Experiments were
performed at least twice in triplicate.
Measurement of PL metabolism.
PL metabolism was monitored in microtiter plates by the incorporation
of 10 µmol/L (3H)choline (1.75 Ci/mmol) or 2 µmol/L
(3H)ethanolamine (2.9 Ci/mmol) into PL constituents as
previously described.14 For comparison and to determine any
specific effects, incorporation of (3H)hypoxanthine (1.2 µCi/well, 6 Ci/mmol) into nucleic acids was monitored concurrently.
Unless otherwise specified, infected erythrocyte suspensions (30 µL,
1 to 5 × 106 infected cells/well, 3% to 10%
parasitemia) were mixed with 20 µL of modified RPMI 1640 without
(control) or with the drug. In some experiments, incubations were
performed at a lower hematocrit level, using twofold less cells in a
final volume of 300 µL. After 5 minutes at 37°C, radioactive
precursors were added (10 µL) either for 1 hour (short period) or 4 hours (long period) using the candle-jar method.19 After
incubation, cells were collected on glass fiber filters (Whatman GF/C)
using a cell harvester and lysed with water. Insoluble material (such
as nucleic acids, proteins, and lipids) retained on the filter was
abundantly washed with water (for 50 seconds). Filters were then dried
for 35 seconds and placed in scintillation vials containing 2 mL of
scintillation cocktail. Final counting was performed after 24 hours at
room temperature. Blank values were obtained by incubating an equal
number of noninfected erythrocytes. When choline was used as
radioactive precursor, filters were presoaked in 0.05%
polyethyleneimine to eliminate nonspecific binding to the filter. Blank
values were deduced from the total radioactivity incorporated by
infected cells.
Measurement of choline transport inhibition.
Pure P knowlesi-infected erythrocytes were isolated after
Percoll/sorbitol fractionation according to Kutner et al21
and then abundantly washed with special RPMI before choline influx measurement, as described previously.8 Briefly, pure
infected erythrocytes (5 to 7 × 107 cells/100 µL,
trophozoites) were preincubated for a short period (5 to 10 minutes) at
37°C in the presence of 750 µL modified RPMI containing the drug
or not containing the drug (control). Choline influx was measured after
the rapid addition of 50 µL (3H)choline (specific
activity, 0.2 Ci/mmol) to give a final hematocrit level of 0.6% to
0.8%. After 6 minutes of incubation at 37°C, the flux was stopped
by adding 2.5 mL of ice-cold modified RPMI. Triplicate 1-mL portions of
cell suspension were immediately overlaid on 400 µL of ice-cold
n-dibutyl phtalate (density, 1.04 g/mL) in polyethylene tubes and
centrifuged in a Beckman 11 Microfuge at 10,000g for 10 seconds
at 4°C. Supernatants were discarded, and 2.5 mL of cold special
RPMI was carefully added on the dibutyl phtalate layer to wash the
walls of the microtubes. After centrifugation, supernatants and dibutyl
phtalate were discarded and cells were lysed and precipitated with 500 µL of 10% (wt/vol) trichloracetic acid. After centrifugation, 400 µL of the supernatant was counted for radioactivity in 10 mL
scintillation cocktail.8 Nonspecific choline transport was
determined under the same conditions but in the presence of 1.2 mmol/L
of choline in the incubation medium.
Measurement of toxicity against a lymphoblastoid cell line (SAR
strain).
Cells were routinely cultured at 37°C in RPMI 1640 medium
complemented with 50 µmol/L  mercaptoethanol, 1 mmol/L glutamine, and 10% fetal calf serum (GIBCO). The effect of drugs on cell viability was measured in microtiter plates after
(3H)thymidine incorporation into nucleic acids of the
lymphoblastoid cell suspension (4,500 cells/well). Cells were first
exposed to various drug concentrations for 24 hours (1 cell cycle) at
37°C and then (3H)thymidine (0.75 µCi/well) was added
for an additional 5 to 6 hours. At the end of incubation, cells were
harvested on glass fiber filters as described above, and filters were
then counted for radioactivity in 2 mL scintillation cocktail. The
results are expressed as the concentration resulting in 50% inhibition (LV50) of thymidine incorporation.
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RESULTS |
Drug effect on P falciparum growth in vitro.
