Combinatorial Chemistry & High Throughput Screening, 2005, 8, 63-79 63

Targeting the Hemozoin Synthesis Pathway for New Antimalarial Drug

Discovery: Technologies for In Vitro β-Hematin Formation Assay
Babu L. Tekwani* and Larry A. Walker

National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of
Pharmacy, University of Mississippi, University, MS 38677, USA
Abstract: Clinical manifestations of malaria primarily result from proliferation of the parasite within the
hosts’ erythrocytes. During this process, hemoglobin is utilized as the predominant source of nutrition. The
malaria parasite digests hemoglobin within the digestive vacuole through a sequential metabolic process
involving multiple proteases. Massive degradation of hemoglobin generates large amount of toxic heme.
Malaria parasite, however, has evolved a distinct mechanism for detoxification of heme through its conversion
into an insoluble crystalline pigment, known as hemozoin. Hemozoin is identical to β-hematin, which is
constituted of cyclic heme dimers arranged in an ordered crystalline structure through intermolecular
hydrogen bonding. The exact mechanism of biogenesis of hemozoin in malaria is still obscure and is the
subject of intense debate. Hemozoin synthesis is an indispensable process for the parasite and is the target for
action of several known antimalarials. The pathway has therefore attracted significant interest for new
antimalarial drug discovery research. Formation of β-hematin may be achieved in vitro under specific chemical
and physiochemical conditions through a biocrystallization process. Based on these methods several
experimental approaches have been described for the assay of formation of β-hematin in vitro and screening of
compounds as inhibitors of hemozoin synthesis. These assays are primarily based on differential solubility
and spectral characteristics of monomeric heme and β-hematin. Different factors viz., the malaria parasite lysate,
lipids extracts, preformed β-hematin, malarial histidine rich protein II and some unsaturated lipids have been
employed for promoting β-hematin formation in these assays. The assays based on spectrophotometric
quantification of β-hematin or incorporation of 14 C-heme yield reproducible results and have been applied to
high throughput screening. Several novel antimalarial pharmacophores have been discovered through these
assays.
Keywords: Malaria, Plasmodium falciparum, hemozoin, beta-hematin, heme, antimalarials, quinolines.

1. INTRODUCTION parasite afford several distinct metabolic characteristics and

unique metabolic pathways, which are different from the
For thousand of years malaria has been a major cause of
human host [2]. These unique metabolic properties (the
human suffering. Despite significant advances in
pathways and the enzymes) may be exploited for therapeutic
understanding the disease and the parasite, malaria still
intervention and new antimalarial drug discovery. The
remains one of the leading causes of morbidity and
malaria parasite mainly grows and proliferates within the
mortality, particularly in the tropics with a staggering 500
host erythrocytes. This is commonly referred to as
million new infections and 1 million deaths annually.
“intraerythrocytic schizogony” and is primarily responsible
Almost half of the world’s population is currently at the risk
for pathogenesis of the disease [3]. Clinical manifestations
for malaria infection. Although malaria incidence is mostly
of malaria result from continuous proliferation of the parasite
centered in tropical regions, the impacts, especially
within the host erythrocytes and with rupture of schizonts
economic, of the disease are global. Recent trends indicate
[3]. The drugs acting on this part of parasite life cycle and
rapid emergence of drug-resistant and more virulent strains
clearance of the parasite from the blood are therefore referred
of the parasite to further intensify the problem [1]. The
to as blood schizontocidal antimalarials [4]. Almost all
choice of therapies currently available for the treatment of
antimalarials currently in use belong to this class; an
malaria is highly limited, and several of these may
exception is the 8-aminoquinoline class which act on
eventually be lost or compromised due to drug resistance.
exoerythrocytic stages of the parasite and are used for
An armament of new antimalarial drugs with proven clinical
treatment of relapsing malaria [5]. During intraerythrocytic
efficacy against current drug-resistant cases of malaria
phase of the malaria life cycle hemoglobin is utilized as a
including Plasmodium falciparum as well as Plasmodium
predominant source of nutrients. The amino acids derived
vivax infections is critical to combat the disease and cope
from digestion of hemoglobin are incorporated into parasite
with the problem of further development of resistance.
proteins and may also be utilized for energy metabolism.
The unique life cycle of the malaria parasite and the Massive degradation of about 5mM hemoglobin releases
resulting microenvironments within the host, vector and the large amount of toxic free heme [6]. Continuous degradation
of hemoglobin and concomitant detoxification of heme are
*Address correspondence to this author at the National Center for Natural absolutely necessary for uninterrupted growth and
Products Research, School of Pharmacy, University of Mississippi, proliferation of the parasite. Therefore, the metabolic
University, MS 38677, USA; Tel: 1-662-915-7882; Fax: -1-662-915-7062; functions related to hemoglobin digestion and heme
E-mail: btekwani@olemiss.edu

