From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
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,
Taı̈b Jei, Pierre Druilhe, and Henri J. Vial
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 1023 to 1027 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
mmol/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.
r 1998 by The American Society of Hematology.
T
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.
HE 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
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.
Submitted August 4, 1997; accepted October 1, 1997.
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9104-0012$3.00/0
1426
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).
(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.
Blood, Vol 91, No 4 (February 15), 1998: pp 1426-1437
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLASMODIUM PHOSPHOLIPID METABOLISM INHIBITION
1427
Table 1. General Structures of Phospholipid Polar Head Group Analogs and Their IC50 and PL50 Against P falciparum In Vitro (Nigerian Strain)
Name
Compounds
IC50 (µmol/L)
PC50 (µmol/L)
PE50 (µmol/L)
NA50 (µmol/L)
800
.2,000
200
300
800
50
80
900
600
2,000
7,750
ND
1,000
9,000
6,500
.8,000
5,750
1,200
ND
47,000
2,300
ND
6,500
275
1,500
1,800
35
3,000
ND
.47,000
6,600
ND
950
8,250
2,800
6,000
5,400
4,200
ND
16,000
Series A: Primary amines
A0
HO-CH2-CH2-CH2-NH2
A1
HO-(CH2)5-NH2
A2
HS-CH2-CH2-NH2
A3
HO-CH2-CH(CH3)-NH2
A4
HO-CH(CH3)-CH2-NH2
A5
(HO-CH2)2CH-NH2
A6*
HO-CH2-CH(C2H5)-NH2
A7
HO-CH2-C(CH3)2-NH2
A8
(HO-CH2)2C(C2H5 )-NH2
A9
H2N-(CH2)5-NH2
Name
R1
R2
IC50 (µmol/L)
PC50 (µmol/L)
PE50 (µmol/L)
NA50 (µmol/L)
1,100
40
0.51
0.61
.1,000
.1,000
2.1
2,600
1,425
42
67
ND
ND
ND
370
1,020
15
36
ND
ND
ND
500
.850
7.5
44
ND
ND
ND
Series B: Secondary amines: R1-NH-R2
B1
HO-CH2-CH2HO-CH2-CH2B2
HO-CH2-CH2C6H5-CH2B3†
HO-CH2-CH2C12H25B4†
HO-CH2-CH(C2H5)
C12H25B5
-CH2-CH(OH)-CH2-CH2B6
-CH2-CH(OH)-CH2-CH2-CH 2B7†
CH3-O-CH2-CH2C12H25Name
R1
R2
Series C: Tertiary amines: R1-N(R2 )-R3
C1
HO-CH2-CH2C2
HO-CH2-CH2C3
HO-CH2-CH2C5
HO-CH2-CH2C6
HO-CH2-CH2C7
HO-CH2-CH2C8†
HO-CH2-CH2C9
pClFOCH2C(/O)O(CH2)2C4
C12
R3
IC50 (µmol/L)
PC50 (µmol/L)
PE50 (µmol/L)
NA50 (µmol/L)
50
420
350
1,300
400
450
1.3
500
370
60
17,500
300
305
210
100
ND
280
380
.20,000
5,200
27,000
.20,000
50
ND
53
2,650
2,750
7,800
2,950
2,200
46
ND
1,200
130
15,000
1,025
.54,000
11,400
4,800
280
-(CH2)2-(CH2)4-(CH2)5CH3CH3C2H5C2H5(CH3)2CH(CH3)2CHCH3C12H25CH3CH3-
HO-CH2-CH2-CH2-CH 2-OH
(CH3)2N-(CH2)6 -N(CH3)2
IC50
(µmol/L)
PC50
(µmol/L)
PE50
(µmol/L)
NA50
(µmol/L)
(CH3)3-N1-CH2-CH 2-Cl
