Review

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Targeting the redox metabolism of

Plasmodium falciparum

Targeting the redox metabolism of Plasmodium falciparum to create a fatal overload of oxidative stress is a route
to explore the discovery of new antimalarial drugs. There are three main possibilities to target the redox metabolism
of P. falciparum at the erythrocytic stage: selective targeting and inhibition of a redox P. falciparum protein or enzyme;
oxidant drugs targeting essential parasite components and heme by-products; and redox cycler drugs targeting the
parasitized red blood cell. Oxidants and redox cycler agents, with or without specific targets, may disrupt the
fragile parasitized erythrocyte redox-dependent architecture given that: redox equilibrium plays a vital role at the
erythrocytic stage; P. falciparum possesses major NADPH-dependent redox systems, such as glutathione and
thioredoxin ones; and the protein–NADPH-dependent phosphorylation–dephosphorylation process is involved
in building new permeation pathways and channels for the nutrient–waste import–export traffic of the parasite.

The impact of malaria & resistance to trials, with among them many combinations of Françoise Nepveu*1,2 &
artemisinin old drugs [203]. From the five species of genus Francesco Turrini3
International programs aimed at controlling Plasmodium (P. falciparum, Plasmodium vivax, 1
Université de Toulouse, UPS;
malaria led to positive results in late 2010 [1,201] Plasmodium ovale, Plasmodium malariae and PHARMA-DEV (UMR 152); F-31062
Toulouse cedex 9, France
through three major distribution actions: insec- Plasmodium knowlesi) responsible for the most 2
IRD, UMR 152, F-31062 Toulouse
ticide-treated nets, rapid diagnostic tests and prominent human infections, P. falciparum is cedex 9, France
3
Department of Genetics, Biology &
combination therapies of artemisinin to deal the most virulent and is responsible for about Biochemistry, University of Turin, Via
with multiple-drug Plasmodium falciparum resis- 80% of human malaria cases and 90% of Santena, 5 bis, 10126 Turin, Italy
*Author for correspondence:
tance. According to the WHO malaria report deaths. The situation of malaria therapy and Tel.: +33 5 6225 6869
2012 [202], there are signs of a slowdown that control is still grave, considering the multidrug Fax: +33 5 6225 9802
could threaten to reverse the recent gains in the resistant P. falciparum, the chloroquine-resistant E-mail: francoise.nepveu@univ-tlse3.fr

fight against malaria. To sustain the improve- P. vivax, the contraindications of primaquine in
ments it is necessary, on one hand, to maintain P. vivax liver-stage infection (only two drugs are
financial and logistical efforts and, on the other available, including bulaquine in India), and the
hand, to tackle the effects of excessive drug resis- need for new products to act against gametocytes
tance to antimalarials. The resistance to arte- to prevent transmission and against hypnozo-
misinins (ARTs) is emerging more and more and ites when dealing with P. vivax and P. ovale, to
has spread to new locations in Asia, out of the avoid recurrence. Global phenotypic screening
Greater Mekong sub-region (Thailand, Myan- for antimalarials has been carried out by aca-
mar, China) [2] and is becoming a major chal- demic and private groups [4,5]. In recent years,
lenge in malaria control because it is the basis of the high-throughput screening of the 2 million-
current treatments. No other antimalarial drugs compound library at GlaxoSmithKline (Brent-
with the same level of efficacy and tolerability of ford, UK)for inhibitors of the P. falciparum
ARTs are available to fight P. falciparum resis- intraerythrocytic cycle revealed thousands of
tance. Moreover, ARTs are also becoming essen- chemical starting points for antimalarial lead
tial for the treatment of severe malaria, decreas- identification (Tres Cantos antimalarial com-
ing mortality rates [3]. No other drugs are ready pounds set) [6]. This intensive screening has gen-
for deployment, and drug development efforts erated new antiplasmodial structures and associ-
are not expected to yield new antimalarial drugs ated databases, encouraging further research to
until the end of this decade [203]. Despite efforts fight malaria. It is important to support ongoing
to discover and develop new antimalarial drugs research on the identified new scaffolds while
during the past 20 years with the support of encouraging research that will adopt innovative
organizations such as Medicines for Malarial concepts, new modes of action and new strate-
Venture (Geneva, Switzerland), there are still gies to discover new antimalarial drugs for the
few drugs that have progressed beyond Phase III different life cycle stages of Plasmodium [7].

10.4155/FMC.13.159 © 2013 Future Science Ltd Future Med. Chem. (2013) 5(16), 1993–2006 ISSN 1756-8919 1993
Review | Nepveu & Turrini

