Download Selective and Potent Inhibitors of the Malaria Parasite Mitochondrial

Proposed Research Work
Project Title:
Selective and Potent Inhibitors of the
Malaria Parasite Mitochondrial bc1
Complex
Introduction:
The World Health Organization (WHO) estimates that 225 million cases of malaria were
reported in 2009 and 85% of deaths worldwide were in children under 5 years of age. [1]
Malaria, a mosquito-borne disease caused by a parasite, occurs throughout tropical and subtropical regions of the world, killing at least a million people annually, most of them young
children in sub-Saharan Africa. Malaria is caused by protozoan parasites of the Plasmodium
genus which are inoculated into humans by female Anopheles mosquitoes (Figure 1).
Figure 1: female Anopheles mosquitoes.
Plasmodium is actually a small, single-cell blood organism or protozoan which originated
from a species of mosquito called Anopheles. This genus has over 100 species, five of which are
capable of infecting human beings: Plasmodium falciparum, P. vivax, P. malariae, P. ovale [2]
and P. knowlesi.[3]. Plasmodium falciparum is responsible for the majority of malaria deaths
globally and is the most prevalent species in sub-Saharan Africa. The remaining species are not
typically as life threatening as P. falciparum, [4] Plasmodium vivax, is the second most
significant species and is prevalent in Southeast Asia and Latin America. P. vivax and
Plasmodium ovale have the added complication of a dormant liver stage, which can be
reactivated in the absence of a mosquito bite, leading to clinical symptoms. P. ovale and
Plasmodium malariae represent only a small percentage of infections. A fifth species
Plasmodium knowlesi – a species that infects primates – has led to human malaria, [5] but the
exact mode of transmission remains unclear.
Life Cycle of the Malaria Parasite (Figure 2):
1. A female Anopheles mosquito carrying malaria-causing parasites feeds on a human and
injects the parasites in the form of sporozoites into the bloodstream. The sporozoites travel to
the liver and invade liver cells.
2. The sporozoites grow, divide, and produce tens of thousands of haploid forms, called
merozoites, per liver cell.
3. The merozoites exit the liver cells and re-enter the bloodstream, beginning a cycle of
invasion of red blood cells, asexual replication, and release of newly formed merozoites from
the red blood cells repeatedly over 1-3 days.
4. Some of the merozoite-infected blood cells leave the cycle of asexual multiplication. Instead
of replicating, the merozoites in these cells develop into sexual forms of the parasite, called
male and female gametocytes that circulate in the bloodstream.
5. When a mosquito bites an infected human, it ingests the gametocytes. In the mosquito gut,
the infected human blood cells burst, releasing the gametocytes, which develop further into
mature sex cells called gametes. Male and female gametes fuse to form diploid zygotes,
which develop into actively moving ookinetes that burrow into the mosquito midgut wall and
form oocysts.
6. Growth and division of each oocyst produces thousands of active haploid forms called
sporozoites. After the oocyst bursts, releasing sporozoites into the body cavity of the
mosquito, from which they travel to and invade the mosquito salivary glands.
Figure 2: Life Cycle of the Malaria Parasite
Cells contain many different types of proteases, which are ubiquitous in all living cells.
As soon as cells are disrupted, proteases are released and can quickly degrade any protein.
Uncontrolled, unregulated, or undesired proteolysis can lead to many disease states including
emphysema, stroke, viral infections, cancer, Alzheimer’s disease, inflammation, and arthritis.
Protease inhibitors thus have considerable potential utility for therapeutic intervention in a
variety of disease states. Therefore, mixtures of different inhibitors are needed for complete
protection of proteins. Proteases or proteolytic enzymes form one of the largest and more
important groups of enzymes. Proteases selectively catalyze the hydrolysis of peptide bonds and
can be divided into four major classes (or groups): aspartic, serine, cysteine, and
metalloproteases.
Origin of the research:
Plasmodium falciparum-parasitized enythrocytes increase their utilization of glucose as
much as 100 times the rate of uninfected host red cells. This increase reflects the need of the
parasites to grow; synthesize RNA, protein, and DNA; and reproduce 12 to 24 new progeny all
in a 48-hour period. The importance of mitochondrial proteins is reflected by the fact that
children keep dying from mysterious illness that has been traced to tiny structures called
mitochondria. Thus it is important to identify or annotate mitochondrial proteins. Mitochondria
in plasmodium parasites have many characteristics that distinguish them from mammalian
mitochondria. Mitochondrial proteins of Plasmodium falciparum are different than human
mitochondrial proteins; this makes Plasmodium falciparum mitochondrial protein as attractive
potential drug targets. Mechanism of Mitochondrial Plasmodium falciparum is unusual
translational machinery as revealed by the highly fragmented mitochondrial ribosomal RNA
genes also appears to have originated at this deflection point. Some of the biochemical properties
of malarial mitochondria also appear to be unconventional. Although tricarboxylic acid cycle
enzymes are encoded by the genome, they do not appear to be involved in the full oxidation of
glucose to fuel mitochondrial ATP synthesis in the blood stages of malaria parasites. A critical
role of the mitochondrial electron transport chain appears to be to serve pyrimidine biosynthesis.
