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Journal of Pharmacy and Pharmacology 3 (2015) 101-115 doi: 10.17265/2328-2150/2015.03.001 D DAVID PUBLISHING

Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of

Solomon Tesfaye, Bhanu Prakash and Prati Pal Singh Center of Infectious Diseases, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, India

Abstract: The emergence of malaria parasite strains resistant to practically all the antimalarial drugs in clinical use is now making it necessary to discover and develop both new antimalarial drugs and treatments. Recent advances in molecular techniques along with the availability of sequence of falciparum may provide a wide range of novel targets in metabolic pathways like isoprenoid biosynthesis, biosynthesis and biosynthesis in the apicoplast of Plasmodium. On the other hand, the combination therapy approach (currently used to retard the selection of parasite strains resistant to individual components of a combination of drugs) has proved to be a success in the combination of sulphadoxine and pyrimethamine, which targets two different steps in the folate pathway of malaria parasite. However, after the success of this therapeutic combination, the efficacy of other combinations of drugs which target different enzymes in a particular metabolic pathway has, apparently, not been reported. Therefore, herein, we review various drug targets so far discovered in apicoplast-related anabolic pathways, especially, with a sharper focus on the possibility to target more than one enzyme at a time in a particular metabolic pathway of malaria parasites.

Key words: Apicoplast, combination therapy, fatty acid, isoprenoid, malaria, .

1. Introduction Other than health-related problems, malaria also incurs enormous economic burden both at national In the era of huge concern on the style diseases, and household levels [5]. The current challenge in the even in the nations where a considerable number of control and treatment of malaria is the emergence, population are suffering from old infectious diseases, spread and consolidation of parasite strains (especially malaria continues to be one of the leading causes of P. falciparum) resistant to conventional antimalarial morbidity and mortality in the tropical and subtropical drugs such as chloroquine and regions of the world. According to global malaria sulphadoxine-pyrimethamine [6]. Moreover, recently estimates, in the year 2013, nearly 3.2 billion people a decline in the effectiveness of artemesinin and its were at the risk, and 198 million clinical cases and derivatives has been reported on Cambodia borders [7, 584,000 deaths occurred. Further, between 2000 and 8]. Artemisnin-resistant P. falciparum strains, as 2013, global expanded malaria interventions resulted monitored by the high prevalence of parasites carrying in nearly 30% reduction in incidence and, malaria K 13 propeller mutations, are now rampant across mortality rates were reduced by an estimated 47% Mayanmar and next to northeastern border of India [9]. worldwide [1]. The largest human population at the The fear is that, if artemesinin-resistant P. falciparum risk of malaria is in Africa, South-East Asia and strains are transmitted to the rest of the world, ACT western Pacific regions with the largest share going to (artemesinin-combination therapy), which has so far Africa [2]. Moreover, the interaction between malaria been the most effective first-line treatment option in and HIV has further aggravated the problem [3, 4]. many countries [10], may also meet the same fate as