(CH3)3-N1-CH2-CH 2-O-PO3
(CH3)3-N1-CH2-CH 2-C(/O)O
(CH3)3-N1-CH2-CH 2-O-C(/O)-CH3
(CH3)3-N1-CH2-
2,000
1,200‡
.8,000
.8,000‡
:200
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
(CH3)3-C-CH2-CH2-OH
(CH3)2-Se1-CH2-CH 2-OH
.2,000
420
ND
ND
ND
ND
ND
ND
Name
Series D
D1
D2
D3
D4
D6
D5
D7
Name
R1
R2
Series E: R1-N1(R2)(R3)-(CH 2)n-1CH3
E1
C2H5C2H5E2†
CH3
CH3
E3†
CH3
CH3
E4*
CH3
CH3
E5*†
CH3
CH3
E6*
CH3
CH3
E7
CH3
CH3
E8
CH3
CH3
E9
CH3
CH3
E10†
C2H5C2H5E13†
C3H7C3H7E20†
CH3
CH3
E30*†
CH3
CH3
E40
CH3
CH3
E41
CH3
CH3
E50
C16H332
R3
n
IC50
(µmol/L)
PC50
(µmol/L)
C2H5CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5C3H7C2H5
C12H25F-CH2F-CH2-
2
7
9
10
11
12
14
16
18
12
12
12
12
14
18
700
300
1.6
0.7
0.5
0.5
0.9
0.8
2.1
0.064
0.033
0.11
0.7
1
0.7
350
700
22
9.5
2
12
18
21
ND
1
3
4.5
ND
22
ND
0.7
5.5
PE50
(µmol/L)
NA50
(µmol/L)
.80,000
.20,000
$200
115
4
25
16
23
ND
22
21
10
ND
27
ND
14,000
5,700
$200
200
5
30
18
44
ND
3.6
2
5
ND
19
ND
2.6
5.5
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1428
ANCELIN ET AL
Table 1. General Structures of Phospholipid Polar Head Group Analogs and Their IC50 and PL50 Against P falciparum
In Vitro (Nigerian Strain) (Continued)
Name
R1
R2
R3
IC50 (µmol/L)
PC50 (µmol/L)
PE50 (µmol/L)
NA50 (µmol/L)
$300
81.3
0.97
0.48
0.6
1.3
0.84
0.34
0.55
0.62
1.5
112
110
250
120
230
4,830
65
32
18
6.5
47
24
8.5
3.6
26
6
40
122.5
44
140
96
120
ND
.8,000
$20,000
78
28
43
48
3
52
.30
7.5
47
.10,000
.9,000
14,500
.20,000
.20,000
ND
.8,000
2,600
90
19
75
28
6.8
16
.30
4.8
52
2,700
1,650
6,700
2,800
.20,000
ND
700
ND
ND
ND
Series F: Quarternary ammonium salts R1-N1(R2)(R3)-CH 2-CH2OH
CH3C2H5F1†
CH3CH3CH3C5H11F2†
CH3CH3C10H21F3*†
F4†
CH3CH3C12H25F5†
CH3CH3C14H29CH3CH3C18H37F6†
CH3C12H25C12H25F7†
C2H5C2H5C12H25F8*†
F9†
C2H5C2H5C14H29F10†
C2H5C2H5C18H37C2H5C12H25C12H25F11†
CH3CH3F-CH2F12†
CH3CH3F-(CH2)2F13†
CH3CH3F-CH2-CH(CH3)F14
CH3CH3(cycl.)C6H11F15†
CH3-(CH2)4F16†
-(CH2)2-O-(CH2)2 F18†
CH3F19
Name
R1
R2
R3
n
IC50 (µmol/L)
PC50 (µmol/L)
PE50 (µmol/L)
NA50 (µmol/L)
700
12
1.7
0.09
650
0.15
1,260
2
630
260
16
6
250
6.5
1,200
50
80,000
.20,000
.200
.20
25,000
.200
800
.20,000
28,000
.20,000
.200
.20
17,200
.200
1,800
14,000
3.4
.200
.200
[R1-N1(R2)(R3)-CH 2)n/2]2
Series G and H: bis quaternary ammonium salts
G1*
CH3
CH3
CH3
CH3
CH3
CH3
G2†
CH3
CH3
CH3
G3*
CH3
CH3
CH3
G4†
CH3
-(CH2)4G20
CH3
-(CH2)4G23†
HO-CH2-CH2C2H5
C2H5
H0*†
HO-CH2-CH2C2H5
C2H5
H3†
HC3
3
CH3
3
HO—CH2—CH2—N1—CH2—C—
\
3
O
CH3
6
8
10
12
5
10
5
10
4
4
2
IC50 and PL50 were measured as described in the Materials and Methods. PL50 correspond to values obtained under high hematocrit level
conditions.
