![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 59, Issue 5, 1298-1306, May 2001
Department of Pharmacology and Therapeutics, The University of Liverpool, Liverpool, United Kingdom (A.M.W.S., P.G.B., I.G.E., P.A.S., S.A.W.); Division of Infection of Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom (H.P.D.); and Molecular Parasitology Group, Institute of Molecular Medicine, Oxford, United Kingdom (B.C.E.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
Conclusions
References
|
|---|
Extensive drug resistance in Plasmodium falciparum
emphasizes the urgent requirement for novel antimalarial agents. Here
we report potent antimalarial activity of a number of diamidine
compounds. The lead compound pentamidine is concentrated 500-fold by
erythrocytes infected with P. falciparum. Pentamidine
accumulation can be blocked by inhibitors of hemoglobin digestion,
suggesting that the drug binds to ferriprotoporphyrin IX (FPIX). All of
the compounds bound to FPIX in vitro and inhibited the formation of
hemozoin. Furthermore, inhibitors of hemoglobin digestion markedly
antagonized the antimalarial activity of the diamidines, indicating
that binding to FPIX is crucial for the activity of diamidine drugs.
Pentamidine was not accumulated into uninfected erythrocytes.
Pentamidine transport into infected cells exhibits an initial rapid
phase, nonsaturable in the micromolar range and sensitive to inhibition
by furosemide and glibenclamide. Changing the counter-ion in the order
Cl
< Br < NO2 < I
<SCN markedly stimulated pentamidine transport. These
data suggest that pentamidine is transported although a pore or ion
channel with properties similar to those of the recently characterized `induced permeability pathway' on the infected red cell membrane. In
summary, the diamidines exhibit two levels of selectivity against P. falciparum. The route of entry and molecular target
are both specific to malaria-infected cells and are distinct from
targets in other protozoa. Drugs that target the hemoglobin degradation pathway of malaria parasites have a proven record of accomplishment. The employment of induced permeability pathways to access this target
represents a novel approach to antiparasite chemotherapy and offers an
additional level of selectivity.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
|
|---|
The
evolution of drug resistance poses significant problems for the
treatment of malaria. The widespread failure of many antimalarial agents, chloroquine in particular, highlights the urgent need for new
drugs (Foley and Tilley, 1998
). Pentamidine and other diamidine
compounds have a long history in the treatment of human protozoal
infections (Ormerod, 1967). Although preliminary studies have shown
that diamidine compounds have activity against P. falciparum (Bell et al., 1990), they have never been used to treat malaria.
Because pentamidine is poorly membrane-permeable, its selective
activity against protozoan parasites is attributed to parasite-specific uptake mechanisms. Active transporters have been described in Leishmania species and trypanosomes that allow pentamidine
to accumulate to high concentrations (Carter et al., 1995; de Koning and Jarvis, 1999). After accumulation within these parasites, pentamidine is thought to attack a number of biological targets (Basselin et al., 1996; Morty et al., 1998; Reddy et al., 1999).
We assume that pentamidine operates at an intracellular site in Plasmodium falciparum. How this highly charged, water-soluble drug manages to penetrate the plasma membrane of the infected red blood cell is unknown, as is the mechanism by which it kills the parasite after accumulation.
In this report, we address both these issues. We demonstrate specific
and extensive accumulation of pentamidine into erythrocytes infected
with P. falciparum. Kinetic and pharmacological
characterization of the initial phase of drug uptake indicates that the
drug penetrates the infected cell through a parasite specific pore with
properties similar to those of the new permeability pathways (NPP)
induced by the parasite on the surface of the infected erythrocyte
(Ginsburg et al., 1983; Ginsburg and Stein, 1988; Kirk et al., 1994;
Upston and Gero, 1995).
Simple penetration through a pore cannot explain the extensive
concentration of the drug by the infected cell. We demonstrate that,
after penetration through the NPP, the accumulation of pentamidine is
largely driven by its binding to ferriprotoporphyrin IX (FPIX) generated during the digestion of hemoglobin (Francis et al., 1997).
Malaria parasites normally crystallize toxic FPIX into nontoxic
hemozoin (malarial pigment). We demonstrate that the diamidines inhibit
the formation of hemozoin from FPIX by interacting directly with FPIX.
Furthermore, we show that this interaction is primarily responsible for
the antimalarial activity.