Key Terms Targeting the redox metabolism how A. gambiae mitochondrial carrier 1 silenc-
Reactive oxygen species: A
Redox systems participate in cell signaling pro- ing could affect mitochondrial respiration and
common term to designate cesses and are critical in maintaining cell homeo- ROS generation, and reported that the consecu-
O2-derived free radicals such as stasis to preserve cellular functions during oxida- tive reduced mitochondrial membrane potential
superoxide radical anion, tive stress and diseases. Inherent to these systems resulted in increased proton-leak, uncoupling of
hydroxyl radical, peroxyl and
alkoxyl radicals, as well as
are reactive oxygen species (ROS), which act oxidative phosphorylation, reduced ROS genera-
O2-derived nonradical species as messengers modulating signal transduction tion and increased A. gambiae susceptibility to
such as hydrogen peroxide. pathways, and in addition inflict direct cellular Plasmodium infection.
Reactive oxygen species have a damage [8]. The major intracellular source of This review will concentrate on the erythro-
relatively short half-life.
ROS is the mitochondrion where the sequential cytic stage of the parasite because the P. falci-
Band 3: Also known as anion reduction of O2 produces 1–2% of ROS. Based parum sensitivity to oxidative stress is amplified
exchanger 1. An important
structural component of the
on the role of ROS and redox mechanisms in by the oxidative events susceptible to occur in
erythrocyte cell membrane, various diseases, therapeutic strategies have a host cell rich in redox entities (oxygen, iron,
making up to 25% of the cell been developed to modulate the redox-activated glutathione, oxido-reductases).
membrane surface. Each red cellular responses. While several therapeutic
blood cell (RBC) contains
approximately 1 million copies approaches, most often with antioxidant char- Erythrocytes & oxidative stress
of anion exchanger 1. acteristics, aimed at reducing the impact of the The red blood cell (RBC) is exposed to oxidative
Protein phosphorylation: deleterious effects of ROS in metabolic diseases, damage. The functionality of RBCs is normally
Important event in malaria are used, the implementation of such strategies kept in check by a number of endogenous redox
infection. There are many more in the case of infectious diseases appear to be enzymes such as superoxide dismutases, catalases,
phosphorylated proteins in the much more complex. Indeed, the host cell, as well glutathione/glutathione peroxidases, glutathione
parasitized RBC than in the
uninfected RBC. The protein– as the pathogen, have signaling pathways and reductases (human [Hs]-GR), methemoglobin
NADPH-dependent redox controls that are interconnected, sharing (metHb) reductases and various oxido-reductases,
phosphorylation– substrates and co-substrates (glutathione [GSH], as well as antioxidants such as membrane-bound
dephosphorylation process is
glutathione disulphide, thioredoxin [Trx], Trx alpha-tocopherol and reduced GSH, which is
involved in building new
permeation pathways and disulphide [Trx(S)2], NADP and NADPH+) one of the major antioxidants in RBCs (2 mM).
channels between the RBC on homologous proteins where the two distinct Erythrocytes use the pentose phosphate pathway
membrane and the parasite for redox systems – the glutathione and the Trx redox to create NADPH, necessary for the formation of
the nutrient–waste import–
export traffic. The protein
systems – comprise a cascade of redox active pro- glutathione, because the other classical pathway,
phosphorylation– teins [9]. Quinones, NAD and NADH are also involving mitochondria, does not exist in RBCs.
dephosphorylation process substrate and co-susbtrates of Hs- and Pf-quinone Uncontrolled oxidative stress and depletion in
requires the NADP/NADPH oxido-reductases. Conventional strategies includ- GSH results in irreversible membrane injury
redox system as well as various
enzymes. These redox
ing target identification, drug design and phar- and cell death (hemolysis). The major source of
co-substrates and enzymes macomodulation are often ineffective because of ROS in RBCs is hemoglobin (Hb), which may
contribute to the redox this nesting. The complexity of malaria makes undergo autoxidation to produce O2·- and can
activation of redox antimalarials the discovery of targeted drugs difficult. lead to the formation of H2O2 and HO·. Since
drugs, which may disrupt the
redox homeostasis of the RBC To complete their development in the mos- the intra-erythrocyte concentration of oxygen-
and lead to premature lysis. quito, Plasmodium parasites have to pass through ated Hb is 5 mM, even a small amount of autox-
Plasmodium digests the midgut epithelium and avoid destruction by idation can produce substantial levels of ROS.
hemoglobin: Source of amino the mosquito immune system, steps that are also Denatured Hb byproducts, particularly free heme
acids for the synthesis of its own controlled by redox mechanisms. Though it is and hemichromes, can promote membrane dam-
proteins, which produces not the subject of this review, it is interesting to age and hemolysis through mechanisms includ-
reactive oxygen species and iron
byproducts. The ferrous and cite some recent results demonstrating the strong ing lipid peroxidation, oxidation of protein thiol
ferric ions potentiate redox sensitivity of P. falciparum to ROS and oxidative groups and oxidation of cytoskeleton proteins.
reactions with reactive oxygen stress in the mosquito. ROS are required for mos- The RBC is thus a special cell in its exposure and
species. quitos to get effective immune responses against sensitivity to oxidative stress. Given the sensitivity
bacteria and Plasmodium [10] with the example of the RBC to oxidative stress, the P. falciparum
of an Anopheles gambiae strain, refractory to erythrocyte stage is appropriate to strategies to
Plasmodium infection due to elevated ROS lev- target its redox mechanisms.
els [11]. Recently, Gonçalves et al. demonstrated
that mitochondrial ROS modulate mosquito Traffic in & out of the P. falciparum-
susceptibility to Plasmodium infection [12]. The infected erythrocyte & redox
A. gambiae mitochondrial carrier 1 is required reactions
to maintain mitochondrial membrane potential Developing inside RBCs, the parasite grows
in mosquito midgut. The authors investigated in a non-nucleated cell, devoid of intracellular