1386-2073/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

64 Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1
detoxification pathways may be potential targets for new
antimalarial drug discovery [6]. Protease inhibitors, which
can inhibit hemoglobin digestion, have shown promising
antimalarial properties in vitro as well as in vivo [7].
However, involvement of multiple proteases and gaps in the
knowledge of the specific roles of each in hemoglobin
degradation, have limited their clinical applications. Broad
substrate specificity of the enzymes and poor selectivity of
the inhibitors will need further study before successful
development of protease inhibitors as antimalarials.
Discovery of a unique heme detoxification process in malaria
parasites, which involves incorporation of heme into a
crystalline complex called hemozoin or the malarial
pigment, and identification of this pathway as a target for
action of several known antimalarials have generated
significant interest in this pathway for new antimalarial drug
discovery [8]. A few recent review articles have highlighted
the mechanism of formation of hemozoin, a crystalline
pigment and the major product of heme detoxification
pathway in malaria parasite [9-12]. However, the exact
mechanism of formation of hemozoin within the parasite
food vacuole, the physiological site of hemoglobin
degradation and hemozoin synthesis, is still a subject on
intense debate. This review focuses primarily on different
experimental approaches used for the assay of hemozoin
formation in vitro. High throughput screening (HTS)
approaches for in vitro hemozoin assays may be useful for
identification of novel inhibitors of this parasite-specific
pathway as potential antimalarial agents.
2. HEMOGLOBIN DIGESTION BY MALARIA
PARASITE: ROLE OF MULTIPLE PROTEASES
The malaria parasite depends on the host to fulfill its
requirements of amino acids for synthesis of proteins and
other metabolic functions. Hemoglobin is the most abundant
protein in the erythrocytes cytoplasm (5 mM) and provides
the major source of amino acids [13]. The malaria parasite,
during intra-erythrocytic development and proliferation,
ingests more than 75% of the hosts hemoglobin. As an
estimate, at 20% parasitemia about 110 g of hemoglobin
would be consumed by the parasite during 48 hours. The
parasite ingests hemoglobin by the process of pinocytosis
via the cytostome [14]. Ingested hemoglobin is degraded
inside the acidic digestive vacuoles through a specific semi-
ordered sequential process, which involves multiple
proteases [15]. Digestive vacuoles in the parasite are acidic
organelles with a pH estimated at 5.0-5.4 [16]. Different
aspartic proteases (plasmepsins), cysteine proteases
(falcipains) and a metalloprotease (falsilysin) have been
characterized from the digestive vacuoles of P. falciparum
[17]. Both plasmepsin I and plasmesin II are able to cleave
native non-denatured hemoglobin [18]. The degradation of
hemoglobin is initiated by plasmepsin I, which hydrolyses
the 33Phe-34Leu bond in the hinge region, that appears to
be vital for integrity of the hemoglobin tetramer. Eight
additional plasmepsin genes have been identified in the P.
falciparum genome. A histo-aspartate protease (HAP) and
plasmepsin IV are homologous to plasmepsin I and II. HAP
and plasmepsin IV were also found to be active in P.
falciparum food vacuole and may be the additional aspartate
proteases involved in hemoglobin digestion [18]. As soon as
Tekwani and Walker
the hemoglobin molecule is relaxed the plasmepsins (I, II)
and the falcipains can further cleave at other sites of the
protein. Four genes coding different falcipains have been
identified in P. falciparum genome. Falcipain 2 and
possibly falcipain 3 have been identified as a major
trophozoite food vacuole cystein proteases [19]. Multiple
orthologs of the falcipains have been identified in other
plasmodial species. The corresponding cysteine proteases
identified in P. vivax have been termed as vivapain 2 and
vivapain 3 [20]. Falcipain 2 and falcipain 3 localize to the
food vacuole, are active at acidic pH of the food vacuole, and
readily hydrolyze native and denatured hemoglobin [19]. P.
falciparum contains two nearly identical copies of the
falcipain 2 gene. Falcipain 2 and 3 are 66.6% identical in
their catalytic domains, but they differ in stage specificity,
with maximal expression of falcipain 2 in early trophozoites
and expression of falcipain 3 is more mature parasites [19].
A multispecificity protease substrate was used to profile the
proteolytic activities in P. falciparum in a stage dependent
manner using a combination of fluorescence and MALDI
mass spectrometry [21]. Cysteine protease activity was
shown to be dominating at neutral pH, whereas aspartic
protease activity contributed predominantly to the
proteolytic repertoire at acidic pH. Maximum proteolysis
was observed at the trophozoite stage followed by the
schizonts and the rings.
It appears that the actions of plasmepsins and falcipains
within the digestive vacuoles, produce small peptides
instead of amino acids [22]. The resulting globin peptides
serve as the substrate for the zinc metalloprotease, falcilysin
(FLN) [23]. FLN prefers to degrade small globin peptides up
to 20 amino acids in length at pH optimum of 5.2,
consistent with the acidic environment of the food vacuole.
The temporal expression pattern of FLN is similar to that of
other globinases, yet it differs in the absence of proteolytic
processing during biosynthesis. Random peptide substrate
analysis has confirmed that FLN is highly active at acidic
pH, which is consistent with its role in hemoglobin
degradation. However, the enzyme was found to be robustly
active at neutral pH, but with substantially different
substrate specificity. FLN is localized to the parasite food
vacuole and is also associated with the vesicular structures
elsewhere within the parasite. It has been proposed that FLN
may have expanded role beyond globin catabolism and may
function as two different proteases in its two locations [24].
Further hydrolysis of the digested hemoglobin by FLN
action still generates small peptides, not the free amino acids
for utilization by the parasite. As no exopeptidase activity
has been detected in the parasite digestive vacuole, it has
been assumed that these peptides translocate in parasite
cytosol where cytosolic neutral exo-peptidases complete the
digestion of peptides to individual amino acids for their
subsequent utilization for parasite growth and maturation
[22]. A neutral aminopeptidase with apparent molecular
mass of 80+10 kDa has been identified in several species of
malaria parasite [25]. A gene encoding for an aminopeptidase
belonging to the M1 family of zinc-metallopeptidases has
been identified in P. falciparum [26]. The deduced amino
acids sequence corresponds to a 1056 residue protein with a
predicted molecular mass of 122 kDa. The antisera raised
against a peptide encoded by this gene labelled two schizont
proteins with molecular mass of 96 and 68 kDa, which may
Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery
be the processed forms of the aminopeptidase. The protein
purified from extracts of P. falciparum schizont culture
displayed aminopeptidase activity. The mechanism of
translocation of small peptides to the parasite cytoplasm is
still not clear. It has been proposed that Pfmdr 1, which has
been localized to the food vacuole membrane and is also a
member of ATP-binding cassette transporter family, might
be involved in pumping out the peptides from the food
vacuoles into the cytoplasm [27].
Massive degradation of hemoglobin by the parasite
generates large amount of free heme as a toxic by-product.
As soon as heme is free from its protein scaffold it attains
toxic characteristics [28]. The biochemical consequences of
accumulation of non-sequestered heme include membrane
lysis and inhibition of parasitic proteases [29-31].
3. HEME DETOXIFICATION PATHWAYS OF THE
MALARIA PARASITE
The intra-erythrocytic asexual stage of the malaria
parasite resides in environment rich in hemoglobin and is
exposed to toxic amounts of heme when hemoglobin
undergoes degradation, as occurs spontaneously in hemolytic
anemia with the production of Heinz bodies [32]. It was
shown that concentrations of heme as low as 20 µM will
lyse malaria parasites within 10 minutes; this amount of
heme can be produced by the destruction of only 0.1% of
total hemoglobin present in erythrocytes [33]. The
trophozoite stage of malaria parasite consumes almost 75%
hemoglobin of erythrocyte, with concomitant release of free
heme. With heme as a prosthetic group of hemoglobin, the
iron is in the ferrous state, but once heme is free from its
protein scaffold it tends to loose one electron and assumes
the ferric state [34]. This ferric heme could promote
membrane damage via its peroxidative properties [35] and
may also interfere with the hemoglobin degradation
pathway. The cysteine protease, falcipain has been shown to
be very sensitive to free heme [6, 36].
To avoid the damaging properties of free heme it is
necessary for the parasite to convert it to non toxic
metabolites. Several properties of hemozoin, which is
structurally similar to β-hematin, make it an ideal excretory
product of heme detoxification. First it is dense and
insoluble under physiological conditions and this may be an
irreversible link between hemoglobin degradation and release
of heme. Due to the coordinated nature of heme in, β-
hematin it does not contribute to oxygen radical stress and
auto-oxidation.
Detoxification of heme occurs through different
pathways. The most important and the predominant
mechanism is sequestration of heme into “hemozoin”, also
known as the malaria pigment. Heme is incorporated into a
crystalline black brown pigment which is accumulated
within the parasite food vacuole. Hemozoin may be
visualized as regular fibrillar crystals by electron microscopy
[37]. Appearance of hemozoin crystals may be used as the
evidence for presence of the malaria parasite within blood
and may have importance in diagnosis of the disease [38].
After schizont maturation when the erythrocyte ruptures,
hemozoin is released into the circulation. It is initially
ingested by macrophages, where it appears to diminish
Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1 65
phagocytic capacity and also alter cytokine secretion
pathways. Hemozoin has been shown as a potent
proinflammatory agent in vivo, which could contribute to the
immunopathology related to malaria [39]. The hemozoin is
predominantly accumulated in liver and spleen of the host,
where amount of hemozoin increases with the progress of
parasitaemia [40]. The molecular structure and mechanism of
hemozoin synthesis have been discussed in more details in
the subsequent sections.
Other pathways proposed for detoxification involve non-
enzymatic [41, 42] as well as enzymatic [43] degradation of
heme to non-toxic metabolites. Figure 1 illustrates different
pathways suggested for detoxification of heme by the
malaria parasite. As an estimate only about one third of the
heme released due to hemoglobin degradation may be
sequestered as hemozoin. The remainder may be detoxified
by alternate pathways [41-43]. A possible route has been
proposed through degradation of heme by a non-enzymatic
hydrogen peroxide mediated process (Fig. 1, pathway 2)
which may occur within the parasite food vacuole. When
oxyhemoglobin is released into the food vacuole of the
parasite at pH 5.2, it is rapidly converted to methemoglobin.
Release of an electron and oxygen as a result of this reaction
generate superoxide anion. The superoxide anion will
dismutate to hydrogen peroxide at pH 5.0. The food
vacuoles may be transiently protected from the oxidative
stress by the antioxidant enzymes viz., superoxide dismutase
and glutathione reductase, acquired from the erythrocyte
cytoplasm. These enzymes may be rapidly degraded by
vacuolar proteases and the lipids and proteins within the
food vacuoles would be susceptible to the oxidative damage.
Free heme may react with hydrogene peroxide displaying
both catalase and peroxidase-like activities that regenerate the
heme molecule. Alternatively, heme may also undergo
peroxidative reaction that causes destruction of porphyrin
ring. It has been shown that under the physiological
conditions similar to that of the parasite food vacuole, free
heme undergoes rapid peroxidative decomposition [41].
Another non-enzymatic mechanism proposed for degradation
and detoxification heme is through its glutathione mediated
degradation [42]. Presence of heme oxygenase, which
converts heme to biliverdine, has been reported in the rodent
species of malaria parasites (43), but no heme oxygenase has
been found in P. falciparum. Glutathione-mediated and
enzymatic degradation have been proposed to occur out side
the parasite food vacuole. However, no evidence has been
given for translocation of the heme across the food vacuoles.
Part of the free heme has been found bound to the parasite
membranes. Possibly, both hemozoin synthesis as well as
heme degradative pathways operate simultaneously in the
malaria parasite and are inhibited by the antimalarials known
to interfere with heme detoxification functions of the
parasite. Egan et al. [44] have shown that 92% of the iron
present in P. falciparum infected erythrocytes was located
within the parasite food vacuole and 88% of this was in the
form of hemozoin. 57Fe-Mossbauer spectroscopy shows that
hemozoin was the only detectable iron species in
trophozoites, these findings were further confirmed by
electronic microscopic imaging [44]. Hemozoin synthesis
pathway may be of greater interest for new antimalarial drug
discovery due to its uniqueness.
66 Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1 Tekwani and Walker

Fig. (1). Different heme detoxification pathways suggested in malaria parasite. Pathway (1) Biocrystallization of heme to hemozoin,
an insoluble black brown pigment, is the most predominant mechanism of heme detoxification [44]; Pathway (2) Peroxidative
degradation of heme [41]- Under acidic conditions of food vacuoles FP-Fe(II), released from hemoglobine is instantly oxidized to Fe-
(III). Transfer of electron to oxygen generates a superoxide radical, which is spontaneously converted to hydrogen peroxide.
Peroxidative cleavage reaction destroys the porphyrine ring; Pathway (3) Glutathione mediated heme degradation [42]; Pathway(4)
Enzymatic degradation of heme to bilirubin [43]. The later two mechanisms require translocation of free heme from digestive vacuole
to the cytosol, which is yet to be established.