We have just reported the in vitro antimalarial activity of 77 compounds, analogs to ethanolamine or choline (listed in Table 1),
along with structure activity relationships, to define the general
structural features involved in the activity so as to be able to design
highly efficient antimalarial compounds.13 All compounds
contained at least one amino group (substituted or not), as in the
ethanolamine or choline molecule. We distinguished eight different
groups based on the degree of substitution of the amine function: group
A corresponded to compounds with primary amines; group B with secondary
amines, group C with tertiary amines; and groups D, E, F, G, and H with
quaternary ammonium groups (the last 4 series are described in more
detail below). The present study was aimed at thoroughly studying the
mechanism of action of these compounds to draw up the pharmacologic
target characteristics. Regarding the antimalarial activity, results
reported in our previous study showed that all of the compounds were
lethal to P falciparum in vitro in a dose-dependent manner,
with IC50 ranging from 4.8 mmol/L to 0.033 µmol/L (Table
1). Amine compounds in groups A, B, and C (except N-substituted
compounds with a long alkyl chain, see Discussion) exhibited far lower
antimalarial activities (IC50 ranging from
103 to 105 mol/L) than compounds
containing one (group E or F) or two (group G, H) quaternary ammonium
groups, with IC50 ranging from 104 to
107 mol/L. The most active compounds were quaternary
mono- or bis-ammonium salts with small polar head groups, eg,
trimethyl, dimethyl (or diethyl) hydroxyethyl, triethyl or tripropyl, N
substituted analogs or methyl pyrrolidinium, possessing a long
lipophilic alkyl chain constituted of at least eight methylene groups
(see Calas et al13 and Discussion).
Ten compounds belonging to groups A, E, F, and G (noted in Table 1)
were then tested both against another chloroquine-sensitive strain
(NF54) and one chloroquine-resistant strain (T23; IC50 against chloroquine = 0.03 µmol/L and 0.6 µmol/L,
respectively; personal observation, 1996), in addition to the
Nigerian strain (IC50 = 0.02 µmol/L; personal
observation, 1996) that was routinely used in this study.
Regardless of the compound and the group to which it belonged, the
IC50 against the three strains were very similar,
differing by less than threefold (these values are thus not
reported in Table 1). These compounds could thus also be efficient
against pharmacoresistant strains.
Drug effect on macromolecule biosyntheses.
The effect of 56 of the most active compounds on PC and PE biosynthesis
was evaluated by measuring the incorporation of radioactive choline
into PC as well as the incorporation of radioactive ethanolamine into
PE using a cell harvester for rapid serial determination.14 Incubations were performed for 4 hours or less (1 hour) to determine an
early effect of the compounds. We also measured the effects of the
compounds on the incorporation of radioactive
hypoxanthine* to determine any possible
specificity toward PL metabolism versus biosynthesis of other
macromolecules such as nucleic acids. The results were expressed as
PL50 and NA50 (corresponding to the drug
concentration that reduced the amount of synthesized PL or nucleic
acids by 50%, respectively).
Figure 1 shows the typical sigmoid
dose-response curves obtained with dimethyl-n-pentyl (2-hydroxyethyl)
ammonium (F2) when tested on choline, ethanolamine, and hypoxanthine
incorporation into macromolecules. This compound inhibited PE and
nucleic acid biosynthesis at very high concentrations
(NA50, 2.6 mmol/L; PE50,  20 mmol/L), which
probably corresponded to an alteration of parasite viability. By
contrast, it specifically inhibited choline incorporation into PC at
much lower concentrations (PC50, 32 µmol/L) and the PC50 was very close to the IC50 (81.3 µmol/L). Fifty-five other compounds were also tested for their
effects on macromolecule biosynthesis. Their PL50 and
NA50 are listed in Table 1. It should be noted that all
group A compounds (containing a primary amine) that specifically
impaired the metabolism of ethanolamine (except A7, which impaired
choline metabolism) required longer incubation (>1.5 hours) to exert
their effects. We have previously shown that A6, which has a free
hydroxyethyl group, was incorporated by the parasite into an unnatural
PL, ie, phosphatidyl-2-amino-1-butanol, that accumulated at the expense
of PE.9 The need to accumulate sufficient amounts of false
PL could explain the relatively long time required for these compounds
to exert their effects. In contrast, specific compounds containing a
tertiary amine or quaternary ammonium (groups C, E, F, G, and H)
impaired choline incorporation into PC as early as 15 to 30 minutes
(Ancelin and Vial11 and data not shown), probably due to
choline transport inhibition (see below). PL50 and
NA50 values given in Table 1 correspond to values obtained
after 4 hours of incubation, ie, the time needed for both types of
analogs to exert a substantial effect.
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| Fig 1.
Effect of F2 on radioactive choline, ethanolamine, and
hypoxanthine incorporations into the macromolecules of P
falciparum-infected erythrocytes. Infected cells (10% hematocrit,
3.6% parasitemia) were incubated for 4 hours at 37°C in the
absence (control) or presence of the indicated concentrations of F2 and
in the presence of 10 µmol/L (3H)choline (1.2 µCi)
( ), 2 µmol/L (3H)ethanolamine (0.3 µCi) ( ), and
trace concentrations of (3H)hypoxanthine (1 µCi) ( ).
The reaction was stopped by cell filtration as described in the
Materials and Methods. Results are expressed as a percentage of the
control (without drug) ± SEM.
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In group A, most compounds except A9, ie, cadaverin, a putrescine
homologue, and A2, a sulfhydryl compound, with a wide variety of
biologic effects22 appeared to specifically act on the
biosynthesis of one PL, whereas biosyntheses of the other PL and of
nucleic acids were affected at much higher drug concentrations. They
generally acted on ethanolamine incorporation, except for A7, which was more PC-specific. In addition, their PL50 were generally in
the same range as their IC50.**
This was not the case for A5, which can be considered as a serine analog probably acting on serine metabolism (as an amino acid or PL
polar head analog).
