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Novel antimalarial drug

targets: hope for new
antimalarial drugs
Expert Rev. Clin. Pharmacol. 2(5), 469–489 (2009)

Athar Alam, Manish
Goyal, Mohd Shameel
Iqbal, Chinmay Pal,
Sumanta Dey, Samik
Bindu, Pallab Maity
and Uday
Bandyopadhyay†
†
Author for correspondence
Division of Infectious Diseases
and Immunology, Indian
Institute of Chemical Biology, 4,
Raja SC Mullick Road, Jadavpur,
Kolkata-700032,
West Bengal, India
Tel.: +91 332 473 3491
Fax: +91 332 473 5197
ubandyo_1964@yahoo.com
Malaria is a major global threat, that results in more than 2 million deaths each year. The
treatment of malaria is becoming extremely difficult due to the emergence of drug-resistant
parasites, the absence of an effective vaccine, and the spread of insecticide-resistant vectors.
Thus, malarial therapy needs new chemotherapeutic approaches leading to the search for new
drug targets. Here, we discuss different approaches to identifying novel antimalarial drug
targets. We have also given due attention to the existing validated targets with a view to develop
novel, rationally designed lead molecules. Some of the important parasite proteins are claimed
to be the targets; however, further in vitro or in vivo structure–function studies of such proteins
are crucial to validate these proteins as suitable targets. The interactome analysis among
apicoplast, mitochondrion and genomic DNA will also be useful in identifying vital pathways
or proteins regulating critical pathways for parasite growth and survival, and could be attractive
targets. Molecules responsible for parasite invasion to host erythrocytes and ion channels of
infected erythrocytes, essential for intra-erythrocyte survival and stage progression of parasites
are also becoming attractive targets. This review will discuss and highlight the current
understanding regarding the potential antimalarial drug targets, which could be utilized to
develop novel antimalarials.
Keywords : antimalarials • apicoplast • drug resistance • drug target • food vacuole • malaria • mitochondrion
• Plasmodium falciparum • target identification

Malaria is a major cause of morbidity and mortal-
ity across the globe [1] . The increasingly serious
problem of malaria parasites to currently used
antimalarials such as chloroquine and sulfadox-
ine/pyrimethamine [2] has led to an urgent need
to develop new drugs against existing validated
targets, as well as to search for new targets as an
effective vaccine is not readily available. Several
enzymes, channels, transporters, interacting mol-
ecules on the parasites for red blood cell (RBC)
invasion and molecules responsible for oxidative
stress in the human malaria parasite Plasmodium
falciparum have been identified and suggested as
potential drug targets [3–5] . Although a few have
been validated [6] , several still need to be validated
to establish a specific antimalarial target. Many
conventional approaches to discover new therapies
against malaria include optimization of current
drug treatments and formulations, developing ana-
logs of existing drugs, testing of compounds from
natural products, identifying resistance-reversal
agents, exploiting combination chemotherapeutic
approaches and utilizing drugs indicated for other
uses [7] are in use and discovering new antima-
larial drug against malarial parasites. While these
approaches have been successful in producing
clinically useful drugs, systematic approaches are
warranted in order to discover new antimalarial
drugs against rapidly mutating malarial para-
sites. Several modern computer- and assay-based
approaches including evolutionary patterning [8] ,
functional genomics [9] , system biology [10] , gene
network [11], interactome studies [12] and structure-
based drug design [13] should help in the search for
new targets. The potential drug targets can finally
be validated by using chemical or genetic methods,
for example gene-expression profiling following
drug treatment, genetic knockout techniques and
by structure–function analysis. In this review, we
will focus principally on well known and newly
discovered potential antimalarial drug targets.
Existing targets & theirs limitations
The advantages of the major metabolic differ-
ences of Plasmodium with its host have been
considered in the design of antimalarial drugs.

www.expert-reviews.com 10.1586/ECP.09.28 © 2009 Expert Reviews Ltd ISSN 1751-2433 469

 Review Alam, Goyal, Iqbal et al.
Major pathways of P. falciparum, such as nucleic acid metabolism,
heme detoxification, oxidative stress and fatty acid biosynthesis,
are known targets for antimalarial design. Most of the antima-
larial drugs available currently have been in use for decades, but
their use is now limited by the emergence and spread of drug resis-
tance. Analysis of antimalarial-resistant Plasmodium by genetic,
molecular and pharmacological approaches has shown that dif-
ferent targets are resistant due to mutations on their key enzymes
or transporters. Chloroquine resistance is caused by mutations of
the Pfmdr1 [14] , Pfcg2 [15] and Pfcrt [16] genes. Atovaquone resis-
tance is associated with mutations at positions 133, 144 and 248
of cytochrome b [17] . Resistance to antifolates (pyrimethamine
and proguanil) is caused by point mutations at positions 51, 59,
108 and 164 of dihydrofolate reductase (DHFR). Sulfonamides
and sulfones become resistant owing to mutations at 436, 437,
540, 581 and 613 of dihydropteroate synthase (DHPS) [18] .
Artemisinin and their derivatives, the safest treatment against
multidrug-resistant P. falciparum malaria is also not an exception
to drug resistance. Artemisinins interact and selectively inhibit
PfATPase6, the only SERCA-type Ca 2+ -ATPase in the P. falci-
parum genome. Malaria parasites become resistant to artemisnins
due to the mutation of PfATPase6 at S769N, A623E and E431K
[19] . An in vitro study in French Guyana documented that P. fal-
ciparum with higher IC50 values for artemisinins were associated
with point mutations at codon S769N of the ATPase6 locus [19] .
Thus, drug resistance resulting from mutations is a major concern
and the identification of new targets is mandatory to design new
drugs against resistant malarial parasites.
Approaches to identify new antimalarial drug targets
The advent of genomics and proteomics including microarray
analyses of gene expression, comparative proteomic analyses, bio-
informatics, evolutionary patterning approaches, system biology
approaches, interactome studies and gene network approaches
have made possible a whole synergistic approach to identifying
novel drug targets. We will discuss some of the unique approaches
in brief to identify antimalarial drug targets.
Evolutionary patterning (EP) is a novel approach utilized to iden-
tify suitable drug target sites that would minimize the emergence
of parasite resistance. This technique identifies amino acids that
are under the most extreme evolutionary constraints [8] . These sites
are predicted to be the most appropriate drug targets since they are
structurally and functionally essential and are least likely to undergo
possible mutations, thus limiting resistance and escalating the lifes-
pan of a drug. Like EP, system biology and gene network approaches
are also among the most advanced approaches that use bioinformat-
ics to identify putative drug targets. Systems biology is a science that
combines numerous biological data streams to enable an insightful
understanding of interdependent biological processes [10] . All the
‘omics (e.g., genomics, transcriptomics, proteomics, interactomics,
metabolomics, cellomics) datasets can be integrated by system biol-
ogy into an interconnected network in order to probe deeply to
understand the mechanisms of disease or drug effects. Similar to sys-
tem biology, the gene network approach is a network-based strategy
for drug target identification. It attempts to reconstruct endogenous
470
metabolic, regulatory and signaling networks with which potential
drug targets interact. Once this information is provided by gene
networks or protein networks, the interaction relationships between
potential drug targets could be unambiguously revealed, so it could
be easily determined which one of these potential drug targets is
most suitable [20,21] . This technique holds the promise of providing
a conceptual framework for theanalysis of the profusion of biologi-
cal data being generated on potential drug targets and providing
insights to understand the biological regulatory mechanisms in dis-
eases, which are playing an increasingly important role in searching
for novel drug targets from the information contained in genomics
[10] . The protein interaction network (PIN) strategy for drug target
identification helps to rapidly interpretate results of hundreds to
thousands of protein expression changes in model systems following
perturbation by drug and protein–protein interactions. These pro-
vide insights into the function of important genes, elucidate relevant
pathways and facilitate the identification of potential drug targets.
Another computer based approach to identify putative antimalarial
drug targets is interactome study. Interactome deals with whole set
of molecules in cells and usually displays as a directed graph of the
interaction between the molecules. Molecular interactions can occur
between molecules within a given family called protein–protein
interaction network (PPI) or PIN, refers proteomics interactome
or belonging to different biochemical families (such as, proteins,
nucleic acids, lipids, carbohydrates, etc,) called the protein–DNA
interactome (network formed by transcription factors and DNA
or chromatin regulatory proteins). Signal transduction pathways
and transcriptional regulation are typical examples of such bio-
logical processes mediated by protein–protein interaction, and the
MI database [301] could be a good source to identify the putative
target that is designed to collect experimentally verified PPIs in a
binary or complex representation [22] . Besides this computer-based
approach, forward genetic approaches, reverse genetic approaches
and cell lysate-based assays are very useful to identify antimalarial
drug targets. Forward genetics approaches are ‘phenotype-driven’
approaches aimed at identifying the genetic basis of phenotypes
that are of biological interest. Genetic complementation is good
example of this approach, which greatly enhances the functional
analysis of the gene [23] . Reverse genetics is an approach to discover
the function of a gene that proceeds in the opposite direction of
so-called forward genetic screens of classical genetics and it seeks
to find possible phenotypes that may derive from a specific genetic
sequence obtained by DNA sequencing. Reverse genetics attempts
to connect a given genetic sequence with specific effects on the
organism. There are several different methods of reverse genetics
that have proved useful in the search for novel targets; it includes
random deletions, insertions point mutations, directed deletion (by
site-directed mutagenesis) [24,25] , and interference using transgenes.
Another strong technique for this approach is RNA interference,
but unfortunately it could not be applied in the case of malaria
because of the lack of enzyme Dicer and RNA-induced silencing
complex, the essential apparatus of RNA interference in the genome
sequence of Plasmodium [302] . Hence, these modern approaches
could be vital in identifying novel and effective drug target against
the malaria parasite.
Expert Rev. Clin. Pharmacol. 2(5), (2009)