Corresponding author: Prati Pal Singh, Ph.D., professor, the other drugs. Therefore, the discovery and research field: microbial and parasite chemotherapy: E-mail: development of new, novel and affordable [email protected]. 102 Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria antimalarial drugs and of new treatments is an urgent (dihydrofolate reductase) [21], and cystein and necessicity. aspartic proteases [22]. Nevertheless, despite the Apicoplast, which is derived from an engulfed red clinical success of these drug combinations, and in the alga, is present in most of apicomplexan parasites like face of the fact that potential apicoplastic antimalarial Plasmodium [11]. Due to its evolutionary distance drug targets/enzymes are evolutionary far distant from from the host, apicoplast is now considered as an various putative targets in the human metabolome, no important for the targeted drug discovery other drug combination(s) has been reported, which [12-14]. Apicoplast houses prokaryotic machinery, targets different enzymes in one particular anabolic presumably to replicate its circular 35-kb genome and pathway of apicoplast of malaria parasite. In this to transcribe and translate the that it possesses review, we discuss various drug targets discovered so [15]. Treatment of malaria parasites with antibiotics far in the apicoplast anabolic pathways, with special such as ciprofloxacin, an inhibitor of the bacterial focus on the possibilities of combining novel DNA gyrase, and blocking antibiotics inhibitors or drugs to target them. including azithromycine, chloramphenicol, 2. Isoprenoid Biosynthesis Pathway clindamycin, and rifampicin results in the so called “delayed death” effect. During antibiotic Isoprenoid biosynthesis is one of the pathways in treatment, which is not recommended for acute malaria parasite which partakes enzymes which are malaria [16], the arrest of growth occurs in the found in more than one compartment to produce its next asexual cycle, whereas the parasites in the current final products, isoprenoids. Isoprenoids are a diverse cycle, apparently remained relatively unaffected [17, range of compounds which are assembled from two 15, 18]. In addition to the study of housekeeping common five-carbon precursors known as IPP processes, apicoplast also offers opportunities to study (isopentenyl pyrophosphate) and DMAPP various metabolic machineries such as isoprenoid, (dimethyl-allyl diphosphate). Some of the known fatty acid and heme biosynthetic pathways alongwith isoprenoids are (a) steroids which are involved in their respective enzymes. Surprisingly, unlike the membrane structure, (b) heme A and ubiquinone “delayed death” effect which has been observed as a which are believed to have significant contribution in consequence of the action(s) of some antibiotics, to electron transport, (c) dolichol that is required for target enzymes involved in these anabolic pathways glycoprotein synthesis, and (d) isopentyladenine that resulted in defaying delayed death effect in P. is present in some transfer [23]. The falciparum [15]. Moreover, the known absence of biosynthesis of isoprenoids depends on the some of the enzymes which constitute these pathways condensation of different numbers of IPP units which in eukaryotic hosts, makes them even more attractive, are synthesized via two different biosynthetic routes novel and ideal targets [19]. That is why, currently, viz. the mevalonate and non-mevalonate pathways investigations of the metabolic processes which occur [24]. In mammals, cytoplasm of , fungi, some in apicoplast constitute new prospects for new and several , isoprenic units are antimalarial drug discovery [20]. derived from the typical mevalonate pathway [23]. In The concept of multipoint inhibition approach, of plants, several , eubacteria, which targets different enzymes in the same metabolic cyanobacteria and , the MEP (methyl pathway, has been evidenced to be a success of erythritol phosphate) pathway or DOXP different drug combinations which inhibit enzymes (1-deoxy-D-xylulose 5-phosphate) pathway produces DHPS (dihydropteroate synthetase) and DHFR IPP and DMAPP [25].

Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria 103

Mevalonate pathway is an important metabolic poorly understood mechanisms, IPP and DMAPP are pathway that provides cells with essential bioactive then condensed to GPP (geranyl diphosphate) by molecules, which are vital for multiple cellular FPPS (farnesyl diphosphate synthase) [30]. GPP then processes. Additionally, this metabolic pathway condenses with the second IPP molecule to form FPP converts mevalonate into sterol isoprenoids, such as (farnesyl diphosphate). Finally, FPP is converted to cholesterol, lipoproteins, steroid hormones and several GGPP (geranylgeranyl diphosphate) by GGPPS intermediate molecules [26]. These intermediates play (geranylgeranyl diphosphate synthase, Fig. 1). important roles in the post-translational modification One of the drugs that targets the isoprenoid of a multitude of proteins involved in intracellular pathway is the antibiotic fosmidomycin (Fig. 2) that signaling, cell growth/differentiation, expression, was isolated from Streptomyces lavendulae as an apoptosis, , protein glycosylation and antibacterial agent in 1970’s [31]. In contrast to other cytoskeletal assembly [27, 28]. This pathway depends antibiotics which act on housekeeping processes on the condensation of three molecules of acetyl CoA located in apicoplast and cause the classic “delayed (coenzyme A) into HMG-CoA death” effect to the malaria parasite, fosmidomycin (3-hydroxy-3-methylglutaryl coenzyme A), which is and its analogs cause a rapid parasite and fever reduced to mevalonate by HMGCoA reductase. clearance mainly due to the immediate ceassation of Mevalonate is further converted into IPP with isoprenoid supply required for a variety of cellular mevalonate 5-phosphate as an intermediate. An functions of malaria parasite [32]. Initially, important difference between cytoplasmic fosmidomycin was reported as a potent inhibitor of acetate/mevalonate pathway and the DOXP the DXR enzyme that is essential in the pathway is that the former uses starting compounds of non-mevalonate pathway, which eventually blocks the two-carbon building blocks, whereas the latter uses biosynthesis of isopentenyl diphosphate and the both two-carbon and three-carbon building blocks subsequent formation of isoprenoids in P. falciparum [28]. [20]. However, recently, a second target of In apicoplast, triose phosphate importers and the fosmidomycin, MCT (methylerythritol phosphate subsequent modifying enzymes generate pyruvate cytidyltransferase) has been reported [33]. The along with glyceraldehydes-3-phosphate (GA3P) to be attempts of these authors to validate the biological used by the first enzyme of the DOXP pathway, effects of fosmidomycin in both P. falciparum and DOXP synthase (DXS; Fig. 1). DXS condenses Escherchia coli using mass spectroscopy have shown pyruvate and GA3P to DOXP [29]; DOXP the inhibition of the growth of both the at reductoisomerase (DXR) then catalyzes the the level of MCT. intramolecular rearrangement and reduction of DOXP A metabolite of known clomazone, to MEP. Subsequent reactions which incorporate 5-ketoclomazone, was also reported to block another different enzymes convert MEP to IPP and DMAPP, enzyme DXS in the same biosynthesis pathway [34]. which are used as intermediates in the production of DXS, a thiamin pyrophosphate-dependent enzyme, isoprenoids such as dolichol and ubiquinones. was cloned in E. coli and the inhibition assay was DOXP pathway, in addition to its hypothesized role done using radiolabelled pyruvate. Further, the in the formation of isopentenylated tRNAs inside authors made it clear that the parent compound apicoplast, also gives IPP and DMAPP as substrates (clomazone) has no effect on the activity of this for the downstream production of isoprenoids in enzyme; rather its metabolite (5-ketoclomazone) cytoplasm. After their shipment to cytoplasm by inhibited recombinant DXS of Chlamydomonas with an

104 Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria

Fig. 1 The DOXP (above) and the cytoplasmic (below) parts of isoprenoid biosynthesis pathway in P. falciparum. 5-ketoclomazone, fosmidomycin and bisphosphonates inhibit DOXP synthase (DXS), [DOXP reductoisomarase (DXR) and methylerythritol phosphate cytidyl- transferase (MCT)] and farnesol diphosphate synthase (FPPs), respectively. GA3P, glyceraldehydes-3-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methyl-D-erythritol; CDP-MEP, 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate; MEcPP, 2-C-methyl-D-erythritol-2,4 cyclodiphosphate; HMBPP, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate; IPP, isopentenyl diphosphate; FPPs, farnesyl diphosphate synthase; GPP, geranyldiphosphate; GPPs, geranyldiphosphate synthase; FPP, farensyl diphosphate; GGPP, geranylgeranyl diphosphate; GGPPs, geranylgeranyl diphosphate synthase.

Fig. 2 Inhibitors of isoprenoid biosynthesis pathway, general structure of fosmidomycin and bisphosphonates (R1 and R2 indicate the positions of variable side chains).

Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria 105

approximate IC50 value of 0.1 mM. Similarly, in form a toxic ATP analogue known as another study, spectrophotometric assays showed that O-isopentenyl-ATP (ApppI) which inhibits the fluoropyruvate also inhibited DXS enzyme from mitochondrial adenine nucleotide translocase and

