Review Article Exploring Drug Targets in Isoprenoid ...downloads.hindawi.com/journals/bri/2014/657189.pdfcomplexan protozoa such as malaria parasites produce their isoprenoids by utilizing

Review ArticleExploring Drug Targets in Isoprenoid Biosynthetic Pathway forPlasmodium falciparum

Tabish Qidwai1 Farrukh Jamal2 Mohd Y Khan3 and Bechan Sharma4

1 Department of Biotechnology Faculty of Engineering amp Technology Raja Balwant Singh Engineering Technical CampusAgra 283105 India

2Department of Biochemistry Dr Ram Manohar Lohia Avadh University Faizabad 224001 India3 Department of Biotechnology Babasaheb Bhimrao Ambedkar University Lucknow India4Department of Biochemistry University of Allahabad Allahabad India

Correspondence should be addressed to Farrukh Jamal farrukhrmlaugmailcom

Received 24 December 2013 Revised 7 February 2014 Accepted 7 February 2014 Published 23 April 2014

Academic Editor Andrei Surguchov

Copyright copy 2014 Tabish Qidwai et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Emergence of rapid drug resistance to existing antimalarial drugs in Plasmodium falciparum has created the need for prediction ofnovel targets as well as leads derived from original molecules with improved activity against a validated drug target The malariaparasite has a plant plastid-like apicoplast To overcome the problem of falciparum malaria the metabolic pathways in parasiteapicoplast have been used as antimalarial drug targets Among several pathways in apicoplast isoprenoid biosynthesis is one of theimportant pathways for parasite as its multiplication in human erythrocytes requires isoprenoids Therefore targeting this pathwayand exploring leads with improved activity is a highly attractive approach This report has explored progress towards the study ofproteins and inhibitors of isoprenoid biosynthesis pathway Formore comprehensive analysis antimalarial drug-protein interactionhas been covered

1 Introduction

Falciparum malaria is a well-known major killer causingapproximately one million deaths per year and 300ndash500 mil-lion clinical cases [(WHO 2010)Worldmalaria report WorldHealth Organization Geneva] The malaria parasite belongsto apicomplexan phylum and has a plastid-like structureldquoapicoplastrdquoThemetabolic pathways in apicoplast differ fromthe host and therefore apicoplast opens up new possibilitiesof targeting P falciparum The isoprenoid metabolic pathwayis crucial for the P falciparum In plants more than 30000 iso-prenoids are known However the number of isoprenoids inmalaria parasites is low Isoprenoid includes cholesterol bileacids steroid hormones dolichol ubiquinone prenylatedproteins and a wide variety of plant terpenoids [1] The iso-prenoid compounds are widespread in the three domains ofarchaebacteria eubacteria and eukaryotes The ldquocyclisationreactionsrdquo are unknown in malaria parasites however rear-rangements and oxidation of the carbon skeleton are respon-sible for the enormous structural diversity Although they are

produced from the condensation of the same precursors inall organisms (isopentenyl pyrophosphate and dimethylallyldiphosphate) the evolutionary origin of their biosynthesisremains controversial Currently two different routes havebeen identified to biosynthesize isopentenyl pyrophosphate(IPP) and dimethylallyl pyrophosphate (DMAPP) The well-known mevalonate pathway in most eukaryotes includemammals higher plants and archaea and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway also known as 1-deoxy-D-xylulose-5-phosphate (DOXP) or nonmevalonatepathway in bacteria and plant plastids especially in severalpathogenic microorganisms [2]

Antimicrobial drug resistance is the most common prob-lem in the control and treatment of many serious infectionsincludingP falciparummalaria tuberculosis and other infec-tious diseases The nonmevalonate pathway of isoprenoidbiosynthesis is essential in eubacteria (not all) and P falci-parum As this pathway is absent in humans consequentlythere is a great interest in targeting the enzymes of non-mevalonate metabolism for antiparasitic drug development

Hindawi Publishing CorporationBiochemistry Research InternationalVolume 2014 Article ID 657189 12 pageshttpdxdoiorg1011552014657189

2 Biochemistry Research International

Fosmidomycin is a broad-spectrum antimicrobial agent andhas been used in clinical trials of combination therapiesfor the treatment of malaria [3] In vitro fosmidomycin isknown to inhibit the deoxyxylulose phosphate reductoiso-merase (DXR) enzyme of isoprenoid biosynthesis from mul-tiple pathogenic organisms Isoprenoid metabolism proceedsthrough DXR even in the presence of fosmidomycin but isinhibited at the level of the downstream enzyme methylery-thritol phosphate cytidylyltransferase (IspD)Overexpressionof IspD in E coli conferred fosmidomycin resistance andfosmidomycin was found to inhibit IspD in vitro [3] Underin vivo conditions fosmidomycin may inhibit IspD directlyor may cause other changes within the cell that reduceIspD activity [3]Mycobacterium tuberculosis synthesizes iso-prenoids via the nonmevalonate or DOXP pathway Previouswork had demonstrated that three enzymes namely DxrIspD and IspF are all required for growth in vitro TheDOXP biosynthetic pathway ofM tuberculosis is specific andessential and represents an attractive potential target for thedesign of new antimycobacterial agents The work by Brownet al (2010) demonstrated that three enzymes in the pathway(Dxr IspD and IspF) are all required for in vitro growth ofM tuberculosis [4] Most eubacteria (not all) synthesize theirisoprenoids using themethylerythritol-4-phosphate pathwaywhereas a minority uses the unrelated mevalonate pathwayand only a few have both [5] Isoprenoids are a large andhighly diverse group of natural products withmany functionsand their synthesis is essential for the parasitersquos survival [6]This paper attempts to cover the different enzymes involved inisoprenoid biosynthesis and their importance in P falciparumand antimalarial drug against these enzymes In additionan insight into host-parasite genetic polymorphisms is alsopresented