In spite of their minimal nature, Plasmodium mitochondria are attractive targets for antimalarial
drugs. [7]
MITOCHONDRIAL ELECTRON TRANSPORT CHAIN: A TARGET OF ANTIMALARIAL DRUGS:
In most eukaryotic cells, the mtETC is required for generation of the protonmotive force
(∆p or∆µH), which is central to oxidative energy metabolism.[8] However, in organisms or
under conditions favoring a glycolytic metabolism, which includes the blood stages of malaria
parasites and some other parasite species, the role of the mtETC is often reduced, and
mitochondria may even become consumers rather than producers of energy.[9] In higher
eukaryotes the mtETC is generally composed of four integral membrane enzyme complexes in
the mitochondrial inner membrane: NADH:ubiquinone oxidoreductase (Complex I),
succinate:ubiquinone oxidoreductase (Complex II), ubiquinol:cytochrome c oxidoreductase
(Complex III, or cytochrome bc1), and cytochrome c oxidase (Complex IV), with ubiquinone
(Coenzyme Q, or Q) and cytochrome c functioning as electron carriers between the complexes
and Complexes I, III, and IV functioning as sites that generate protonmotive force. Plasmodium
spp. have lost the large multisubunit Complex I of the mitochondrial inner membrane but have
retained electron transfer Complexes II through IV (Figure 2a). They do possess a singlesubunit, non-proton-pumping NADH dehydrogenase (NDH) that reduces ubiquinone. The
apparent subunit composition of the mtETC complexes [10] deduced from the genome data (31)
suggests that mtETC complexes of the malaria parasite have a much simpler subunit composition
than their counterparts in mammals and yeast. Complex III in Plasmodium has 7 identifiable
subunits instead of 10 as seen in yeast, and Complex IV has only 6 recognizable subunits instead
of the 12 seen in yeast.
Figure 2:
Mitochondrial electron transport chain (mtETC) of malaria parasites and Complex III reactions targeted by drug candidates. (a) The enzyme
complexes of the mtETC are embedded in the inner membrane of the mitochondrion. The reactions at Complex III and Complex IV result in
translocation of protons from the matrix to the intermembrane space. Also shown are additional dehydrogenases that provide electrons to the
mtETC via ubiquinone (yellow structures labeled “Q”; malate:ubiquinone oxidoreductase and glycerol-3-phosphate dehydrogenase are not
shown). Complex V is shown in two sections. The enzymes are drawn as ribbon diagrams of the structures of orthologues available in the Protein
Data Bank. (b) The schematic depicts Complex III, with the substrates ubiquinol (QH2) and ubiquinone (Q) interacting with their reaction sites
[ubiquinol oxidation site (Qo) and ubiquinone reduction site (Qi), respectively]; the electron and proton transfers of the ubiquinol:cytochrome c
oxidoreductase reaction, according to the Q-cycle mechanism; and the five classes of drug or drug candidates that block the reaction at the Qo
site.
However, it is possible that in these deep-branching organisms, the mitochondrial
complexes may possess additional highly divergent subunits that may not yield easily to
bioinformatic identification. Subunit II of Complex IV is split, and the genes for the two parts are
not found in the mitochondrial DNA but have migrated to two different chromosomes.
Verification of the in silico compositions by biochemical studies is still largely lacking owing to
the difficulty of purifying a meaningful amount of the complexes [11], which is exacerbated by
the apparent low levels and/or activities of the respiratory enzymes in the blood stages of the
parasites. The low activities of the mtETC complexes are consistent with the largely glycolytic
carbon and energy metabolism of the blood stage parasites. Despite the relatively low activity of
the mtETCin malaria parasites, it still appears to be the primary source of the mitochondrial
proton electrochemical gradient. Furthermore, the sensitivity of parasites to inhibitors of the
mtETC indicates that it is indispensible to the parasites. Hydroxynaphthoquinone inhibitors of
Complex III are lethal to apicomplexan parasites including Plasmodium spp., which lead to the
development of the antimalarial drug atovaquone. Atovaquone targets the ubiquinol oxidation
site (Qo) of cytochrome b with high selectivity [12]. Unfortunately, high-level resistance to
atovaquone occurred at a relatively high frequency and correlated with mutations at position 268
(Tyr) of cytochrome b. This position lies near a highly conserved motif and participates in
forming the Qo site. Similar resistance mutations at this site and other nearby positions in
cytochrome b also arose in P. yoelii raised in mice treated with suboptimal levels of atovaquone.