4. MOLECULAR CHARACTERISTICS OF the structure of hemozoin. The IR spectrum of hemozoin

HEMOZOIN: AN ORDERED CRYSTALLINE was significantly different from that of hematin or heme and
STRUCTURE contained additional peaks at 1664 and 1210 cm-1, a
characteristic of Fe-carboxylate bond. Solubility properties
Hemozoin is not identical to hematin
of hemozoin are similar to a derivative of heme called β-
(Ferriprotoporphyrin IX hydroxide), as was speculated
hematin. Hemozoin and β-hematin show similar elemental
initially [45] since they show very distinct solubility
composition, infrared spectra, and also extended x-ray
properties. Hemozoin is insoluble in weakly basic
absorption fine structure spectra [46]. The x-ray powder
bicarbonate buffer and aprotic solvents such as dimethyl
diffraction pattern obtained from β-hematin was also
sulfoxide and pyridine [46]. In contrast hematin is readily
indistinguishable from that of hemozoin [46]. Laser Raman
solubilized in both types of solvents, either by solvation of
spectroscopy, which has been used as a powerful technique
carboxylate side chains or by solvent coordination with the
for elucidation of both structural and functional information
ferric iron. It was also proposed that hemozoin is composed
on heme and other metalloporphyrins [50], also showed that
of some parasite-derived proteins or peptides, which might
the spectrum of hemozoin is identical to the spectrum of β-
have a role in the sequestration of heme [47, 48]. However
hematin at all applied excitation wavelengths [51]. An
hemozoin is resistant to non-specific proteolysis and its
unexpected observation of dramatic band enhancement at the
elemental composition was found to be very similar to heme
excitation wavelength of 780 nm could be employed for
[46, 49]. Proteins associated with hemozoin might have
Raman imaging of hemozoin in the parasite food vacuole
bound to the pigment non-specifically during the process of
using intact P. falciparum infected erythrocytes. This
isolation. Hemozoin isolated from parasite extracts or tissues
technique enabled investigation of hemozoin within its
(liver and spleen) of malaria infected animals employing
natural environment. These observations indicate that
non-denaturing sucrose density gradient exhibit totally
hemozoin and β-hematin both are structurally similar and
different spectrum of proteins associated with it
posses an ordered crystalline structure. Based on solubility
(unpublished observations). Hemozoin isolated from the
characteristics, infra-red (IR) spectroscopy and x-ray
parasite lysate and that accumulated in the liver and spleen
absorption pattern, the crystalline structure of hemozoin or
has shown similar elemental composition, solubility
β-hematin was initially proposed as a polymer of heme [46,
properties and infrared spectroscopic characteristics [40].
49]. Heme (Ferriprotoporphyrin IX, FP IX) units (Fig. 2A)
Slater et al. [46] used infrared spectroscopy, x-ray diffraction
in β-hematin are linked to each other through a coordinate
absorption spectroscopy and chemical synthesis to determine
Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1 67

Fig. (2). (A). Free ferriprotoporphyrin IX, (B). Reciprocal Fe-carboxylate ferriproporphyrin IX dimer, the basic units of β-hematin or
hemozoin, as per the structure suggested by Pagola et al. [53]. (The pictures provided by Dr Simon J. Hocart).