2 Scope of Apicoplast as AntimalarialDrug Target

The parasites such as Plasmodium spp Babesia spp Tox-oplasma gondii Cryptosporidium spp Isospora belli andCyclospora cayetanensis infect humans [9] With the excep-tion of Cryptosporidium spp these parasites possess api-coplast a nonphotosynthetic plastid-like organelle The api-coplast genome houses only 68 open reading frames (ORFs)for rRNAs tRNAs ribosomes RNA polymerase transla-tional elongation factor (EF-Tu) ClpC chaperone and Fe-Scluster protein (SufB) [1 9] Most of the proteins involved inthe apicoplastmetabolic pathways are encoded in the nucleussynthesized in the cytoplasm and subsequently importedinto the apicoplast [2]

The apicoplast contains metabolic pathways critical forliver-stage and blood-stage development During the bloodstages parasites lacking an apicoplast can grow in thepresence of isopentenyl pyrophosphate demonstrating thatisoprenoids are the only metabolites produced in the api-coplast which are needed outside of the organelle Two ofthe isoprenoid biosynthesis enzymes are predicted to rely oniron-sulfur (FeS) cluster cofactors however little is knownabout FeS cluster synthesis in the parasite or the roles that FeS

cluster proteins play in parasite biology [10] In P falciparumthe protein SufS and its partner SufE were found exclusivelyin the apicoplast and SufS was shown to have cysteinedesulfurase activity in a complementation assay IscS andits effector Isd11 were solely mitochondrial suggesting thatthe Isc pathway cannot contribute to apicoplast FeS clustersynthesis The Suf pathway was disrupted with a dominantnegative mutant resulting in parasites that were only viablewhen supplemented with IPP These parasites lacked theapicoplast organelle and its organellar genomemdasha phenotypenot observed when isoprenoid biosynthesis was specificallyinhibited with fosmidomycinThese results demonstrate thatthe Suf pathway is essential for parasite survival [10]

Fatty acid isoprenoid haem biosynthesis Fe-S clustersand DNA transactions are prominent pathways in api-coplast The most important metabolic functions and themevalonate independent 1-deoxy-D-xylulose-5-phosphate(DOXP) pathway of isoprenoid synthesis and the type IIfatty acid synthesis system operate inside the apicoplastClassical antibacterial drugs such as ciprofloxacin tetra-cycline doxycycline clindamycin and spiramycin inhibitthe apicoplast-located gyrase and translation machineryrespectively and are currently used for the treatment ofinfections with apicomplexan parasites Fosmidomycin aninhibitor of isoprenoid synthesis was proven to be effectiveagainst acute falciparum malaria in clinical phase II studiesFosmidomycin alone or in combination with clindamycinwas evaluated for the treatment of acute uncomplicatedfalciparum malaria Monotherapy using fosmidomycin ledto a fast parasite clearance but was inefficient in radicalelimination of the parasites [11] Triclosan an inhibitor offatty acid synthesis was active in a malaria mouse model Invitro antimalarial activity was shown for inhibitors of peptidedeformylase and the import of apicoplast-targeted proteinsIn the drug discovery against P falciparum the presence ofapicoplast has been a milestone [12]

3 Isoprenoid Biosynthesis

It is required for the production of isopentenyl pyrophosphateand dimethylallyl pyrophosphate that plays a role in thebiosynthesis of molecules used in protein prenylation cellmembrane maintenance hormones protein anchoring andN-glycosylation Plants and apicomplexan protozoa such asmalaria parasites have the ability to produce their isoprenoidsby means of an alternative pathway (nonmevalonate path-way) which takes place in their plastids Interestingly mostbacteria including important pathogens such as M tuber-culosis synthesize IPP and DMAPP via the nonmevalonatepathway

31 Nonmevalonate (MEPDOXP Pathway) Plants and api-complexan protozoa such as malaria parasites produce theirisoprenoids by utilizing an additional alternative pathwaycalled the methylerythritol phosphate (MEP) or nonmeval-onate pathway which takes place in their plastids (Figure 1)

Higher plants possess the MEV pathway in the cytosolin addition to the MEP pathway in the plastids In con-trast the MEV pathway has not yet been detected in

Biochemistry Research International 3

Apicoplast

35kb Doxycycline

Isoprenoid precursor biosynthesis

IPPDMAPP Isoprenoids

prenylated proteins

Parasite

Erythrocyte

IPPOPP

Fosmidomycin

Pyruvate+

G3PDOXP MEP

IPPOPP OPP

+

DMAPP

MEP isoprenoid precursor biosynthesis

Figure 1 The role of the apicoplast in production of isoprenoid precursors IPP and DMAPP which are exported into the cytoplasm andused to synthesize small molecule isoprenoids and prenylated proteins P falciparum is unable to synthesize isoprenoid precursors either dueto inhibition of the biosynthetic pathway by fosmidomycin or loss of the apicoplast following doxycycline inhibition which can be chemicallyrescued by addition of exogenous IPP The exogenous IPP enters the host cell through unknown membrane transporters and fulfills themissing biosynthetic function [7]

the cytosol of apicomplexan parasites The nonmevalonatepathway or 2-C-methyl-D-erythritol-4-phosphate1-deoxy-D-xylulose-5-phosphate pathway (MEPDOXP pathway) ofisoprenoid biosynthesis is an alternative metabolic path-way leading to the formation of IPP and DMAPP [7]The MEP pathway consists of 8 steps and 7 enzymes(Figures 2(a) and 2(b))