Here we explain the antimalarial action of diamidine drugs, based on selective transport through a parasite specific pore and subsequent binding to FPIX. This unique approach to highly selective antimalarial action is based on the targeted exploitation of two parasite-specific pathways. Diamidines such as pentamidine represent a very promising starting point for the design of a new class of antimalarial agent.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
|
|---|
Reagents. All reagents were obtained from Sigma unless otherwise stated. Silicon oil 550 was obtained from Dow Corning limited. [3H]Pentamidine isethionate [specific activity, 3.63 TBq/mmol (98 Ci/mmol), 5 mCi/ml in ethanol; 99.9% pure by high-performance liquid chromatography], unlabeled stilbamidine, propamidine, and berenil were kindly donated by Prof Mike Barrett, Division of Infection of Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK. The [3H]pentamidine isethionate was originally custom synthesized and purified by Amersham Pharmacia Biotech UK, Ltd. (Little Chalfont, Buckinghamshire, UK).
Culture of P. falciparum and Drug Sensitivity
Assays.
The 3D7, HB3,and K1 strains of P. falciparum
used were obtained from Prof. D. Walliker, Edinburgh University
(Edinburgh, UK) and recloned in house. The TM6 strain was obtained from
Dr. P. Tan-Ariya (Mahidol University, Bangkok, Thailand). The
chloroquine resistant isolates K1 and TM6 originate from Thailand and
have recently been fully characterized in terms of the chloroquine phenotype and genotype (Mungthin et al., 1999). Parasites were maintained in continuous culture and synchronized using standard techniques. When free parasites were required, they were released from
the host cell using the method of Elford (1993). The sensitivity of the
infected erythrocytes to various drugs was determined by measuring the
ability of serial dilutions of drugs to inhibit the incorporation of
radiolabeled [3H]hypoxanthine into parasite
nucleic acids (Desjardins et al., 1979). In each case, the lowest
feasible inoculum size was used (0.5% parasitemia, 1% hematocrit).
IC50 values were calculated for each assay using
the four-parameter logistic method (Grafit program; Erithacus Software,
Kent, UK). The effect of the combination of diamidines and Ro 40-4388 on parasite growth was tested by titration of the two drugs at fixed
ratios proportional to their IC50 values. The
fractional inhibitory concentrations of the resulting IC50 values were plotted as isobolograms
(Berenbaum, 1978).
The Effect of Furosemide on the in Vitro Activity of Pentamidine. Synchronous trophozoite stage (approximately 36 h) cultures were washed twice in growth medium without serum. Cultures were suspended at an inoculum size of 1 (inoculum size = percent parasitemia × percent hematocrit) in growth medium without serum, containing the following concentrations of pentamidine; 0, 10, 30, 100, 300, 1,000, 3,000, and 10,000 nM. Parallel cultures contained the same concentrations of pentamidine plus 100 µM furosemide, which was diluted into prewarmed medium (37°C) from a 10 mM stock in dimethyl sulfoxide. After incubation for 6 h at 37°C, the cultures were washed three times in complete growth medium to remove unbound drug. The cells were then resuspended in complete growth medium at an inoculum size of 1. [3H]Isoleucine was added (1 µCi/ml) and the cultures were gassed and returned to the incubator for a further 24 h. After incubation, the cells were pelleted and exposed to 0.15% saponin at room temperature for 10 min to lyse the host cells and uninfected erythrocytes. Free parasites were pelleted, the supernatant was discarded, and the cell samples were taken and processed for scintillation counting. Growth was assessed in the absence of pentamidine and in the presence or absence of 100 µM furosemide, as appropriate. For each pentamidine concentration in the presence or absence of 100 µM furosemide, the results were expressed as percentage of control growth in the absence of pentamidine.
Measurement of the Uptake of [3H]Pentamidine. Uninfected erythrocytes or erythrocytes infected with synchronized trophozoites of Plasmodium falciparum were suspended in the appropriate buffer containing [3H]pentamidine at a concentration of 50 nM. Preliminary experiments established that there was no significant difference in pentamidine accumulation of the different clones.