The few compounds in group B, regardless of their activity, were
nonspecific with respect to PE and PC metabolism. Furthermore, most
PL50 and NA50 values differed markedly from the
IC50 and there was no correlation between the effects on PL
(or nucleic acid) metabolism and the effects on parasite growth. This
was especially true for N-substituted compounds with a long alkyl chain
(12 carbon atoms, B3 and B4; see below). It should be noted that very
few compounds were tested in this series, making it difficult to draw
any conclusions for this group.
Concerning group C, PL-specific compounds that showed good
PL50/IC50 correlations were the only ones in
which N was not included in a cyclic structure and was substituted with
a short alkyl chain (C5, C6, and C7). As in group B, when C8 was
N-substituted by a long alkyl chain (12 carbon atoms), its action was
neither PL-specific nor growth-correlated. When nitrogen was included
in the ring structure (C1, C2, C3, and C4), the effects were dependent
on the number of atoms in the heterocycle. The actions of molecules with a 6-atom heterocycle (C3 and C4) were not PL-specific or growth-correlated; C2, which has a ring of four carbon atoms, was
PL-specific and its PE50 was more correlated with its
IC50 than with the PC50, which was lower.
Lastly, C1, which contained an aziridine group, acted on PL metabolism
but also acted on nucleic acid synthesis at lower concentrations.
Compounds of group E (quaternary ammonium salts with 4 alkyl chains)
were specific to PC metabolism provided that they were N-substituted
with an alkyl chain having less than 12 carbon atoms (E1, E2, E3, E4,
and E5). With longer alkyl chains (12, 14, or 16 atoms, ie, E6, E7,
E10, E13, E20, E40, E8, and E50), the compounds acted almost
identically and at similar concentrations on PC and nucleic acid
metabolism. We have previously shown that compounds such as E4, E6, E7,
or E8 acted very quickly, ie, as early as 30 minutes, but the earliest
effect was on PL metabolism.10 After 1 hour of incubation,
the drug can thus also affect nucleic acid metabolism, which may
explain the absence of any differential effect on PL and nucleic acids
for these very rapid acting drugs. Nevertheless, the long alkyl chain
also seems to be responsible for the absence of correlation with the
IC50.
In group F (quaternary ammonium with 3 alkyl and 1 hydroxyethyl
N-substitution), we observed the same pattern of effects according to
the length of the alkyl chain. Indeed, N-substituted compounds with
short alkyl chains (up to 5 atoms, F1 and F2) were PC-specific and
their effect on PC metabolism was also correlated with parasite growth.
The presence in the short alkyl chain of a ring structure with six
carbon atoms (aromatic [F12, F13, and F14] or nonaromatic [F15 and
F16]) also led to compounds that were very PC-specific, with
PL50 highly correlated with the IC50. Compounds
with 10 and 12 carbon atoms showed PC-specific inhibition, but this was
not correlated with growth inhibition and increasing the length of the
alkyl chain (up to 18 carbon atoms) produced compounds that were
neither PC-specific nor correlated (F5, F6, F9, and F10), as already
shown for group E compounds, and this was also the case when two long
alkyl chains were present as N substitutions (F7 and F11).
All compounds of group G and H with two quaternary ammonium groups were
tested for their effect on PL metabolism. Except for H0, which was only
slightly more specific to PL (especially PE) than to nucleic acid
metabolism, all acted very specifically on de novo PC biosynthesis,
regardless of the distance between the two nitrogens, from 5 to 12 methylene groups. In this case also, a short alkyl chain between the
two nitrogens resulted in a high PL50-IC50
correlation (see G1 or G20), whereas for 10 to 12 methylenes, the
compounds showed a slightly lower PL50-IC50
correlation (see G3, G4, G23, or H3). On the other hand, HC3 (whose
alkyl chain between the two N contained two aromatic rings) was
PC-specific and showed a high PL50-IC50
correlation.
Effect of hematocrit level.