Abbreviation: ND, not determined.
*Correspond to compounds that were tested both against two chloroquine-sensitive strains (Nigerian and NF54) and also against a
chloroquine-resistant strain (T23).
†Correspond to molecules that were synthesized for this study.13 Underlined bold compounds correspond to the 50 compounds considered for
IC50/PL50 correlation (Fig 3) and compounds in italics are those whose PL50 depended on the hematocrit level (Table 2).
‡Value that is probably too low because of partial hydrolysis to choline during the experiment.
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 knowlesiinfected 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
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLASMODIUM PHOSPHOLIPID METABOLISM INHIBITION
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 3
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 knowlesiinfected 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 3 107 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 b 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.
1429
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 1023 to 1025 mol/L) than compounds containing
one (group E or F) or two (group G, H) quaternary ammonium
groups, with IC50 ranging from 1024 to 1027 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 chloroquinesensitive strain (NF54) and one chloroquine-resistant strain
(T23; IC50 against chloroquine 5 0.03 µmol/L and 0.6 µmol/L,
respectively; personal observation, 1996), in addition to the Nigerian
strain (IC50 5 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
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
*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).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1430
Fig 1. Effect of F2 on radioactive choline, ethanolamine, and
hypoxanthine incorporations into the macromolecules of P falciparuminfected 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 mmol/L (3H)choline (1.2 mCi) (W), 2 mmol/L (3H)ethanolamine (0.3
mCi) (Q), and trace concentrations of (3H)hypoxanthine (1 mCi) (M).
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) 6 SEM.
(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.
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
ANCELIN ET AL
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
*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.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLASMODIUM PHOSPHOLIPID METABOLISM INHIBITION
[F15 and F16]) also led to compounds that were very PCspecific, 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.
1431
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 hematocritdependent 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.
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).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1432
ANCELIN ET AL
Choline transport inhibition. We checked the effect of
some compounds shown to specifically inhibit PC metabolism
on choline transport into infected erythrocytes. P knowlesiinfected 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
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.
DISCUSSION
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 mL final volume (5 3 106
parasites) (solid symbols) or at low hematocrit level (1.3%) in a final
volume of 300 mL (containing 2.5 3 106 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 mmol/L (3H)choline at 1.92 Ci/mmol, 2 mmol/L
(3H)ethanolamine at 2.74 Ci/mmol, and trace concentration of (3H)hypoxanthine at 4 mCi/well. The results are derived from a typical
experiment performed in triplicate.