Potential antimalarial drug targets
In this section we discuss the reported potential antimalarial drug
targets as claimed by the corresponding workers (Table 1) . However,
careful research will be necessary to functionally validate these
currently available new targets before moving forward to discover
new antimalarials for chemotherapy using these targets.
Invasion of the parasite to the erythrocyte & its release
The intra-erythrocytic stage of the malaria parasite actually causes
the pathology and clinical manifestations in the host. Therefore,
invasion of RBCs by merozoites released from the infected liver
or infected erythrocyte (i.e., reinvasion of the erythrocyte) are
the route of host pathology. The Plasmodium infects RBC by
rapidly gaining entry to cells using their own invasion apparatus
to penetrate and establish themselves [3] . To invade RBCs, the
parasite first disrupts its vacuolar home, known as the parasi-
tophorous vacuole and then the RBC membrane. P. falciparum
cell membrane aspartyl protease, known as signal peptide pepti-
dase (PfSPP) is one of the vital proteases for merozoite invasion
[26] . PfSPP binds to the erythrocyte band 3 for invasion, and
L-685, an inhibitor of mammalian SPP, leads to the inhibition of
parasite invasion as well as its growth in erythrocytes [26] . Release
of merozoites from erythrocytes is the next most important step
to reinfect host cells. Proteases involved in this process include
members of the serine repeat antigen (SERA) family that local-
ize to the parasitophorous vacuole [27,28] and cysteine protease
in the cytosol of infected RBCs [29,30] . Cysteine protease dipep-
tidyl peptidase 3 (DPAP3) and subtilisin family serine prote-
ase PfSUB1 may mediate the cascade of final events of parasite
release from the infected RBCs [31,32] . Inhibition of DPAP3 and
PfSUB1 by peptide vinyl sulfones JCP405 and JCP410, and the
chloroisocoumarin JCP104, respectively, hamper parasite release
[31] . Therefore, inhibition of proteases that have important roles in
the general rupture process can be an effective target against the
parasitic life cycles. Cysteine protease inhibitor E-64, cysteine/
serine protease inhibitors leupeptin and calpeptin, and a serine
protease inhibitor SBP-1 specific to SERA-5, offer antimalarial
activity by inhibiting parasite release, further validating these pro-
teases as potential antimalarial drug targets (Table 1) [30,33] . MSP-1
is vital to mediate initial contact to the host erythrocyte, and
monoclonal antibody against MSP-1 has been shown to immu-
nize against P. falciparum [34,35] . Targeting these proteins will help
to inhibit the primary interaction of the malaria parasite with the
RBCs. To activate the invasion process, secondary interactions
are involved that are mediated by the Duffy binding-like protein
family (EBA-175, EBA-140 and EBA-181) [3] and P. falciparum
reticulocyte binding protein homolog (PfRh) and Plasmodium
vivax reticulocyte binding protein 1 [36–39] . P. falciparum expresses
four members of the Rh family, PfRh1, PfRh2a, PfRh2b and
PfRh4 [36–39] . These proteins are upregulated during infection
and binds to reticulocytes but not normocytes, suggesting that
they are responsible for the host-cell preference and could be
a potent target for the development of antimalarial drugs [40] .
Rhoptry-associated proteins 1 (RAP1) and RAP2 are localized to
the rhoptries, specialized organelles of the invasive P. falciparum
www.expert-reviews.com
Antimalarial drug targets Review
merozoite. These proteins are also involved in erythrocyte inva-
sion. Both RAP1 and RAP2 are important vaccine candidates
because it has been shown that antibodies to RAP1 are able to
block merozoite invasion in vitro [41–44] . Other parasite molecules,
such as the anion transporter protein (AE)-1, are also assumed
to be putative antimalarial drug targets. Sulfated cyclodextrins,
which interact with the AE-1, inhibit parasite growth by pre-
venting the entry of Plasmodium into RBCs [45] . Examination of
cultures treated with sulfated cyclodextrin indicates that intra­
cellular forms of the parasite were unaffected; however, increased
numbers of extracellular merozoites were present. It has also been
found to inhibit Plasmodium berghei merozoite entry and could
reduce the parasitemia of P. berghei infection in a mouse model.
Thus, results suggested that the compounds inhibit a common
step in the merozoite invasion process in P. falciparum in vitro and
P. berghei in vivo [45] . In conclusion, preventing malaria by inhibit-
ing parasite invasion and release might be an important strategy
to combating antimalarial resistant Plasmodium. SERA 5 is the
most promising target since specific inhibitors are available that
offer antimalarial activity by inhibiting parasite release (Table 1) .
Digestive food vacuole
The Plasmodium digestive food vacuole (FV) is central to the
metabolism of the parasite. In the FV, hemoglobin is digested,
hemozoin is formed, amino acids are transported, oxygen radi-
cals are detoxified, drugs are accumulated and acidification is
sustained [46] . Since these are essential processes for parasite sur-
vival, they represents unique chemotherapeutic targets against the
malaria parasite (Table 1) .
Hemoglobin digestion & aminopeptidases
For its survival and growth, the malaria parasite creates space
inside the RBC by endocytosing and catabolizing up to 75%
of the hemoglobin of the RBC to constituent amino acids
[4,47,48] . This process makes large quantities of free amino
acids available [49] and modulates the osmotic environment of
the host cell [50] . Endo- and exo-peptidase are key enzymes to
catabolize the hemoglobin and are therefore potential targets
for antimalarial drug development. Endopeptidases (plasmep-
sin [PM] I, II, and IV, histo-aspartic protease, falcipain-2,
-2´, and -3, and falcilysin) contribute to the hydrolysis of the
a- and b-globin chains of hemoglobin to oligopeptides [51,52] .
These oligopeptides are further hydrolyzed to dipeptides by
the FV exopeptidase DPAP1 [53] . Recently, a model has been
proposed for hemoglobin catabolizm whereby amino acids are
generated from globin-derived oligopeptides in the FV lumen
through the activities of P.  falciparum aminopeptidase N
(PfA-M1) and P.  falciparum aminopeptidase P (Figur e  1) [49] .
Inhibition of pm by statine and allophenylnorstatin-based
inhibitors [54] , falcipain by phenylurenyl chalcone deriva-
tives [55] and PfA-M1 by bestatin and hPheP[CH 2 ]Phe [56–58]
have been shown to block hemoglobin degradation and kill
the parasites (Table  1) . Statin-based inhibitors inhibit growth
of P.  falciparum in  vitro (IC50 = 0.1 µM). The most active
derivative of phenylurenyl, chalcone 1-[3´-N-(N´-phenylurenyl)
471
 Review Alam, Goyal, Iqbal et al.

Table 1. Antimalarial drug targets.

Target location Pathway Target Inhibitors/candidate molecule Refs.
Parasite invasion, Merozoite invasion AMA1 N-methylated 20 residue peptide, R1 [195]
release and PfSPP L-685 [26]
gametocytogenesis RAP Monoclonal antibody [44]
MSP-1 Monoclonal antibody [35]
AE1 Sulfated cyclodextrin [45]
Merozoite release SERA5 Disulfide-bounded cyclic peptide (SBP1) [33]
PfSUB1 Chloroisocoumarin JCP104 [31]
Cysteine protease E64, Leupeptin A [30]
DPAP3 Peptide vinyl sulfones JCP405 [31]
and JCP410
Food vacuole Hemoglobin degradation Plasmepsin Allophenylnorstatine-based inhibitor [196]
Falcipain Phenylurenyl chalcone derivatives [55]
Aminopeptidase N Bestatin, hPheP[CH2]Phe [56]
Heme detoxification Hemozoin Chloroquine [197,198]

Parasite Glucose uptake PfHT O-3 hexose derivatives [91] .

transporters and Phospholipid synthesis Choline transporter G25 [88]
channels Anion channel PSAC Dantrolene [76]
Ion homeostasis V-Type ATPases Bafilomycin A1 [199]
Na + /H + antiport Amiloride [92]
H + /K+ pump Omeprazole [93,94]
Chloroquine accumulation PfCRT Verapamil [200]
Nucleotide biosynthesis Folate-biopterin transport Probenecid [95]

Nucleus Nucleic acid regulation Helicase Actinomycin, daunorubicin [201]

PfHDAC-1 (Histone deacetylase-1) Apicidin, WR301801 [202,203]
DNA Adozelesin, centanamycin [204,205]
Histone acetyltransferase Curcumin [206]
Pfmrk 1,3-diaryl-2-propenones [13,154]

Cytosol Glycolysis Lactate dehydrogenase Gossypol derivatives [207]