The relatively high number of compounds with long alkyl chains in amine
groups (B or C) and quaternary ammonium salts (group E, F, or G) that
showed no PL50/IC50 correlation prompted us to investigate whether the difference between the hematocrit level used in
the growth inhibition assay (0.5% to 1.5%) and the hematocrit level
used in the PL inhibition assay (8% to 20%) could account for the
difference between IC50 and PL50. For some
compounds, notably chloroquine, such a hematocrit-dependent effect of
the IC50 has already been reported.23 We thus
compared the effects of several drugs on macromolecular biosyntheses
(PL and nucleic acids) at two different hematocrit levels, generally
differing by 10-fold. Figure 2 shows that,
with tetradecyltrimethyl ammonium (E7), a 10-fold increase in the
hematocrit level led to a 5- to 10-fold increase in the
PL50 and in the NA50. It is particularly interesting that, at a low hematocrit level (ie, 1.3%), the
PC50 (1.8 µmol/L) was in close agreement with the
IC50 (0.9 µmol/L). Twelve other compounds were tested on
PL metabolism at two hematocrit levels
(Table 2). The same hematocrit level effect
was observed with E40 and E8, compounds containing 14 and 16 carbon
atoms, respectively. This was also the case for F5, F9, and F6, with 14, 14, and 18 carbon atoms, respectively. By contrast, for compounds with an alkyl chain containing less than 12 carbon atoms, regardless of
the group to which they belonged, ie, E, F or G (see E4, F3, or G3),
hematocrit level variations caused no marked difference in the
PL50. Compounds with 12 carbon atoms had intermediary
results, depending on the group and thus on nitrogen substitution
(secondary amine, N-trialkyl or N-dialkyl hydroxyethyl monoquaternary
ammonium, or bis quaternary ammonium compounds): B4 showed a marked
hematocrit level effect, E6 and F4 showed a less pronounced effect
(2.5- to 4-fold), and no effect was observed with G4. By contrast,
decreasing the hematocrit level did not modify the specificity pattern,
regardless of the group and the methylene number of the long
hydrophobic alkyl chain.
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| Fig 2.
Effect of hematocrit level on the inhibition of PL and
nucleic acid biosyntheses by E7. Experiments were performed for 4 hours at 37°C with the same infected cell suspensions (5.6% parasitemia) either under the high hematocrit level (13%) conditions described in
the Materials and Methods, ie, in 60 µL final volume (5 × 106 parasites) (solid symbols) or at low hematocrit level
(1.3%) in a final volume of 300 µL (containing 2.5 × 106 parasites) (open symbols), resulting in a 10-fold
difference in hematocrit level. In both cases, the concentrations and
the specific activities of the radioactive precursors were similar, ie,
10 µmol/L (3H)choline at 1.92 Ci/mmol, 2 µmol/L
(3H)ethanolamine at 2.74 Ci/mmol, and trace concentration
of (3H)hypoxanthine at 4 µCi/well. The results are
derived from a typical experiment performed in triplicate.
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Parasite growth and PL metabolism inhibition.
Lastly, we studied the correlation between the inhibition of PL
metabolism (expressed as PL50) and parasite growth
(IC50) for 50 compounds, which are underlined in Table 1.
In this study, we excluded compounds that more likely possess other
mechanisms of action, such as A2 (a widely acting sulfhydryl reagent),
A9 (putrescine analog), A5 (which more likely acts as a serine analog), C1 (whose aziridin ring confers alkylating properties24),
and C3, C4, and C12 (which were found to inhibit more specifically, and
in a more growth-correlated way, nucleic acid metabolism than PL
biosynthesis). B2 was also not considered in this study due to the
absence of specific action and of correlation with growth inhibition
(IC50 was more than 20-fold lower than PL50 or
NA50). For this study, PL50 values obtained
under high hematocrit level conditions (see above) are reported; there
were not enough data obtained under low hematocrit level conditions (in
which only a few compounds were investigated) to make general
systematic comparisons.
As expected, the effects of these compounds on the two responses were
linearly correlated (Fig 3), the slope of
the regression line was 0.51, the ordinate at the origin was 1.82, and
the correlation coefficient was 0.89 (which corresponds to a risk of
well below 0.1%). The dotted (bisecting) line in Fig 3 shows the
theoretical correlation that would be obtained if PL50
equalled IC50. The molecules clearly segregated into two
groups: one whose IC50 was greater than 50 µmol/L (21 molecules), appearing well distributed along the bisecting line,
whereas the second group (including 29 molecules with IC50
lower than 50 µmol/L) seemed to deviate from this theoretical line,
with IC50 substantially lower than PL50. Among
this latter group, eight compounds (see Fig 3 legend) possessing more
than 12 methylene groups in their hydrophobic alkyl chain were shown to
have a hematocrit-dependent effect (Table 2). Nine other compounds also
probably possess the same property considering their alkyl chain
lengths. Four compounds were shown to have no hematocrit-dependent
effect, which was probably also the case for three others considering
the methylene number present in their hydrophobic chain. This group
thus mainly contains molecules (about two-thirds) with a
hematocrit-dependent effect, and at equal hematocrit level, the
PL50 would probably be more closely correlated with
IC50. Overall, this could explain the observed deviation
for this group of active compounds.
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| Fig 3.
Correlation between the inhibition of PL metabolism and
the inhibition of parasite growth. PL50 and
IC50 (concentration producing 50% inhibition of PL
synthesis and parasite growth, respectively) measured as described in
the Materials and Methods were from Table 1. PL50 values
correspond to high hematocrit level conditions (Table 2). The 50 compounds considered for this correlation are in bold characters and
underlined in Table 1. Squares correspond to compounds for which a
hematocrit-dependent effect on PL metabolism was demonstrated () or
probable ( ). Diamonds indicate compounds for which no
hematocrit-dependent effect was demonstrated ( ) or probable ( ),
and (×) stands for compounds of group H for which a hematocrit level
effect was not determined. The dotted line corresponds to the
theoretical line when PL50 = IC50 (slope = 1, ordinate at the origin = 0).