bond, between ferric iron of one unit to propionate carboxyl
group of another [46]. Further, in situ analysis of hemozoin
in intact desiccated malarial trophozoites by synchronized x-
ray powder diffraction supported the polymeric nature of the
malaria pigment [52]. This study showed that two chains of
hemozoin are also associated by intermolecular hydrogen
bonding between uncoordinated carboxyl side chains. A later
study, which used simulated annealing technique to analyze
the powder diffraction data, suggested that the crystals of β-
hematin were constituted of cyclic heme dimers [53]. The
heme units in these dimers are linked to each other through
reciprocal iron-carboxylate bonds (Fig. 2B) and different
dimers are also linked with each other through hydrogen
bonds between remaining carboxylate groups [54] providing
the characteristic crystalline structure to the pigment (Fig.
3A, B). Analysis of phase homogeneity and crystal
morphology of β-hematin confirms the ordered crystalline
structure of the pigment [55].
Synthetic β-hematin formed by different chemical
methods has shown marked morphological differences when
analyzed by scanning electron microscopy [55]. The β-
hematin crystallites formed from the acid precipitation
reaction have few distinguishable faces and have upper
68 Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1
estimate of 0.2 µm for the largest visible crystal dimension.
In contrast, the crystallites resulting from base-mediated
reaction have maximum lengths about 20 fold larger. These
crystallites have more ordered morphology and are of regular
size throughout the samples than those formed under acid
precipitation reaction. The pigment isolated from the human
malaria parasites viz., P. falciparum, P. vivax, P. ovele and
P. malariae, the primate malaria parasite P. knowlesi and
the rodent parasite P. yoelii have a regular, flate-faced
cuboidal morphology with modest size differences as
analyzed by scanning electron microscopy [56]. However,
the hemozoin crystals isolated from the avian malaria
parasite P. gellinaceum exhibit regularly irregular barrel
shape with a waffle surface. Homozoin isolated from
different malaria parasite species also shows variable
susceptibility to degradation by hydrogen peroxide. These
morphological differences have been attributed to the
different biochemical environments within the parasites. The
different shapes and sizes of hemozoin crystals in different
malaria parasites may distinguish diverse malaria species
[56].
5. MECHANISM OF HEMOZOIN FORMATION IN
THE MALARIA PARASITE: PERSISTING DEBATE
This is the most important and intriguing aspect related
to hemozoin synthesis pathway, but is still under extensive
debate. Several theories have been proposed to explain the
mechanism of hemozoin formation by the malaria parasite.
The concept of mechanism of hemozoin synthesis has been
shifting along with the more accurate understanding of the
structure of the hemozoin. The very early idea of formation
of the malaria pigment was simply the association &
sequestration of free heme, released as a result of
hemoglobin degradation, with a protein (either a partially
digested peptide derived from hemoglobin or a parasite
derived protein) [47]. Later, when hemozoin was
characterized as a heme polymer [46], the proposal was made
of an enzymatic reaction termed as ‘heme polymerization’
and a parasite derived proteinaceous factor named “heme
polymerase” was proposed to catalyze this reaction [57, 58].
Formation of a product similar to hemozoin could be
demonstrated in the presence of various factors related and
unrelated to malaria parasites and based on these reports
different mechanisms of formation of malaria pigment have
been proposed. The reports demonstrating in vitro synthesis
of β-hematin may be mainly categorized into four groups
viz., chemical synthesis and spontaneous formation of β-
hematin; role of different proteins in initiation of β-hematin
formation; formation of hemozoin by autocatalysis and
finally initiation of heme polymerisation by different lipids.
5.1. Chemical Formation of β-Hematin: A Non-
Physiological Process
High resolution X-ray crystallographic studies and other
analysis have conclusively proved that hemozoin (the
pigment isolated from the malaria parasite lysate) and β-
hematin (the synthetic pigment) are identical [46, 53]. This
has facilitated the investigations on understanding the
mechanism of pigment formation. The monomeric heme
tends to aggregate in acidic solutions (pH<4.5) as
determined by ultra-centrifugation, 1H NMR, and electron
Tekwani and Walker
absorption spectroscopy [59]. However, these oligomeric
aggregates are different from β-hematin and may be separated
from it by their differential solubility in NaHCO3/Na2CO3
buffer solutions. The micellar aggregates of hematin dissolve
in this solution while β- hematin does not. In aqueous
solutions conversion of heme to β-hematin may be
chemically achieved only under supraphysiological
conditions. Chemical synthesis of β-hematin may be
achieved under aqueous as well as non-aqueous conditions.
Incubation of hematin in glacial acetic acid at 70-80OC for
about 18 hours yields formation of β-hematin. About 40-
50% of hematin is polymerized into β- hematin by this
process. The process may be termed as the thermal
dehydration [59]. β-Hematin has also been prepared
chemically in high yield by abstraction of HCl from hemin
with a non-coordinating base in strictly anhydrous
conditions [59]. Egan et al. [60] proposed formation of β-
hematin in malaria parasite as a spontaneous chemical
reaction. They proposed that formation of β-hematin can
occur spontaneously between 6 and 65 OC in 0.1-4.5M
acetate and pH 4.2-5.0. They also demonstrated that in vitro
synthesis of β-hematin was essentially complete within 30
min at 600C, 2 hours at 37 OC, and 8 days at 6 OC in
absence of any parasitic material, in 4.5 M acetate buffer of
pH 4.8. In low acetate buffer (0.5M) the rate of reaction is
slow and it is 50% complete after 7 days of incubation at 37
OC. In buffers of higher ionic strength the concentration of
solubilized hematin is greater and this may be the reason of
higher reaction rate in high acetate medium. This reaction
was also reported to proceed in a mixture of 0.1M glutamate
and 0.4 M glycine, pH 4.5. By mossbaur spectroscopy, this
reaction was determined as zero order, instead of
autocatalytic [61]. Incorporation of β-hematin, in reaction
mixture does not show any effect on the amount of
hemozoin formation. The products formed under above
conditions were characterized by IR spectroscopy, but were
not washed with the solvents, such as bicarbonate buffer and
2.5% SDS or DMSO, which distinguish heme aggregates
and β-hematin. However, formation of β-hematin does not
occur spontaneously under physiological conditions, similar
to those prevailing within the malarial digestive vacuoles
[62]. Later it was reported that, β-hematin can be
spontaneously synthesized, if monomeric hematin is allowed
to incubate for several days at 37 OC at pH-4.8, without any
parasitic material. This product was characterized by washing
with bicarbonate buffer and by IR spectra [63]. Still, these
studies could not explain that how synthesis of a material
identical to β-hematin occurs so efficiently in the malaria
parasite under mild physiological conditions. The parasite
digestive vacuole, the physiological site for hemozoin
synthesis, has acidic pH, along with presence of organic
acids, lipids and proteins. In vitro formation of β-hematin
under aqueous acidic conditions and physiological
temperature may be achieved only in the presence of some
biological factors viz., the malaria parasite lysate, organic
solvent extracts of the parasite lysate, histidine rich protein
II or III, preformed hemozoin or β-hematin and also some
unsaturated lipids [9, 10, 63-66]. These in vitro methods of
β-hematin formation, which simulate physiological
environment of the parasite digestive vacuole, along with
other cell biological studies have provided useful tools for
gaining insight into this unique function of malaria parasite
Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery
and understanding the physiological mechanism
hemozoin biogenesis.
5.2. Lipids Catalyzed β-Hematin Formation:
Physiochemical Oxidative Mechanism
Hemozoin biosynthesis within the digestive vacuoles of
the malaria parasite may be a lipid-catalyzed physiochemical
reaction. Fractionation studies suggest that lipid is the only
active substance in malaria erythrocyte which may catalyze
β-hematin in vitro. All the factors promoting β-hematin are
recovered in the chloroform extract of P. berghei infected
erythrocytes and none is in the remaining dilapidated
hemozoin or protein fractions [9]. The factors catalyzing β-
hematin formation are also recovered into the acetonitrile
extract of the P. falciparum [67]. The acetonitrile extract
catalyzes β-hematin formation in the absence of any pre-
formed hemozoin. The acetonitrile extract from synthetic β-
hematin prepared from normal erythrocytes extracts,
trophozoite lysate and the purified malarial hemozoin also
promote β-hematin formation in vitro [65, 68]. The factors
catalyzing β-hematin formation in acetonitrile extract of P.
falciparum have been identified as methyl esters of oleic,
palmitic and stearic acids. The acetonitrile extract of normal
erythrocytes also promotes β-hemetin formation [68]. The
phospholipids viz., phosphatidyinositol, phosphatidyl
serine, sphingomyelin and phospatidyl choline also catalyze
β-hematin formation [63]. The commercially available
unsaturated fatty acids viz., arachidonic acid, linoleic acid,
oleic acid and palmitoleic acids, and 1-mono and
dioleoylglycerol are able to promote β-hematin formation [9,
66]. Tri-oleoylglycerol, cholesterol, dioleoyl
phosphatidylethanolamine and saturated fatty acids viz.,
stearic acid and palmitic acid do not promote this reaction.
The unsaturated fatty acids and their mono- and diglycerides
serve to concentrate monomeric heme and keep it in a
favorable state for dimerization [9]. In vitro β-hematin
formation promoted by Plasmodium yoelii homogenate, the
lipid extracts, and linoleic acid were blocked by ascorbic
acid, reduced glutathione, sodium dithionite, beta-
mercaptoethanol, dithiothreitol, and superoxide dismutase
[69]. Oxidized glutathione did not show any effect.
Preoxidized preparations of the lipids extracts or the P.
yoelii homogenate failed to catalyze β-hematin formation.
Depletion of oxygen in the reaction mixtures also inhibited
the lipid-catalyzed β-hematin formation. β-Hematin
formation was also inhibited by p-aminophenol, a free
radical chain reaction breaker. An oxidative mechanism has
been proposed for lipid-mediated β-hematin formation,
which may be mediated by generation of some free radical
intermediates of heme [69]. These reports suggest that the
factors responsible for hemozoin formation are not specific
to malaria parasite. Leinoleic acid or the derivatives of
linoleate may be the principal lipids that catalyze β-hematin
formation within the digestive vacuoles as the parasite is
rich in linoleic acid which may be derived from inner
membranes of hemoglobin laden endocytic vesicles [9, 70].
Heme released as a result of hemoglobin degradation
immediately co-precipitate with the unsaturated lipids. This
process and the microenvironment within the parasite
digestive vacuoles or the endocytic vesicles produce the
necessary physiochemical environment for dimerization of
heme and the unsaturated lipids undergo oxidation as a
Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1 69
of result of this process. Accumulation of oxidative products of
polyunsaturated lipids further supports this hypothesis [9].