Protein synthesis Heat-shock protein 90 Geldanamycin [208]
Signal transduction Protein kinases Oxindole derivatives [209]
Gs protein Propranolol [210,211]
Nucleic acid biosynthesis PfPNP Immucillins [9,103]
Carbon dioxide metabolism Carbonic anhydrase Acetazolamide, sulfanilamide [212]
Oxidative stress Glutothione reductase Methylene blue [126]
g-glutamylcysteine synthetase Buthionine sulfoximine [118]
Thioredoxin reductase 5,8-dihydroxy-1,4-naothoquinone [125,213]
Reactive oxygen species (ROS) Curcumin, artemisinins [206,214]
Glutathione S-transferase Protoporphrin IX, cibacron blue, [127]
S-nitrosoglutathione
Cell cycle regulation PfMetAP2 Fumarranol, Fumaggillin [216]
PfMetAP1b XC11 [216]
Polyamine metabolism Ornithine decarboxylase DFMO [217]
Biological methylation SAH-hydrolase Nucleoside analogs [218]
Pyrimidine metabolism Thymidylate synthase 5-fluoroorotate [219]
Choline biosynthesis Phosphocholine cytidylyltransferase Choline analog T3 [88]
Coenzyme-A biosynthesis Pantothenic acid Pantothenic acid analogues [220]
Purine metabolism Hypoxanthine-guanine Immucillin-H [13]
phosphoribosyl transferase
AE1: Anion transporter protein; AMA1: Apical membrane antigen 1; DFMO: Difluoromethylornithine; DOXP: 1-deoxy- d -xylulose-5-phosphate; DPAP3: Cysteine
protease dipeptidyl peptidase 3; FabH: b-keto-ACP synthase III; FabI: Enoyl ACP reductase; MECP: 2C-Methyl- d -erythritol 2,4-cyclodiphosphate; PDF: Peptide
deformylase; P.f: Plasmodium falciparum; PfALAD: P. falciparum d-aminolevulinic acid dehydratase; PfALAS: P. falciparum d -aminolevulinic acid synthase;
PfCRT: P.f chloroquine resistant transporter; PfDHOD: P.f dihydroorotate dehydrogenase; PfMetA 1b: P.f methionine aminopeptidase 1b; PfHT: Hexose transporter;
Pfmrk: P.f mitogen related kinase; PfPNP: P.f purine nucleoside phosphorylase; PfSPP: P f signal peptide peptidase; PfSub1: P.f subtilisin-like protease;
PSAC: Plasmodial surface anion channel; RAP: Rhoptry associated protein; SAH: S-adenosyl-l-homosysteine; SERA5: Serine repeat antigen 5.

472 Expert Rev. Clin. Pharmacol. 2(5), (2009)

 Antimalarial drug targets Review

Table 1. Antimalarial drug targets (cont.).

Target location Pathway
Mitochondrion Electron transport system
Pyrimidine biosynthesis
Folate biosynthesis
Target Inhibitors/candidate molecule Refs.
Cytochrome c oxidase Licochalcone A, atovaquone [221,164]
Alternative NADH:dehydrogenase Diphenylene iodonium chloride, [222]
(PfNDH2) Diphenyl iodonium chloride
PfDHODH Phenyl-substituted triazolopyrimidines [223]
Dihydrofolate reductase Pyrimethamine, cycloguanil [224]
Dihydropteroate synthase Dapsone, sulphamethoxazole [225]

Apicoplast Protein biosynthesis 16s, rRNA Doxycycline, tetracycline [226]

Elongation factor Tu Amythiamicin [227]
RNA polymerase b subunit Rifampicin [228]
PDF Hydroxamates [229]
DNA replication DNA topoisomerase II Ciprofloxacin [230,231]
Type II fatty acid FabH, Thiolactomycin [232]
biosynthesis Fab1 4-hydroxyanthecotulide [175]
Isoprenoid bioynthesis DOXP reductoisomerase Fosmidomycin [233]
Heme biosynthesis PfALAS Ethanolamine [184]
PfALAD Succinylacetone [185]
Protein farnesylation Farnesyl transferase Peptidomimetics [234]
Amino acid biosynthesis 5-enopyruvyl shikimate 3-phosphate Glyphosate [235,236]
synthase
AE1: Anion transporter protein; AMA1: Apical membrane antigen 1; DFMO: Difluoromethylornithine; DOXP: 1-deoxy- d -xylulose-5-phosphate; DPAP3: Cysteine
protease dipeptidyl peptidase 3; FabH: b-keto-ACP synthase III; FabI: Enoyl ACP reductase; MECP: 2C-Methyl- d -erythritol 2,4-cyclodiphosphate; PDF: Peptide
deformylase; P.f: Plasmodium falciparum; PfALAD: P. falciparum d-aminolevulinic acid dehydratase; PfALAS: P. falciparum d -aminolevulinic acid synthase;
PfCRT: P.f chloroquine resistant transporter; PfDHOD: P.f dihydroorotate dehydrogenase; PfMetA 1b: P.f methionine aminopeptidase 1b; PfHT: Hexose transporter;
Pfmrk: P.f mitogen related kinase; PfPNP: P.f purine nucleoside phosphorylase; PfSPP: P f signal peptide peptidase; PfSub1: P.f subtilisin-like protease;
PSAC: Plasmodial surface anion channel; RAP: Rhoptry associated protein; SAH: S-adenosyl-l-homosysteine; SERA5: Serine repeat antigen 5.

phenyl]-3(3,4,5-trimethoxyphenyl)-2-propen-1-one, inhib-
its falcipain (IC50 = 1.76 µM) and P.  falciparum growth
in  vitro. Bestatin inhibits P.  falciparum growth in culture
(IC50 = 8–14 µM), and the PfA-M1 inhibitor, hPheP[CH 2 ]
Phe, offers antimalarial activity in vivo with no toxicity [56–58] .
Many antiretrovirus protease inhibitors (e.g., HIV protease
inhibitors) have also been established in clinical use as antima-
larials [59] . For example, saquinavir and ritonavir in combina-
tion with chloroquine and mefloquine have been established
as an antimalarial [59] , although their primary target and mode
of action is not clear and further studies will be necessary to
ascertain the primary target and mode of action of these drugs.
Therefore, PfA-M1 may be the most promising antimalarial
drug target as evident from in vitro and in vivo pharmacologi-
cal data on antimalarial effects owing to specific inhibition by
specific inhibitors.
Heme detoxification
After the digestion of hemoglobin by different endo- and exo-
peptidases in the FV of the malaria parasite, very high quanti-
ties of redox active free heme is produced, which is very toxic
[60,61] . Plasmodium has adopted a unique process to detoxify free
heme through the formation of its insoluble crystalline polymer
(hemozoin or malaria pigment) [62] by mechanisms that are not
fully understood [63] . Various mechanisms/factors have been pro-
posed to detoxify the free heme [60] . The factors for hemozoin
formation include, histidine-rich proteins [64] , nonenzymatically
in vitro at acidic pH [65,66] , lipids [67–69] and heme detoxification
protein (HDP) (Figure  1) [70] . HDP is highly conserved across
the Plasmodium genus, which can efficiently produce hemozoin.
HDP is delivered to the FV, the site of hemozoin formation, via
a unique trafficking route [70] . Other factors to detoxify the free
heme are reduced glutathione [5] , heme-binding protein [71] and
H2O2 [72] . Heme detoxification or hemeozoin formation is a vali-
dated antimalarial target (Table 1) . Chloroquine, the most widely
used antimalarial, binds to heme [64,73] and prevents its detoxifi-
cation, leading to heme toxicity and death of the parasite. ([Aryl]
arylsufanylmethyl)pyridine inhibits hemeozoin formation and
offers antimalarial activity [74] . Methylene blue (MB) inhibits
heme polymerization (as well as P. falciparum glutathione reduc-
tase) within the parasite’s FV and prevents methemoglobinemia,
and is under clinical trials [75] . Recently, the safety and efficacy
of MB combined with artesunate, and of MB combined with
amodiaquine, were studied in a controlled trial in Burkina Faso
and was found to potentially accelerate parasite clearance when
used in artemisinin combination therapy (ACT). However, fur-
ther studies are needed to define the possible roles of MB in drug
combination therapy for malarial treatment. Many other com-
pounds, such as azoles, isonitriles and xanthone, inhibit heme
polymerization and exhibit antimalarial property [60] . Since heme
polymerization is a perfectly validated antimalarial drug target,
synthesis of a new heme polymerization inhibitor or identifica-
tion of a heme polymerization inhibitor from natural products
will be helpful to develop novel antimalarials. It is worth men-
tioning that HDP is a most promising new target, which requires
extensive research to validate it as a novel antimalarial target.
As HDP has no homology to any of the known heme-binding
proteins, designing a specific inhibitor against HDP that will
inhibit heme polymemerization and offer antimalarial activity,
will be a rational approach to discovering novel antimalarials.

www.expert-reviews.com 473
 Review Alam, Goyal, Iqbal et al.

PVM

PPM

Cytosome

FV
FV
V-type H+ pump

ATP
pH 5–5.4
Oxyhemoglobin Methemoglobin ADP + iP
(Fe2+) (Fe3+)
O2- O2 H+
Ubiquitinated proteins
Plamepsin I,II,IV
Falcipain 2, 2´, 3

Heme Haem PfMDR1?

(Fe3+) SOD Transporter
Globular peptides
HBP HDP
GSH HRP II and III H2O2 Histo-aspartic proteases Proteasome
Lipid Falcilysin
Detofication Catalase
Unknown proteins ATP
Autopolymerization H2O + O2 Oligopeptides Oligopeptides
Hemozoin Degradation ADP + iP

Amino-peptidases
DPAP1
X-Pro-Xn Di-peptides Xn
PfLAP
PfAPP
PfPAP PfA-M1 PfA-M1

Pro-Xn + Xn-1 +
1 amino acid Amino acid 1 amino acid
Free amino acids

Food vacuole

Amino acid transporter?