32 Enzymes ofMEPPathway Cassera et al (2004) suggestedthat two genes encoding the enzymes of MEP pathwaynamely 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase play a rolein isoprenoid biosynthesis in P falciparum Fosmidomycincould inhibit the activity of 1-deoxy-D-xylulose-5-phosphatereductoisomerase [13]Themetabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathwayfor the biosynthesis of isopentenyl diphosphate but is alsoinvolved in the biosynthesis of thiamin (vitamin B1) andpyridoxal (vitamin B6) in plants and many microorgan-isms An added advantage of targeting 1-deoxy-D-xylulose-5-phosphate synthase is its influence on vitamins B1 and B6biosynthesis in malaria parasite [14]

Most of the downstream intermediates (1-deoxy-D-xy-lulose-5-phosphate 2-C-methyl-D-erythritol-4-phosphate4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol4-(cyti-dine-5-diphospho)-2-C-methyl-D-erythritol-2phosphateand 2-C-methyl-D-erythritol-24-cyclodiphosphate) of theMEP pathway in the three intraerythrocytic stages of P fal-ciparum have been unfolded [15]The effect of fosmidomycinon the biosynthesis of each intermediate of this pathway

and isoprenoid biosynthesis (dolichols and ubiquinones)has been explored MEP pathway is functionally active inall intraerythrocytic forms of P falciparum and de novobiosynthesis of pyridoxal in a protozoan has been reported[16] Its absence in the human host makes both pathwayspotentially very attractive targets for antimalarial drugdevelopment

Another enzyme of the nonmevalonate pathway 1-deoxy-D-xylulose-5-phosphate reductoisomerase which is involvedin the transformation of 1-deoxy-D-xylulose 5-phosphateto 2-C-methyl-D-erythritol-4-phosphate DXR protein fromthe human malaria parasite P falciparum (PfDXR) wasoverproduced in Escherichia coli and crystallized using thehanging-drop vapour-diffusion method in the presence ofnicotinamide adenine dinucleotide (NADPH) [16] Casseraet al (2007) reported that in P falciparum the forma-tion of isopentenyl diphosphate and dimethylallyl diphos-phate intermediates in the biosynthesis of isoprenoids andoccurs via the methylerythritol phosphate pathway [17] Fos-midomycin is a specific inhibitor of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase The effect of fosmidomycinon the levels of each intermediate and its metabolic require-ment for the isoprenoid biosynthesis such as dolichols andubiquinones throughout the intraerythrocytic cycle of Pfalciparum have been analyzed Fosmidomycin treatmentresulted in a decrease of the intermediate levels in the MEPpathway as well as in ubiquinone and dolichol biosynthesisThe MEP pathway associated transcripts were modestlyaltered by the drug indicating that the parasite is not stronglyresponsive at the transcriptional levelThis was the first study


DOXP synthase

CDP MEP synthase

Pyruvate + glyceraldehyde

1-Deoxy-D-xylulose-5-phosphate (DOXP)

DOXP reductoisomerase (fosmidomycin inhibitor)2-C-Methyl-D-erythritol-4-phosphate (MEP)

4-(Cytidine-5-diphospho)-2-C-methyl-D-erythritol (CDP-ME)

4-(Cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP-2P)

2-C-Methyl-D-erythritol-24-cyclodiphosphate (MEP-24-cPP)

Dimethyl allyldiphosphate DMAPP

Geranyl diphosphateGPP C10

Isopentenyl diphosphate IPP

(a)

H3C

H3C

H3C

H2C

H3C H3C

H3C

H3C

O O

COOH+ H

O

O

O OO

O

O

O

O

CMPO

P

P P

P P P

OH

OHOH

OH OH

OH OHOH

OH OH

OH

OH OH

OPO32minus OPO3

2minus

OPO32minus

OPO32minus

CO2

DXS

MEP

CMS

CMP

CMPMCS

CTP

PPi

NADPHH+ NADP+

DXR

Ominus Ominus Ominus

Ominus

ADP ATP

CMK

HDS

2 Fdred

2 Fdox

H2OH2O

OH

CH3

CH3

CH3O

O

O

O

O

O

O

O O

O

O

O

Ominus Ominus

Ominus

Ominus OminusOminus

OminusOminus

Ominus

NAD(P)HH+ NAD(P)+

HDR

P

P P

P DMAPP

IPP

CDP-MECDP-MEPMEcPP

HMB-PP

DXPG3PPyr

(b)

Figure 2 (a) Synthesis of isoprenoid compound by nonmevalonate pathway a flow chart (b) chemical reactions involved in biosynthesis ofisoprenoid via nonmevalonate pathway


that compares the effect of fosmidomycin on the metabolicand transcript profiles in P falciparum which has only theMEP pathway for isoprenoid biosynthesis

33 Dolichol Phosphate Mannose Synthase The intraerythro-cytic stages of P falciparum use dolichol and its phospho-rylated derivatives as carrier lipids in biosynthesis of severalglycoconjugates Anchors andN-linked glycoproteins requiredolichyl phosphate and dolichyl pyrophosphate as carriers ofdifferent monosaccharide constituents Kimura et al (1996)demonstrated the effect of N-linked glycoproteins on dif-ferentiation of intraerythrocytic stages of P falciparum [18]The reaction between dolichol phosphate (Dol-P) and guano-sine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man) is catalyzed by dolicholphosphate mannose synthase (DPM) enzyme [19] Thismolecule acts as mannose donor forN-glycosylation and gly-cosylphosphatidylinositol (GPI) biosynthesis P falciparumDPM1 (Pf dpm1) possesses a single predicted transmembraneregion near the N-terminus but not the C-terminus Pf dpm1was unable to complement a mouse mutant deficient in DPMbut efficiently complemented the Schizosaccharomyces pombefission yeast mutant indicating a difference between fissionyeast and mammalian DPM genes