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We investigated the potential reversal of inhibition of PC metabolism
by a high choline concentration (200 µmol/L) to further ascertain
whether these molecules act by impairing choline phospholipidic metabolism in Plasmodium-infected erythrocytes. The experiment was performed with F13, which specifically inhibited PC metabolism (PC50, 44 µmol/L). Figure 4
clearly shows that a 20-fold excess of choline caused a substantial
shift in the dose-response curve of PC metabolism inhibition, leading
to a specific 12-fold increase in the PC50 when choline was
used as radioactive precursor. By contrast, under the same conditions,
no modification in ethanolamine or hypoxanthine incorporation was
observed (data not shown). Similar results were obtained with G3, which
led to a specific shift (>14-fold) in the PC50 towards
higher concentrations (data not shown).
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| Fig 4.
Effect of high choline concentration on the inhibition of
PC metabolism by F13. Infected erythrocytes (5 × 106
infected cells) were preincubated for 5 minutes in the presence of 10 µmol/L () or 200 µmol/L ( ) choline. The drug was then added
at the indicated concentration and after 5 minutes, and the PC
biosynthesis assays were initiated by the addition of 10 µCi
(3H)choline and pursued for 5 hours at 37°C.
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Choline transport inhibition.
We checked the effect of some compounds shown to specifically inhibit
PC metabolism on choline transport into infected erythrocytes. P
knowlesi-infected erythrocyte suspensions that can be obtained in
great quantities and at very high parasitemia (>95%) were used for
this purpose. Figure 5 shows the effects on
choline transport of four alkyltrimethyl ammonium compounds (group E)
in which the saturated alkyl chain had from 10 to 16 carbons. Similar
action patterns were obtained for all compounds. They all competitively inhibited choline influx (under zero trans-influx measurement conditions8). The Ki values for E4 (Fig 5A), E6 (Fig 5B),
E7 (Fig 5C), and E8 (Fig 5D) were 1.8, 1.1, 7.2, and 3.5 µmol/L, respectively. All of these values closely agreed with previously reported IC50 and PC50 values (Table 1). In a
separate set of experiments, these compounds were tested over a wider
range of concentrations. Up to 20 µmol/L, they all showed competitive
behavior with similar Ki values (1.9, 0.5, 5.6, and 1.8 µmol/L,
respectively), but at higher concentrations (80 and 240 µmol/L) they
acted in a noncompetitive manner, with Ki values around 30 to 40 µmol/L, depending on the compound (data not shown). This distinct
drug concentration-dependent behavior is probably related to the
inhibition site of these alkyltrimethyl ammonium salts, depending on
whether they act at the outer (competitive) or inner side
(noncompetitive) of the membrane, as described for normal
erythrocytes.25,26
View larger version (29K):
[in this window]
[in a new window]
| Fig 5.
Choline influx into P knowlesi-infected
erythrocytes as a function of choline concentration in the presence of
E4 (A), E6 (B), E7 (C), or E8 (D). Infected erythrocyte suspensions
(98% parasitized, trophozoite stage) with 5.2 × 107
cells/100 µL (0.7% final hematocrit) were preincubated for 5 minutes
at 37°C in the presence of 750 µL special RPMI containing the
drug (solid symbols) at 3 µmol/L (A and B) or 6 µmol/L (C and D) or
no (open symbols) drug. Choline influx measurement was initiated by the
rapid addition of 50 µL (3H)choline (specific activity,
0.2 Ci/mmol) at the indicated concentration. After 6 minutes of
incubation at 37°C, the flux was stopped by adding 2.5 mL of
ice-cold special RPMI, followed by centrifugation of a 1-mL aliquot
through n-dibutyl phtalate at 4°C as described in the Materials and
Methods. Nonspecific choline transport was determined in the presence
of 1.2 mmol/L choline under the same conditions. The results are
expressed by the double-reciprocal plot of the initial velocity of
choline influx into infected erythrocytes (expressed as nanomoles per
1010 infected cells per minute) ± SEM. Each point is the
mean of triplicate determinations in one typical experiment.
|
|
Toxicity of some polar head analogs.
To investigate the specificity against other rapidly dividing
eucaryotic cells, 12 compounds whose IC50 against
Plasmodium ranged from 0.8 to 110 µmol/L were tested on the
viability of a lymphoblastoid cell line (SAR). After 24 hours (1 cell
cycle) of contact of lymphoblastoid cells with various drug
concentrations at 37°C, incorporation of (3H)thymidine
into nucleic acids was monitored for 5 to 6 hours at 37°C. The
results are expressed as LV50, resulting in 50% inhibition of lymphocyte viability as reflected by the inhibition of thymidine incorporation (Table 3). The
LV50 was from 2.5-fold (F8) to 16,600-fold (G23) higher
than the IC50, confirming the absence of any correlation between the effects on parasite growth and on lymphoblastoid cell viability.