Expert Rev. Clin. Pharmacol. © Future Science Group (2009)

Figure 1. Hemoglobin uptake, degradation and heme detoxification in an intra-erythrocytic malaria parasite. After parasite
invasion, the double membrane cytostome is formed due to an invagination of the plasma and parasitophorous membrane, which
intakes erythrocyte cytoplasm containing Hb in a pinocytosis fashion and bud off as TVs. TVs transport Hb to the FV of the parasite.
Inside the FV, Hb is degraded to globular peptides by plasmepsin I, II and IV and falcipain 2, 2 and 3, which are then further degraded to
oligopeptides by histo-aspartic protease and falcilysin endopeptidase activities. The oligopeptides can either be shortened by removal of
a dipeptide by DPAP1 or a single amino acid by PfA-M1 in the FV, or transported to the cytoplasm where further proteolytic cleavage
leads to the production of free amino acids. The details of the transporter that transports the oligopeptides is not clear, however, it is
assumed that PfMDR1 is associated with this transport. Peptides with an N-terminal X-Pro sequence will be substrates for PfAPP (X
represents any amino acid). Dipeptides are hydrolyzed to amino acids by PfA-M1. The majority of the released free heme is converted
into HZ by the action of HDP, HRP II and III, lipids, and by autopolymerization. Some of the heme is transported to the cytosol of the
parasite where it is detoxified by the action of GSH and HBP. Superoxide produced during the conversion of oxyhemoglobin to
methemoglobin is converted to H2O2 by SOD, which can be broken down by catalase. Some of the heme is also degraded by the
hydrogen peroxide. An ATP-dependent V-type H + pump is responsible for vacuole acidification.
DPAP: Dipeptidyl aminopeptidase; FV: Food vacuole; GSH: Glutathion; HBP: Heme binding protein; HDP: Heme detoxification protein;
HRP: Histidine rich protein; HZ: Hemeozoin; PfA-M1: P. falciparum aminopeptidase N; PfAPP: Plasmodium falciparum aminopeptidase;
PfMDR1: P. falciparum multidrug resistant 1; PPM: Parasite plasma membrane; PVM: Parasitophorous vacuolar membrane;
SOD: Superoxide dismutase; TV: Transport vesicle.

474 Expert Rev. Clin. Pharmacol. 2(5), (2009)


Parasite channels & transporters
For rapid growth and multiplication inside RBCs, the malaria
parasite requires a sufficient amount of key substrates to fuel the
vigorous metabolism of the parasite. Therefore, the parasite pre-
pares the host RBC by introducing specialized alterations for the
uptake and disposal of materials for its own survival [76] . Two
unusual ion channels have been identified within the infected
RBC; the Plasmodial surface anion channel (PSAC), which is
induced on the RBC membrane several hours after invasion of
the RBC [76,77] , and parasitophorous vacuolar membrane (PVM),
which is distinct from the PSAC [76,78] . These channels provide a
sequential diffusive pathway of nutrient acquisition to the intra-
cellular parasite [76] . PSAC allows the diffusion of various nutri-
ent solutes (e.g., hypoxanthine, cysteine, glutamine, glutamate,
isoleucine, methionine, proline, tyrosine, pantothenic acid and
choline) down their concentration gradients from serum into the
RBC cytosol. Once these nutrients enter the vacuolar space, these
are readily taken-up through the parasite’s plasma membrane via
specific carrier-type transporters [79–81] . In addition to anions, it
is permeable to sugar, amino acids, purines, some vitamins, some
organic cations and some halide anions such as SCN-, I- , Br-, and
Cl- [76,77] . In contrast to other anionic channels, PSACs maintain
a low Na+ and K+ permeability ratio of no more than 0.001 rela-
tive to Cl- [82] . This is a very low maintenance of Na+ permeability
despite the high permeability of Cl-, making it unique amongst
anion channels.
Compared with the detailed study on PSAC, the PVM channel
remains less well studied. One basic reason for the limited number
of studies on the PVM channel is the technical difficulty of patch-
clamp of freed parasites; however, several recent advances in the
technology give hope to the obtaining of further details on this
channel, and could also help the ion channel experts correlate
these channels with the drug target. It is encouraging that the
Plasmodium genome does not reveal any clear homology between
the PSAC and PVM channel with known host channel genes,
and has functional differences from other anion channels [76] .
Therefore, these features of the P. falciparum-induced channels
may be an excellent target for the development of highly specific
blockers and effective antimalarial drugs (Table 1) . A functional
mutation of PSAC was derived by treating parasite with blasti-
cidin S in vitro, and blasticidin S-resistant parasite was selected.
Resistance to blasticidin S during culture correlated with sig-
nificant reductions in permeability to multiple solutes [83] . The
channel’s selectivity profile and pharmacology also significantly
change and slower growth of mutant parasites suggests that PSAC
serves as an important role in intracellular parasite survival [83] .
Furthermore, malarial parasites are rapidly killed by dantroline
derivatives specific to PSAC, suggesting that it may be a good
template to design new and more specific PSAC inhibitors [84] for
development of future antimalarial drugs. PSAC is the most prom-
ising target among other channels because it plays a critical role in
various types of nutrient acquisition to the intracellular parasite,
no clear homology with known host channel genes, and its func-
tional difference from other anion channels. Although, PSAC is
a promising target, it requires careful in vivo pharmacological
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Antimalarial drug targets Review
validation before adopting a chemotherapeutic strategy. In addi-
tion to these channels, two additional K+ channels have been iden-
tified in the P. falciparum genome [85] and targeted disruption of
the gene encoding PbKch1, which has 82% identity with PfKch1,
shows that PbKch1-null parasite-infected mice survived longer
than the mice infected with wild-type parasite [85] ; however, the
validation of this K+ channel as a potent drug target is required.
Intra-erythrocytic stages of P. falciparum are wholly depen-
dent on host glucose for energy. Glucose uptake is mediated by
a P. falciparum-encoded facilitative hexose transporter (PfHT).
O-3 hexose derivatives (compound 3361) inhibit uptake of glu-
cose and fructose by PfHT and selectivity of these derivatives for
PfHT is confirmed by lack of inhibition of hexose transport by
the major mammalian glucose and fructose transporters (Gluts)
1 and 5. Compound 3361 also inhibits glucose uptake medi-
ated by the P. vivax orthologue of PfHT. Compound 3361 kills
P. falciparum in culture and significantly reduces multiplication
of P. berghei in a mouse model. The aforementioned data vali-
date PfHT as a novel drug target and as an important step in
the development of novel antimalarials directed against mem-
brane transport proteins [86] . During asexual development within
erythrocytes, malaria parasites synthesize considerable amounts of
membrane. Transport of choline is thought to play a significant
role in phosphatidylcholine biosynthesis to generate membrane.
Compounds that inhibit phosphatidylcholine biosynthesis de novo
from choline have been shown to be potent antimalarial drugs
[87] . Transport of choline is thought to play a significant role
in phosphatidylcholine biosynthesis in membrane generation.
Compounds that inhibit phosphatidylcholine biosynthesis de novo
from choline have been shown to be potent antimalarial drugs.
The lead compound, G25, potently inhibited in vitro growth of
the human malaria parasites P. falciparum and Plasmodium vivax
and was 1000-fold less toxic to mammalian cell lines. Very low
dose G25 therapy completely cured monkeys infected with P. fal-
ciparum and P. cynomolgi [88] . Although choline transport has
been suggested to be the target of G25, the exact mode of action
of this compound is not clear. However, G25 specifically tar-
gets the pathways for synthesis of the two major phospholipids,
phosphatidylcholine and phosphatidylethanolamine to exert its
antimalarial activity [89] . Since Plasmodium channels and trans-
porters have been the targets of choice for antimalarial drug
development for many years, many channels and transporters
have been targeted, such as, P. falciparum equilibrative nucleoside
transporter 1(PfENT) [90] , PfHT [91] , choline transporter [88] ,
V-Type ATPases [79] , Na+/H+ antiport [92] , H+/K+ pump [93,94] ,
P. falciparum chloroquine-resistant transporter PfCRT [4] , and
folate-biopterin transporter (Table 1) [95] . Based on available phar-
macological data on channels and transporters, PSAC, PfHT
and choline transporter will be promising antimalarial targets
for further work exploring novel antimalarials.
Nucleic acid biosynthesis
Nucleotides are precursors of DNA and RNA and participate in
many biochemical processes in the mammalian host, as well as
in the parasite. Contrary to mammalian cells, intra-erythrocytic
475
 Review Alam, Goyal, Iqbal et al.
stages of the malaria parasite are incapable of synthesizing purines
de novo [96,97] , thus relying on preformed host purine precursors.
On the other hand, Plasmodia cannot salvage either pyridine
bases or nucleosides and must synthesize pyrimidine nucleotides
de novo [96,97] . Thus, purine salvage and pyrimidine synthetic
pathways are potential drug targets (Table 1) . The parasite-specific
enzymes for salvage are hypoxanthine-guanine phosphoribosyl-
transferase (HGPRT), inosine dehydrogenase and adenylsucci-
nate synthase [98] . HGPRT may be essential for parasite survival,
since antisense polynucleotides that target HGPRT mRNA kill
parasites in vitro [99] . The malaria parasite can also take up ade-
nosine and convert it to inosine by adenosine deaminase or take
up inosine directly from the host [96,97] . It is then converted to
inosine monophosphate (IMP) by purine nucleoside phosphory-
lase. IMP is converted to adenylosuccinate by adenylosuccinate
synthetase and finally to AMP by adenylosuccinate lyase [100] .
IMP is also converted to xanthosine 5´-monophosphate (XMP)
by IMP dehydrogenase and XMP is then converted to GMP
by GMP synthetase [101] . Biochemical characterization suggests
that GMP synthetase differs from its mammalian counterpart
in several aspects [102] . Hypoxanthine production is mediated by
phosphorolysis of inosine to hypoxanthine, catalyzed by P. falci-
parum purine nucleotide phosphorylase (PfPNP). Immucillin is
a transition-state analog that has been shown to kill the parasite
through inhibition of PfPNP in vitro [9,103] . Thus, a structure-
based compound designed for high-throughput screening would
be helpful in discovering and developing new antimalarials by
targeting HGPRT and PfPNP enzymes. Since Plasmodium lacks
the parasite-specific uridine and thymidine kinase, it is con-
cluded that Plasmodia lack pyrimidine salvage and must synthe-
size pyrimidines via the de novo pathway [96,97,104] . The enzymes
of the de novo pyrimidine biosynthesis pathway are carbamoyl
phosphate synthase, aspartate transcarbamylase, dihydroorotase,
dihydroorotate dehydrogenase (PfDHODH), orotate phosphori-
bosyl transferase and orotidine 5-phosphate decarboxylase. Last,
three enzymes differ significantly from the host enzyme [105–107] .
DHODH, the single redox reaction in the pyrimidine de novo
synthetic pathway that catalyzes the reaction from dihydroorotate
to orotate, bridges pyrimidine synthesis and the mitochondrial
electron transport system, and is also the major source of elec-
trons for the mitochondrial electron transport chain of intra-
erythrocytic malaria parasites [108] . DHODH and the electron
transport chain may also be the site of inhibition by certain anti-
malarial drugs: atovaquone, a naphthoquinone, inhibits parasite
DHODH, although its primary target appears to be the blockade
of pyrimidine synthesis by the inhibition of the respiratory chain
of malarial mitochondria at complex III [15] . The next most impor-
tant pathway for nucleic acid metabolism is the folate pathway.
Tetrahydrofolate, a carrier of activated one-carbon groups, is a key
coenzyme in amino acid and nucleotide metabolism and can be
synthesized either de novo or by a salvage pathway [109,110] . The
two bifunctional key enzymes for the folate pathway are dihy-
dropteroate synthase (DHPS), 2-amino-4-hydroxy-6-hydroxy
methyl-dihydropteridine pyrophosphokinase (PPPK) (DHPS-
PPPK), DHFR and thymidylate synthase (TS) (DHFR-TS) [111] .
476
Both PPPK and DHPS intervene in reactions in a de novo folate
pathway that generate H2folate, while DHFR with TS catalyzes
the downstream reaction by NADPH-dependent reduction of
H2folate to H4folate, a necessary cofactor for the biosynthesis of
thymidylate, purine nucleotides and certain amino acids [104] .
TS could be a potential drug target as this enzyme is one of the
most conserved in nature [112] , and it is targeted by 5-fluorooro-
tate (Table 1) [113] . DHFR is a valid antimalarial drug target, but
development of resistance against DHFR-specific antimalarial
drugs owing to mutations in the DHFR gene is a problem. The
Medicines for Malaria Venture (MMV) has claimed that they
have designed and synthesized inhibitors of thewild-type as well
as resistant DHFR and developed a series of compounds through
in vivo models of efficacy and obtained data on the pharmacoki-
netics of the best compounds [114] . Thus, DHFR could again be a
good antimalarial drug target against resistant malaria parasites.
Oxidative stress
Hemoglobin degradation by the malaria parasite in an acidic FV
results in the production of redox active by-products, toxic free
heme and reactive oxygen species (ROS), conferring oxidative
stress on the host cell [115] . To avoid this disaster, most of the
free heme is detoxified by different mechanisms as described in
the heme detoxification section. Free heme may cause oxidative
damage to host proteins and membranes, inhibit parasite enzymes
and lyze erythrocytes [115] . Apart from this metabolically derived
oxidative stress, the production of ROS by the host adds oxida-
tive burden to the parasitized cell. In order to sustain a redox
equilibrium to survive, malarial parasites contain a variety of
low molecular-weight antioxidants, such as tripeptide glutathione
(GSH), various antioxidant enzymes including glutathione- and
thioredoxin-dependent proteins [116,117] and superoxide dismutase.
The key enzymes of P. falciparum, which are directly or indirectly
involved in redox metabolism, are superoxide dismutase, g-gluta-
myl-cysteine synthetase, glutathione synthetase [118] , glutathione
reductase (GR) [119] , glutathione-S-transferase (PfGST) [120] , glu-
tamate dehydrogenase, thioredoxin reductase, thio­redoxins [121] ,
thioredoxin-dependent peroxidases [122,123] and 1-cys-peroxire-
doxin [124] . Since GSH is synthesized by the consecutive action
of g-glutamylcysteine synthetase and glutathione synthetase, and
it is reported that inhibition of g-glutamylcysteine synthetase by
l-buthionine-(S, R)-sulphoximine leads to the death of the para-
site [125] , these enzymes may be a potential antimalarial drug target.
GR maintains the redox state of the cell by keeping the GSH in its
reduced state. Inhibition of GR by methylene blue [126] attracted
much attention to Plasmodium GR as a potential therapeutic tar-
get against malaria (Table 1) . GST, another enzyme linked to the
antioxidant role of GSH, catalyzes the conjugation of glutathione
with a wide variety of hydrophobic compounds and, as a result,
nontoxic products are formed that can be readily eliminated. GST
can be inhibited by cibacron blue, S-hexylglutathione and proto-
porphyrin IX [127] . Two compounds, 5,8-dihydroxy-1,4-naphtho-
quinone and 5-nitro-2-furanacrolein, have plasmodicidal effects
in vitro by inhibiting thioredoxin reductase [125] . Many drugs and
compounds exhibit antimalarial activity by promoting oxidative
Expert Rev. Clin. Pharmacol. 2(5), (2009)