A cause and effect relationship between a mutation introduced at Tyr-268 and the resulting twoorders-ofmagnitude reduction in the susceptibility of cytochrome bc1 to inhibition by
atovaquone was demonstrated using a bacterial system. Experiments with this system also
provided additional information on the drug’s mode of action. To obviate the resistance problem,
atovaquone is sold commercially as MalaroneTM, a synergistic combination of atovaquone and
proguanil. In the presence of proguanil, the effective dose of atovaquone is significantly reduced;
however, once a malaria parasite acquires the resistance mutation in cytochrome b, the
synergistic effect of proguanil is lost. The precise molecular mechanism by which it potentiates
the action of atovaquone (and other inhibitors of cytochrome bc1) has yet to be elucidated. The
demonstrated that proguanil lowers the concentration of atovaquone required to significantly
reduce the mitochondrial membrane potential in P. yoelii by six- to eightfold, while qualitative
measurements of P. falciparum–infected erythrocytes indicated that the degree of membrane
potential reduction is increased when atovaquone is combined with proguanil. Atovaquone is an
effective drug, but is expensive and subject to relatively facile development of resistance. Drug
resistance is a major problem for malaria treatment and prevention, and the search for new,
effective, and inexpensive drugs is a continuing and urgent need. Even though Complex III of
the mtETC is essential to both the human host and the malaria parasite, it continues to be a
promising target for the development of antimalarial drugs. Extensive structural and genetic
evidence suggests that the Qo site of Complex III is large and capable of accommodating two
ubiquinol molecules. Various subclasses of Complex III inhibitors bind to nonidentical but
overlapping regions of the Qo site. In agreement with this view of the Qo site, several classes of
chemicals provide promising leads with antimalarial activities.
4-Pyridone analogs of the clopidol class that selectively inhibit Plasmodium Complex III
are at an advanced stage of development [13]. Winter et al. have synthesized [14]
haloalkoxyacridone derivatives that appear to target the bc1 complex and inhibit human malaria
parasite growth with inhibitory concentrations in the picomolar range. Quinolones related to the
early antimalarial candidate endochin that also seem to target Complex III while exhibiting
minimal atovaquone cross-resistance have been developed. [15] The dihydroacridinedione
WR249685 is a selective inhibitor of the cytochrome bc1 complex [16]. Thus, at least five
different structural entities have the potential to be developed as antimalarials by having the
capacity to selectively inhibit Plasmodium Complex III (Figure 2b).
Past Work:
Atovaquone is a new anti-malarial agent that specifically targets the cytochrome bc1 complex
and inhibits parasite respiration. A growing number of failures of this drug in the treatment of
malaria have been genetically linked to point mutations in the mitochondrial cytochrome b gene.
Because the yeast bc1 complex is also inhibited by atovaquone. [17] To better understand the
molecular basis of atovaquone resistance in malaria, [18] we introduced five of these mutations,
including the most prevalent variant found in Plasmodium falciparum (Y268S), into the
cytochrome b gene of the budding yeast Saccharomyces cerevisiae and thus obtained
cytochrome bc1 complexes resistant to inhibition by atovaquone. By modeling the variations in
cytochrome b structure and atovaquone binding with the mutated bc1 complexes, we obtained
the first quantitative explanation for the molecular basis of atovaquone resistance in malaria
parasites Fig 3.
O
HO
O
Cl
Atovaquone
Figure 3. View of the atovaquone-binding pocket of
the yeast cytochrome bc1 complex showing the
location of mutations conferring resistance to
atovaquone in Plasmodium.
A team at Drexel University College of Medicine in Philadelphia led by Akhil Vaidya [19]
looked at internal structures called mitochondria in Plasmodium falciparum, the deadliest of the
four types of the parasite that cause malaria in people. Series of diaryl ether substituted 4pyridones have been identified as having potent antimalarial activity superior to that of
chloroquine against Plasmodium falciparum in vitro and murine Plasmodium yoelii in vivo.