View this table:
[in this window]
[in a new window]
|
Table 3.
Comparative Effect of 12 Polar Head Analogs on P
falciparum Growth (IC50) and Lymphoblastoid Cell
Viability (LV50)
|
|
| |
DISCUSSION |
PL are absolutely necessary for parasite membrane
biogenesis,3-5 and we showed that impairment of PL
metabolism was lethal to P falciparum in vitro.9-12
One interest of the present systematic study was to use a large series
of polar head analogs, with a wide range of in vitro antimalarial
activities to determine whether antimalarial activity is mediated by PL
metabolism inhibition. The results could be used to establish
structure-activity relationships, potentially useful for drawing up
general rules for modeling new therapeutic molecules.
Generally speaking (see for more detail our recent
report13), the amine compounds in groups A, B, and C
(except for N-substituted compounds with a long alkyl chain) exhibited
far lower antimalarial activities (IC50 ranging from
103 to 105 mol/L) than compounds
containing one (group E or F) or two (groups G and H) quaternary
ammonium groups, with IC50 from 104 to
108 mol/L. Most quaternary ammonium compounds of
groups E, F, and G exhibited good antimalarial activity, especially
those with a long alkyl chain (containing more than 8 carbon atoms);
their IC50 ranged from 2.1 µmol/L to 33 nmol/L. Compounds
in group F differed from those in group E by the replacement of a
methyl by a hydroxyethyl group as in choline, but their
IC50 did not differ substantially. The antimalarial
activities of group E, F, G, and H compounds increased as a function of
the alkyl chain length, up to 10 to 12 methylene groups. Increasing the
length of the alkyl chain did not further improve efficacy, at least for groups E and F. The presence of another quaternary ammonium did not
significantly modify the efficiencies, except when 12 carbon atoms were
present between the two quaternary ammonium groups, for which there was
a fivefold decrease in IC50, whereas compounds with a short
alkyl chain were found to be less efficient than the corresponding
monoammonium molecules. Ten compounds belonging to group A, E, F, G, or
H tested against a chloroquine resistant strain (T23) possessed the
same activity, which indicates that PL metabolism inhibitors could be
very useful when dealing with pharmacoresistant strains of P
falciparum.
Regarding mechanism of action, compounds in group A acted specifically
on PL metabolism, except for those substituted at the  position of
the nitrogen with hydroxymethyl that are rather close analogs of
serine. Analogs monosubstituted with methyl or ethyl groups at or
position acted specifically on PE metabolism, whereas A7,
substituted with two methyls at the position of the nitrogen, acted
more specifically on PC metabolism. This highlights the importance of
the steric volume close to the N, which probably determines whether a
compound acts as an ethanolamine or choline analog.
Concerning the compounds in groups E and F that specifically acted on
PL metabolism, some possessed PC50 about 10-fold higher than their IC50. This concerns compounds having a long
alkyl chain containing more than 10 carbon atoms, whose activity was
highly dependent on the hematocrit level, ie, on the number of drug
molecules per cell. At low hematocrit level, ie, under conditions
similar to those used for antimalarial activity assessment,
PL50 and IC50 were in close agreement (Table
2). By contrast, for compounds with an alkyl chain with less than 8 to
9 carbon atoms, the hematocrit level had no effect on the
PL50, regardless of the group (E, F, or G), and the
IC50 and PL50 remained roughly correlated. The presence of a second quaternary ammonium (group G or H) led to high
specificity, but there was a slightly weaker correlation, except for
HC3.
When considering the correlation between PL metabolism impairment and
parasite growth inhibition, molecules appeared to segregate into two
groups (Fig 3). The first one, whose IC50 was higher than
50 µmol/L (21 molecules), appeared to be well distributed along the
bisecting line (PL50 = IC50). By contrast, the
second group, including 29 molecules that had a long alkyl chain and an
IC50 of less than 50 µmol/L, showed a PL50
that was higher than expected as compared with the IC50,
likely due the hematocrit level effect related to the long hydrophobic
alkyl chain (see above). For all 50 compounds, there was thus a close
correlation between PL metabolism impairment and parasite growth
inhibition.
Other lines of evidence that these compounds act by PL metabolism
inhibition were provided by the reversal of PC metabolism inhibition by
an excess of choline (Fig 4). Nevertheless, choline excess did not lead
to a parallel shift in IC50 (data not shown). One possible
explanation could be that these compounds could enter infected
erythrocytes.
Finally, four compounds were shown to be potent inhibitors of choline
transport into erythrocytes, with Ki values in the micromolar range (as
compared with a Kt for choline of 8.5 µmol/L8). The Ki values were in very close
agreement with the PC50, indicating that PC50
provides an adequate index of the inhibitory effect on the choline
transporter.