stress especially the endoperoxide antimalarials [74,128,129] , such as
artemisinin, and several of its semisynthetic derivatives including
artemether, arteether and artesunate, appear to act by increasing
oxidant stress and peroxidic compounds by alkylation of heme
or protein [130,131] . Artemisinins became a crucial part of most
recommended regimens as they work against otherwise resistant
parasites. However recent signs of in  vitro resistance to some
artemisinins and their derivatives make the development of new
treatments an even more urgent priority [132] . Vennerstrom et al.
synthesized several synthetic trioxolane derivatives incorporating
the critical endoperoxide pharmacophore of artemisinin such as
RBx-11160 (OZ277) [133] . They obtained synthetic peroxides with
similar or enhanced antimalarial properties and improved the
pharmacokinetics compared with those of semisynthetic arte-
misinin derivatives. OZ277 is highly active against laboratory-
adapted P. falciparum strains and rodent parasites in vivo and is
currently in a clinical development program [133] . However, the
testing of novel antimalarials against nonculture-adapted field
isolates of P. falciparum is important for assessing the variability of
drug activity in areas where parasites are resistant to other classes
of antimalarials. Several other antimalarial endoperoxides such
as 1,2,6,7-tetraoxaspiro and nonadecane (N-89) have also been
reported as potent antimalarials in vitro [134] . However, further
validations of these drugs are warranted for the clinical develop-
ment programs. Thus, oxidative stress and peroxidic compounds
represents a most promising rationale for antimalarial chemo-
therapy (Table 1) . PfGST is thought to be a highly attractive target
for antimalarial drug development since the architecture of the
PfGST substrate-binding site varies significantly from its human
counterparts and only this isoenzyme is present in the parasite.
Membrane phospolipid biosynthesis
Growing and dividing malaria parasites require large amounts of
phospholipids to produce the membranes that encircle the para-
sitophorous vacuole, cytosol and numerous subcellular sections
that are synthesized from plasma fatty acids [104] . Phospholipid
biosynthesis is a potential antimalarial target (Table 1) . The amount
of lipid in infected erythrocytes is therefore significantly higher
than that of normal erythrocytes. In Plasmodia, synthesis of this
phospholipid occurs through two major metabolic pathways, the
de novo choline pathway and the serine decarboxylation-phos-
pho-ethanolamine methylation pathway [135] . Cytidine diphos-
phate (CDP)-choline pathway (Kennedy pathway) has been
suggested for the biosynthesis of phosphatidylcholine (PC) in
P. falciparum [136] . Choline kinase (CK) is the first enzyme in
the Kennedy pathway and inhibition of this pathway prevents
parasite growth [137] . Choubey et al. has cloned, overexpressed
and purified a human homolog of CK [138] , P. falciparum choline
kinase (PfCK), and identified an inhibitor of PfCK, hexadecy-
ltrimethylammonium bromide, which has a very close struc-
tural resemblance to hexadecylphosphocholine (miltefosin), a
well-known antiproliferative and antileishmanial drug [136,138] .
Bisquaternary ammonium salts, the structural analogue of phos-
pholipid precursor choline, have been shown to target membrane
biogenesis in P. falciparum by blocking the biosynthesis of PC.
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Antimalarial drug targets Review
The first generation of these compounds consisted of mono- and
biquaternary ammonium salts [139,140] , and their lead compound
G25 proficiently inhibited the growth of drug-resistant strains of
P. falciparum in vitro [141] , and abolished Plasmodium infection
without recrudescence in rodent [142] and primate models [88] at
very low doses. Bis-thiazolium compounds T3 and T4 exerted
a highly rapid cytotoxic effect against malaria parasites in vitro
at very low doses [143] and recently using a proteomic approach
(MS/MS) Le Roch et al. detected a downregulation of the cho-
line/ethanolamine-phosphotransferase protein and validated
it as a potent antimalarial drug, a key enzyme involved in the
final step of biosynthesis of phosphatidylcholine (PC) [135] . In
eukaryotic cells, CTP:phosphocholine cytidylyltransferase is the
rate-limiting enzyme in the de novo synthesis of PC, catalyzing
the formation of CDP-choline pyrophosphate by phosphocholine
and CTP [144] . The cytidylyltransferase of P. falciparum has been
isolated and overexpressed [145] and presents significant differences
with respect to its mammalian host, both in terms of sequence
and regulation of the enzymatic activity. This cytidylyltransferase
could be a putative antimalarial drug target, however, synthesis
of a specific inhibitor and the in vitro and in vivo validation as a
drug target is a prerequisite. The new compounds that have been
discovered against phospholipid biosynthesis are exciting leads
for new antimalarial drug discovery.
Cyclin-dependent protein kinases & cell-cycle regulation
The intracellular multiplication cycle of the malaria parasite dif-
fers from the common cell cycle of the host [146] . The multiple
cycles of DNA replication and nuclear division without cytokinesis
causing the generation of single syncytial cells containing 8–32
nuclei are the major differences between the Plasmodium and its
host cell division cycle [147] . Cyclin-dependent protein kinases
(CDKs) are a family of kinases that require cycline for their activ-
ity and are thought to be the heart of the cell-cycle machinery that
regulate the cell [147] . The susceptibility of the malaria parasite
to known CDK inhibitors, suggests that CDKs are vital to the
growth and development of the parasite [147] . The amino acid
alignments of the Plasmodial and human CDKs identify unique
residue among the kinases, especially in the active site that can be
exploited to develop selective antimalarial compounds [147] . Based
on a sequence homolog with the mammalian CDKs, several CDKs
have been identified in the malaria parasite; however, only P. falci-
parum protein kinase (PfPK)-5 and -6, and P. falciparum mitogen
related kinase (Pfmrk) have been characterized in P. falciparum
[148] . PfPK5 phosphorylates histone H1, casein and the C-terminal
domain of RNA polymerase II in vitro and regulates the S-phase
of the parasitic cell cycle [149] , although their endogenous substrate
have not been identified [147] . Flavopiridol and olomoucine inhibits
PfPK5, and parasite growth in vitro indicates that modification of
these compounds may produce potent antimalaria [149] . PfPK6,
which shares 38 and 33 sequence identity with human CDK2 and
p38 mitogen-activated protein kinase, respectively, are thought
to span from G1 to mitosis and phosphorylates both histone H1
and the small subunit (R2) of the malarial ribonucleotide reduc-
tase [150] . Although PfPK6 is sensitive to the CDK inhibitors,
477
 Review Alam, Goyal, Iqbal et al.