Pseudomonas aeruginosa and E. coli with IC50 values induces apoptosis [40]. of 400 μM and 80 μM, respectively [35]. Several independent works have reported the Fluoropyruvate is believed to bind covalently to the activities of several drugs, stand-alone, on various active site of DOXP synthase [28]. enzymes of non-mevalonate isoprenoid biosynthesis. The last enzyme of DOXP pathway, Lyt B, has However, our literature search, using PubMed search been reported to be involved in the catalysis of engine for published articles from 1996 onwards, on Pi⁄Sigma (π/σ) metallacycle intermediates [36]. These the combination of these drugs to target more than one authors showed the inhibition of Lyt B by a potent enzyme at the same time, did not result in much inhibitor, propargyl diphosphate, and suggested that evidence in this regard except only one study on E. formation of matallacycle complex (between the coli [41]. This particular study reported the alkyne group of the compound and the fourth Fe of synergestic effect of a combination of fosmidomycin the Fe4SO4 complex of the enzyme) as a possible and bisphosphonates in the inhibition of the growth of mechanism for inhibition. This particular work, which E. coli K12. The enzymes which were targeted in E. was done by using ENDOR (electron-nuclear double coli isoprenoid biosynthesis pathway were similar to resonance) spectroscopy, gave an insight for the those in P. falciparum. However, apparently, there are development of new inhibitors. Bisphosphonates is no reports concerning the screening of these drugs for another group of drugs which acts on isoprenoid their synergistic activity against malaria parasites. biosynthesis pathway (Fig. 2). They are 3. Fatty Acid Biosynthesis Pathway pyrophosphate analogs in which the oxygen bridge between the two phosphorus atoms has been replaced , which are highly abundant components of by a carbon substituted with various side chains. all organisms, serve as building blocks of membranes, These compounds, because of their inhibitory activity depots for energy storage and post-translational towards FPPS of osteoclast cells in bone, are clinically modifications which regulate the localization and used as anti-boneresorptive drugs. Current studies function of proteins. Lipids have also been identified regarding this group of drugs have reported that as pathogenetic factors for various infectious diseases lipophilic bisphosphonates have better efficacy in the [42]. Fatty acid biosynthesis is fundamental to cell inhibition of FPPS as compared to the commercially growth, differentiation as well as homeostasis. Almost available polar bisphosphonates [30, 37]. In contrast all living organisms including apicomplexan parasites to the above mentioned studies, Jordao et al. (2011) such as P. falciparum synthesize fatty acids. Before [38] have reported that risedronate (hydrophilic the discovery and characterization of several P. bisphosphonate) had an IC50 of 20.3 ± 1.0 µM against falciparum FAS (fatty-acid synthesis) enzymes, it was P. falciparum, in vitro, and caused 88.9 % inhibition believed that P. falciparum lacks a de novo FAS of malaria parasite P. berghei in a rodent model, on pathway [43]. However, this belief was disproved the seventh day of treatment. Bisphosphonates also based on the results of the genomic sequencing of P. known to stimulate human γδT cells [39] and, thus, falciparum. The results confirmed that P. falciparum may elicit antiparasitic immune response. Moreover, is capable of synthesizing its own fatty acids. Further, the inhibition of FPPS also results in the accumulation all the enzymes of FAS pathway are known to be of the substrate IPP, which conjugates to AMP to present in apicoplast [44].

106 Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria

FAS pathway in apicomplexan parasites is different the action of acetyl-CoA synthetase or from pyruvate from that of their eukaryotic hosts in a number of by PDHC (pyruvate dehydrogenase complex) [43]. In aspects. For instance, unlike their eukaryotic hosts, malaria parasite, apicoplast-localized PDHC is FAS pathway of apicomplexan parasites is catalyzed thought to be the primary source of acetyl-CoA [46]. by discrete enzymes, and is known as type II pathway The first enzyme in the pathway, ACCase (acetyl CoA or “dissociative” pathway [43]. On the other hand, carboxylase), converts acetyl-CoA to malonyl-CoA mammalian FAS pathway (type I or associative which is later changed to malonyl-acetyl carrier pathway) takes place in , and all steps of this protein (malonyl-ACP) by malonyl-CoA: ACP pathway are catalyzed by fatty acid synthase, a single transcylase (MCAT) [46] (Fig. 3). β-ketoaceyl-ACP giant polypeptide enzyme [16]. synthase III catalyzes the condensation of FAS pathway is one of the best studied pathways in malonyl-ACP with other acetyl-CoA to form a apicoplast. This pathway is the main source for de 3-oxoacyl-ACP product [47]. In the elongation novo synthesis of fatty acids and lipoic acid [42]. process, OAR (3-oxoacyl-ACP reductase), after Lipoic acid is synthesized in both mitochondria and removing carbonyl group, reduces 3-oxoacyl-ACP to apicoplast by lipoic acid synthase enzyme. This acid is 3-hydroxyacyl-ACP that enters in dehydration cycle a potent antioxidant that may have an important role [48]. Once produced, 3-hydroxyacyl-ACP is in protecting the parasite against oxidative stress converted into enoyl-ACP by β-hydroxy-acyl-ACP during erythrocytic stage of its life cycle [45]. The dehydratase and enters into the second reductive step main carbon source for this pathway is acetyl-CoA, (catalyzed by enoyl-ACP reductase) to complete the which can either be generated from acetate through first round of elongation cycle (Fig. 3).