For the initial rate of uptake studies, the infected cells were enriched to approximately 70% parasitemia using Plasmagel. The cells were suspended in HEPES-buffered RPMI medium without bicarbonate, pH 7.4. The suspension (typically 108 cells/ml) was prewarmed to 37°C in a water bath. The pentamidine and furosemide were added at time 0 (the final pentamidine concentration was adjusted to 20 µM using unlabeled drug). At the required times, 200-µl samples were removed and transferred to chilled microcentrifuge tubes containing 800 µl of ice-cold RPMI, layered over 400 µl of silicon oil followed by immediate centrifugation (15,000g for 2 min). The tube tip containing the cell pellet was cut off and the cells were lysed with 100 µl of distilled water. The lysate was solubilized and decolorized by adding 100 µl of a cocktail containing five parts quaternary ammonium hydroxide, two parts H2O2, and two parts glacial acetic acid. The samples were then counted by liquid scintillation counting. Subsequent influx experiments to test the effect of counter-ion substitution (buffers as used by Kirk and Horner, 1995b) and to test the concentration-dependence of furosemide
were performed at a single time point of 1 min. Initial surface binding
of [3H]pentamidine was assessed by fast
diluting labeled pentamidine into ice-cold cell suspensions and
centrifuging immediately. Counts attributable to surface binding were
subtracted from the pellet counts. For the remainder of the
accumulation experiments, reactions were terminated by layering 1 ml of
the cell suspension over 400 µl of silicon oil and centrifuging and
processing as described above. Binding was measured after 2 h in
50 nM [3H]pentamidine and various
concentrations of unlabeled pentamidine, in the presence or absence of
10 µM Ro 40-4388.
Nonspecific uptake was determined by measuring and subtracting the
counts attributable to an equal number of uninfected erythrocytes. The
cellular accumulation ratio is the ratio of the amount of pentamidine
in the infected cell to the amount of pentamidine in a similar volume
of buffer after incubation. The volume of the infected cell was assumed
to be the same as that of the uninfected cell (Kirk et al., 1994). The
intracellular drug concentration was calculated by multiplying the
cellular accumulation ratio by the buffer drug concentration after incubation.
UV/Visible Spectral Scans. FPIX or protoporphyrin IX (PIX) (Porphyrin Products) solutions were prepared fresh for each experiment. Solutions (3 mM) were prepared in 0.1 M NaOH. Immediately before each scan, samples were fast diluted into 0.2 M HEPES buffer, pH 7.0, to give a final concentration of 3 µM. in the presence or absence of 3 µM pentamidine. Samples (400 µl) were placed in quartz cuvettes and scanned against the appropriate blank in a Hewlett Packard 8452A diode array spectrophotometer (Hewlett Packard, Palo Alto, CA).
Binding of Pentamidine to FPIX or PIX in Vitro.
The binding
of [3H]pentamidine to FPIX or PIX adsorbed to
erythrocyte ghost membranes was measured as described previously for
[3H]chloroquine (Bray et al., 1999)
Determination of the Effect of Diamidines and Chloroquine on the
Production of Hemozoin in Vitro.
Assays were performed as
described in Bray et al. (1999). Briefly, an aliquot of trophozoite
lysate (100 µl) and FPIX (100 µl of 3 mM in 0.1 M NaOH) were mixed
with an aliquot of 1 M HCl (10 µl) and sodium acetate (500 mM, pH
5.2) was added to give a volume of 900 µl in each tube. A series of
drug concentrations were prepared in water and 100 µl of each added
to the appropriate samples. Samples were mixed and incubated for
12 h at 37°C, with occasional mixing. After incubation, samples
were centrifuged (15,000 g, 15 min, 21°C) and the hemozoin pellet
repeatedly washed with 2% w/v SDS in 0.1 M sodium bicarbonate, pH 9.0, with sonication (30 min, 21°C, bath sonicator; Decon FS100
Ultrasonics Ltd, UK) until the supernatant was clear (usually 3-4
times). After the final wash, the supernatant was removed and the
pellet was resuspended in 1 ml of 0.1 M NaOH and incubated for a
further 1 h at room temperature. Samples were then mixed by
aspiration with a pipette. The hemozoin content was determining by
measuring the absorbance at 400 nm (DU640 spectrophotometer; Beckman
Coulter, Fullerton, CA) using a 1-cm quartz cuvette. The amount of
hemozoin formed during the incubation was corrected for preformed
hemozoin (the amount of preformed hemozoin in the parasite extract was
determined from a sample containing extract, but no substrate, which
was incubated and repeatedly washed with 2% SDS as stated previously). The concentration of drug required to produce 50% inhibition of hemozoin production (IC50) was determined
graphically as described for the drug sensitivity assays.