1994 Future Med. Chem. (2013) 5(16) future science group

 Targeting the redox metabolism of Plasmodium falciparum | Review
organelles, incapable of de novo synthesis and aggregates and enhances opsonization by anti-
not having any endocytic machinery. The para- band 3 antibodies: events that explain the
site must therefore create its own molecules and phagocytic removal of erythrocytes in thalas-
proteins, including new membranes, sub-cellular semia [27]. These reported studies illustrate that
compartments and new transporters needed for band 3 phosphorylation and oxidative events are
the import/export traffic of nutrients and waste connected in pRBC. Similar phosphorylation
products [13,14]. To assure the in and out traffic, of band 3 and membrane destabilization are
the parasite has developed a complex vesicular observed in G6PD-deficient erythrocytes that
system that extends into the RBC cytoplasm to exhibit resistance to parasites [28]. All these data
the plasma membrane. Membrane properties are suggest that redox activation of band 3 phos-
strongly dependent on oxidative events. Band 3, phorylation and its biological sequelae might be
the major integral protein of the erythrocyte a way to accelerate the disruption of the pRBC,
membrane, plays several important functions stopping the maturation of the parasite. This
in RBC metabolism and morphology for nor- constitutes, in the authors opinion, another way
mal anion exchange activity, cytoskeletal struc- to target the redox mechanisms of P.  falciparum
ture, cell shape and glycolytic activity. Oxida- to discover new antimalarial drugs.
tive events increase tyrosine phosphorylation of
band 3, which derives from both an oxidative The erythrocyte stage of P. falciparum
activation of Syk kinase [15–17] and an oxidative & the redox targets
inhibition of a major tyrosine phosphatase [18]. After a bite, sporozoites injected from the mos-
Protein phosphorylation is an important event quito salivary glands migrate to the liver to
in malaria infection. The P. falciparum infection mature into schizonts, which will subsequently
leads to a dramatic increase in the phosphory- release merozoites that infect RBCs and start
lation level of erythrocyte proteins: 170 P. fal- the intra-erythrocyte stage of the parasite. Ring
ciparum proteins and 77 human proteins have stage trophozoites mature to schizonts, and
been reported as phosphorylated proteins in the merozoites released from infected RBCs restart
parasitized RBC (pRBC), while only 48 human a new blood cycle every 48 h. Within eryth-
proteins were identified in the corresponding rocytes, the Plasmodium digests hemoglobin,
fractions from uninfected RBCs [19]. The pro- mainly in the parasite acidic digestive vacuole
tein phosphorylation–dephosphory­lation pro- (DV), as a source of amino acids for the syn-
cess requires the NADP/NADPH redox system, thesis of its own proteins, but also to induce
kinases and phosphatases, as well as enzymes a progressive reduction in the RBC volume to
that catalyze the formation of NADPH. prevent premature lysis [29]. This digestion pro-
Some of these protein phosphorylation– duces ROS byproducts, free heme and ferripro-
dephosphorylation partners may be putative toporphyrin (FPIX [Fe(III)]), a toxic byproduct
drug targets. The increase in band 3 tyrosine that is detoxified by the parasite via the forma-
phosphorylation leads to significant changes in tion of the malarial pigment hemozoin in the
the RBC membranes properties, altering both food vacuole [30], which is insoluble in biologi-
rates of glucose metabolism and multiple mem- cal conditions. Loria et al. have also suggested
brane protein interactions [20,21]. Band 3 tyrosine that (FPIX [Fe(III)]) can undergo a peroxida-
phosphorylation regulates the structural stability tive decomposition in the vacuole to form free
of the RBC membrane in decreasing its affinity iron [31]. A significant degradation of the heme
to ankyrin, inducing membrane blebbing and might also take place in the cytosol where glu-
vesiculation [22] and its hemorheological proper- tathione participates in the biotransformation
ties [23]. Hemoglobinopathies have been demon- of nonpolymerized heme through a cascade of
strated to be accompanied by increased band 3 redox reactions as demonstrated by Ginsburg’s
tyrosine phosphorylation [24,25]. Sepsis is also team [32,33].
accompanied by increased band 3 tyrosine phos- The autoxidation of Hb (Fe(II)) occurs spon-
phorylation and alterations in band 3 membrane taneously in normal RBCs. MetHb can also
organization [26]. It has been reported in the be produced by oxidants and ROS. In normal
thalassemic RBC model, which is also a model conditions the reaction is reversible and produc-
to understand the disruption of the pRBC, that tion of metHb is in balance with the conversion
hemichrome located in the membrane binds of metHb back to Hb. In pathological condi-
to band 3, mediates oxidation of band-3 cyto- tions, increased oxidative stress and/or impaired
plasmic domains, generates high-MW band 3 antioxidant defense enhance the production of