Fig. 3 II in apicoplast of P. falciparum. ACC, acetyl-CoA carboxylase; ACP, acyl carrier protein; FabD, ACP transacylase; FabH, β-ketoacyl-ACP synthase III; FabG, β-ketoacyl-ACP reductase; FabA, β-hydroxydecanoyl-ACP dehydratase/isomerase; FabZ, β-hydroxyacyl-ACP dehydatase; FabI, enoyl- ACP reductase; FabB, β-ketoacyl-ACP synthase I; FabF, β-ketoacyl-ACP synthase II.

Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria 107

Several studies on FAS II have reported the enzymes in P. falciparum samples collected from inhibition of the growth of the blood-stage of patients in Senegal [55], recent reports have suggested Plasmodium by blocking various enzymes [49, 50, 16]. that fatty acid biosynthesis is critical only for the late However, recent gene deletion experiments have liver stage development of malaria parasite. Taking proved that apicoplastic FAS II is only essential in the into account the importance of evaluating the reported late liver stage of the life cycle of malaria parasite [51]. inhibitors for pre-erythrocytic activity and checking It has been demonstrated that the deletion of two their specific binding to their respective enzymes, we, critical enzymes of FAS II, Fab B/F and Fab Z, did the authors of this review, believe that it is imperative not affect asexual blood stage replication [52]. Rather, to discuss a few of the reported compounds. the late liver stage of these parasites was unable to Aryloxyphenoxypropionate (fops) such form exo-erythrocytic merozoites. In agreement with as clodinafop (Fig. 4) and cyclohexanediones (dims) the above results, hexachlorophene (Fig. 4), a known have been reported to inhibit the rate-limiting enzyme inhibitor of Fab G [53], was accounted to inhibit liver of the pathway (ACCases) with IC50 values of stage growth in vitro [54]. Even more, it was reported 100-200 Μm [56]. It has been shown that these that the growth of Fab I deficient P. falciparum in its herbicides particularly inhibit carboxyltransferase blood-stage was not affected [51]. domain of the apicoplastic form of ACCase, whereas Despite the expression of FAS II enzyme genes, the cytosolic form of this enzyme is not affected both in blood and mosquito stages and the [57]. Despite the fact that these compounds have upregulation of genes which encode type II FAS been reported to inhibit ACCase of P. falciparum with

Fig. 4 Different inhibitors of fatty acid synthesis.

108 Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria

a concentration > 100 µM [58], and that there might IC50 of 1–4 µg/ml [64]. The interesting feature of these be safety-related issues, they still have the potential to macrophytes that even makes them a good lead serve as a good lead. KAS III (β-ketoacyl-ACP compound is that, they are not cytotoxic to human L6 synthase III) catalyzed the second step of chain cell line. Acetylenic fatty acids, 2-, 5-, 6-, and initiation in this pathway (decarboxylative 9-HADs (hexadecynoic acids), were also evaluated for condensation of malonyl-ACP and acetyl-CoA, the inhibition of the growth of blood-stage P. forming a β-ketoacetyl–ACP product) and the natural falciparum and liver stage P. yoelii [65]. Among these products ceruline[58] and thiolactomycin (Fig. 4) [59] compounds, 2-HAD was the only one that arrested the are well characterized inhibitors of this enzyme. In liver stage growth of P. yoelii, and also exhibited the another study Lee et al. (2009) [60] have shown that best inhibitory activity against Fab I and Fab Z with