These were derived from the anticoccidial drug clopidol through a systematic study of the effects
of varying the side chain on activity. Relative to clopidol the most active compounds show >500fold improvement in IC50 for inhibition of P. falciparum in vitro and about 100-fold
improvement with respect to ED50 against P. yoelii in mice. These compounds have been shown
elsewhere to act selectively by inhibition of mitochondrial electron transport at the cytochrome
bc1 complex Table 1. [20]
Table 1: In Vitro and in Vivo Antimalarial Activity of
Phenoxyaryl-4(1H)-pyridones
O
O
R
X
H 3C
CH 3
Antimycin A1 (1, Figure 1, antimycin) is a dilactone salicylamide that was isolated from
Streptomyces sp.1 It is a potent inhibitor of the Qi site of complex III (bc1 complex) in the
mitochondrial respiratory chain, with a dissociation constant with bovine heart mitochondrial
particles of 32 pM. [21]
Figure 1. Structure of antimycin A1 (1) showing binding interactions
with the bc1 complex.[22]
A series of azole-fused salicylamides were prepared as analogues of antimycin and assayed for
activity at complex III of the mitochondrial respiratory chain. The activity of these compounds
approached that of antimycin in inhibitory potency and some showed growth reduction of
Septoria nodorum in vitro. Compounds was shown to bind at the Qi site of complex III by redshift titration of the bc1 complex. [23]
Cl
H
N
O
OH
O
O
H
N
N
H
Antimycin eqyivalent
Cl
A
OH
O
O
N
H
A
formamidosalicylamide
Cl
Twenty-six novel naphthoquinone aliphatic esters were synthesized by esterification of 1,4naphthoquinone alcohols with various aliphatic acids. The 1,4-naphthoquinone alcohols were
prepared from 1-hydroxy-2-naphthoic acid in nine steps with excellent yields. Twenty-four of
the novel synthetic naphthoquinone esters showed significant antimalarial activity with IC50
values in the range of 0.03-16.63 µM. The length of the aliphatic chain and the presence of C-20
substituents on the propyl chain affected the activity. Interestingly, compounds showed potent
inhibition against P. falciparum 3D7 cyt bc1 and no inhibition on rat cyt bc1. They showed IC50
values in the nanomolar range, providing full inhibition of cyt bc1 with one molecule inhibitor
bound per cyt bc1 monomer at the Qo site. [23]
Proposed Research Plan:
Cheaper antimalarial drugs are needed to be developed on a continuous basis for us to keep up
with the emergent resistant strains. A better understanding of the mitochondrial physiology
would guide efforts to derive new antimalarial drugs," wrote Vaidya, a professor of microbiology
and immunology.
The aim of this research is to investigate two series of indole chloroquinole analogues where the
4′-hydroxyl function of amodiaquine is replaced by either fluorine or chlorine; the intention, as in
previous work, was to produce analogues incapable of producing quinoneimine metabolites by
P450 oxidation. The array of target molecules is highlighted in Chart 1. For comparison
purposes, we have also examined the antimalarial profile of two 4′-dehydroxy analogues of
amodiaquine. Following completion of the syntheses and appropriate antimalarial assessment
and preclinical pharmacological assessment, we aimed to select suitable back-up compounds to
1e with appropriate properties suitable for drug development. Chemistry Although the original
reported route to 4-fluoroamodiaquine was relatively efficient, we reasoned that shortening the
number of synthetic steps and developing a route where a point of diversity appears in the
penultimate step of the synthesis could achieve a significant improvement to the synthesis of
target molecules in Chart 1. The alternative route adopted for the synthesis of 4′-chloro and 4′fluoro analogues is shown in Scheme 2. For the synthesis of 4′-chloro analogues, nitro reduction
of 3a was easily achieved using iron powder and HCl in refluxing aqueous ethanol. The resulting
amine 3b was then allowed to react with 4,7-dichloroquinoline to provide 3c in excellent yield as
a yellow powder following standard workup. Quinoline 3c proved to be quite insoluble in
dichloromethane, so a combination of dichloromethane, DMF, and a small amount of
diisopropylethylamine was used as solvent to dissolve the alcohol for MnO2 mediated oxidation.
After 18 h of reflux, precipitates derived from the oxidant MnO2 were filtered off. In many runs,
significant losses occurred at this stage and a more satisfactory workup procedure involved the
use of a precolumn with ethyl acetate prior to flash column chromatography using MeOH/
dichloromethane as eluent. The key aldehyde was obtained in moderate to good yields following
silica gel chromatography. The final step involved reductive amination of the aldehyde 13 with a
variety of different amines to produce a small array of target molecules depicted in Chart 1. In all
cases the reductive amination procedure worked well and column chromatography gave the
products 3d-m in good yields (Table 1).
HN
N
Cl
N
Interdisciplinary relevance
• This research can be useful for all the parasites of Plasmodium falciparum.
• It is in fact convergence of technology and education.
• It deals with synergism of computer technology and TELL.
• This research project has profound relevance for interdisciplinary teaching and
learning. Its outcome can bring paradigm shift in the way things are taught and will be
taught.
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