future science group www.future-science.com 1995

Review | Nepveu & Turrini

Key Term metHb, the generation of ROS and consequently n Targeting specific parasite components, heme
Redox compound: Can
the generation of unstable Hb and irreversible and ROS byproducts for increasing oxidative
undergo reversible redox Hb oxidation products, giving hemichrome stress;
reactions on at least one of its forms. Hemichromes release their heme into
redox moieties. An oxidant
n Introducing a redox compound into the redox
the erythrocyte membrane with consequent
agent may present any cycles of the host cell and/or of the parasite,
reversibility in its reduction
lipid peroxidation, biomolecule oxidation and
perturbing the redox defenses of the parasite
reaction. Some drugs may target membrane destabilization [34]. A chain of events
and/or disrupting the fragile architecture of
a redox protein or enzyme and is induced by the generation of hemichromes
also be redox cyclers, which is the infected RBC (Figure 1).
during the intra-erythrocyte parasite develop-
the case of atovaquone.
ment [35] that finally determine band 3 phos-
phorylation and clustering observed in infected Targeting a redox protein of
erythrocytes [36]. It has been previously observed P. falciparum
that hemichromes binding to the membrane are Since the sequencing of the genome of P. falci-
capable of quite specifically oxidizing two Cys parum in 2002 [41], knowledge of the redox tar-
residues located in the cytoplasmic domain of gets in the parasite has grown. Many studies in
band 3 [27]. It has been subsequently demon- this area, and the recent report of Kehr et al. [39],
strated that the oxidation of the two Cys residues have enabled a representation of the subcellular
allows the phosphorylation of band 3 through compartmentalization of the major redox-related
Syk [17] and that band 3 phosphorylation plays a proteins in P. falciparum. The redox protein and
permissive role in its clustering [22]. enzyme targeting has already produced promising
The probability of iron-catalyzed redox reac- pharmacophores and results (Figure 1).
tions is then strongly increased in parasitized P. falciparum seems to maintain an active
RBCs, with the different iron species issued mitochondrial electron transfer chain to serve
from Hb degradation (metHb, hemichrome just one metabolic function: regeneration of ubi-
aggregates, iron at the crystal hemozoin surface, quinone required as the electron acceptor for Pf-
free heme, free iron) and ROS produced. In con- DHODH. This protein, validated as a new drug
clusion, free iron, heme byproducts and pulses of target, is a flavin mononucleotide-dependent
ROS may be encountered in the DV and cyto- mitochondrial enzyme that catalyzes the oxida-
sol of the parasite, and in membranes leading tion of dihydroorotate to produce orotate, the
to ROS byproducts (oxidized biomolecules). fourth step in de novo pyrimidine biosynthesis
P. falciparum is highly adapted to cope with [42]. Other targets are located in the mitochon-
these oxidative species and redox reactions, and drion and in the cytosol: Pf-NDH2; G3PDH;
is endowed with a complete network of enzymes MQO; SDH; the cytochrome bc1 complex, cyto-
and proteins controlling the redox status of its chrome c, cytochrome c oxidase; and the F1F0-
environment [37–40]. Central to this antioxidant ATPase (complex V). The presence of enzymes
battery are the two major NADPH-dependent in P. falciparum mitochondrion that are absent
redox systems with the complete glutathione in mammalian cells, such as the MQO and the
system and the complete Trx system. The gluta- P. falciparum-NDH2, provides interesting tar-
thione system also includes the two enzymes that gets for drug design [43,44]. Targeting the P. fal-
mediate the de novo synthesis of GSH, g-GCS and ciparum mitochondrion is a proven drug target
GS. In addition to these antioxidant systems, the as demonstrated with the drug atovaquone [45], a
parasite also has superoxide dismutases, various 1,4 naphtoquinone inhibiting the bc1 complex,
oxido-reductases, kinases and dehydrogenases. which has been clinically successful, especially in
Redox targets essential for parasite survival are combination with proguanil (Malarone®), for the
also localized in the mitochondrion. Considering treatment of chloroquine-resistant infections [46].
the intensive nested redox reactions occurring in The P. falciparum mitochondrion is behind the
infected RBCs, targeting the redox metabolism discovery of new active entities in the last couple
of P. falciparum appears feasible to identify new of years giving major results. Potent antimalarial
drugs (although the notion that molecules may inhibitors of the P. falciparum bc1 complex have
have multiple biological targets should always be been obtained with 4(1H)-pyridones [47] and ami-
considered and this might be of advantage with nomethyl-3,5-di-tert-butylphenol, which is in pre-
regard to the development of resistance): clinical stage drug trials [48]. Factors influencing
the specificity of inhibitor binding to the human
n Targeting and inhibiting a redox-specific and Pf-DHODH have recently been reported
P. falciparum protein or enzyme; [49] and Pf-DHODH inhibitors with excellent

1996 Future Med. Chem. (2013) 5(16) future science group

 Targeting the redox metabolism of Plasmodium falciparum | Review

Red blood cell architecture

and metabolism
– Protein phosphorylation
– Junctionnal complexes
– HsGR
– Oxido-reductases
– QR2 (NR/NRH) Pf-redox proteins
– Redox cofactors (NAD/NADH) – PfGR
– Glutathion – PFTrxR
– Hemichrome – PFDHODH
Redox cycling drugs, – PFNDH2
bioreducible drugs, ligands – bc1 complex
prodrugs Selective inhibitors
Chloroquine, primaquine Atovaquone
Heme by product DSM265; P218;
Methylene blue
Naphthoquinones MK-4815; 4(1H)-pyridones;
Indolone-N-oxides quinolones; chalcones

Proteins

Junction
protein

DV
N bc1
M

Parasite
Specific parasite components
– Heme and heme by-products; iron
Key – PfGR
Targets – Redox cofactors (NAH/NADH)
Drug types – Glutathione system
Drugs Oxidant drugs, redox cycling drugs, iron ligands
Candidates and new leads Aminoquinolines, artemisinines
M: Mitochondria Naphtoquinones, quinolines, peroxides and ferroquines
DV: Digestive vacuole 1,2,4,5-tetraoxane (RKA 182); trioxolanes (FBEG100
N: Nucleus and OZ439)

Future Med. Chem. © Future Science Group (2013)

Figure 1. Redox targets in Plasmodium falciparum-infected red blood cells.