Molecular Modeling Studies. All molecular modeling was carried out on an O2 R5000 silicon graphics workstation within the Cerius2 molecular modeling environment. Simulation studies were carried out on pentamidine and heme. Each molecule was energy minimized from the `as constructed' conformation using the universal force field. The universal force field is able to paramatize a wider range of atom types than earlier force fields.
Energy minimization alone is able to find only the nearest energy minimum to the starting conformation of a given system. To generate a wider representative set, we have employed the method of simulated annealing similar to that employed by Milne (1997). Molecular dynamics
(MD) runs at 298K for a simulation time of 5 ps were performed for each
drug molecule, and the resulting structure was energy minimized to gain
a low-energy conformation. This process was repeated until 10 structures per molecule had been generated. For each MD calculation,
the last minimized structure in the set was used as the starting
conformation for the next MD simulation.
The aims of the modeling study were to investigate the potential of
pentamidine for interacting with heme, the proposed drug receptor site
within the parasite food vacuole. Having generated a set of
conformations for pentamidine, an investigation into its interaction
with haem was carried out. A model of heme (ferriprotoporphyrin IX) was
constructed with a single hydroxyl group ligated to the Fe atom at an
axial coordination site above the ring. This unit was then energy
minimized with the Universal force field potential set. From earlier
studies, it was found that the conformation in which both carboxylic
acid groups were orientated on the same side of the porphyrin ring
(
-hematin) was lower in energy that when the groups were on opposite
faces (
-hematin).
For the pentamidine model obtained after energy minimization-dynamics
simulation, the lowest energy (folded) conformation was chosen to
generate a complex with haem in the following manner. We have found,
from previous studies, that antimalarial agents containing aromatic
rings can 
-stack with the delocalized -system of the porphyrin
ring. With this restriction, the porphyrin model was intercalated
between the two aromatic rings of pentamidine in such a way that both
benzene rings could potentially form a -stacking interaction with
the planar haem ring. From this starting point, the dimers were then
energy minimized using the universal parameter set.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
|
|---|
In Vitro Activity of Diamidine Compounds.
The diamidine
compounds were tested in vitro against a panel of P. falciparum clones that have varying susceptibility to the standard
antimalarial drugs chloroquine, quinine, and pyrimethamine (Table
1).
|
Pentamidine Is Transported through the NPP. In order to probe the antimalarial mode of action of the diamidines, the uptake of radiolabeled pentamidine was investigated
Pentamidine uptake into erythrocytes infected with Plasmodium falciparum displays two distinct phases: a period of rapid uptake is observed over the first 5 min, followed by a slower phase of drug accumulation lasting several hours (Fig. 1A). After 3 h, pentamidine accumulation is extensive, reaching levels 500-fold greater than the incubation medium. Pentamidine is not significantly accumulated by uninfected erythrocytes, indicating that the pathways responsible for drug accumulation are parasite-specific.
|
; Upston and Gero, 1995). Furthermore, the
IC50 value of inhibition of pentamidine uptake
(approximately 10 µM) is similar to the IC50
value for the inhibition of transport of known NPP substrates by
furosemide (Kirk and Horner, 1995b)
Pentamidine uptake is also blocked by other NPP inhibitors, such as
glibenclamide, phloridzin, and NPPB (Kirk et al., 1993; Kirk and
Horner, 1995a; Upston and Gero, 1995; data not shown). By
contrast, pentamidine uptake is not inhibited significantly by
millimolar concentrations of arginine, adenine, inosine, putrescine, or
spermidine (data not shown). These compounds are inhibitors of
pentamidine transport systems known to be present in
Leishmania species and trypanosomes (Carter et al., 1995;
Basselin et al., 1996; de Koning and Jarvis, 1999). Transport of
pentamidine into isolated parasites seems to be much less sensitive to
furosemide compared with intact infected cells
(IC50 values of 300 µM versus 10 µM; Fig.
2). These data suggest that the
furosemide-sensitive pathway is on the host erythrocyte membrane,
consistent with the location of the NPP.
|
). Similarly, we show that substituting chloride
with thiocyanate, nitrate, bromide, or iodide results in a marked
stimulation of pentamidine uptake (Fig.