60% of submicromolar inhibitors of KAS III among IC50 values of 0.38 and 0.58 µg/mL, respectively. sulfides, sulfonamides and sulfonyl groups screened 4. Heme synthesis pathway by using pharmacophore of PfKASIII, inhibited the in vitro growth of malaria parasites. Even if there are Besides detoxifying heme that comes from host structural differences between KAS III and KAS I/II heamoglobin degradation, P. falciparum also (elongate β-acyl-ACP through condensation reaction), synthesizes its own heme [66]. Heme is an important these enzymes share some of the inhibitors like prosthetic group in many proteins such as plastencin [61]. Moreover, it was also found out that cytochromes [67]. It has been demonstrated that heme some derivatives of thiolactomycine and 1, biosynthetic pathway in P. falciparum is shared 2-dithiole-3-one (known KAS I/II inhibitors) block among apicoplast, and cytosol of the

KAS III with IC50 values of 5-10 µM and 0.53–10.4 parasite [68]. From a target discovery point of view, µM, respectively [49]. the idea to discover the localization of different The other lead compounds, NAS-21 and NAS-91 enzymes which participate in this pathway is (Fig. 4), were brought to light as inhibitors of PfHAD indispensable. Moreover, because there is an overlap with IC50 values of 100 µM and 7.4 µM, respectively between mammalian and parasitic enzymes, those of [62]. Based on the homology model of PfHAD, endosymbiotic origin are thought to be expedient to docking studies were carried out on these compounds. target [16]. The results adumbrated that both these compounds are Heme synthesis pathway starts in mitochondria of competitive for crotonoyl-CoA and malaria parasite with the synthesis of δ-ALA β-hydroxybutyryl-CoA and they inhibited FAS in (aminolaevulinic acid) from glycine and succinyl-CoA cell-free extracts of malaria parasites [18]. Another that is catalyzed by ALA synthase [69]. It has been enzyme of FAS II is Fab I. This enzyme is inhibited reported that, iron chelators in addition to their ability by the linear sesquiterpene lactones; anthecotulide, to form toxic complex with iron, can also withhold 4-hydroxyanthecotulide and 4-acetoxyanthecotulide iron from heme synthesis and cause a down-regulation [63]. From these compounds, the oxygenated in the synthesis of ALA synthase [70]. Iron chelators derivatives, 4-hydroxyanthecotulide and like deferiprone and desferrioxamine, known for their 4-acetoxyanthecotulide, were found to inhibit both unsuitable adsorption and pharmacokinetic properties,

Fab G and Fab I with an IC50 value of 20-75 µg/mL. were tried as adjunctive treatments in different The extract of Turkish freshwater macrophytes, controlled trials and showed positive results [71]. But Cladophroa glomerata and Ulva lactuca also showed considering the fact that the data from these studies antiplasmodial activity by inhibiting Fab I with an were not statistically significant, these drugs are

Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria 109 expensive and they have to be administered regarding its use in combination therapy. parentrally, they do not seem to be the ideal drugs to After the synthesis of ALA in mitochondrion, heme be used in malarious areas. On the other hand, biosynthetic pathway relocates itself to the apicoplast FBSO701, an oral iron chelator with good which contains enzymes like, δ-aminolaevulinate pharmacokinetic properties, showed a better potency dehydratase (Hem B), porphobilinogen deaminase

(IC50 = 5 μM) as compared to deferiprone (IC50 = 15 (Hem C) and uroporphyrinogen III decarboxylase

μM) and deferoxamine (IC50 = 30 μM) [72]. In this (Hem E) (Fig. 5) [68]. An interesting feature about study, in addition to in vitro activity on P. falciparum Hem B is that in addition to its de novo synthesis, the culture in sybrgreen drug inhibition assays, a single parasite imports this enzyme from the host into the oral dose of this compound also cured the lethal P. cytosol during its intraerythrocytic stage (Bonday et yoelii infection in mice. FBSO701 is now in Phase II al., 2000) [73]. This feature is presumed to take about human trials for the treatment of transfusional iron 75%-90 % of total ALAD activity in the parasite overload. Although, apparently, this compound is a [74, 75], and is considered as one of the attractive potential candidate for combination therapy with other chemotherapeutic targets in the pathway. Even if there antimalarial compounds, no reports are available yet is a possibility that the parasite may backup, the reduced