Erythrocyte

Glucose

Hexose
transporter
Erythrocyte
ALA
HemB
Glucose Glycolysis PEP Pyruvate Lactate
Pyk-2

Mitochondrion
Pyk-1
PEP Pyruvate
Glycine MTET
TCA cycle decarboxy Ch2•THF CH2•THF fmet•tRNA PDH
enzymes -lase DXS
Serine
SHMT

Folate carrier? reaction

Acetyl
Glycine DOXP
Glycine -CoA
Ch2•THF THF ACCase
ISpC Maloyl
HemB Porpho-
ALAS ALA -CoA
e- Succenyl-CoA ALA bilinogen ISpD ACP FabD
Transporter ? Maloyl
HemC ISpE ACP
CoQH2 CoQ
FabH
(Many steps) Hydroxy-methylbilane
Pyrim ISpF
Pyrimidine Orotate Dehydro FabG
-idine -orotate HemD ?
Cytochrome 8-10 IPP ISpG FabZ/A
Cytochrome + Hydroxy- Urobilinogen III ACS Transporter
assembly benzoate ISpH FabI
Heme Coq1 Fatty acyl-CoA
HemE C-IS
HemH IPP IPP
ProtoP IX Coprobilinogen III Transported
to ER
HemG HemF ?
Protobilin Coprobilin Transporter ?
-ogen IX HemF -ogen III Protobilinogen IX

Transporter ? Transporter ?
Protobilin HemF Transporter ?
HemG -ogen IX

Apicoplast
Location of these steps Transporter ?
are not known, it may be in
mitochondria or cytosol

DNA synthesis

THF
dTMP
TS Cytosol
dUMP
Ch2•THF
Glycine
SHMT
Serine
THF

Expert Rev. Clin. Pharmacol. © Future Science Group (2009)

478 Expert Rev. Clin. Pharmacol. 2(5), (2009)

 Antimalarial drug targets Review

Figure 2 (left). Overview of the mitochondrial and apicoplast metabolism and their crosstalk. Heme biosynthesis starts in the
mitochondrion, where ALAS reacts with succinyl-CoA (which may be generated by the incomplete TCA cycle) with glycine (produced by
glycine decarboxylase reaction) to produce ALA, which is then transported into the apicoplast. In the apicoplast, a series of enzymatic
reactions catalyzed by HemB, HemC, HemD and HemE lead to the formation of urobilinogen III, which is transported to the peri-
mitochondrial membrane. Urobilinogen III in apicoplast is then further catalyzed to protobilinogen by HemF, which is then transported to
the cytosol of the parasite by an unknown transporter. There is no known homologue of the HemD enzyme in the P. falciparum
genome. The location of the HemF and HemG enzymes is unknown, but is probably in either the cytosol or mitochondrion. In the final
step of this pathway, proto-heme is produced, which is catalyzed by HemH in the mitochondrion. Proto-heme can then be further
modified before being used as a prosthetic group in proteins such as cytochromes of the electron transport chain. The glycine
decarboxylase system functions in either the breakdown of glycine and production of CH2-tetrahydrofolate, or in the reverse direction for
the synthesis of glycine. The mitochondrion contains a homologue of SHMT, which utilizes CH2-tetrahydrofolate for the production of
serine or in the reverse direction to produce glycine and CH2-tetrahydrofolate. CH2-tetrahydrofolate is also transported outside the
mitochondrion by folate carrier for the synthesis of dTMP, which is catalyzed by TS in the cytosol and to the apicoplast for the synthesis
of formylmethionine tRNA by MTFT. In the apicoplast the isoprenoid tail of coenzyme Q is synthesized as IPP units via the DOXP
pathway. This is then transported to the mitochondrion, where Coq1 adds eight or nine IPP units to 4-hydroxybenzoate. This
4-hydroxybenzoate ‘head’ group is modified by a series of reactions ultimately to produce coenzyme Q. This coenzyme Q by their
reduction from five different mitochondrial dehydrogenases and reoxidation by type-2 DHOD leads to the formation of pyrimidine for
the synthesis of nucleic acids. The carbon substrate for plastid fatty-acid synthesis (i.e., acetyl Co-A), is generated from pyruvate by
PDHC, which further produces fatty acid by a series of reactions. The fatty acid is then transported outside the apicoplast by ACS
transporter. Pyruvate is probably generated from imported PEP by pyruvate kinase.
ACCase: Acetyl-CoA carboxylase; acetyl CoA: Acetyl coenzyme A; ACP: Acyl carrier protein; ACS: Acyl CoA synthetase;
ALA: g-aminolevulinic acid; ALAS: ALA synthase; DHOD: Dihydroorotate dehydrogenase; DOXP: 1-deoxy-D-xylulose-5-phosphate;
dTMP: Deoxy thymidine monophosphate; DXS: 1-deoxy-D-xylulose-5-phosphate (DOXP) synthase; FabB/F: b-ketoacyl ACP synthase I/II;
FabD: Malonyl-CoA transacylase; FabG: b-ketoacyl ACP reductase; FabH: b-keto-ACP synthase III; FabI: Enoyl ACP reductase;
FabZ: b-hydroxyacyl-ACP dehydratase; HemB: porphobilinogen synthase; HemC: Porphobilinogen deaminase; HemD: Uroporphyrinogen
III synthase; HemE: Uroporphyrinogen III decarboxylase; HemF: Coproporphyrinogen oxidase; HemG: Protoporphyrinogen oxidase;
HemH: ferrochelatase; IPP: Isopentenyl pyrophosphate; IspC: DXP reductoisomerase; IspD: 4-diphosphocytidyl-2C-methyl-D-erythritol
synthetase; IspE: 4-diphosphocytidyl-2C-methyl-Derythritol kinase; IspF: 2-C-methyl- d-erythritol 2,4-cyclodiphosphate synthase;
IspG: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; IspH: 1-hydroxy-2-methyl-2- (E)-butenyl 4-diphosphate reductase;
MTFT: Methionyl-tRNA formyl transferase; PDHC: Pyruvate dehydrogenase complex; PEP: Phosphoenolpyruvate; Pi: Inorganic phosphate;
PP: Pyrophosphate; PPT: Phosphoenolpyruvate/phosphate translocator; PYK: Pyruvate kinase; TPT: Triose phosphate transporter;
TS: Thymidylate synthase.