3).
|
; Kleyman et al.,
1995; Navas et al., 1996; de Koning and Jarvis, 1999). It is possible
that pentamidine may exert its antimalarial effect by inhibiting the
transport of essential nutrients or metabolites across the host
erythrocyte membrane. In this case, the extensive uptake of pentamidine
by the infected cell may have little relevance in the mode of action of
the drug. However, coincubation with furosemide significantly protects
the parasite from the antimalarial effect of pentamidine, increasing
the pentamidine IC50 value by at least an order
of magnitude (Fig. 4). These data
suggest that the extensive uptake of pentamidine through the
furosemide-sensitive pathway is required for antimalarial activity.
|
Pentamidine Binds to FPIX in the Malaria-Infected Erythrocyte and
Inhibits the Crystallization of FPIX into Hemozoin.
Pentamidine
accumulation over the second slower phase is saturable Scatchard
analysis (Fig. 5A) reveals at least two
classes of binding site in malaria-infected erythrocytes that exhibit Michael-Menten kinetics: a high affinity site
(Kd value of 2.5 µM) and a lower
affinity site (Kd value of
approximately 200 µM). These data indicate that malaria parasites
possess saturable intracellular receptors for pentamidine. Malaria
parasites continuously generate FPIX in large quantities from the
action of parasite proteases on host cell hemoglobin and FPIX is known
to act as a receptor for several antimalarial drugs (Francis et al.,
1997; Bray et al., 1998, 1999; Mungthin et al., 1998). Pentamidine has
structural features that suggest it may bind to FPIX (see below).
|
). This inhibitor allows
hemoglobin digestion to be blocked reversibly in situ. Consequently,
the release of FPIX in is also blocked (Bray et al., 1999). Because
FPIX exists only transiently in viable cells, this maneuver permits the
measurement of the contribution of FPIX binding in the mechanism of
drug accumulation of intact infected erythrocytes (Bray et al., 1998,
1999; Mungthin et al., 1998). A 10 µM concentration of Ro 40-4388 is
sufficient to block the generation of FPIX by approximately 70% (Bray
et al., 1999). Under the same conditions, the high-affinity
accumulation of pentamidine is reduced by 60% (Fig.
6).
|
|
; Pagola et al., 2000). Chloroquine is a good example of a drug
that works in this way. All of the diamidines are able to inhibit the
formation of hemozoin in vitro at concentrations comparable with
chloroquine (Fig. 8). Taken together, all
of our data provide good evidence that the diamidines kill parasites by
binding avidly to FPIX, inhibiting the formation of hemozoin and
causing a build-up of toxic FPIX or FPIX-drug complex.
|
Molecular Modeling of Pentamidine and Its Interaction with
FPIX.
Energy-minimized structures generated by the molecular
modeling program are presented in Fig. 9,
A and B. Rather than adopting a linear conformation, the intramolecular
interactions seem to colocalize the two charged groups of pentamidine
(Fig. 9A).
|
, the potential repulsive
effects of the charged region presumably would be less severe than if
the two charged groups were at either end of the molecule.
Alternatively, this conformation may favor ion pairing with the
permeant anion. At a maximum of just over 6 Å in diameter, the
molecule is comfortably within the 10-12 Å upper size limit proposed
for substrates of the NPP (Staines et al., 2000).
This conformation of pentamidine also facilitates a proposed
interaction with FPIX, in which an aromatic phenyl ring of the pentamidine molecule stacks with the aromatic porphyrin ring (Fig.
9B). Other antimalarial drugs that disrupt the formation of hemozoin
also pi stack with FPIX in this manner. The drugs are thought to
sterically hinder the formation of a bond between the iron of one
porphyrin and a carboxylate group of an adjacent porphyrin (O'Neill et
al., 1997). This bond is required for the formation of the hemozoin
crystal (Pagola et al., 2000). In the configuration depicted in Fig.
5C, the phenyl ring of pentamidine is positioned optimally for stacking (approximately 3.5 Å from the face of the porphyrin ring).
Consequently, the amidine nitrogens are greater than 3.5 Å away from
the porphyrin iron. At this distance, an electrostatic bond is not
favored. This is supported by the demonstration that strong drug
binding also takes place when the iron is removed from FPIX.