A 5.3. Histidine Rich Proteins : The Nucleation Sites for β-
Hematin Formation
A unique class of malarial histidine rich proteins (HRP)
with characteristic multiple tripeptide (ala-his-his) repeats
have been found to be involved in heme detoxification
function of malaria parasite [64]. Screening of a P.
falciparum lambda gt11 cDNA expression library with the
antiserum against hemozoin, identified the clones of HRP II
which was subsequently found to initiate formation of β-
hemetin crystals in vitro at acidic pH [64]. HRP III, the
analog of HRPII, also catalyzes similar reaction. The HRP II
and HRP III consist of repetitive hexa-peptide sequences
containing histidine and alanine pairs. Ala-His-His-Ala-Ala-
Asp, is the major repeat hexa-peptide in Pf HRP II, which
appears 30 times. Pf HRP II also contains 18 repeats of tri-
peptide, Ala-His-His. HRP III contains two distinct regions
of tandem repeats separated by a gap of 26 amino acids. The
first region contains three repeats of Ala-His-His, two of
Ala-His- His-Val-Ala-Asp and 13 repeats of Ala-His-His-
Ala-Ala-Asn and one Ala-His-His-Ala-Ala-Asp. Second
repeat unit in Pf HRP III is Asp-(Asp/Gly)-Ala-His-His,
which is different from Pf HRP II. Both proteins contain a
hydrophobic leader peptide, which may have some role in
translocation of these proteins [71]. The hexa-peptide repeat
has been identified as the heme binding motif in HRPII
[72]. However linear monomers, dimmers and trimers of the
hexapiptide (Ala-His-His-Ala-Ala-Asp) failed to promote β-
hematin formation. They rather inhibited the reaction,
probably through competing for heme [72]. In an effort to
understand the mechanism of β-hematin formation by HRP
II it was demonstrated that some peptide dendrimers,
designed on the basis of tandem tripeptide (ala-his-his)
repeat motif of PfHRPII, were able to bind heme and also
promoted β-hematin formation in vitro. Tandem
organization of tripeptide, the heme binding site, may be
necessary for initiating formation of β-hematin by PfHRP-II
[73]. Presence of HRPII in P. falciparum digestive vacuole
has been demonstrated by con-focal microscopy [64].
However, major amount of HRP II produced by the malaria
parasite is secreted out of the parasite and even out of the
infected erythrocytes [74]. Immunochemical detection of
HRP II in plasma or serum of the infected patients is used in
diagnosis of malaria [75]. It was assumed that HRP II is
internalized inside the digestive vacuole with the
hemoglobin ingestion, where it binds with the heme released
after hemoglobin degradation and converts it into hemozoin.
In another immunolocalization studies HRP II was mainly
localized in the cytosol of infected erythrocytes. However a
small sub-population of HRP II was co-localized with a food
vacuole associated protein, p-glycoprotein homologue (Pgh-
1) [76]. Both native as well as recombinant Pf HRP II
promote in vitro synthesis of hemozoin, whereas many other
proteins with heme binding characteristics, including human
histidine rich glycoproteins or bovine serum albumin, do
not convert heme to β-hematin [64]. It has been recently
hypothesized that HRPII initiates formation of β-hematin as
a probable nucleation site, and the further growth of
hemozoin crystal involves unsaturated lipids [77]. However,
some researchers failed to observe a synergistic action of
HRPII and mono-oleoyl glycerol in formation of hemozoin
70 Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1
[9]. Further investigations are needed to ascertain the role of
HRP in hemozoin synthesis.
5.4. A Cytostomal Pathway of Hemozoin Biogenesis:
Role of Inner Membrane of Endocytic Vesicles
Hampelmann et al. [78] have recently proposed a unique
cytostomal pathway of bio-crystallization of heme to form
hemozoin. The pathways is based on the perception that a
nucleation center or template is essential for initiation of the
Fig. (3). (A). Hemozoin, (malaria pigment) is a crystalline
macromolecule consisting of repeating Fe-O reciprocal dimers
linked through van der Waals and hydrogen bonding
interactions [53, 54]. (B). A space filling representation of the
hemozoin crystal made in SYBYL from the unit cell of
hemozoin [53], colored for depth to illustrate the grooves on
the growing surface where the interaction are thought to occur
between aminoquinolines and the crystal. (The pictures
provided by Dr Simon J. Hocart).
Tekwani and Walker
crystal formation [64, 65], which may subsequently grow
further through an auto catalytic mechanism [65].
Intraerythrocytic schizogony of malaria parasite and
ingestion of hemoglobin exhibit a unique cell biological
characteristic. Malaria parasite ingests host cell cytoplasm
through cytostome, a unique mouth like sub-cellular
structure [79]. This process results into formation of unique
double membrane transport vesicles, which ultimately fuse
with the parasite food vacuole and act as the carriers for the
transport of host derived nutrients, mainly hemoglobin [80].
This hypothesis considers that non-selective nutrient
permeable channels present on parasitophorous vacuolar
membrane (PVM) [81] generate an acidic environment
within the transport vesicles and digestion of hemoglobin is
initiated in these vesicles, even before they are fused with
the parasite digestive vacuole [79]. The lipids and proteins
present in PVM, which is the inner transport vesicle
membrane, provide the nucleation site for initiation of
hemozoin crystal formation. The electron microscopic
observation that hemozoin crystals present in side the
parasite digestive vacuoles are associated with membrane
fragments that are distinct from the food vacuole membrane
provided supporting evidence in favor of this hypothesis
[78]. Ultrastructural analysis of P. falciparum has shown
presence of hemozoin in digestive vacuoles as well as small
vesicles of the parasite. An earlier observation by Orjih [82]
that normal erythrocyte ghosts treated with NaOH or HCl or
acetic acid, termed as activated membranes, convert 70% of
heme to β-hematin in a cell free reaction system, may also
be presented in support of this hypothesis. The cytostomal
pathway involving lipids-catalyzed mechanism presents a
convincing hypothesis for biogenesis of hemozoin. Release
of unsaturated lipids from the inners membranes of
hemoglobin laden transport vesicles results in co-
precipitation of heme and its dimerization. Formation of
hemozoin in axenically grown malaria parasite trophozoites,
without erythrocytic membranes poses a question against
this hypothesis [83]. The medium used in axenic cultivation
of malaria parasite contains erythrocyte sonicate. The
erythrocyte membranes ingested from the medium may
provide the template and also generate the lipids necessary
for hemozoin formation. More conclusive experimental
evidences would be required to convince formation of
hemozoin through this interesting and unique mechanism.
5.5. Biomineralization or Biocrystallization Mechanism
Soon after rediscovery of structure of hemozoin as a
heme dimer [53, 55], and confirmation of hemozoin
formation as a non-enzymatic but still a biocatalytic reaction
[10, 12, 62], the process of hemozoin formation in malaria
parasite was redefined as biomineralization [84] or
biocrystallinzation [85]. The crystalline structure of
hemozoin (heme dimers crystallized through intermolecular
hydrogen bonding) (Fig. 3) may be compared with the ice
and sugar crystals. Hemozoin was therefore also termed as a
bio-crystal [12]. Egan et al. in a controlled study on
mechanism of β-hematin formation determined that the
process occurs via rapid precipitation of amorphous hematin,
followed by its slow conversion to the crystalline β-hematin.
They also showed that formation of iron-carboxylate bond
(generation of heme dimers) and formation of crystalline β-
hematin through intermolecular hydrogen bonding, are
Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery
essentially simultaneous [84]. These authors concluded that
formation of β-hematin parallels many biomineralization
processes and also suggested that hemozoin synthesis in
malaria parasite may also be a unique biomineralization
process. Biomineralization describes the deposition of
minerals within or outside the cells of living organisms
[86]. Biomineralization is the process by which
microorganisms mediate and catalyze inorganic reactions so
as to form new mineral assemblages. The goal of
biomineralization as a remediation tool is to control
microbial processes and their environment so as to sequester
and immobilize specific heavy metals within exceptionally
strong bonds of neo-formed mineral assemblages. In this
way, the bound metals are no longer mobile within a
geological time-scale. Formation of β-hematin though fits
well in this definition but biomineralization refers mainly to
the formation of inorganic salts under controlled biological
conditions. Definitive role of proteins and/or lipids has been
suggested in hemozoin biosynthesis and the process seem to
follow at least two step mechanism i.e initiation or
nucleation of hemozoin formation followed by growth of the
biocrystal. Biocrystallization may be suggested as more
appropriate term to describe the hemozoin formation in
malaria [78]. Biocrystallization, like any other
crystallizations, is a process involving the steps of
nucleation and growth, where molecules have to be brought
into a supersaturated, dynamically unstable state [87].
Nucleation is a prerequisite and the first step to crystal
growth, yet excess nucleation causes the production of a
large number of small crystals instead of a smaller number of
useful ones. Variation in the size and shape of hemozoin
crystals isolated from different species of malaria parasite has
been attributed to variable intracellular environment [56].
Significant advancements have been in determining
chemical and molecular structure of the malaria pigments.
Similarly, considerable knowledge has been generated with
regard to determination of optimal physical, chemical and
biochemical conditions and the microenvironment necessary
for formation of β-hematin, particularly simulating the
biochemical environment of the physiological site of
hemozoin synthesis and accumulation. However, exact
mechanism of hemozoin formation within the parasite
digestive vacuoles and pin pointed role of specific parasite
and/or host derived proteins and/or lipids in initiation and
growth of hemozoin crystals are yet to be determined.
6. EXPERIMENTAL APPROACHES FOR IN VITRO
β-HEMATIN FORMATION ASSAY: APPLICATION
IN HIGH THROUGHPUT SCREENING
Several experimental approaches have been describes for
determination of formation of β-hematin in vitro and
evaluation of different antimalarials as well as other test
compounds for inhibition of this reaction. The important
difference between monomeric heme and β-hematin is their
differential solubility in organic and aprotic solvents and
also in SDS and mildly alkaline bicarbonate solutions [46,
49]. This property has been used for isolation and
quantification of hemozoin from the malaria parasite lysate
[46], tissues of malaria infected animals [40] and also in in
vitro β-hematin formation assays. Various groups have
employed different methods for the assay of β-hematin
Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1 71
formation. The major variation in hemozoin formation
assays performed by various labs has been at the level of use
of different reaction cocktails including the different catalytic
factors in the assay and also at the level of processing of
assay mixtures at the end of the reaction for quantification of
β-hematin. Spectrophotometric [88-94], radioisotopic [95],
fluorometric [96] and high performance liquid
chromatography (HPLC)–based [97] assays have been
described for quantification of β-hematin. Monomeric heme