Pf: Plasmodium falciparum.

safety profiles are in preclinical trials [203]. New major redox systems, Pf-GR and the P. falci-
heterocyclic quinolones [50] with potent antima- parum-Trx receptor may not be the provision of
larial properties have the capacity to inhibit both NADPH for reductive reactions [55]. Identifica-
Pf-NDH2 and the bc1 complex. Chalcones from tion of inhibitors for putative malaria drug tar-
natural and synthetic sources present antimalarial gets among novel antimalarial compounds did
potential and are thought to act against malarial not confirm hits against GDHa [56]. These data
cysteine proteases [51] or cyclins [52,53]. Some cause imply that GDHa is not a suitable drug target.
a loss of the mitochondrial membrane potential The search for new molecules with atovaquone-
[54], which may be connected with their quinoid like structures has generated various naphthoqui-
structure suggesting that they could enter redox none series and the most recent results illustrate
cycles activated by oxido-reductases. that many of them are redox cyclers and modula-
Recent studies have demonstrated that the tors of P. falciparum-GR, P. falciparum-TrxR and
GDHa, which was hypothesized as one of the also of Hs-GR with potent antimalarial properties
major sources of NADPH required by the two [57,58]. It is proposed that atovaquone has no effect

future science group www.future-science.com 1997

Review | Nepveu & Turrini
on these two disulfide reductases due to its too under the specific conditions, acidic and oxidiz-
high reduction potential [57], this physicochemi- ing, of the parasite digestive vacuole [69]. Forma-
cal observation is relevant to discriminate bc1 tion of the ferriquinium salt is accompanied by the
complex inhibitors from GR and TrxR inhibi- production of hydroxyl radicals. Second, a series
tors. In recent years, nitroaromatic compounds of FQ conjugates with either glutathione reductase
and organic nitrates were demonstrated as potent inhibitors or glutathione depletors via a hydrolyz-
GR inhibitors [59,60]. Screening efforts at Glaxo- able amide linker have been built to target differ-
SmithKline and Novartis (Basel, Switzerland) ent pathways and compare CQ and FQ in their
on different putative targets, including the redox antimalarial properties [70]. The results evidenced
enzymes TrxR and GR, confirmed antimalarial a unique redox mode of action of FQ and ferroce-
hits with no clear evidence that the antimalarial nyl analogs distinct of those of CQ and nonfer-
activity of those compounds could be attributed rocenic 4-aminoquinoline analogs. Recent stud-
to the inhibition of these targets [4–6,61]. ies on FQ derivatives report cross-susceptibility
with quinolone-based drugs on fresh isolates [71].
Oxidant drugs targeted to specific Moreover, these studies have revealed that certain
parasite components side chains, for example, the g-lactam unit, pos-
The ROS and iron byproducts present in the sess promising antimalarial activities, thus open-
pRBCs, which are rare in healthy RBCs, remain ing the field to the discovery of new antimalarial
the typical pRBC targets. Aminoquinolines such pharmacophores for the future [63].
as chloroquine (CQ) and ARTs are thought to ART derivatives are currently the drugs of
target these redox-active heme byproducts gen- choice for the treatment of malaria due to the
erating an oxidative stress fatal to the parasite. multidrug-resistant P. falciparum [72]. The asexual
Considering that pRBC are selectively damaged blood stage is the primary target of ARTs. Semi-
by oxidants, the interest for new oxidant drugs synthetic and synthetic endoperoxides have
with antimalarial properties began more than been developed over the past 20 years. In 2004,
30 years ago [62]. At this point, it is important to Vennerstrom et al. designed stable antimalarial
clarify that there are two types of oxidants, some trioxolanes [73]. OZ277 (Figure 2), also known as
are intrinsically oxidizing (e.g., quinones and arterolane maleate, was the first synthetic ozon-
quinoid compounds, peroxides), while others do ide to be evaluated clinically. Arterolane maleate
not have apparent redox properties but indirectly has completed Phase III clinical trials [203] and
induce oxidation reactions by their mechanisms is now on the market in India as a combination
of action (e.g., aminoquinolines). In this view, product with piperaquine phosphate (Synriam®).
artemisinin-like endoperoxides are oxidant while However, when administered to malaria patients
the oxidative stress generated by chloroquine is as monotherapy, OZ277 displayed a reduced half-
more a downstream event arising from its strong life compared with the one observed in healthy
interaction with heme. volunteers, which was only two- to threefold lon-
A current strategy to overcome the resistance of ger than that of dihydroartemisinin, the active
the parasite to chloroquine and to 4-aminoquino- metabolite of ARTs. Vennerstrom and coworkers
lines is to develop analogs, notably by alteration of successfully designed a new series of ozonide with
the CQ side chain [63–67]. Ferroquines (FQ) are an a longer half-life and better blood exposure pro-
ingenious molecule; built to target heme byprod- file. They identified a new drug candidate with
ucts and at the same time act as a Trojan horse, exceptional pharmacokinetic and antimalarial
bringing the enemy inside, the redox iron. Inves- properties, the drug candidate OZ439 (Figure 2)
tigations to understand the differences of activity [74], which is currently undergoing Phase IIa tri-
between CQ and FQ have been carried out [68] als in malaria patients [203]. OZ439 holds a great
and a multifactorial mechanism of action has been deal of promise for malaria therapy as it has the
proposed by its capacity to targets lipids, to inhibit potential for a one-dose cure and a treatment for
the formation of hemozoin and to generate ROS. artemisinin-resistant strains. O’Neill and cowork-
Two experimental approaches have been decisive ers identified a water-soluble 1,2,4,5-tetraoxane
in understanding this mechanism. First, the real drug candidate, RKA 182 (Figure 2), which has
contribution of the redox properties of the ferro- outstanding antimalarial activity, stability, low
cene in FQ has been considered through experi- toxicity and adequate dimethyl ether properties
mental and theoretical approaches. Unlike CQ, (distribution, metabolism and excretion) as a
FQ demonstrates a reversible one-electron redox promising drug candidate that is now under pre-
reaction (iron (II)/ferricinium/iron (III) system) clinical studies [75,203]. This work establishes the