Many eukaryotic cells such as yeast and a number ofmammalian cells are unable to incorporate more complexisoprenoid precursors such as FPP and GGPP In contrastintraerythrocytic forms of P falciparum easily metabolisethese compounds when they are added to the culturemedium permitting the subsequent identification of higherisoprenoids Walter (1986) reported that dolichol kinase arate limiting enzyme for the supply with dolichyl monophos-phate as glycosyl carrier lipid in P falciparum and inhibitionof this enzyme (dolichol kinase) occurred bymefloquine [20]The enzyme was found to be associated with the pellet frac-tion and to depend on cytidine triphosphate as phosphoryldonor Couto et al (1999) identified dolichol dolichyl phos-phate and dolichyl pyrophosphate of 55 and 60 carbons (1112isoprenic units) [21] Biochemical studies in P falciparumindicated that in addition to the pathway for synthesis ofphosphatidylcholine from choline (CDP-choline pathway)the parasite synthesizes this major membrane phospholipidvia an alternative pathway named the serine-decarboxylase-phosphoethanolamine-methyltransferase (SDPM) pathwayusing host serine and ethanolamine as precursors [22]

The pathogenic stages of P falciparum are those thatinvade mature erythrocytes which are devoid of internalorganelles and incapable ofde novo lipid biosynthesisDespitethis P falciparum undergoes dramatic morphological andmetabolic developmental changes and asexually divides toform new daughter cells within a human erythrocyte [23]This rapid multiplication of P falciparum within host ery-throcytes entails the active production of new plasma mem-branes in which phospholipids are the major architecturaland functional components Phosphatidylcholine (PtdCho)2and phosphatidylethanolamine (PtdEtn) comprise 40ndash50and 35ndash45 respectively of the parasitersquos total plasmamembrane phospholipid content [24] Genome data predicts

that P falciparum possesses enzymatic pathways for the syn-thesis of all the necessary phospholipids from precursorstransported fromhostmilieu such as serine choline inositolglycerol and fatty acids [24 25] The PfPMT gene in Pfalciparum encodes the phosphoethanolamine methyltrans-ferase that specifically methylates phosphoethanolamine tophosphocholine (p-Cho) via the SDPM pathway [26] Thestudies show that PfPMT is important formembrane biogen-esis development survival and propagation of the parasiteCompounds that target PfPMT could thus be combinedwith those that specifically target choline transport cholinephosphorylation or other steps of the CDP-choline pathwayin order to completely block PtdCho biosynthesis and kill theparasite

In P falciparum the biosynthesis of ubiquinone orcoenzyme Q involves two major steps synthesis of thebenzoquinone by the shikimate pathway and synthesis of theisoprene side chain by the MEP pathway The presence ofdolichol and isoprenylated proteins has been detected in Pfalciparum but no studies are available about the biosynthesisof the isoprenic side chain attached to the benzoquinone ringof coenzyme Q De Macedo et al (2002) showed that P fal-ciparum synthesizes different homologs (coenzyme Q(8) andcoenzyme Q(9)) depending on the given intermediate [27]The authors also demonstrated that nerolidol treatment of Pfalciparum parasites resulted in a reduced ability to synthesiseCoQ and inhibited P falciparum growth in vitro Treatmentwith nerolidol arrested development of the intraerythrocyticstages of the parasites indicating that the drug may have anantimalarial potential

4 Antimalarial Compounds againstIsoprenoid Biosynthetic Pathway

Biosynthesis of several isoprenoids in P falciparumwas stud-ied and terpenes (molecules with a similar chemical structureto the intermediates of the isoprenoids pathway) as potentialantimalarial drugswere evaluated [28ndash30] Different terpenesand S-farnesylthiosalicylic acid were tested on cultures ofthe intraerythrocytic stages of P falciparum and the 50inhibitory concentration for each one was found Farnesolnerolidol limonene and linalool terpenes have been usedagainst P falciparum [30] All the terpenes tested inhibiteddolichol biosynthesis in the trophozoite and schizont stagesFarnesol nerolidol and linalool showed stronger inhibitoryactivity in the biosynthesis of the isoprenic side chain ofthe benzoquinone ring of ubiquinones in the schizont stageThe inhibitory effect of terpenes and S-farnesylthiosalicylicacid on the biosynthesis of both dolichol the isoprenic sidechain of ubiquinones and the isoprenylation of proteins inthe intraerythrocytic stages of P falciparum appears to bespecific because overall protein biosynthesis was not affected

A variety of proteins undergo posttranslational mod-ification such as prenylation near the carboxyl terminuswith farnesyl (C15) or geranylgeranyl (C20) groups Proteinfarnesyltransferase (PFT) transfers the farnesyl group fromfarnesyl diphosphate to the SH of the cysteine near the C-terminus of proteins such as Ras PFT inhibitors (PFTIs)


have been extensively developed as anticancer agents becauseof their ability to block tumor growth in experimentalanimals PFTIs have been explored as antimalarial andantitrypanosome agents because these compounds are muchmore toxic to these parasites than to mammalian cells andthere are a large number of lead compounds which have beenexplored as part of the antiparasite drug discovery program[31] MEP pathway inhibition with fosmidomycin reducesprotein prenylation confirming that de novo isoprenoidbiosynthesis produces the isoprenyl substrates for proteinprenylation One important group of prenylated proteins issmall GTPases such as Rab family members which mediatecellular vesicular trafficking [32]