Pentamidine also quenches the Soret peak of protoporphyrin IX (PIX)
(Fig. 10). Furthermore, the binding of
pentamidine to PIX adsorbed to erythrocyte ghost membranes displays an
affinity very similar to the binding of pentamidine to FPIX in the same
system (Fig. 5C).
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
|
|---|
Here we demonstrate potent in vitro antimalarial activity of several diamidine compounds. The activity of the diamidines against P. falciparum compares favorably and without cross-resistance with that of the standard antimalarial agents chloroquine, quinine, and pyrimethamine (Table 1). The antimalarial selectivity of these compounds can be attributed to the targeting of at least two distinct parasite-specific pathways.
Transport of Diamidines into the Malaria-infected Erythrocyte. We have shown that pentamidine is essentially impermeable to normal uninfected erythrocytes but enters malaria infected erythrocytes rapidly, via a furosemide-sensitive and nonsaturable pathway (Fig. 1, A and B). This pathway shows a striking similarity to the well-characterized NPP induced by the intracellular parasite in the host erythrocyte membrane.
The membrane of the human erythrocyte normally has very low cation permeability. However, after infection with P. falciparum, the permeability to K+ and several organic cations is markedly increased (Kirk and Horner, 1995b; Staines
et al., 2000). The NPP are anion-selective; like a number of other
anion-selective channels (Franciolini and Petris, 1990), they are known
to have significant permeability to cations (Kirk and Horner,
1995b).
The transport of cations through the NPP is stimulated by the
substitution of Cl with other counter-ions
(Kirk and Horner, 1995b). Similarly, we demonstrate that the
transport of pentamidine into malaria-infected erythrocytes is markedly
stimulated by the substitution of Cl with
SCN,
NO3,
Br, or I (Fig. 3).
This phenomenon seems to operate at the level of the host cell membrane
or the parasitophorous vacuole membrane, because the transport of
pentamidine into free parasites is not influenced significantly by
counter-ion substitution (data not shown). Furthermore, the transport
of pentamidine into free parasites is much less sensitive to inhibition
by furosemide, with an IC50 value of 300 µM
versus 10 µM for intact infected erythrocytes (Fig. 2). Taken together, our data suggest that pentamidine can gain access to the
intracellular parasite via a pathway exhibiting all of the functional
characteristics of the NPP.
The Inhibition of FPIX Crystallization by Diamidine Compounds.
Pentamidine is concentrated by the infected cells 500-fold within
3 h (Fig. 1A). Pore systems such as the NPP will allow the equilibration of substrates across a membrane barrier but will not
themselves allow the substrate to be concentrated. Therefore, additional factors such as intracellular drug binding or parasite active transporters are required to concentrate the drug. One potential
parasite drug receptor that is present in the appropriate quantity is
FPIX. The ability of the diamidines to quench the Soret peak of FPIX
shows that a binding interaction takes place in vitro (Fig. 5B). Energy
minimization modeling indicates that the most stable conformation
effectively intercalates the porphyrin within the diamidine molecule,
with the aromatic phenyl ring of the amidine group stacking on the
surface of the porphyrin ring (Fig. 9B). In this conformation, the
amidine nitrogens are unlikely to coordinate with the iron of the
porphyrin. In support of our model, we provide strong evidence that the
porphyrin iron is not required for pentamidine binding.
; Foley and Tilley, 1998). All of the
diamidines were able to inhibit the crystallization of FPIX in vitro
and with similar efficiency to chloroquine (Fig. 8). Importantly,
inhibition was observed at micromolar concentrations that are easily
achieved inside the parasite within 3 h (Fig. 1A). These data
reveal that the interaction between FPIX and the diamidine drugs is a
critical event in parasite killing and pinpoint FPIX as the likely
molecular target of this class of antimalarial.
| |
Conclusions |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
|
|---|
Diamidine drugs are impermeable to normal human erythrocytes but are taken up rapidly into P. falciparum-infected erythrocytes via NPP induced by the parasite in the host cell membrane. Once inside the infected cell, the drugs bind avidly to FPIX and kill the parasites, probably by inhibiting FPIX crystallization.
Currently available diamidines are highly toxic and are very poorly
absorbed orally. However, recent reports suggest that it may be
possible to overcome both of these problems by using relatively simple
chemical substitutions (Francesconi et al., 1999). Here we show that
diamidines are likely to share a common mode of action with chloroquine
but are not recognized by the chloroquine resistance mechanism.