1998 Future Med. Chem. (2013) 5(16) future science group

 Targeting the redox metabolism of Plasmodium falciparum | Review

O
O O O O
O N
O O
NH NH2
O

Trioxolane (OZ277) Trioxolane (OZ439)

N
N

O O N

O O O

Tetraoxane (RKA 182)

Figure 2. Peroxide derivatives as antimalarials on the market (OZ277 in combination with

piperaquine phosphate [73] ) or as drug candidates in preclinical and clinical studies (OZ439
[74] ; RKA 182 [75] ).

potential of the tetraoxane as a new antimalarial regenerated by two NADPH-dependent disul-

pharmacophore. fide oxido-reductases (GR and TrxR). Among
Due to the necessity of artemisinin-based the most effective ligands of these enzymes, the
combination therapies to fight resistance, hybrid most potent antiplasmodial effects are observed
drug species combining artemisinin and quinoline for redox cyclers acting as subversive substrates.
skeletons have been built [72]. The combination The first redox cycler reported is methylene blue
of artemisinin derivatives with a second drug (Figure 3), which has been part of the malaria
having a different mode of action was proposed pharmacochemistry arsenal for more than a cen-
to increase efficacy and prevent drug resistance. tury. Guttman and Ehrlich reported the first trial
New chimeric molecules combining a potentially of methylene blue in 1891 for the cure of malaria
alkylating trioxane moiety to a 4-aminoquinoline [80], which was the first synthetic antimalarial
group were designed to give the trioxaquine series drug used in human medicine at the beginning
[76] with the PA1103/SAR116242 that progressed of the 20th century, and then abandoned due
until preclinical studies [77,203]. However, there are to the reversible side effects of turning urine
some limits in these hybrid molecular approaches green and sclera (white parts of the eyes) blue,
given, first, the difficulty to assess the benefit or perceived as toxic effects by the patients, and
the risk (toxicity) of this hybridization and, sec-
ond, the difficulty for a single molecule to interact
with two targets at the same time when they are
-
not really in close proximity. An example is the +
CI
N S N
hybrid molecule combining ART and FQ, the tri-
oxaferroquine, for which high doses are necessary
N
to cure in vivo mice infected with Plasmodium
vinckei petteri [78]. The current trend is to develop Methylene blue (MB)

fragmenting hybrid molecules using biotrans- Atovaquone

formable linkers. The hybrid molecule becomes
the prodrug of the two entities after fragmenta-
tion, as recently reviewed by Renslo et al. [79].
O
An advantage of this strategy is the possibility to O
mask a partner drug’s intrinsic bioactivity until it CI
reaches the parasite inside the RBC. O N+
O-

Redox cycler drugs targeting the 1,4-napthtoquinone (benzylNQ) Indolone-N-oxides (INOD-1)

parasitized RBC
P. falciparum prevents oxidative damage
Figure 3. Redox-active antimalarial compounds.
by a cytosolic antioxidant thiol pool that is

future science group www.future-science.com 1999

Review | Nepveu & Turrini
also the launch of CQ (in the 1940s). Observing continuous redox cycle. Studies on a fluorinated
redox cycling between methylene blue, NADPH suicide substrate indicate that the GR-catalyzed
and metHb(Fe(III)) [81], and understanding bioactivation of naphtoquinones is essential for
the G6PD and GR deficiencies were the start- the observed antimalarial activity. In conclusion,
ing points to designing new redox cyclers with the antimalarial naphtoquinones are believed to
antimalarial properties [82]. 1,4-naphtoquinones perturb the major redox equilibria of the tar-
(Figure  3), are redox cyclers with antimalarial geted infected RBC, which results in develop-
properties [83]. These compounds are reduced ment arrest and death of the malaria parasite at
via one or two electron transfer reactions and the trophozoite stage [85–87].
regenerated in the presence of major oxidants Indolone-N-oxide derivatives (INODs;
in the pRBC. Recently, Davioud-Charvet et al. F igure  3; INOD-1) exhibit a potent activity
have developed new naphtoquinones with potent against the blood stage of P. falciparum [88] and
antimalarial properties and used an interdisci- do not present hemolytic effects on healthy
plinary approach to understand the antima- RBCs. The P. falciparum ring stage has been
larial mode of action of the 1,4-naphtoqui- found to be more vulnerable to INODs than the
nones (Figure 3; benzylNQ) [84]. It is proposed late trophozoite stage [89]. The main structural
(Figure 4) that active napthoquinones are taken feature of INODs is the redox heterocyclic core
up by the pRBC to be reduced by the Hs-GR (redox pharmaco­phore), in which the nitrone
in the RBC cytosol and then transported in the moiety is conjugated to the ketone function
DV. They are then cycled between the acidic giving the molecule the capability to undergo
compartments and the cytosols and reduced several reversible oxidation–reduction reactions
by the Hs-GR or by the P. falciparum-GR in a [90]. The INODs IC50 values are dependent on