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 structureactivity 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 1023 to 1025
mol/L) than compounds containing one (group E or F) or two
(groups G and H) quaternary ammonium groups, with IC50 from
1024 to 1028 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
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLASMODIUM PHOSPHOLIPID METABOLISM INHIBITION
1433
Table 2. Effect of Hematocrit Level on the PL50 and NA50
Compounds
n
IC50 (µmol/L)
PC50 (µmol/L)
B4
C12H25-NH-CH(C2H5 )-CH2OH
12
0.61
7.2
67
5
36
7.5
44
Yes
E4
C10H21-N1(CH3) 3
10
0.7
C12H25-N1(CH3) 3
12
0.5
E7
C14H29-N1(CH3) 3
14
0.9
E40
C14H29-H1(CH3) 2-CH2F
14
1
E8
C16H33-N1(CH3) 3
16
0.8
80
115
8
25
2.9
16
4.4
27
2.2
23
145
200
8.5
300
4.6
18
2
19
6.6
44
No
E6
5
9.5
3.2
12
1.8
18
5.5
22
2.6
21
F3
C10H21-N1(CH3) 2-(CH2)2OH
10
0.97
C12H25-N1(CH3) 2-(CH2)2OH
12
0.48
F5
C14H29-N1(CH3) 2-(CH2)2OH
14
0.6
F9
C14H29-N1(C2H5 )2-(CH2)2OH
14
0.55
F6
C18H37-N1(CH3) 3
18
1.3
33
78
12
28
5.1
43
5.7
.30
7.5
48
62
90
8
19
11.5
75
6.8
.30
5.2
28
No
F4
7
18
2.2
6.5
5.5
47
3.7
26
6
24
G3
(CH3)3N1-C10H 21-N1(CH3)3
10
1.7
G4
(CH3)3N1-C12H 25-N1(CH3)3
12
0.09
12
16
4.8
6
PE50 (µmol/L)
.200
.200
.20
.20
NA50 (µmol/L)
.200
.200
.20
.20
Hematocrit Level Effect
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
No
The PL50 and NA50 were measured and expressed as in Table 1. Experiments were performed as described in Fig 2, ie, either at low hematocrit
level (2.5 3 106 infected cells/300 µL final volume) or at a 10-fold higher hematocrit level (5 3 106 infected cells/60 µL final volume). Values in italics
correspond to measurements performed at the lowest hematocrit level (usually between 1.1% and 3%). Yes indicates an effect of hematocrit level
on metabolic activity inhibition and no is the opposite; n corresponds to the number of carbons present in the long alkyl chain.
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 a position of the nitrogen with hydroxymethyl that are
rather close analogs of serine. Analogs monosubstituted with
methyl or ethyl groups at a or b position acted specifically on
PE metabolism, whereas A7, substituted with two methyls at the
a 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 5 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
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1434
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 (M) or probable (N). Diamonds indicate compounds for which no hematocrit-dependent effect was demonstrated
(U) or probable (V ), and (3) 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 5 IC50 (slope 5 1,
ordinate at the origin 5 0).
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
Fig 4. Effect of high choline concentration on the inhibition of PC
metabolism by F13. Infected erythrocytes (5 3 106 infected cells) were
preincubated for 5 minutes in the presence of 10 mmol/L (W) or 200
mmol/L (X) 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 mCi (3H)choline and pursued for 5
hours at 37°C.
ANCELIN ET AL
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 5 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 a or b 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 highaffinity 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
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLASMODIUM PHOSPHOLIPID METABOLISM INHIBITION
1435
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 3 107 cells/100 mL (0.7% final hematocrit) were preincubated
for 5 minutes at 37°C in the presence of 750 mL special RPMI containing the drug (solid symbols) at 3 mmol/L (A and B) or 6 mmol/L (C and D) or no
(open symbols) drug. Choline influx measurement was initiated by the rapid addition of 50 mL (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) 6 SEM. Each point is the mean of
triplicate determinations in one typical experiment.
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
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1436
ANCELIN ET AL
Table 3. Comparative Effect of 12 Polar Head Analogs on P
falciparum Growth (IC50) and Lymphoblastoid Cell Viability (LV50)
Compounds
IC50 (µmol/L)
LV50 (µmol/L)
E3
E8
F2
F3
F4
F8
F13
G3
G4
G23
H3
HC3
1.6
0.8
81.3
0.97
0.48
0.34
110
1.7
0.09
0.15
2
4
6.6
14
18,000
85
3.7
0.85
4,700
9,500
95
2,500
9,500
7,000
LV50 were measured after 24 hours of drug contact with cells as
described in the Materials and Methods. IC50 against P falciparum are
from Table 1.