Therefore, our studies define a novel antimalarial pharmacophore with
considerable advantages over conventional FPIX-binding antimalarial drugs.
| |
Acknowledgments |
|---|
We would thank M. P. Barrett for the generous gift of radiolabeled pentamidine.
| |
Footnotes |
|---|
Received October 19, 2000; Accepted February 2, 2001
This work was supported by Wellcome Trust grants to P.G.B. and S.A.W.
Send reprint requests to: Dr. Patrick G. Bray, Department of Pharmacology and Therapeutics, The University of Liverpool, Liverpool L69 3BX, United Kingdom. E-mail: p.g.bray{at}liv.ac.uk
| |
Abbreviations |
|---|
NPP, new permeability pathway; FPIX, ferriprotoporphyrin IX; PIX, protoporphyrin IX; MD, molecular dynamics.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  A. E. Purfield, R. R. Tidwell, and S. R. Meshnick Interactions of DB75, a Novel Antimalarial Agent, with Other Antimalarial Drugs In Vitro Antimicrob. Agents Chemother., June 1, 2008; 52(6): 2253 - 2255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Leepin, A. Studli, R. Brun, C. E. Stephens, D. W. Boykin, and A. Hemphill Host Cells Participate in the In Vitro Effects of Novel Diamidine Analogues against Tachyzoites of the Intracellular Apicomplexan Parasites Neospora caninum and Toxoplasma gondii Antimicrob. Agents Chemother., June 1, 2008; 52(6): 1999 - 2008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. Biagini, N. Fisher, N. Berry, P. A. Stocks, B. Meunier, D. P. Williams, R. Bonar-Law, P. G. Bray, A. Owen, P. M. O'Neill, et al. Acridinediones: Selective and Potent Inhibitors of the Malaria Parasite Mitochondrial bc1 Complex Mol. Pharmacol., May 1, 2008; 73(5): 1347 - 1355. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Rodenko, A. M. van der Burg, M. J. Wanner, M. Kaiser, R. Brun, M. Gould, H. P. de Koning, and G.-J. Koomen 2,N6-Disubstituted Adenosine Analogs with Antitrypanosomal and Antimalarial Activities Antimicrob. Agents Chemother., November 1, 2007; 51(11): 3796 - 3802. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Midgley, K. Fitzpatrick, L. M. Taylor, T. L. Houchen, S. J. Henderson, S. J. Wright, Z. R. Cybulski, B. A. John, A. McBurney, D. W. Boykin, et al. Pharmacokinetics and Metabolism of the Prodrug DB289 (2,5-Bis[4-(N-methoxyamidino)phenyl]furan Monomaleate) in Rat and Monkey and Its Conversion to the Antiprotozoal/Antifungal Drug DB75 (2,5-Bis(4-guanylphenyl)furan Dihydrochloride) Drug Metab. Dispos., June 1, 2007; 35(6): 955 - 967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Choubey, P. Maity, M. Guha, S. Kumar, K. Srivastava, S. K. Puri, and U. Bandyopadhyay Inhibition of Plasmodium falciparum Choline Kinase by Hexadecyltrimethylammonium Bromide: a Possible Antimalarial Mechanism Antimicrob. Agents Chemother., February 1, 2007; 51(2): 696 - 706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. L. Lew A choline "vacuum cleaner" Blood, November 15, 2004; 104(10): 3006 - 3007. [Full Text] [PDF]
|
|
|
|
|
|
G. A. Biagini, E. M. Pasini, R. Hughes, H. P. De Koning, H. J. Vial, P. M. O'Neill, S. A. Ward, and P. G. Bray Characterization of the choline carrier of Plasmodium falciparum: a route for the selective delivery of novel antimalarial drugs Blood, November 15, 2004; 104(10): 3372 - 3377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Klenke, M. P. Barrett, R. Brun, and I. H. Gilbert Antiplasmodial activity of a series of 1,3,5-triazine-substituted polyamines J. Antimicrob. Chemother., August 1, 2003; 52(2): 290 - 293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||
) and
erythrocytes infected with the HB3 strain of P.
falciparum (
). Data are means ± S.E. from six
individual experiments. B, time course of the initial uptake of 20 µM
[3H]pentamidine into HB3 infected erythrocytes in the
presence (
), stilbamidine (
) and chloroquine (
). Data are means ± S.E. from five individual experiments.