Substituted tetrahydroquinolines (THQs) have been pre-viously identified as inhibitors of mammalian protein farne-syltransferase Fletcher et al (2008) designed and synthesizeda series of inhibitors that are selective for P falciparumfarnesyltransferase (PfPFT) Several PfPFT inhibitors havebeen found to inhibit the malarial enzyme with IC50 valuesdown to 1 nM and that blocks the growth of P falciparum ininfected whole cells (erythrocytes) with ED50 values downto 55 nM Potent Plasmodium-selective farnesyltransferaseinhibitors that arrest the growth of malaria parasites havebeen explored [33] A new synthetic pathway was devisedto reach tetrasubstituted 3-arylthiophene 2-carboxylic acidsin a three-step solid-phase synthesis This very efficientmethodology provided more than 20 new compounds thatwere evaluated for their ability to inhibit protein farnesyl-transferase from different species as well as Trypanosomabrucei and P falciparum proliferation [34]

Strong inhibition of P falciparum PFT (PfPFT) bypeptidomimetics illustrated the potential of targeting theseenzymes in developing drug therapy for malaria Ohkandaet al (2001) have recently demonstrated the potency of avariety of other peptidomimetics as inhibitors ofP falciparumgrowth and PfPFT activity [35] Moura et al (2001) havealso shown that themonoterpene limonene inhibits parasitedevelopment and prenylation of P falciparum proteins [36]The enzymes of the nonmevalonate pathway for isoprenoidbiosynthesis are attractive targets for the development ofnovel drugs against malaria and tuberculosis

Fosmidomycin and FR900098 (an N-acetyl derivativeof fosmidomycin) are inhibitor of DOXP reductoisomerasewhich showed antimalarial activity in vitro and in vivo[15 29] (Figure 3)

Reverse hydroxamate-based inhibitor for IspC enzymewas evaluated by Behrendt et al (2011) [37] Fosmidomycinhas been proven to be efficient in the treatment ofP falciparum malaria by inhibiting 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) an enzyme of the non-mevalonate pathway which is absent in humans (Figure 4)

1-Deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr)represents an essential enzyme of the mevalonate-inde-pendent pathway of the isoprenoid biosynthesis Using fos-midomycin as a specific inhibitor of Dxr this enzyme waspreviously validated as target for the treatment of malariaand bacterial infections The replacement of the formylresidue of fosmidomycin by spacious acyl residues yieldedinhibitors active in the micromolar range As predicted by

flexible docking evidence was obtained for the formation ofa hydrogen bond between an appropriately placed carbonylgroup in the acyl residue and the main-chain NH of Met214located in the flexible catalytic loop of the enzyme [38 39]Specific inhibition of enzymes of the nonmevalonate pathwayis a promising strategy for the development of novel antiplas-modial drugs 120572-Aryl-substituted 120573-oxa isosteres of fos-midomycin with a reverse orientation of the hydroxamic acidgroup were synthesized and evaluated for their inhibitoryactivity against recombinant 1-deoxy-d-xylulose 5-phosphatereductoisomerase (IspC) of Plasmodium falciparum and fortheir in vitro antiplasmodial activity against chloroquine-sensitive and resistant strains ofP falciparumThemost activederivative inhibits IspC protein of P falciparum (Pf IspC)with an IC

50value of 12 nM and shows potent in vitro

antiplasmodial activity [40]The structure-activity relationships for 43 inhibitors

of 1-deoxyxylulose-5-phosphate (DOXP) reductoisomerasederived fromprotein-based docking ligand-based 3DQSARand a combination of both approaches as realized by AFMoC(adaptation of fields for molecular comparison) have beenpresented by Silber et al (2005) [41] A series of novel31015840-amido-31015840-deoxy-N(6)-(1-naphthylmethyl) adenosines wassynthesized applying a polymer-assisted solution phase(PASP) protocol and was tested for antimalarial activityversus theDd2 strain of Plasmodium falciparum Further thisseries and 62 adenosine derivatives were analyzed regard-ing 1-deoxy-D-xylulose-5-phosphate reductoisomerase inhi-bition Biological evaluations revealed that the investigated31015840N(6)-disubstituted adenosine derivatives displayed mod-erate but significant activity against the P falciparum parasitein the low-micromolar range [42]

5 Genetic Polymorphism in P falciparum

P falciparummalaria is an example of evolutionary selectionBoth host and parasite show the phenomenon of naturalselection Several polymorphisms in human host and parasitehave been found Table 1 shows details of genes involvedin isoprenoid metabolism and their polymorphisms in Pfalciparum In the drug discovery process the polymorphismsin parasite genes encoding the target protein should beconsidered In P falciparum the genetic polymorphisms at 10loci are considered potential targets for specific antimalarialvaccines [43] The polymorphism is unevenly distributedamong the loci loci encoding proteins expressed on thesurface of the sporozoite or the merozoite (AMA-1 CSPLSA-1 MSP-1 MSP-2 and MSP-3) are more polymorphicthan those expressed during the sexual stages or insidethe parasite (EBA-175 Pfs25 PF4845 and RAP-1) Com-parison of synonymous and nonsynonymous substitutionsindicates that natural selection may account for the poly-morphism observed at seven of the 10 loci studied Thisinference depends on the assumption that synonymoussubstitutions are neutral The authors obtained evidence foran overall trend towards increasing A+T richness but noevidence of mutation Although the neutrality of synony-mous substitutions is not definitely established this trend


H3N+

H2PO3

PO3H2

OH

Pamidronate

NHO

OH

OHPO

O

O

H

N

OH

OHOH

P

HO

O

NOH

OHOH

P

H

O

O

N HO

HO

OHOH

OH

P

P

O

O

NHO

OH

OHP

O

O

CH3

OHOH

OH

N P

O

OCH3

P OHOHN

HOO O

O

CH3

Actonel Fosmidomycin

FR900098

Figure 3 Structures of diphosphate and bisphosphonates (inhibitors of pathway) [8]