Fig. 5 Heme biosynthesis pathway in P. falciparum. Succinyl acetone and atovaquone inhibits Hem B and Hem H respectively. ALA, δ- aminolevulinic acid; ALAS, ALA synthase; HemB, porphobilinogen synthase; PBG, porphobilinogen; HemC, porphobilinogen deaminase; HMB, hydroxymethylbilane; HemD, uroporphyrinogen III synthase; UROGEN III, uroporphyrinogen III; HemE, uroporphyrinogen III decarboxylase; COPROGEN, coproporphorinogen III; HemF, coproporphorinogen oxidase; PROTOGEN IX, protoporphyrinogen IX; HemG, protoporphyrinogen oxidase; ProtoP IX, protoporphyrinogen protein X; HemH, ferrochelatase.

110 Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria

Fig. 6 Inhibitors of heme synthesis. catalytic efficiencies of its heme biosynthetic enzymes inhibitor of electron transport chain (ETC)-dependent during intraerythrocytic stage by importing enzymes enzyme dihydroorotate dehydrogenase, was tested from the host [68], it is still controversial to the against this enzyme. The results showed that it could established fact that liver-stage schizogony needs inhibit the growth of P. falciparum in culture [78]. more metabolic substrates than that of blood stage The combination of atovaqone with proguanil [76]. It has also been reported that succinyl acetone (malarone) is currently in use in some parts of the has the ability to inhibit both parasite and host ALAD, world to reverse resistance against proguanil. and can kill the parasite when added to the culture However, the mechanism of synergism is not by [77]. In addition to succinyl acetone, it has been inhibition of heme biosynthesis enzymes, rather it is described that N- and C-terminal deleted fragment of due to the inhibition of primidine synthesis which is host ALAD can compete with the enzyme for binding coupled to electron transport system [80]. to P. berghei membrane [73]. Thus, ALAD- NC was Ferrochelatase (Hem H) is the last enzyme in the found to decrease the enzyme’s activity and heme heme biosynthetic pathway which is localized in synthesis when added to a culture of P. falciparum, mitochondrion. It is responsible for insertion of and ultimately led to the death of the parasite. ferrous iron into protoporpherin IX [81]. Heme H is Recently, the localization of coproporphyrinogen also imported from the host to the cytosol of the oxidase (Hem F) and protoporphorinogen oxidase IX parasite in a bigger proportion as compared to its de (Hem G), which, for some time remained a mystery in novo synthesis in the parasite. As per our literature this pathway, has been discovered by enzyme bioassay survey, apparently, no inhibitor of Hem H, has been and immunoflorecence microscopy [78, 79]. The reported so far. Further, there are no published reports results of these studies have proved Hem F is regarding the combination of the above or other localized in cytosol and Hem G is localized in compounds, which target different enzymes in the mitochondrion. Evaluation of herbicides like heme biosynthetic pathway. This literature position oxadiazon, aclonifen and oxyfluorfen (Fig. 6) against thus suggests the need to the above mentioned this enzyme showed the inhibition of in vitro growth compounds or their analogs in combination, to inhibit of P. falciparum. [16] In addition to the reported different enzymes in heme synthesis pathway, which herbicides, atovaquone (Fig. 6), a well known ultimately might lead to the discovery of novel