roscovitine and olomoucine, further in vivo and in vitro valida- Mitochondria

tion as an antimalarial target is required [150,151] . Pfmrk shares
46% identity with the mammalian CDK-7, which functions as
a CDK-activating kinase (CAK) [152,153] . Even if Pfmrk failed to
either phosphorylate or activate PfPK5 and is not considered as the
functional homologue of CDK-7, the possibility exists that it may
possess CAK activity either in the presence, or a diverse cycline
subunit or towards, a CDK other than PfPK5 [147] . 1,3-diaryl-2-
propenones (chalcone derivatives) selective inhibition of Pfmrk
may be an additional mechanism of antimalarial activity [154] .
Based on structural data of human CDKs bound to the inhibitor,
these same compounds can be modified to decrease their binding
affinities for the human CDKs, while increasing specificity for the
plasmodial CDKs, thus, could be a good approach to develop-
ing inhibitors against drug-resistant malaria parasites (Table 1) . In
this sense, roscovitine, and 1, 3-diaryl-2-propenones, inhibitors
of PfPK6 and Pfmrk, respectively, may be further developed as
potent and selective inhibitors since they contain numerous amino
acid variations within the active pocket of these CDKs compared
with the human CDKs. CDKs are being considered as a potent
antimalarial drug target by various authors based on their in vitro
data, but owing to lack of physiological substrates for CDKs and
the in vivo data, it is hard to consider it as a potent target at pres-
ent. CDKs may be explored as a potential drug target if research
is directed to search for its physiological substrate with the view to
develop analogs to inhibit CDK activity and Plasmodium growth
in vitro and in vivo.
Plasmodium parasites have undeveloped mitochondria during
their asexual blood-stage life-cycle, lacking criste of the inner
membrane [155] and the fully functional TCA cycle [156,157] . There
is no evidence that this leads to the formation of acetyl-CoA
and Plasmodium derives its energy through glycolysis (Figure 2)
[157] . Plasmodia contain a genome of 6 kb that is found closely
associated with the apicoplast at all stages of parasite development
[157,158] . Even though it acquires several nuclear gene-coded pro-
teins through import mechanisms involving tim–tom complexes,
it also encodes three mitochondrial proteins (cytochrome b and
two subunits of cytochrome oxidase), as well as two fragments
of rRNA [158] , which help to form functional ribosomes [159] .
Electron transport chains of Plasmodia appear to function via the
FAD-linked TCA enzymes, malate-quinone oxidoreductase and
succinate dehydrogenase. Dihydroorate dehydrogenase converts
dihydroorate to orotate in the mitochondrion by transferring the
electrons to Coenzyme Q (ubiquinone) (Figure 2) [160] . Recently,
Painter et al., has shown that the malarial parasite maintains
an active electron transport chain to regenerate the ubiquinone
required as the electron acceptor for dihydroorotate dehydro-
genase [161] . Since contribution of electrons donated from cyto-
solic and mitochondrial NADH and NAD(P)H to the electron
transport chain is not clear, understanding the localization and
function of NAD(P)H dehydrogenase and glycerol-3-phosphate­
dehydrogenase is significant. The NAD(P)H dehydrogenase in
P. falciparum differs from the human counterpart, and is therefore

www.expert-reviews.com 479
 Review Alam, Goyal, Iqbal et al.
a unique drug target [160] . Conversion of orotidine monophos-
phate to uridine monophosphate, which is catalyzed by oroti-
dine decarboxylase, has also been investigated as a unique drug
target [162] . It is also assumed that the parasite mitochondrion
plays possible roles in folate, Fe–S cluster and heme biosynthesis,
and with experimental validation many steps in these pathways
could become drug targets are already on the market (Figure 2) [163] .
Several drugs that act on Plasmodium mitochondria. Atovaquone,
a hydroxynaphthoquinone, acts on the ubiquinol oxidation site
(Qo) of cytochrome b with high affinity and depolarizes malarial
mitochondria [164,165] . Structural features of the redox centers
of cytochrome b differ from mammalian cytochrome b, thus
possibly explaining the selectivity of antimalarial action [166] .
These distinctive features of Plasmodia mitochondria have yet
to be exploited and the discovery of a new antimalarial agent is
anticipated (Table 1) .
Apicoplast
Apicomplexan parasites, including P. falciparum, contain a plastid-
like organelle termed the apicoplast. The idea was obtained from
the swallowing up of photosynthetic red algae in ancient times.
The apicoplast genome of P. falciparum contains a 35-kb DNA
and is much smaller than its plastid ancestor [167] . Although the
majority of proteins of this organelle are encoded in the nuclear
genome and are subsequently transported to the apicoplast, it also
encodes a full set of tRNAs, some ribosomal proteins, three genes
for the subunits of an oligomeric RNA polymerase, a gene for the
elongation factor PfTu and a gene contributing to the Fe–S path-
way [163] . Since the apicoplast contains unique metabolic pathways
such as fatty acid, isoprenoid and heme synthesis, which are not
found in the human host [168] , it could be an ideal source of drug
targets (Figure 2 & Table 1) .
Fatty acid biosynthesis
Fatty acid synthesis (FAS) is a major function of the apicoplast.
There are two major pathways of fatty acid biosynthesis, FAS I
and FAS II. Eukaryotes mostly rely on FAS I enzymes, which
are large multifunctional enzymes [169] . Plasmodium do not con-
tain FAS I, and rely on FAS II for the de novo production of
fatty acids [170] ; thus FAS II of Plasmodium may be a potential
drug target for the development of antimalarials (Figure 2 & Table 1)
[171] . From the antimalarial drug development point of view, two
important enzymes of the FAS II pathway are b-Ketoacyl-ACP
synthase III (Fab H) and enoyl-ACP reductase (FabI). Fab H
catalyzes the condensation of malonyl-ACP and acetyl-CoA,
forming a b-ketoacetyl-ACP product [172] and FabI catalyzes the
NADH-dependent reduction of enoyl-ACP into a saturated acyl
chain [169] . The Fab H inhibitors thiolactomycin, clodinafop and
quizalofop exhibit inhibition of P. falciparum growth in in vitro
cultures [170,173] . Enoyl-ACP reductase, the next most important
target among the FAS II type fatty acid biosynthetic enzyme of
late liver stage development of malaria parasite [174] , has been
claimed to be inhibited by 4-hydroxyanthecotulide, the com-
pounds obtained from the Anthemis auriculata plant with IC50
values of 20–75 µg/ml [175] . Enoyl-ACP reductase was previously
480
claimed to be essential for blood stage fatty acid biosynthesis
and is inhibited by triclosan [176] . In conclusion, Fab H and FabI
have a vital role for apicoplast type II fatty acid biosynthesis and
could be a potent antimalarial drug targets [170] . Nevertheless,
further in vivo validation of these drugs is warranted prior to
clinical trials.
Isoprenoid & heme biosynthesis
Repeating units of isopentenyl pyrophosphate (IPP) builds iso-
prenoids. It forms prosthetic groups of some enzymes and is also
used in the synthesis of ubiquinone and dolichol. To generate the
intermediate isopentyl diphosphate molecule, both mammals and
fungi depend on mevalonate. The presence of the nonmevalon-
ate pathway for IPP biosynthesis is also evident in the malaria
parasite genome [163] . Bacteria as well as P.  falciparum utilize
1-deoxy-d-xylulose-5-phosphate (DOXP) as a precursor molecule,
which is referred to as the mevalonate-independent pathway. This
plastid-encoded mevalonate-independent pathway of isoprenoid
biosynthesis has been considered as a potential target for anti-
malarial chemotherapy (Figure 2 & Table 1) [177] . Fosmidomycin and
its derivative FR900098 inhibit DOXP reductoisomerase, the
key enzyme of the DOXP pathway, which is absent in humans.
These phosphonic acid derivatives have low toxicity and dem-
onstrate significant antiparasitic activity, which facilitates the
explanation of this pathway as a chemotherapeutic target [178] . In
P. falciparum, a mixed pathway for heme biosynthesis utilizing
the parasite genome-coded enzymes localized in the apicoplast,
mitochondrion and cytosol with the final formation of heme in
the mitochondrion has been reported (Figure 2) [160,167,179] . The
committed precursor for heme biosynthesis is d-aminolevulinic
acid (ALA), which is synthesized from glycine and succinyl-CoA
by P. falciparum ALA synthase (PfALAS). ALA is then converted
to porphobilinogen, one of the important intermediates of heme
biosynthesis by ALA dehydratase (ALAD) [180] . It has been found
that P. falciparum imports host ALAD from the red cell [181] . It
is also found that the biochemical properties of PfALAS [182] and
PfALAD [183] are different from those of the corresponding host
enzymes and that both enzymes are needed for parasite survival.
Inhibition of the heme-biosynthetic pathway by ethanolamine and
succinylacetone inhibition of PfALAS and PfALAD leads to death
of the parasite [184,185] . Hence, the complex heme biosynthetic
pathway of the malaria parasite would represent an ideal drug
target (Table 1) . Phase II clinical studies show that fosmidomycin
alone or in combination with clindamycin cure 100% on day 28
with treatment durations of 5 and 4 days and cure 90% with a
treatment duration of 3 days [186] . Thus, DOXP reductoisomerase
also appears to be a good antimalarial target.
Expert commentary
The major problem of current malarial chemotherapy is the emer-
gence of drug resistance against the available antimalarial drugs.
This situation warrants the identification of functionally vali-
dated antimalarial drug targets to discover new novel chemother-
apeutically viable molecules to treat resistant malaria. Although
the MMV, a not-for-profit organization, is trying their best to
Expert Rev. Clin. Pharmacol. 2(5), (2009)