(a) (b)

(c) Fosmidomycin (d) FR900098

Figure 4 (a) Interaction of fosmidomycin with PfDXR FR900098 complex (b) The carbon atoms of the FR900098 molecule are shown inmagenta (c) fosmidomycin and (d) FR900098


Table 1 Details of genes involved in isoprenoid metabolism and their polymorphisms in P falciparum 3D7

Gene ID Previous ID Chromosome Product description Protein length NonsynonymousSNPs in all strains

SynonymousSNPs in allstrains

PF3D7 0104400 PFA0225w 1 4-Hydroxy-3-methylbut-2-enyldiphosphate reductase (LytB) 535 3 3

PF3D7 0106900 PFA0340w 12-C-Methyl-D-erythritol4-phosphate cytidylyltransferaseputative (IspD)

734 6 2

PF3D7 0209300 PFB0420w 22-C-Methyl-D-erythritol24-cyclodiphosphate synthase(IspF)

240 0 0

PF3D7 0318800 PFC0831w 3 Triosephosphate isomerase putative 357 0 0

PF3D7 0503100 PFE0150c 54-Diphosphocytidyl-2c-methyl-D-erythritol kinase (CMK)putative

537 4 0

PF3D7 0508300 PFE0410w 5 Triose phosphate transporter(oTPT) 342 0 0

PF3D7 0530200 PFE1510c 5 Triose phosphate transporter(iTPT) 524 1 0

PF3D7 0623200 PFF1115w 6 Ferredoxin NADP reductase (FNR) 371 1 1

PF3D7 1022800 PF10 0221 10 4-Hydroxy-3-methylbut-2-en-1-yldiphosphate synthase (GcpE) 824 1 0

PF3D7 1037100 PF10 0363 10 Pyruvate kinase 2 (PyKII) 745 2 1PF3D7 1318100 MAL13P195 13 Ferredoxin putative 194 2 0

PF3D7 1337200 MAL13P1186 13 1-Deoxy-D-xylulose 5-phosphatesynthase 1205 13 2

PF3D7 1439900 PF14 0378 14 Triosephosphate isomerase (TIM) 248 0 0

PF3D7 1467300 PF14 0641 14 1-Deoxy-D-xylulose 5-phosphatereductoisomerase (DOXR) 488 2 2

towards an A+T rich genome cannot explain the accumula-tion of substitutions at least in the case of four genes (AMA-1CSP LSA-1 and PF4845) because the GrarrC transversionsare more frequent than expected Predicted polymorphismsin genes encoding isoprenoid biosynthesis may play animportant role in drug response (Table 1)

Nonsynonymous single nucleotide polymorphisms in 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB)2-C-methyl-D-erythritol 4-phosphate cytidylyltransferaseputative (IspD) 1-deoxy-D-xylulose 5-phosphate synthase1-deoxy-D-xylulose 5-phosphate reductoisomerase (DOXR)and 4-diphosphocytidyl-2c-methyl-D-erythritol kinase(CMK) proteins may change the amino acids Such changemay affect the proteins and interaction with antimalarialdrugs

51 DNA Sequence Variation(s) and Evolution Mutationsor genetic variations that have ability to alter the activityor availability of transcription factors as well as mutationsthat alter the cis-regulatory sequences (transcription factorbinding sites) to which they bind can change expressionof gene and both types of changes contribute to evolutionSeveral studies have suggested that mutations affecting cis-regulatory activity (transcription factor binding site) are

the predominant source of expression divergence betweenspecies [44 45] Changes in gene expression often alterphenotypes mutations that affect gene expression can affectfitness and contribute to adaptive evolution DNA sequencevariations reported in genes involved in dolichol pathway ofP falciparum were shown

52 DNA Sequence Variation and Drug Response In thegenome different types of variations are reported such as copynumber variations (CNV) microsatellite repeats (SSR) andsingle nucleotide polymorphisms (SNP) which are known toplay role in regulation of gene expression Presence of suchvariations in the transcription factor binding site (TFBS)mayinfluence the binding of transcription factor to their respec-tive binding sites and is responsible for the alteration of geneproduct in the cell The presence of several polymorphismsin the upstream region gives rise to different haplotypesThese haplotypes in the transcription factor binding sitesalter the interaction of transcription factor (TF) to TFBS andmay cause transcriptional modification of gene altering thelevel of protein Genetic variation associated with increasedsusceptibility to complex diseases can elucidate genes andunderlying biochemical mechanisms linked to disease onset


and progression It has been suggested that when gene showsassociation with disease the most common condition is thatgenetic variation or causalmutations alter an encoded proteinsequence But a significant number of undiscovered causalmutations may alter the regulation of gene transcriptionHowever it remains a challenge to separate causal geneticvariations from linked neutral variations One of the mostimportant applications of genetic polymorphisms is the drugresponse Earlier we have detected that peoples from dif-ferent ethnic backgrounds have shown different susceptibil-ityresistance to falciparummalaria [46 47] It has been clearfrom several studies that polymorphisms are important factorfor determining the drug responseThemost studied exampleof polymorphisms and drug response in the P falciparum isthe multidrug resistance gene (pfmdr-1) that contributes tovariability and is known to play role in drug response Thepeoples varying in the genotype at a particular locusmay havedifferent responses to a particular drug which is illustrated inFigure 5