and hemozoin/β-hematin may also be differentiated by
fourier transformed infra-red (FT-IR) spectroscopy [46]. Use
of quantitative FT-IR has been also shown in determination
of β-hematin [60]. Formation of hemozoin occurs inside the
digestive vacuole of malarial trophozoite which has an acidic
environment. Heme tends to aggregate under acidic pH. The
aggregates of heme can be separated from β-hematin by their
differential solubility in alkaline bicarbonate solutions [46].
The miceller aggregates of heme dissolve in this solution
while β-hematin does not. For assay of β-hematin formation,
these differential physical and chemical characteristics of
monomeric heme/heme aggregates and β-hematin may be
utilized. Most of the assays require separation of monomeric
heme and β-hematin for quantification. Estimation and
quantification of hemozoin or β-hematin in presence of
monomeric heme or the heme aggregates has also been
described recently [98]. Different protocols employed for β-
hematin formation assays and their applicability for routine
and high throughput screening have been discussed.
6.1. Necessary Physicochemical Conditions for In Vitro
β-Hematin Formation Assays
Formation of β-hematin in vitro may be achieved in
several ways. The in vitro β-hematin formation assays are
founded on a basic protocol which involves incubation of
monomeric heme (hemin HCl or hematin) in an appropriate
buffer at acidic pH (pH 3.0 -5.5). Formation of β-hematin is
initiated by addition of an appropriate nucleation or a
catalytic factor. The test compounds are also added to the
reaction mixtures. After incubation for 12-24 hours the
reactions are processed in different ways and the resulting β-
hematin is quantified by different methods. Most of the
protocols use separation of the β-hematin pellet by
centrifugation or filtration and sequential washing of the
pellet with water or Tris-HCl/SDS solution and alkaline
bicarbonate solution or DMSO. Dorn et. al., [63] have done
a systematic comparison of different chemical and
biochemical factors which nucleate or initiate or catalyze
formation of β-hematin. The trophozoite lysate mediated
reaction has been found unsuitable for β-hematin formation
inhibition assay due to presence of membranes, which may
bind with the test compounds. Presence of preformed
hemozoin may also compromise with sensitivity of the
assay. The acetonitrile extract of parasite trophozoite lysate
has been found suitable for in vitro screening. Later, some
commercially available unsaturated lipids particularly
unsaturated fatty acids and mono-olyeol glycerol have also
been found more suitable catalytic factors, than the parasite
lipids extracts, for use in these assays [66]. The pH below
5.5 is absolutely essential for formation of β-hematin.. Most
of the assays use the solutions with low salt concentration
particularly 50-200 mM sodium acetate (pH 3.0 -5.5) and
also use a suitable catalytic or nucleating factor for
72 Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1
promoting β-hematin formation. The assays based on
spontaneous formation of β-hematin, the biomineralization
tests, have also been described for in vitro screening and
evaluation of the compounds as inhibitors of β-hematin
synthesis [ 89, 91, 92].
6.2. Spectrophotometric Assays
Different versions of spectrophotometric assay of β-
hematin formation are available. All these assays are based
on differential solubility characteristics of heme and β-
hematin. For spectrophotometric quantification of β-hematin
it is necessary to dissolve the pigment formed during the
reaction. The only solvent, which can dissolve β-hematin, is
NaOH (0.1- 1N) solution. However, this results into
breaking of iron-carboxylate bond and conversion β-hematin
to heme. β-Hematin, therefore may be quantified
spectrophotometrically as heme equivalents. Different assays
vary at the level of use of different reaction cocktails. A low
throughput assay based on the use of lysate of rodent malaria
parasite, P. yoelii, for promoting β-hematin formation has
been described [88]. This assay involves use of
centrifugation for pelleting the heme aggregates and β-
hematin and washing of the pellet sequentially with Tris-
HCl and Sodium bicarbonate/SDS solutions. This assay,
which is performed in test tubes with a total reaction volume
of 1 ml, can not be performed in microplates and is not
suitable for large scale or high throughput screening. Some
laboratories have also used spontaneous chemical formation
of β-hematin, as described by Egan et al. [60]. The
spectrophotometric assay described by Basilico et al. [89]
uses 96-well U bottomed microplates. Each well receives
200 µl of the assay mixture consisting of 400 nmol of heme
(dissolved in 0.1M NaOH), 50 µl of the test compound
(compound:heme ratio between 1:1 to 1:8) and 50 µl of
acetic acid (0.8 mmol). Final pH of the reaction mix is 3.00
and plates are incubated at 37˚C for 24 h. The plates are then
centrifuged at 3300 g for 15 min, the pellet is washed with
DMSO and finally dissolved in 0.1M NaOH. The assay has
been validated with standard antimalarials which are known
to inhibit β−hematin formation. This assay uses non-
physiological pH and is sensitive to chloride and phosphate
salts. The assay was further modified by Parapini et al. [92]
to overcome these short comings. At higher pH (above 6.0)
heme tends to exist predominantly in the form of µ-oxo
dimers. This is likely to prevent formation of iron-
carboxylate bond and formation of β-hematin. Under acidic
pH this equilibrium is shifted to monomeric form, which
facilitates the formation of β-hematin. Use of hemin (heme-
Cl) instead of hematin (heme-OH) which is dissolved in
DMSO in place of NaOH could overcome these problems.
Recovery of β-hematin is significantly increased by keeping
the pH of the reaction mixture at 5.00. However, even the
modified assay [92] may not be suitable for high throughput
screening due to use of multiple centrifugation steps, which
may not be compatible with the automation. The assay was
further modified as “ferriprotoporphyrine IX
biomineralization” assay to make it suitable for application
to HTS [91]. The modified assay, which is performed in
flat-bottomed 96-well microplate, uses the reaction mixture
containing 50 µl of 5 mg/ml hemin-Cl (freshly dissolved in
DMSO), 100 µl of 0.5M Sodium acetate buffer (pH 4.4) and
50 µl of the test compound. This yields final pH of the
Tekwani and Walker
reaction mixture as 5-5.2. It is recommended to adhere to a
particular order of addition of the reagents as hemin-Cl,
acetate buffer and finally the test compound solution. For
estimation of β-hematin, after incubation for 18-24 h at
37οC, all the contents of the master incubation plate are
transferred to a 96-well multiscreen filtration plate with the
use of a multi-channel pipette. The filtration plate is kept on
a vacuum manifold and washed with 200 µl of DMSO. The
β-hematin pellet retained on the filters is dissolved in 0.1N
NaOH. Finally, 150 µl from each well is transferred into a
fresh clear 96-well plate. The plates are read on a microplate
reader at 405 nm for quantification of β-hematin. The
spectrophotometric assays described above do not use any
catalytic or nucleation factor for promoting β-hematin
formation. These assays are based on biomineralization
mechanism of β-hematin formation [84]. Another version of
filtration-based microplate assay has been described recently
by us [94]. The reaction conditions used in this assay are
close to the physiological environment of the parasite
digestive vacuoles, where biogenesis of hemozoin really
occurs. This assay uses lipids for promoting β-hematin
formation. The physiological mechanisms suggesting the
role of lipids, particularly associated with the inner
membranes of the endocytic vesicles, have an edge over
other mechanism suggested for hemozoin biogenesis [10].
Use of assay conditions, which are closer to physiological
environment, are likely to provide better results. Initial
validation of this assay indicates that sequential washing of
the pellet or the retenate on the filters, which contains heme
aggregates as well as β-hematin, with Tris/SDS and alkaline
bicarbonate solutions is necessary for complete removal of
monomeric heme aggregates. No spontaneous formation of
β-hematin occurs when 100 µM of hemin-Cl (dissolved in
0.1N NaOH) suspended in 100 mM acetate buffer (pH 5.0)
is incubated at 37οC for 15-20 h. Both, the pellet of the
purified β-hematin and the pellet containing the mixture of
heme aggregates and β-hematin, show IR spectra
characteristic of β-hematin. Pure preparations of heme and β-
hematin, when analyzed separately, may be differential by
FT-IR. But, IR spectroscopic analysis can not differentiate
between monomeric heme aggregates and β-hematin in a
mixture. Additional analysis by x-ray diffraction absorption
spectroscopy and scanning electron microscopy may be
important [55]. The filtration-based spectrophotometric assay
is directly performed in Millipore Multiscreen® plates. This
reduces an additional step of transfer of the contents from the
reaction plate, as used in biomineralization assay [91].
Reaction mixture, in a total volume of 200 µl, contains
heme (100 µM), acetate buffer (100 mM, pH 5.0), and the
test compounds dissolved in DMSO. The assay may tolerate
up to 5% DMSO. The reaction is initiated by addition of 1
µg of mono-oleoyl glycerol to each well. Use of mono-oleol
glycerol provided more reproducible results than acetonitrile
extracts of the parasite lysate or other unsaturated fatty acids.
The controls without catalytic factor are also set-up. The
plates are incubated at room temperature (24-28C) for 14
hours with constant shaking. At the end of incubation the
plate is put on the filtration manifold and vacuum is applied
to stop the reaction and remove all the soluble contents.
This step also removes the colored compounds or the
extracts, which may interfere with spectrophotometric
quantification of β-hematin. The plate is sequentially washed
with Tris-HCl/SDS solution and alkaline bicarbonate buffer
Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery
pre warmed at 37oC. For dissolving final β-hematin pellet
20 µl of 1N NaOH is added to each well and the plates are
incubated at 37 C for 10 to 30 min. 180 µl Tris-HCl (100
mM, pH 7.4) containing 2.5 % SDS is added to each well.
Dissolution of β-hematin pellet directly in 0.1N NaOH, as
suggested earlier in radiometric assay [95] and also in
spectrophotometric biomineralization assays [89, 91, 92],
may not dissolve the β-hematin pellet completely or would
require extended incubation or extensive vortexing.
Quantification of heme in NaOH/Tris-HCl/SDS solution
provides better sensitivity than in 0.1N NaOH alone [58].
The contents (150 µl) are transferred to a fresh flat bottomed
clear microplate and the plates are read at 405 nm on a
microplate reader for quantification of β-hematin as heme
equivalents. Spectrophotometric assays are robust,
inexpensive and provide sensitivity almost comparable to
the tedious, cumbersome, more expensive radioisotopic and
HPLC based assays. These assays are also suitable for
screening colored and fluorescent compounds and also the
crude extracts, which may normally interfere with
quantification of heme. Several alternate approaches for in
vitro β-hematin formation assays and quantification of β-
hematin have also been discussed to provide comprehensive
information available of this subject and also to present a
comparative picture of different assays.
6.3. Quantitative Infra-Red Spectroscopic Assay
Formation of β-hematin may be achieved spontaneously
under non-physisological conditions (4.5 M sodium acetate
and 60oC) without any nucleating factor or catalytic agents
[60]. Monomeric heme and β-hematin may be differentiated