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 a or b 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.
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.
REFERENCES
1. Wernsdorfer WH, Payne D: The dynamics of drug resistance in
Plasmodium falciparum. Pharmacol Ther 50:95, 1991
2. Van Deenen LLM, De Gier J: Lipids of the red cell membrane, in
Surgenor G (ed): In the Red Blood Cell. New York, NY, Academic,
1975, p 147
3. Holz GG: Lipids and the malaria parasite. Bull WHO 55:237,
1977
4. Sherman L: Biochemistry of Plasmodium (malarial parasites).
Microbiol Rev 43:453, 1979
5. Vial HJ, Ancelin ML, Philippot JR, Thuet MJ: Biosynthesis and
Dynamycs of lipids in Plasmodium-infected mature mammalian erythrocytes. Blood Cells 16:531, 1990
6. Vial HJ, Ancelin ML: Malarial lipids, in Avila JL, Harris JR (eds):
Subcellular Biochemistry, vol 18: Intracellular Parasites. New York,
NY, Plenum, 1992, p 259
7. Ancelin ML, Vial HJ: Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes. Biochim Biophys Acta
1001:82, 1989
8. Ancelin ML, Parant M, Thuet MJ, Philippot JR, Vial HJ:
Increased permeability to choline in simian erythrocytes after Plasmodium knowlesi infection. Biochem J 273:701, 1991
9. Vial HJ, Thuet MJ, Ancelin ML, Philippot JR, Chavis C:
Phospholipid metabolism as a new target for malaria chemotherapy.
Mechanism of action of D-2-amino-1-butanol. Biochem Pharmacol
33:2761, 1984
10. Ancelin ML, Vial HJ, Philippot JR: Inhibitors of choline
transport into Plasmodium-infected erythrocytes are effective antiplasmodial compounds in vitro. Biochem Pharmacol 34:4068, 1985
11. Ancelin ML, Vial HJ: Quaternary ammonium compounds efficiently inhibit Plasmodium falciparum growth in vivo by impairment of
choline transport. Antimicrobial Agents Chemother 29:814, 1986
12. Beaumelle BD, Vial HJ: Correlation of the efficiency of fatty
derivatives in suppressing Plasmodium falciparum growth in culture
with their inhibitory effect on acyl-CoA synthetase activity. Mol
Biochem Parasitol 28:39, 1988
13. Calas M, Cordina G, Bompart J, Ben Bari M, Jei T, Ancelin ML,
Vial HJ: Antimalarial activity of molecules interfering with Plasmodium falciparum phospholipid metabolism. Structure-activity relationship analysis. J Med Chem 40:3557, 1997
14. Ancelin ML, Vialettes F, Vial HJ: An original method for rapid
serial determination of phospholipid biosynthesis. Applications to
mammalian lymphocytic cells and a lower eucaryote, Plasmodium
falciparum. Anal Biochem 199:203, 1991
15. Moore GE, Gerner RE, Franklin HA: Culture of normal human
leukocytes. JAMA 199:87, 1967
16. Richards WH, Maples BK: Studies on Plasmodium falciparum
in continuous culture. 1. The effects of chloroquine and pyrimethamine
on parasite growth. Ann Trop Med Parasitol 73:99, 1977
17. Ponnudurai T, Leeuwenberg ADEM, Meuwissen JHET: Chloroquine sensitivity of isolates of Plasmodium falciparum adapted to in
vitro culture. Trop Geogr Med 33:50, 1981
18. Fidock DA, Sallenavesales S, Sherwood JA, Gachihi GS,
Ferreiradacruz MD, Thomas AW, Druilhe P: Conservation of the
Plasmodium falciparum sporozoite surface protein gene, STARP, in
field isolates and distinct species of Plasmodium. Mol Biochem
Parasitol 67:255, 1994
19. Jensen JB, Trager W: Plasmodium falciparum in culture: Use of
outdated erythrocytes and description of the candle-jar method. J
Parasitol 63:883, 1977
20. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD: Quantitative
assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16:710, 1979