Overall, these results (good IC50/PC50/Ki of
choline transport correlation, reversal of de novo PC biosynthesis by
choline excess) show that choline entry and incorporation into PC is
probably the target of these analogs in infected erythrocytes. In
particular, the effect on the second enzymatic step of the de novo PC
biosynthesis (choline kinase) was ruled out, because substantial
inhibition of choline kinase by E4, G3, or HC3 only occurred at
millimolar concentration, ie, three orders of magnitude higher than
their IC50 or PC50.11,27
In P falciparum-infected erythrocytes, choline is incorporated
via a specific carrier showing high affinity for choline
(Kt = 8.5 µmol/L) and whose characteristics are
close to those of normal erythrocytes, except for a 10-fold increase in
Vm.8 A probable absence of stereoselectivity of
this carrier was suggested using or methyl choline
stereoisomers.28 On the other hand, there is an induced
permeability pathway with very broad specificity to various unrelated
solutes, preferably anions, but also neutral or to a less extent,
cationic solutes, because this pathway could incorporate choline at
very high concentrations (millimolar).29 This latter
pathway has been described as a stereoselective channel protein
containing a hydrophobic region and a positive charge or dipole to
provide anion selectivity.30 The pharmacologic effect
observed in our study, notably with quaternary ammonium salts,
concerned inhibition of high-affinity choline carrier of infected
erythrocytes, considering, for instance, the imperative cationic charge
requirement for antimalarial activity and PC metabolism inhibition (see
the total inefficiency of D5 in which the quaternary ammonium was
replaced by the tetrasubstituted carbon) or also specificity of
inhibition of PC metabolism versus PE, nucleic acid (Table 1), or
protein synthesis (Ancelin et al10 and data not shown).
This latter characteristic as well as the permanent charge of
quaternary ammonium also rule out any lysosomotropic effect previously
reported for some N-dodecyl substituted tertiary amines that impaired
parasite protein synthesis.31
As for the hematocrit level effect, there is also a pivotal point for
the choline transport inhibition pattern as a function of chain length,
because the lowest Ki values for choline transport inhibition
correspond to 10 to 12 carbons (Fig 5). The carbon chains of these
compounds probably form hydrophobic associations with the transporter
so that the alkyl group extends from the trimethyl ammonium moiety over
a length of approximately 12 carbon atoms. The increase in the Ki
values at 14 carbon chain length suggests that the end of the
hydrophobic alkyl group may butt against a hydrophilic domain and be
repelled by it.
Potent antimalarials are therefore substances having a small quaternary
ammonium group with a long hydrophobic chain, which could likely
combine with an anionic site and a long hydrophobic region
(corresponding to 10 to 12 methylenes) of the choline transporter. Substances with two quaternary ammonium groups are as potent as (up to
10 methylenes) or slightly more potent (12 methylenes) than the
corresponding monoquaternary compounds.
Nevertheless, a crucial problem in drug development is the specificity
required to achieve an acceptable therapeutic index. The usually much
higher toxic effect on the malarial parasite than on the lymphoblastoid
cell line, as well as the total absence of correlation between
IC50 and LV50 (Table 3), more likely indicates that the structural requirements for inhibition of PL metabolism are
highly specific to infected cells and/or that the malarial parasite is more dependent on this metabolism.
A second potential problem with choline analogs concerns involvement of
choline as precursor of the neurotransmitter, acetylcholine. Some of
the present data indicate that the structural requirements to inhibit
choline entry into infected erythrocytes differ from that to inhibit
high-affinity choline transport (HACT) in
synaptosomes.32,33
First, the cholinergic compound hemicholinium (HC3) showed a far lower
IC50 in synaptosomes (0.02 µmol/L) than the bisquaternary compound G3 (1.5 µmol/L).33 By contrast, against P
falciparum, the activities of both of them are very close (4 µmol/L and 1.7 µmol/L, respectively). Because the distance between
the two nitrogen atoms in HC3 is very close to that spanned by the 10 methylene groups of G3, the presence of aromatic rings between the two
quaternary ammonium groups and/or the possibility of
hydrogen-bonding through the HC3 hydroxyl group to the binding site do
not seem to be involved in its antimalarial effect on P
falciparum, contrary to its cholinergic effect on
synaptosomes.32
Secondly, substantial differences between the choline carrier in
erythrocytes and synaptosome HACT (see Fisher and Hanin34 for review) also concern the steric fit of analogs at the active site.
For small choline analogs, slightly increasing the steric hindrance of
the polar head (N substitutions) is dramatic for the erythrocyte
facilitated choline transport system but not for the HACT
system.35 N-ethylcholine (F1) is bound almost as well as
choline by the HACT, but 100-fold more weakly than choline through
erythrocyte choline transport,35 and it was also weakly active against P falciparum (IC50, >300
µmol/L). Finally, using methyl choline stereoisomers, we have also
shown other characteristics distinct from that of
synaptosomes,36 such as the absence of stereospecificity
and the opposite effect of methyl substitution at the or position of the nitrogen.28 The structural requirements for
inhibition of choline incorporation into P falciparum-infected erythrocytes are thus very specific and differ from the requirements for inhibition of choline uptake into synaptosomes and also into many
other animal cells and tissues (see Lerner37 for review).