Apicoplast Biosynthetic Pathways as Possible Targets for Combination Therapy of Malaria 111 antimalarial drugs. support. B.P. is grateful to NIPER for financial assistance. This is NIPER communication No. 496. 5. Future Prospects References Combination therapy, the current choice to retard and roll-back the resistance crisis, is probably the only [1] Fact Sheet World Malaria Report. 2014. http://www.who.int/malaria/media/world_malaria_report option available to extend the working life of drugs, _2014/en/. which are on the front-line in the battle against [2] Snow, R. W., Guerra, C. A., Noor, A. M., Myint, H. Y., malaria. Apart from delaying the emergence of and Hay, S. I. 2005. “The Global Distribution of resistance, it’s a bare fact that a combination of two Clinical Episodes of Plasmodium falciparum Malaria.” Nature 434 (7030): 214-17. drugs, which act on different enzymes (targets) in the [3] Goselle, O. N., Onwuliri, C. O., and Onwuliri, V. A. 2009. same metabolic pathway, may also give good efficacy “Malaria Infection in HIV/AIDS Patients and its and toxicological profiles [82, 2]. So far, the search Correlation with Packed Cell Volume (PCV).” Journal of Vector Borne Diseases 46 (3): 205-11. for the targets to discover new antimalarial drug has [4] Skinner-Adams, T. S., McCarthy, J. S., Gardiner, D. L., been made by the identification of new potential and Andrews, K. T. 2008. “HIV and Malaria targets in apicoplastic metabolic pathways, which are Co-infection: Interactions and Consequences of known to be evolutionarily distant from those of the Chemotherapy.” Trends in Parasitology 24 (6): 264-71. [5] Cibulskis, R. E., Bell, D., Christophel, E. M., Hii, J., humans. Following this further, more than a few Delacollette, C., Bakyatia, N., and Aregawi, M. W. 2007. known and new lead compounds have been screened “Estimating Trends in the Burden of Malaria at Country for their inhibitory activity against metabolic enzymes Level.” American Journal of Tropical Medicine and like DXR, DXS and FPPS in isoprenoid pathway; Hygiene 77 (6): 133-37. [6] Davis T. M., Karunajeewa, H. A., and Llett, K. F. 2005. ENR, the KAS family, KAR and HAD in FAS II; and “Artemisinin-Based Combination Therapies for ALAD and PPO in heme synthesis pathways. Uncomplicated Malaria.” Medical Journal of Australia However, after the sulphadoxine-pyrimethamine 182 (4): 181-85. [7] Dondorp, A. M., Nosten, F., Yi, P., Das, D., Phyo, A. P.; (inhibitor of dihydropteroate synthetase and Tarning, J., Lwin, K. M., Ariey, F., Hanpithakpong, W., dihydrofolate reductase, respectively, in folate Lee, S. J., Ringwald, P., Silamut, K., Imwong, M., pathway) combination, combination approaches which Chotivanich, K., Lim, P., Herdman, T., An, S. S., Yeung, target different metabolic enzymes in the same S., Singhasivanon, P., Day, NP., Lindegardh, N., Socheat, D., and White, N. J. 2009. “Artemisinin Resistance in pathway have apparently not been tried against Plasmodium falciparum Malaria.” New England Journal malaria parasite. Thus, taking in to consideration the of Medicine 36: 455-67. recent advances in the in vitro antimalarial screening [8] Na-Bangchang, K., Ruengweerayut, R., Mahamad P., assays using whole parasites, we do hope that there Ruengweerayut, K., and Chaijaroenkul, W. “Declining in Efficacy of a Three-day Combination Regimen of remains much to be done in the pursuit of new Mefloquine-Artesunate in a Multi-drug Resistance Area combinations of drugs which target different enzymes along the Thai-Myanmar Border.” Malaria Journal 9: belonging to one particular metabolic pathway. 273-77. [9] Tun, K. M., Imwong, M., Lwin, K. M., Win, A. A., Hlaing, Acknowledgments T. M., Hlaing, T., Lin, K., Kyaw, M. P., Plewes, K., Faiz, M. A., Dhorda, M., Cheah, P. Y., Pukrittayakamee, S., We are grateful to Prof. K. K. Bhutani, Officiating Ashley E. A., Anderson, T. J., Nair, S., McDew-White, M., Director, National Institute of Pharmaceutical Flegg, J. A., Grist, E. P., Guerin, P., Maude, R. J., Smithuis, Education and Research (NIPER), for his help and F., Dondorp, A. M., Day, N. P., Nosten, F.,White, N. J., and Woodrow, C. J. 2015. “Spread of encouragement. S. T. thanks Government of the Artemisinin-Resistant Plasmodium falciparum in Federal Democratic Republic of Ethiopia for financial Myanmar: A Cross-Sectional Survey of the K13

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