Red blood cell

6-phosphogluconolactone

Glucose-6-phosphate Plasmodium falciparum

GSSG G6PDH
Drugox
HMS FPIX
degradation
NADPH FPIX(Fe )
II

NADP+ GSH HO
NADPH O2
NADP+ Fenton
GSSG SOD
Drugox HsGR O2 - FeIII
GSH
PfGR FPIX(FeII) H2O2
Drugox
Drugred
Drugred

Hb metHb Proteases
(FeII) (FeIII) FPIX
Amino (FeIII)
acids
O2
Hemoglobin O2 - Hemozoin
Food
H2O2 vacuole

Future Med. Chem. © Future Science Group (2013)

Figure 4. The model for lead 1,4-naphtoquinone (benzylNQ) bioactivation affecting the redox homeostasis of the
Plasmodium falciparum-infected red blood cell.

2000 Future Med. Chem. (2013) 5(16) future science group

 Targeting the redox metabolism of Plasmodium falciparum | Review
1
INODox
2
INODred 4
Several reduced
and oxidized forms, Activation of
including radical Syk kinase
intermediates 5
3
Redoxcyling: Band 3
oxidoreductases, phosphorylation
redox co-factors, and clustering
reducers ? 6

Membrane
destabilization
and vesiculation
Parasite

Stop of parasite 7
DV growth

Future Med. Chem. © Future Science Group (2013)

Figure 5. Antimalarial indolone-N-oxide derivatives (1), redox bioactivation (2,3) in red

blood cells, and cascade events (4–7) occurring only in parasitized red blood cells [88–93] .
DV: Digestive vacuole.

their reduction potential [88]. The electron trans- n Accelerated formation of membrane aggregates
fer recorded during the first reduction process containing hyperphosphorylated band 3, Syk
corresponds to the reduction of the N-oxide kinase and hemichromes;
to a nitroxide radical-anion [91]. The redox
homeostasis of the infected RBC is affected n Dose-dependent release of microvesicles
by the INODs redox cycling properties via a containing membrane aggregates;
cascade of reactions, not yet fully elucidated.
The bioactivation of these compounds in RBCs
gives an active reduced metabolite (dihydro- A
INODs), which has been isolated and identified
in healthy and P. falciparum-infected RBCs [92].
B
The bio­reduction is thiol- and enzyme-depen-
Phosphorylated band 3

dent (Figure  5; steps 1–3). This bioreduction

(arbitrary units)

process activates a Syk kinase (Figure 5; step 4)

which results in a dramatic rise in band 3 tyro-
sine phosphorylation in pRBCs (Figure 5; step
5) and has been identified as the major cova-
lent modification induced by INODs [93]. This
triggers complete dissociation of ankyrin from
band 3 leading to membrane vesiculation and C
loss of membrane surface area until the explo-
sion of the P. falciparum-infected RBCs and
parasite death (Figure 5; steps 6 and 7) [22,93]. <100 100 < IC < 500 > 500
The correlation between the IC50 of INODs IC50 (nM)
and the induction of band 3 phosphorylation is
illustrated in Figure 6. These results were obtained Figure 6. Relation between band 3
by a proteomics approach by treating pRBCs at phosphorylation and IC50 of indolone-N-
early parasite maturation stages with INODs at oxide derivatives against
their IC50 concentrations, whereas no effect was Plasmodium falciparum. (A) seven
observed in controlled RBCs. The following was compounds; (B) ten compounds; and
(C) five compounds [88,93] .
also observed:

future science group www.future-science.com 2001

Review | Nepveu & Turrini
n A dose-dependent increase in band 3 phosphory­ to the reduced enzyme and that primaquine
lation and a parallel increase in vesicle release in binds exclusively to the oxidized enzyme [98].
a concentration range corresponding to the IC50; Because QR2 accepts modulators of different
types, quinones and pseudo-quinones, this
n Reduction in band 3 phosphorylation, P. falci-
human enzyme may contribute to increased
parum-infected RBC vesiculation, and anti­
oxidative stress in pRBCs. Recently, Leung
malarial effects of INODs upon addition of Syk
et al. demonstrated that QR2 functions as a
Kinases inhibitors.
chloroquine-dependent f lavin redox switch,
These results suggest that INODs cause a which could explain off-target effects of chlo-
strong destabilization of the P. falciparum-infected roquine and other bioactive molecules [99].
RBC membrane through a mechanism apparently The cellular source of the reduced form of
triggered by the activation of a redox signaling the QR2-coenzyme, NRH, is not known, but
pathway rather than by direct oxidative damage. what is known is that NR is involved in NAD
INODs inhibit parasite maturation at the metabolism [100].
ring stage as do artemisinine derivatives. Late
trophozoites and schizonts are less susceptible Future perspective
to the INODs antimalarial action [89]. In vitro, Targeting the redox metabolism of P. falciparum
ARTs have been demonstrated to select P. falci- has produced new antimalarial drug candidates in
parum clones with increased antioxidant response recent years. Consequent to the genome sequence
[94], while this effect has not been observed with of the human malaria parasite in the 2000s, tar-
INODs. In conclusion, INODs interfere with the geting P. falciparum redox proteins became pos-
regulatory mechanisms of the host cell membrane sible with computational approaches and has
without affecting nonparasitized erythrocytes, produced drug candidates that are now in clini-
which constitutes a new mechanism of action. cal phases. Empiric and phenotypic approaches
Taking the pRBC as a redox target, it targeting the specific parasite components such
becomes interesting to mention quinone reduc- as the ROS and heme byproducts generated sev-
tase-2 (NQO22; QR2) as a protein involved in eral original scaffolds and hybrid molecules with
redox cycling. QR2, a cytolosic enzyme, has a excellent leads. The authors believe that the whole
key role in various pathological situations [95] parasitized RBC is also a redox target. We suggest
and is now becoming a target for several thera- that band 3 tyrosine phosphory­lation resulting
peutic agents. QR2 catalyzes the two-electron from the interaction between the oxidative stress
reduction of menadione (and quinones) via exerted by the intracellular parasites and some
the oxidation of the co-enzyme, the nicotin- reversible redox drugs lead to premature lysis of
amide roboside (NR). QR2 prevents oxidative the cell, which is fatal to Plasmodium. Multiple
stress in RBCs by reducing the intracellular nested redox targets co-exist between the host cell
concentration of quinones and decreasing and the parasite, redox enzymes and coenzymes,
the concentration of semiquinones, and their antioxidants and ROS, the combination of which
subsequent contributions to the generation of may recycle reversible redox molecules until RBC
ROS. Consequently, quinones that are physi- breakdown. Thus, there are possibilities for the
ological substrates of QR2 may contribute to future in the development of these redox recyclers
oxidative stress in RBCs by different mecha- to build potent antimalarials.
nisms [96]: direct oxidation of thiols (glutathi-
one); generation of metHb by direct oxidation Acknowledgements
of oxyhemoglobin; and formation of semiqui- The authors thank THY Nguyen and E Najahi for
nones that auto-oxidize to form ROS such as assistance with the figures.
superoxide. Graves et al. demonstrated that
QR2 (and not QR1) is present in human RBC Financial & competing interests disclosure
and that quinolones can inhibit QR2 activity The authors have no relevant affiliations or financial involve-
[96]. The authors recently demonstrated that ment with any organization or entity with a financial interest
the recognition of many different substrates as in or financial conflict with the subject matter or materials
modulators by QR2 may explain its capacity to discussed in the manuscript. This includes employment, con-
propagate the toxicity of quinones or pseudo- sultancies, honoraria, stock ownership or options, expert
quinones through the production of ROS [97]. t­estimony, grants or patents received or pending, or royalties.
Kwiek et al. reported that the QR2 inhibitors No writing assistance was utilized in the production of
quinacrine and chloroquine bind exclusively this manuscript.

2002 Future Med. Chem. (2013) 5(16) future science group

 Targeting the redox metabolism of Plasmodium falciparum | Review

Executive summary
The impact of malaria & the resistance to artemisinin
„„The situation for the control and therapy of malaria is still alarming considering the multidrug-resistant Plasmodium falciparum. The
resistance to artemisinins has spread to new locations in Asia. Innovative concepts and new modes of action of antimalarial agents
are needed to develop new drugs.
Targeting the redox metabolism
„„Given the role of reactive oxygen species (ROS) and redox mechanisms in various diseases, therapeutic strategies have been
developed to modulate the redox-activated cellular responses.
Erythrocytes & oxidative stress
„„Given the sensitivity of the red blood cells (RBCs) to oxidative stress, the P. falciparum erythrocytic stage is appropriate to strategies
targeting its redox mechanisms.
Traffic in & out of the P. falciparum-infected erythrocyte & redox reactions
„„ The parasite creates its own molecules and proteins, including new membranes, sub-cellular compartments and new transporters
needed for the import/export traffic of nutrients and waste products.
„„ P. falciparum infection leads to a dramatic increase in the phosphorylation level of erythrocyte proteins.
„„ The protein phosphorylation–dephosphorylation process requires the NADP/NADPH redox system, kinases and phosphatases, as well
as enzymes that catalyze the formation of NAD.
„„Phosphorylation of RBC proteins and oxidative events are connected in parasitized RBCs.
The erythrocyte stage of P. falciparum & the redox targets
„„Considering this locus of intensive nested redox reactions in an infected RBC, attempts to define drugs targeting the redox
metabolism of P. falciparum may be proposed:
- Targeting and inhibiting a redox-specific P. falciparum protein or enzyme;
- Targeting specific parasite components, heme and ROS byproducts for increasing oxidative stress;
- Introducing a redox agent into the redox cycles of the host cell and/or of the parasite perturbing the redox defenses of the
parasite and/or disrupting the fragile architecture of the infected RBC.
Targeting a redox protein of P. falciparum
Redox protein and enzyme targeting has already produced promising pharmacophores.
„„

Oxidant drugs targeted to specific parasite components

„„The ROS and iron byproducts present in the parasitized RBC remain the typical parasitized RBC target. Aminoquinolines and
artemisinins are thought to target these redox-active heme and ROS byproducts generating an oxidative stress fatal to the parasite.
Many new molecules have been developed in this field.
Redox cycler drugs targeting the parasitized RBC
„„ Multiple nested redox targets co-exist between the host cell and the parasite, redox enzymes and coenzymes, and antioxidants and
ROS, the combination of which may recycle reversible redox molecules until the RBCs break down. Thus, there are perspectives for
the future in the development of these redox recyclers to build potent antimalarials.
„„ Among the most effective ligands of the oxido-reductases (GR, TrxR, QR2), the most potent antiplasmodial effects are observed for
redox cyclers acting as subversive substrates (quinones [naphtoquinones] and pseudo-quinones [indolone-N-oxides]).

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