Hence, the antimalarial activity of the 77 polar head analogs is
mediated by inhibition of PL biosynthesis. PL metabolism of P
falciparum-infected erythrocytes, especially de novo PC
biosynthesis from choline, is thus a quite realistic target for a new
malaria chemotherapy, even in cases of polypharmacoresistance. The
highest antimalarial activities measured here were around 0.1 µmol/L
(E10, E20, F8, G4, and G23) and as low as 33 nmol/L for E13. Moreover, the structure/activity relationships already highlighted13
provide general rules for improving this activity and facilitate
modeling of new therapeutic molecules.
| |
FOOTNOTES |
Submitted August 4, 1997;
accepted October 1, 1997.
*
A compound was defined as PL-specific when it affected the
metabolism of only one PL, either PC or PE from choline or
ethanolamine, respectively, without any simultaneous effect on the
metabolism of the other PL, or on nucleic acid synthesis (ie, when the
PL50 was at least 2.5-fold lower than the
NA50).
**
A compound was considered to show a good correlation between PL
metabolism and parasite growth inhibition when its PL50 was in close agreement with its IC 50 , ie, when there was less
than 2.5-fold difference between values.
Supported by the UNDP/World Bank/WHO special program for Research and
Training in Tropical Diseases (Grant No. 950165), the Commission of the
European Communities (INCO-DC, PL-950529:IC18-CT960056), the CNRS (GDR
No.1077, Etude des parasites pathogènes), the Ministère de
l'Enseignement Supérieur et de la Recherche (DPST No. 5 and MENESR-DGA/DSP and AUPELF-UREF ARC no. X/7.10.04/Palu 95), and the
VIRBAC Laboratories.
Address reprint requests to Marie L. Ancelin, PhD, CNRS
UMR 5539, Department of Biologie-Santé, CP 107, UM II, Place E. Bataillon, 34095 Montpellier Cedex 5, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors owe special thanks to Prof J.W. Kosh (University of South
Carolina, Columbia, SC) for providing selenium choline and to F. Vialettes for his skilled technical assistance.
| |
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H. J. Vial, S. Wein, C. Farenc, C. Kocken, O. Nicolas, M. L. Ancelin, F. Bressolle, A. Thomas, and M. Calas
Prodrugs of bisthiazolium salts are orally potent antimalarials
PNAS,
October 26, 2004;
101(43):
15458 - 15463.
[Abstract]
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R. Roggero, R. Zufferey, M. Minca, E. Richier, M. Calas, H. Vial, and C. Ben Mamoun
Unraveling the Mode of Action of the Antimalarial Choline Analog G25 in Plasmodium falciparum and Saccharomyces cerevisiae
Antimicrob. Agents Chemother.,
August 1, 2004;
48(8):
2816 - 2824.
[Abstract]
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S. K. Sharma, M. Kapoor, T. N. C. Ramya, S. Kumar, G. Kumar, R. Modak, S. Sharma, N. Surolia, and A. Surolia
Identification, Characterization, and Inhibition of Plasmodium falciparum {beta}-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ)
J. Biol. Chem.,
November 14, 2003;
278(46):
45661 - 45671.
[Abstract]
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G. A. Biagini, E. Richier, P. G. Bray, M. Calas, H. Vial, and S. A. Ward
Heme Binding Contributes to Antimalarial Activity of Bis-Quaternary Ammoniums
Antimicrob. Agents Chemother.,
August 1, 2003;
47(8):
2584 - 2589.
[Abstract]
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M. L. Ancelin, M. Calas, V. Vidal-Sailhan, S. Herbute, P. Ringwald, and H. J. Vial
Potent Inhibitors of Plasmodium Phospholipid Metabolism with a Broad Spectrum of In Vitro Antimalarial Activities
Antimicrob. Agents Chemother.,
August 1, 2003;
47(8):
2590 - 2597.
[Abstract]
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M. L. Ancelin, M. Calas, A. Bonhoure, S. Herbute, and H. J. Vial
In Vivo Antimalarial Activities of Mono- and Bis Quaternary Ammonium Salts Interfering with Plasmodium Phospholipid Metabolism
Antimicrob. Agents Chemother.,
August 1, 2003;
47(8):
2598 - 2605.
[Abstract]
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J. Wiesner, D. Henschker, D. B. Hutchinson, E. Beck, and H. Jomaa
In Vitro and In Vivo Synergy of Fosmidomycin, a Novel Antimalarial Drug, with Clindamycin
Antimicrob. Agents Chemother.,
September 1, 2002;
46(9):
2889 - 2894.
[Abstract]
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K. Wengelnik, V. Vidal, M. L. Ancelin, A.-M. Cathiard, J. L. Morgat, C. H. Kocken, M. Calas, S. Herrera, A. W. Thomas, and H. J. Vial
A Class of Potent Antimalarials and Their Specific Accumulation in Infected Erythrocytes
Science,
February 15, 2002;
295(5558):
1311 - 1314.
[Abstract]
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Abstract
Introduction
Abstract