encourage early development of the antimalarial drugs by com-
bination therapy such as DacartTM (chlorproguanil–dapsone–
artesunate), Eurartesim® (dihydroartemisinin–piperaquine),
Pyramax® (pyronaridine–artesunate) or individual compounds
such as tafenoquine, isoquine and 4 (1H)-pyridone [114] , most of
them are restricted to old targets. Therefore, identification of new
antimalarial drug targets will provide extra room for researchers
and organizations such as the MMV to combat resistant malaria
by developing novel antimalarial drugs. The Malaria Genome
Project has provided new scope to the search for new targets. For
the identification of suitable targets the application of modern
approaches such as evolutionary patterning, system biology and
gene networks, and forward and reverse genetics are necessary.
These different approaches may help to create a target protein
bank with functional and important proteins from the malaria
parasite to explore potential drug targets. The optimization of
targets for antimalarial screening should be done after genetic
and/or chemical validation, pharmacological testing and struc-
tural characterization, identification of specific inhibitors against
which the parasite will fail to develop resistance [187] . Killing the
malaria parasite at the liver stage could be a preventive approach
against host pathology. The few existing drugs that kill malarial
parasites at the liver stage are primaquine (an 8-aminoquino-
line derivative) and the combination of atovaquone–proguanil.
Toxicity caused by hemolysis of RBCs in patients with some types
of G6PD deficiency, a condition frequent in African populations,
is a disadvantage of the 8-aminoquinolines [188] . Mazier and col-
leagues extracted the antimalarial compounds from the plant
Strychnopsis thouarsii, which has antimalarial activity. The active
compound was found to be a morphinan core, which was chemi-
cally modified to give N-cyclopentyl-tazopsine. N-cyclopentyl-
tazopsine is shown to have a higher selectivity for hepatic para-
sites, compared with the blood stages, and is less toxic to host
cells than the parent compound [189] .
Inhibition of parasite invasion to erythrocytes might be an
important target because invasion of RBC by malaria parasites is
the route of host pathology. The P. falciparum erythrocyte mem-
brane protein 1 (PfEMP1) of P. falciparum has been implicated
in the cytoadhesion of P. falciparum-infected erythrocytes to vas-
cular endothelium [190] . PfEMP1 is composed of multiple cystein-
rich extracellular domains, which show affinity for an array of
host receptors including the glycoasaminoglycan heparan sulfate.
PfEMP1 species of infected erythrocytes of children with severe
malaria frequently bind to the host receptor heparan sulfate present
on both endothelial cells and erythrocytes [191] . Heparin, which is
similar to heparan sulfate in that it is composed of the same build-
ing blocks, was previously used in the treatment of severe malaria
but was discontinued owing to the occurrence of serious side effects
such as intracranial bleeding. Recently, Vogt et al. discovered novel
glycans (dGAG) and validated them in vivo using different models
that depolymerized heparin that lacked anticoagulant activity,
blocked up to 80% of infected erythrocytes from binding in the
micro-vasculature and released already sequestered parasites into
the circulation [191] . Thus, modified heparin could be a promising
candidate for adjunct therapy in severe malaria.
www.expert-reviews.com
Antimalarial drug targets Review
Hemozoin formation is still a unique antimalarial target
that warrants further attention. Although chloroquine, which
inhibits hemozoin formation, is resistant, research should be
directed to find out ways for resistance withdrawal or design
novel heme polymerization inhibitors using different scaffolds
of heme–polymerization inhibitors. Many new aminoquino-
lines syntheized by systematic variation of the branching and
basicity of the side chain of chloroquine yielded a series of new
antimalarial drugs including 7-chloro-4-aminoquinoline and
its derivatives [192] , thiourea, thiazolidinedione and thiopara-
banic acid derivatives of 4-aminoquinoline [193] , exhibiting high
activity in vitro against different chloroquine-resistant strains of
P. falciparum and P. vivax. The newly discovered HDP in P. fal-
ciparum is found to be extremely potent in converting heme into
hemeozoin in vitro. HDP will be a promising antimalarial drug
target because it is functionally conserved across the Plasmodium
genus and its gene locus could not be disrupted [70] ; however,
further in vivo activity and studies such as gene knockout are
warranted to validate it as a choice of antimalarial drug target.
Peroxidic antimalarials are effective against parasites that have
evolved resistance to aminoquinoline, folate antagonists and the
arylamino alcohols.
The most important synthetic endoperoxides in use are arte-
misinin derivatives. They act by inhibition of PfATP6, a sar-
coplasmic endoplasmic reticulum calcium ATPase. RBX11160
(OZ277), a synthetic endoperoxidic antimalarial, which is in
the advanced stages of clinical development, inhibits PfATP6
with relatively low potency (apparent Ki: 7700 nM) compared
with artemisinin [194] . It is anticipated that huge ongoing work
on different metabolic pathways of unique organelles, such as
the apicoplast (absent in the host) and mitochondria (distinct
from the host) may open new avenues for the identification of
suitable antimalarial targets. It is worth mentioning that selec-
tive ion channels of infected erythrocytes (PSAC and PfHT)
have the potential to be promising targets for the development
of new antimlarials. SERA5, which has an important role in the
regulation of the intra-erythrocytic development of late-stage
parasites and enoyl-ACP reductase (FabI), one of the important
enzyme of the FAS II pathway, could also be considered as prom-
ising targets against which to develop novel antimalarial drugs.
Antimalarial screening of huge phytochemicals or medicinal
plants found worldwide may also help to identify antimalarial
targets, since two popular antimalarials – quinine and artemis-
inin – came from plant sources and theirs targets were identified
later on. Several promising targets for drug intervention have
been revealed in recent years. In this review, we have enlisted
almost all the known validated, partially validated and promis-
ing antimalarial drug targets available in the literature (Table
1) with a view to provide an open source of targets for further
study to develop antimalarials. We have tried to incorporate the
maximum numbers of antimalarial molecules but reported lead
molecules may be much higher than indicated in this report. It
can be anticipated that effective research in the near future capi-
talizing on these targets may lead to suitable viable targets against
which to develop novel antimalarials to combat resistant malaria.
481
 Review Alam, Goyal, Iqbal et al.

Five-year view
The availability of genome sequences has triggered continuous
efforts throughout the globe for structural and functional anno-
tation of various important parasite-specific proteins respon-
sible for parasite invasion, survival in the host and pathology.
Research in this direction will produce valuable data to explore
novel treatable targets. Medicinal chemists will capitalize on via-
ble or validated targets to design and synthesize important lead
molecules for the development of novel antimalarials, which will
be forwarded for clinical trial after toxicological safety analysis.
The development of drug resistance is a serious problem with
P. falciparum malaria. A combination therapy comprising dif-
ferent antimalarials targeting different targets will most likely
be the future chemotherapeutic strategy to avoid the parasite
developing resistance the parasite against all of the antimalarial
drugs. New drugs to control at the liver stages are an abso-
lute priority for global malaria control efforts. Targets such as
SERA5, HDP, plasmepsin II and PfATP6 are very promising
antimalarial drug targets where efforts should be focused on the
future development of antimalarial drugs.
Financial & competing interests disclosure
This work was supported by CSIR, New Delhi through the Supra-
Institutional project (SIP 0007). ��������������������������������������
The authors have no other relevant affili-
����
ations or financial involvement with any organization or entity with a
financial interest in or financial conflict with the subject matter or materials
discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Key issues
• Prevention of malaria is difficult owing to the lack of an effective vaccine and the emergence of drug-resistant malarial parasites.
Therefore today, chemotherapeutic intervention is the only option. Insecticide-treated bed nets, known to be highly effective in
reducing malaria morbidity and mortality, are also a good approach.
• To develop new antimalarials, identification of a novel drug target or capitalization of already validated targets for the rational design of
new molecules is warranted.
• Evolutionary patterning, system biology and gene networks, forward and reverse genetics, and cell lysate-based assay approaches can
be adopted critically to identify novel drug targets.
• Many parasite proteins are currently claimed as drug targets based on preliminary studies; however, extensive studies are essential to
functionally and structurally validate these targets in order to confirm whether these are treatable or nontreatable targets.
• Targeting proteins such as SERA 5, which plays a crucial role in intra-erythrocytic proliferation of Plasmodium falciparum, could be a
good target to inhibit reinvasion to the fresh red blood cells by the malaria parasite.
• The food vacuole, although an old target, still has tremendous potential if the newly discovered P. falciparum food vacuolar
aminopeptidases, such as aminopeptidase P and aminopeptidase N, are utilized for rational drug design.
• The interactome studies of three separate genomes (apicoplast, mitochondria and nucleus) will certainly help to discover novel targets
against which to develop antimalarials.
• Last, but not least, the ion channel of infected erythrocytes and intra-erytrocyte biochemistry of the parasite may open new avenues in
the search for a novel drug target.

5 Becker K, Tilley L, Vennerstrom JL, drug target sites in Plasmodium

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