21. Kutner S, Breuer WV, Ginnsgurg H, Aley SB, Cabantchik ZI:
Characterization of permeation pathways in the plasma membrane oh
human erythrocytes infected with early stages of Plasmodium falciparum. J Cell Physiol 125:521, 1985
22. The Merck Index (ed 10). Rahway, NJ, Merck & Co, 1983
23. Elabbadi N, Ancelin ML, Vial HJ: Use of radioactive ethanolamine incorporation into phospholipids to assess in vitro antimalarial
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLASMODIUM PHOSPHOLIPID METABOLISM INHIBITION
activity by the semiautomated microdilution technique. Antimicrobial
Agents Chemother 36:50, 1992
24. Colhoun EH, Rylett RJ: Nitrogen mustard analogues of choline
potential: For use and misuse. Trends Biochem Sci 11:55, 1986
25. Krupka RM: The kinetics of transport inhibition by noncompetitive inhibitors. J Membr Biol 74:175, 1983
26. Devés R, Krupka RM: Apparent noncompetitive inhibition of
choline transport in erythrocytes by inhibitors bound at the substrate
site. J Membr Biol 74:183, 1983
27. Ancelin ML, Vial HJ: Several lines of evidence demonstrating
that Plasmodium falciparum, a parasitic organism, has distinct enzymes
for the phosphorylation of choline and ethanolamine. FEBS Lett
202:217, 1986
28. Vial HJ, Ancelin ML, Elabbadi N, Orcel H, Yeo H-J, Gumila C:
Infected erythrocyte choline carrier inhibitors: Exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition. Mem Inst Oswaldo Cruz 89:91, 1994
29. Kirk K, Horner HA, Elford BC, Ellory JC, Newbold CI:
Transport of diverse substrates into malaria-infected erythrocytes via a
pathway showing functional characteristics of a chloride channel. J Biol
Chem 269:3339, 1994
30. Kirk K, Horner HA: Novel anion dependence of induced cation
1437
transport in malaria-infected erythrocytes. J Biol Chem 270:24270,
1995
31. Cabantchik ZI, Silfen J, Firestone A, Krugliak M, Nissani E,
Ginsburg H: Effects of lysosomotropic detergents on the human
malarial parasite Plasmodium falciparum in in vitro culture. Biochem
Pharmacol 38:1271, 1989
32. Tamaru M, Roberts E: Structure-activity studies on inhibition of
choline uptake by a mouse brain synaptosomal preparation: Basic data.
Brain Res 473:205, 1988
33. Roberts E, Tamaru M: The ligand binding site of the synaptosomal choline transporter: A provisional model based on inhibition
studies. Neurochem Res 17:509, 1992
34. Fisher A, Hanin I: Choline analogs as potential tools in developing selective animal models of central cholinergic hypofunction. Life
Sci 27:1615, 1980
35. Krupka RM: Expression of substrate specificty in facilitated
transport systems. J Membr Biol 117:69, 1990
36. Ferguson SSG, Diksic M, Collier B: Stereospecificity of highand low-affinity transport of choline analogues into rat cortical synaptosomes. J Neurochem 57:915, 1991
37. Lerner J: Choline transport specificty in animal cells and tissues.
Comp Biochem Physiol 93C:1, 1989
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1998 91: 1426-1437
Antimalarial Activity of 77 Phospholipid Polar Head Analogs: Close
Correlation Between Inhibition of Phospholipid Metabolism and In Vitro
Plasmodium Falciparum Growth
Marie L. Ancelin, Michèle Calas, Jacques Bompart, Gérard Cordina, Dominique Martin, Mohammed Ben
Bari, Tai?b Jei, Pierre Druilhe and Henri J. Vial
Updated information and services can be found at:
http://www.bloodjournal.org/content/91/4/1426.full.html
Articles on similar topics can be found in the following Blood collections
Red Cells (1159 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.