6 Discussion

It is very difficult to treat malaria because of the developmentof resistance of the parasite to antimalarial(s) Currently themain drug available is artemisinin (ART) and its derivativesand serious efforts are underway to develop ART-basedcombination therapies The new pharmacophores attackingdifferent targets in the parasite can bring in new strategiesto combat malaria Apart from efficacy toxic side effectspharmacokinetic compatibility and potential to developresistance would all be the major parameters in the eventualdevelopment of a successful drug Therefore all the targetsand molecules discussed in this review are important

Apicoplast itself contains a small circular genome mostof the proteome of this organelle is encoded in the nucleargenome and the proteins are subsequently transported tothe apicoplast It is assumed to contain a number of uniquemetabolic pathways not found in the vertebrate host makingit an ideal ldquoplaygroundrdquo for those interested in drug targetsThe apicoplast contains metabolic pathways critical for liver-stage and blood-stage development During the blood stagesparasites lacking an apicoplast can grow in the presence ofisopentenyl pyrophosphate (IPP) demonstrating that iso-prenoids are the only metabolites produced in the apicoplastwhich are needed outside of the organelle Two of theenzymes involved in isoprenoid biosynthesis are predicted torely on iron-sulfur (FeS) cluster cofactors [48]

Since the nonmevalonate pathway of isoprenoid biosyn-thesis is essential in eubacteria and P falciparum and thispathway is not present in humans there is a great interestin targeting the enzymes of nonmevalonate metabolism forantiparasitic drug development Fosmidomycin is currentlyin clinical trials of combination therapies for the treat-ment of malaria [49] Cell-penetrating peptides such asoligoarginines improved the intracellular delivery of thedrug fosmidomycin [50] Analogues of fosmidomycin thatis substituted in both the C120572 and the hydroxamate positionshave been found more potent than fosmidomycin in terms

of killing P falciparum in an in vitro assay [51] A screenof host cell proteins that might facilitate lipid scavengingby the parasite during the liver stage has pointed towardthe surface-expressed cholesterol ester receptor scavengerreceptor class B type 1 (SR-BI) [42] A decrease or completeloss of SR-BI surface availability via treatment with siRNA(short interfering RNA) antibodies drugs or gene deletionresults in a reduction in parasite invasion and growth inhepatocytes [52 53]

P falciparum mitochondrion presents structural andphysiological characteristics different from mitochondria inother eukaryotes [54] One of the reasons is the adaptationof the parasite to different environments in particular thegreat differences in oxygen tension between the host and themosquito Mitochondrial role in the intraerythrocytic envi-ronment particularly focuses on mitochondrial metabolicpathways that relate to oxidative phosphorylation includingthe tricarboxylic acid cycle de novo pyrimidine biosynthesisvia dihydroorotate dehydrogenase and the particularitiesof the electron transport chain Such unique particularitiesof parasite mitochondria could be promising targets fordevelopment of a new therapy The elucidation of the roleof this organelle in aerophilic respiratory metabolism andthe association of antimalarial drugs with hyperbaric oxygentherapy might provide new treatments for infection by Pfalciparum Howe et al (2013) demonstrate that proteinprenylation depends on de novo isoprenoid biosynthesis in Pfalciparum [32] When isoprenoid biosynthesis is inhibitedthe digestive food vacuole of the parasite is structurallyand functionally disrupted These results suggest that a keyfunction of isoprenoids in malaria is protein prenylation andsupport a role for prenylated proteins such as Rab GTPasesin FV maintenance

Dolichol and its derivatives are the lipids discoveredmost recently in eukaryotic cells They serve as inducers ofmembrane fusion The DOXP pathway provides precursorsfor protein farnesylation Plasmodial farnesyl transferaseactivity has unique biochemical features and inhibitors ofthis process have in vitro antimalarial activity [35 55] Theresistance of malaria parasites to available drugs continues togrow increasingly limiting our ability to control this seriousdisease Recent increases in the pace of progress in thisarea will be helpful in the development of novel antimalarialtherapies

As outlined in this review a number of pathways andinhibitory substances have already been identifiedwhich holdgreat promise for the future With the completion of thePlasmodium genome project new biosynthetic routes in theapicoplast are discovered and efforts are on to explore themas candidate drug targets The development of a number ofgenetic tools to validate these possible targets in transgenicparasites and also for drug screening purposes will have anenormous impetus on the development of drugs against thispathogen The analysis of the utility of isoprenoid biosynthe-sis pathway in P falciparum reveals that the proteins of thispathway might be important drug targets The study of hostgenetic factors and polymorphisms in parasite isoprenoidbiosynthetic genes will be helpful in the analysis of diseasesusceptibility and drug response


Alteration in protein levels may affect antimalarial drug response

Drug-protein interaction is affected and this may alter antimalarial drug response

Prediction of polymorphisms in genes involved in isoprenoid metabolic pathway of P falciparum and host genes involved in malaria pathogenesis

Regulatory polymorphisms may influence the level of protein(s) Coding region polymorphisms may

influence the structure of protein(s)

Figure 5 Diagrammatic representation of role of polymorphisms in drug response

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the Council of Scientific amp Indus-trial Research New Delhi India for providing researchfacility and supportive environment to carry out doctoralresearch work at Central Institute of Medicinal amp AromaticPlants Lucknow India Supportive facilities and conduciveenvironment at Dr Ram Manohar Lohia Avadh UniversityFaizabad India are thankfully acknowledged

References

[1] C D Poulter ldquoBioorganic chemistry a natural reunion of thephysical and life sciencesrdquo Journal of Organic Chemistry vol 74no 7 pp 2631ndash2645 2009

[2] J Lombard and D Moreira ldquoOrigins and early evolution ofthe mevalonate pathway of isoprenoid biosynthesis in the threedomains of liferdquoMolecular Biology and Evolution vol 28 no 1pp 87ndash99 2011