by their infrared spectroscopic characteristics [46, 60]. IR
spectra of β-hematin show sharp bands at 1660 and 1207
cm-1, which are absent in IR spectra of heme. Based on this
property Egan et al. [60] have described a quantitative FT-IR
assay for evaluation of antimalarial compounds in vitro on
formation of β-hematin. The anti-malarial drugs quinine,
chloroquine and amodiaquine were demonstrated to inhibit
β-hematin formation in this assay. Due to non-physiological
assay conditions used in this assay and also due to
limitation of this assay for processing large number of
samples for IR analysis, the FT-IR assay may not be
applicable for routine large scale screening of the
compounds. However FT-IR analysis may be used to obtain
confirmatory results with the lead compounds selected from
the large scale screening.
6.4. HPLC Assay
The HPLC method described for quantification of heme
and β-hematin and also for β-hematin formation assay, is
also based on their differential solubility characteristics [97].
The assay involves ion-pair reverse chromatography,
utilizing tetramethyammonium chloride (TMAC) and
heptane sulfonic acid (HS) as the ion-pair agents in the
presence of 40% acetonitrile, and is performed on a
polymeric-resin-based column with a phenyl bonded phase.
The reaction mixtures with appropriate concentration of
heme are incubated in 167 mM sodium acetate (pH 5.0) and
P. falciparum lysate. Reaction is terminated by addition of
an equal volume of 10 mM TMAC/10 mM HS/40%
CH3CN/60% water/H3PO4 to pH 2.5 and 200 µl is directly
Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1 73
injected onto the HPLC column. Initial elution of
monomeric heme is obtained with the solvent containing 10
mM TMAC, 10 mM HS, 40% CH3CN, 60% water and
H3PO4 to adjust the pH to 2.5. The peak of monomeric
heme could be detected at 400 nm. Subsequent shift of pH
of the mobile phase to 12.0 converts β-hematin to heme
which may be eluted as another peak. Both remaining
monomeric heme as well as β-hematin formed may be
analyzed in this assay. HPLC assay shows linearity in the
range of 78 pmol to 20 nmol of heme. The HPLC assay
though is highly sensitive and specific but is tedious and
therefore may not be used routinely for screening the
compounds.
6.5. Radioactive 14C-Heme Incorporation Assay
Initial experiments on understanding the mechanism of
hemozoin synthesis by malaria parasite used incorporation of
14C-labelled heme into β-hematin [57, 68, 63]. This assay
also is based on differential solubility characteristics of heme
and β-hematin. A high throughput assay has been developed,
which is based on incorporation of 14C-labelled heme into β-
hematin. The assay uses 96-well filtration microplates and
requires a Wallace 1450 MicroBeta liquid scintillation
counter. The semi-automated high throughput screening
assay uses the Zymark bioassay robot system and has been
adopted from an original heme polymerization assay [63].
The reaction is carried out in 96-well microplates. Each well
receives, in a total volume of 100 µl, 500 mM sodium
acetate (pH 4.8), unlabelled heme (10 pmols), 0.56 nCi of
14C-hemin, acetonitrile extract of P. falciparum trophozoit
lysate (10 µl) and the test compound added as DMSO
solution (10 µl). After overnight incubation at 370C
contents of the plates are transferred into a MultiScreen DV
filtration plate, filtered and the contents of each well are
washed once with 400 µl of the solution containing 100
mM Sodium bicarbonate-0.2% SDS and twice with 200 µl
of Tris-HCl (pH 7.5). Thorough washing of the pellet
retained on the filers is necessary to remove non β-hematin
heme aggregates. Incorporation of 14C-heme into β-hematin
pellet retained on the filters may be monitored by
scintillation counting. This is the only β−hematin formation
assay, which has been truly adopted for high throughput
screening. The assay has been applied for screening of
synthetic compounds libraries and also microbial broths.
Some novel non-quinoline antimalarial pharmacophores have
been identified through this assay. At the time of
development of this assay, the role of lipids in β-hematin
formation was still under investigation. Later studies have
proved that lipids are the major components of acetonitrile
extracts of the parasite lysate [35], which are involved in
promoting β-hematin formation. Use of some commercially
available unsaturated fatty acids has also been demonstrated
as the catalysts for promoting β-hematin formation in vitro.
It is therefore likely that this assay may be further simplified
by use of these commercially available lipids in place of
acetonitrile extracts of the P. falciparum lysate. The
radiometric assay is simple and sensitive and may also be
used for screening the colored as well as fluorescent
compounds. The radioactive assay however is still more
expensive (primarily due to high cost of 14C-heme) than
spectrophotometric assay, involves risk of health hazards due
74 Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1
to use of radioactivity and requires proper disposal of the
radioactive material.
6.6. Biocrystal Growth Assay
The crystal growth assay developed by Sullivan and co-
workers [98] is based on spectrophotometric determination
of growth of β-hematin crystal on pre-formed β-hematin seed
crystals. Synthetic β-hematin used as a seed in this assay is
prepared by incubating heme in 4.5 M acetate solution at
60oC as described earlier [60]. Preparation of a large single
batch of β-hematin has been recommended to minimize the
variation in crystal growth due to quality of the seed.
DMSO concentration in these assays is kept below 1%. This
may limit the use of the assay for testing the compounds,
which are soluble in DMSO, at higher concentrations. The
assay mixture contains 5 nmol of preformed β-hematin and
50 µM bovine hemin chloride, in 0.5 ml of 0.1M
ammonium acetate, pH 4.8 and incubated at 37oC for 12
hours. As in earlier assays, in this assay too quantification
of β-hematin crystal growth is based on differential
solubility of free heme and crystalline β-hematin in sodium
bicarbonate solutions. Another version of this assay [98] is
based on differential spectrophotometric properties of heme
and β-hematin crystals [58]. Large difference in absorbancy
of soluble free heme vs. insoluble crystalline β-hemetin at
400 nm allows quantification of free heme in the reaction
mixture without its separation from β-hematin. The reaction
is terminated by addition of concentrated sodium
bicarbonate/SDS solution and the reaction mixtures are
directly read at 400 nm. The absorbance of this mixture is
directly proportional to quantity of remaining free heme,
which is readily soluble in SDS/bicarbonate solution, while
β-hematin remains insoluble and does not interfere with
determination of free heme. The reaction mixtures are again
read at 400 nm after addition of 10N NaOH, which results
into decrystallization of β-hematin back to heme. For high
throughput crystal growth inhibition test the assay may be
performed in 24 well micro-plates with total reaction
mixture volume of 250 µl. The remaining monomeric heme
may be quantified after addition of 0.5 mL of concentrated
sodium bicarbonate/SDS solution. This assay involves no
centrifugation and no changing of plates or tubes. The assay
has been validated with several quinoline and non-quinoline
antimalarials which are known to act through inhibition of
hemozoin synthesis and has been found to be suitable for
high throughput screening. Performance of this assay with
use of other nucleating agents is yet to be determined. For
high throughput screening the assay may still require
validation in 96 well microplates. This assay is unsuitable
for evaluation of test compounds which show significant
absorbance at 400 nm. Sensitivity of this assay may be
compromised due to use of preformed β-hematin. Quantity
of the substrate (heme) and β-hematin should be limited to
keep the final absorbance of the reaction mixture within the
scale of the reader.
7. HEMOZOIN: THE ANTIMALARIAL DRUG
TARGET
Synthesis of hemozoin by the malaria parasite as a non-
toxic metabolite of heme detoxification process is the most
distinct characteristic of the parasite. Malaria pigment was
Tekwani and Walker
discovered even before the discovery of the parasite [99].
Even though several questions, regarding exact mechanism
of hemozoin synthesis by the malaria parasite, still remain
unanswered, convincing evidences have been produced on
inhibition of heme detoxifications functions of the malaria
parasite as a target for action of most of the blood
schizontocidal antimalarials, which are currently in clinical
use [8, 11, 12]. Inhibition of heme detoxification function of
the malaria parasite would lead to accumulation of toxic
heme which would kill the parasite due to membrane lysis
and interference with other vital functions of the parasite [6,
35, 36]. Quinoline antimalarials have been found to be the
most consistent inhibitors of hemozoin synthesis. Several
novel antimalarials also have been found to interfere with the
process of hemozoin formation and some inhibitors of
hemozoin synthesis are being perused as novel lead
antimalarial structures [95] (Fig. 4).
Quinine, the oldest known antimalarial, led to the
discovery of chloroquine, the most widely used antimalarial
drug almost for more than half a century. Chloroquine
though has lost its importance as an antimalarial due to
widely spread emergence of drug resistance [1]. Quinolines
however are still considered as important lead structures and
several labs are working on synthesis of the analogs [100-
102]. Several theories have been proposed for the mechanism
of antimalarial action of chloroquine and related
antimalarials and also have been the focus of discussion of
some recent reviews [103-105]. Interaction of quinolines
with heme has been extensively studied and well established
[106, 107]. Quinolines interact and form a non-covalent
complex with heme and also inhibit the synthesis of
hemozoin. Chloroquine binds non covalently to the growing
face of hemozoin crystal and prevents its further growth
[108, 109] (Fig. 5). A chloroquine induced masking of a
lipid, which promotes hemozoin formation, has also been
suggested as a mechanism of inhibition of hemozoin
synthesis by this drug [110]. Other blood schizontocidal
antimalarial drugs viz., quinine, quinidine, halofantrine, and
mefloquine also inhibit this process [90]. A significant
correlation has been established between antimalarial efficacy
and inhibition of β-hematin formation by these
antimalarials. Incubation of chloroquine with the purified
hemozoin, isolated from the tissues of malaria infected mice,
also causes its dissolution [111]. Primaquine, an 8-
aminoquinoline, which primarily kills malaria parasite
growing in hepatic cells, does not inhibit β-hematin
formation. However, some 8-aminoquinoline antimalarials
including tafenoquine, which also show prominent blood
schizontocidal action, inhibit this process [112].
Bisquinoline analogs (1, 2) have been found to be the potent
inhibitors of β-hemetin formation and also active against
both chloroquine resistant and susceptible strains of P.
falciparum. Therefore, blood schizontocidal antimalarial
action of quinolines may be attributed primarily to their
interaction with heme as well as hemozoin and subsequent
inhibition of hemozoin synthesis.
Artemisinin, which was originally isolated from a
Chinese herb Artemisia annua, and its first generation
derivatives viz., arteether, artemether and sodium artesunate
have provided useful alternatives for the treatment of drug
resistant and complicated cases of malaria [113]. Artemisinin
has been shown to target different parasite functions.
Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1 75

Cl
Cl

N
HN N
HN
N NH HN N

N (CH2)n
Ro 47-7737 (1) N
N
O Bis(4-(4-diethylamino-1-methylbutyl)aminoquinoline (2)

HO N
HN NH
O (CH2) 5 O
HO O OH H2 N NH2
Cl
OH OH Pentamidine (5)

HO
2,3,4,5,6-pentahydroxyxanthone (X5) (3) Clotrimazole (4)

OH

N N

N M N N M N
OH
N N
N HO2C HO
HO2C

Cyproheptadine (6) CO2H CO2 H

Metalloporphyrin IX (7) Metallodueteroporphyrin IX (8)
O
OH
O NC
HO OH
NC
O
HO
NC
O Axisonitrile-3 (11)
Diisocyanoadociane (10)
Ellagic acid (9) Cl
N N
O
HN
HO
N N N

O
Cl
OH Cl
Benzophenone (Ro 22-8014) (13)
Triarylcarbinol (Ro 06-9075 (12)
Fig. (4). Some novel inhibitors of malarial heme detoxification functions and antimalarial pharmacophores.

However, fast and selective antimalarial action of these drugs
has been primarily attributed to the ferrous iron/heme
induced bioactivation of the enoperoxide bridge and
formation of carbon-centered cytotoxic radicals. The reactive
intermediates further cause alkylation of biomolecules such
as heme and other target proteins or enzymes leading to
death of the parasite. Uptake studies using 14C-labelled
artemisinin have shown that the drug taken up by the
malaria parasite is concentrated in hemozoin [114]. Covalent
binding of artemisinin with heme in hemozoin has also been
suggested. Artemisinin has shown inhibition of β-hematin
formation in vitro in some parasite lysate and lipids-based in
vitro assays at high concentrations [115] and subsequent
inhibition of hemoglobin digestion in the malaria parasite.
However, artemisinin and some analogs failed to show
inhibition of β-hematin formation in in vitro
biomineralization assays [116]. The β-hematin formation
assays for evaluation artemisinin and analogs require
additional washing of the β-hemetin pellet with ethanol (90).
The role of heme iron in bioactivation of endoperoxide
atimalarials and hemozoin as the primary target for their
antimalarial action is still under debate.
Hydroxyxanthones have been identified as a novel class
of antimalarial compounds [117]. A xanthone analog, 2, 3,
4, 5, 6, -pentahydroxyxantone (3) has shown antimalarial
action against chloroquine-sensitive and multidrug-resistant
76 Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1
strains of P. falciparum in vitro. A recent study has also
demonstrated antimalarial activity of hydroxyxanthones in
vivo in P. berghei/mouse model [118]. The antimalarial
potency of hydroxyxanthones correlates with their ability to
inhibit β-hematin formation in vitro. Hydroxyxanthones
form soluble complexes with heme and prevent the
precipitation of heme in aqueous solution at acidic pH of
5.2. A structure-activity relationship study has shown that
carboxy-oxygen, the two aromatic systems, 4- and 5-
hydroxyl groups on these hydroxyxanthones are important
for their interaction with heme and inhibition of β-hematin
formation [119].
Fig. (5). The suggested mechanism of inhibition hemozoin
crystal growth by quinoline antimalarials. The mode of binding
or interaction between hemozoin and chloroquine (CQ) is
illustrated with CQ sitting on the growing face of hemozoin
[108, 109].(The pictures provided by Dr Simon J. Hocart).
Clotrimazole (4), an imidazole containing antifungal
drug, has also shown promising antimalarial activity in vitro
[120]. The drug primarily acts by causing changes to the
calcium ion flux [121]. Clotrimazole has a relatively high
Tekwani and Walker
affinity for binding with heme and also inhibits heme
detoxification functions of malaria parasite [122].
Clotrimazole also enhances heme induced membrane damage
and inhibits β-hematin formation [123]. Some other
antifungal azoles viz., ketoconozole and miconazole, which
show significantly lower antimalarial activity as compared to
clotrimazole, also bind to heme with similar affinities.
However, a significant correlation was noticed between
inhibition of in vitro β-hematin formation and antimalarial
activity of the antifugal azoles (Tekwani, B.L., unpublished
observations). Potent antimalarial action of pentamidine (5),
a diamidine drug used for treatment of Pneumocystis carinii
pneumonia and leishmania infections, has also been
proposed due to its binding with heme and interference with
heme detoxification functions of the parasite [124].
Cyproheptadine (6), an antihistaminic antidepressant has
shown significant blood schizontocidal activity in vivo
against a lethal multidrug-resistant (MDR) strain of
Plasmodium yoelii nigeriensis. A combination of
cyproheptadine and chloroquine shows improved
antimalarial activity compared to treatment with either drug
alone. Cyproheptadine inhibits in vitro β-hematin formation
in a parasite lysate-based assay. Treatment of P. yoelli
infected mice with cyproheptadine also causes significant
decrease in hemozoin level in the infected erythrocytes [125].
The mechanism of the antimalarial action of cyproheptadine
and its enhanced antimalarial activity with chloroquine could
be due, in part, to their inhibitory effect on heme
detoxification function.
With the discovery of hemozoin as an antimalarial drug
target, a new class of compounds viz., metalloporphyrines
(7) and metallodueteroporphyrines (8) have emerged as
potential antimalarials. Marked elevation of erythrocytic zinc
protoporphyrine IX occurs in some of the anemias associated
with malaria disease protection. Zinc protoporphyrin IX,
binds heme and also inhibits seed β-hematin crystal
formation as well as crystal extension [125]. Gallium
protoporphyrin IX, gallinium deutereoporphyrine IX and tin
protoporphyrin IX show marked inhibition of β-hematin
formation in trophozite lysate based assay [126, 127]. These
compounds also show antimalarial activity in vitro in P.
falciparum cultures. Fe(II)-Protoporphyrine IX (FP-Fe II),
released as result of hemoglobin digestion, is immediately
oxidized and then incorporated into hemozoin (Fig. 1).
Fe(II)-Protoporphyrine IX has been identified as a novel
endogenous atimalarial which inhibits incorporation of heme
into β-hematin and also kills malaria parasite [128]. FP-Fe II
itself does not incorporate into β-hematin.
In vitro β-hematin formation assays have also been
applied to screening of natural products and also the extracts.
Ellagic Acid (9), an antimalarial compound isolated from
bark of Tristaniopsis calobuxus inhibits β-hematin
formation. [129 ]. Screening of 178 plant extracts from the
pharmacopia of Bolivian ethnia Tacana through in vitro β-
hematin formation assay have identified some plant extracts
with significant inhibition of β-hematin formation and also
promising antimalarial activity in vitro [130]. From a series
of antimalarial terpene isonitriles, isolated from marine
sponges, the two potent compounds viz., diisocyanoadociane
(10) and axisonitrile-3 (11) have been found to interact with
heme by forming a coordinated complex with FP iron and
also inhibit formation of β-hematin [131]. The two
Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery
compounds also inhibit alternate peroxidative and
glutathione mediated heme detoxification functions.
Screening of 42 compounds isolated from nine plants used
in South Africa for the treatment of malaria identified several
antimalarials that interfere with heme detoxification
functions of malarial parasite. Most of the compounds were
found to be isoquinolines, which is a novel class of
compounds acting through binding with heme [132].
High throughput screening of a library of more than 100,
000 compounds, which were randomly selected, through a
radiometric in vitro β-hematin formation assay have
identified some novel inhibitors. Follow up studies with the
lead compounds in in vitro P. falciparum cultures and also
in the Plasmodium berghei murine model further identified
the hemozoin synthesis inhibitors with actual antimalarial
activity. A triarylcarbinol (Ro 06-9075) (12) and a related
benzophenone (Ro 22-8014) (13) that showed oral activity in
the murine model have been identified as novel antimalarial
pharmacophores [95].
CONCLUSIONS
Discovery of unique metabolic functions and pathways in
malaria parasite have provided valuable tools for molecular
target-based new antimalarial drug discovery research [2].
Most of the antimalarials, which are currently in clinical use,
have been identified through random screening and empirical
approaches. Exact mechanism of selective antimalarial action
of these antimalarials is still obscure. However, most of the
antimalarials, which kill blood stages of malaria parasite,
have shown a common property of interaction with heme
and interference with heme detoxification functions of the
malaria parasite. Despite of living in a pool of heme, which
is generated as a result of hemoglobin digestion, the malaria
parasite depends on de novo biosynthesis of this important
co-factor [133]. The parasite therefore depends on some
unique heme detoxification mechanisms, to get rid of the
indigenous toxic heme pool. Hemozoin, the major product
of the malarial heme detoxification pathway, is formed
possibly through a non-enzymatic, physiochemical
biocrytallization mechanism. Formation of hemozoin, which
is identical to β-hematin (a synthetic analog), possibly
requires a nucleation factor for initiation of the reaction and
follows an autocatalytic crystal growth. Availability of in
vitro assays for quantitative determination of β-hematin
formation and use of these assays in in vitro screening has
been useful in identification of several novel antimalarial
pharmacophores. The Spectrophotometric assays, based on
quantification of β-hematin as heme equivalents, and the
radioactive assays, which are based on incorporation of 14C-
heme into β-hematin, may be useful for high throughput
screening. Significant efforts have been made for
standardization of biological and physiochemical conditions
necessary for in vitro β-hematin formation assays. Synthesis
and accumulation of hemozoin occurs inside the parasite
digestive vacuoles. Therefore, the methods which use the
assay conditions closer to the microenvironment within the
parasite digestive vacuole, are likely to generate better
results. The review on comprehensive and comparative
accounts of different in vitro β-hematin formation assay
methods vis-à-vis advantages and limitations of each method
Combinatorial Chemistry & High Throughput Screening, 2005, Vol. 8, No. 1 77
should be helpful for making use of these assays for in vitro
screening and new antimalarial drug discovery research.
ACKNOWLEDGEMENTS
The new antimalarial drug discovery research in the
author’s lab is supported by Center for Disease Control &
Prevention, USA, under Cooperative Scientific agreements
(U50/CCU418839 and UR3/CCU418652) and by United
States Department of Agriculture (USDA)-ARS, under
cooperative scientific agreement no 58-6408-20009. We are
highly thankful to Dr Simon J. Hocart, Tulane University
Health Sciences Center, New Orleans, for providing the
pictures of heme, hemozoin and hemozoin-chloroquine
structure models.
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