[3] B Zhang K M Watts D Hodge et al ldquoA second target of theantimalarial and antibacterial agent fosmidomycin revealed bycellular metabolic profilingrdquo Biochemistry vol 50 no 17 pp3570ndash3577 2011

[4] A C Brown M Eberl D C Crick H Jomaa and T ParishldquoThe nonmevalonate pathway of isoprenoid biosynthesis inMycobacterium tuberculosis is essential and transcriptionallyregulated by Dxsrdquo Journal of Bacteriology vol 192 no 9 pp2424ndash2433 2010

[5] F J Sangari J Perez-Gil L Carretero-Paulet JM Garcıa-Loboand M Rodrıguez-Concepcion ldquoA new family of enzymescatalyzing the first committed step of the methylerythritol4-phosphate (MEP) pathway for isoprenoid biosynthesis in

bacteriardquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14081ndash14086 2010

[6] F M Jordao E A Kimura and A M Katzin ldquoIsoprenoidbiosynthesis in the erythrocytic stages of Plasmodium falci-parumrdquoMemorias do Instituto Oswaldo Cruz vol 106 no 1 pp134ndash141 2011

[7] W Eisenreich A Bacher D Arigoni and F Rohdich ldquoBiosyn-thesis of isoprenoids via the non-mevalonate pathwayrdquo Cellularand Molecular Life Sciences vol 61 no 12 pp 1401ndash1426 2004

[8] E Oldfield ldquoTargeting isoprenoid biosynthesis for drug discov-ery bench to bedsiderdquo Accounts of Chemical Research vol 43no 9 pp 1216ndash1226 2010

[9] J Wiesner A Reichenberg S Heinrich M Schlitzer and HJomaa ldquoThe plastid-like organelle of apicomplexan parasites asdrug targetrdquo Current Pharmaceutical Design vol 14 no 9 pp855ndash871 2008

[10] J E Gisselberg T A Dellibovi-Ragheb K A Matthews GBosch and S T Prigge ldquoThe suf iron-sulfur cluster synthesispathway is required for apicoplast maintenance in malariaparasitesrdquo PLOS Pathogens vol 9 no 9 article e1003655 2013

[11] J Wiesner and H Jomaa ldquoIsoprenoid biosynthesis of theapicoplast as drug targetrdquo Current Drug Targets vol 8 no 1 pp3ndash13 2007

[12] G I McFadden ldquoThe apicoplastrdquo Protoplasma vol 248 no 4pp 641ndash650 2011

[13] M B Cassera F C Gozzo F L DrsquoAlexandri et al ldquoThemethylerythritol phosphate pathway is functionally active in allintraerythrocytic stages of Plasmodium falciparumrdquo Journal ofBiological Chemistry vol 279 no 50 pp 51749ndash51759 2004

[14] V K Singh and I Ghosh ldquoMethylerythritol phosphate pathwayto isoprenoids kinetic modeling and in silico enzyme inhibi-tions in Plasmodium falciparumrdquo FEBS Letters vol 587 no 17pp 2806ndash2817 2013

[15] H Jomaa J Wiesner S Sanderbrand et al ldquoInhibitors ofthe nonmevalonate pathway of isoprenoid biosynthesis asantimalarial drugsrdquo Science vol 285 no 5433 pp 1573ndash15761999


[16] T Umeda N Tanaka Y Kusakabe M Nakanishi Y Kitadeand K T Nakamura ldquoCrystallization and preliminary X-ray crystallographic study of 1-deoxy-d-xylulose 5-Phosphatereductoisomerase from Plasmodium falciparumrdquo Acta Crys-tallographica Section F Structural Biology and CrystallizationCommunications vol 66 no 3 pp 330ndash332 2010

[17] M B Cassera E F Merino V J Peres E A KimuraG Wunderlich and A M Katzin ldquoEffect of fosmidomycinon metabolic and transcript profiles of the methylerythritolphosphate pathway in Plasmodium falciparumrdquo Memorias doInstituto Oswaldo Cruz vol 102 no 3 pp 377ndash383 2007

[18] E A Kimura A S Couto V J Peres O L Casal and A MKatzin ldquoN-linked glycoproteins are related to schizogony of theintraerythrocytic stage in Plasmodium falciparumrdquo Journal ofBiological Chemistry vol 271 no 24 pp 14452ndash14461 1996

[19] H Shams-Eldin C S deMacedo S Niehus et al ldquoPlasmodiumfalciparum dolichol phosphate mannose synthase represents anovel claderdquo Biochemical and Biophysical Research Communica-tions vol 370 no 3 pp 388ndash393 2008

[20] R D Walter ldquoPlasmodium falciparum inhibition of dolicholkinase by mefloquinerdquo Experimental Parasitology vol 62 no3 pp 356ndash361 1986

[21] A S Couto E A Kimura V J Peres M L Uhrig and AM Katzin ldquoActive isoprenoid pathway in the intra-erythrocyticstages of Plasmodium falciparum presence of dolichols of 11 and12 isoprene unitsrdquo Biochemical Journal vol 341 no 3 pp 629ndash637 1999

[22] W H Witola K El Bissati G Pessi C Xie P D Roepe and CBMamoun ldquoDisruption of the Plasmodium falciparum PfPMTgene results in a complete loss of phosphatidylcholine biosyn-thesis via the serine-decarboxylase- phosphoethanolamine-methyltransferase pathway and severe growth and survivaldefectsrdquo Journal of Biological Chemistry vol 283 no 41 pp27636ndash27643 2008

[23] S Krishna ldquoMalariardquo British Medical Journal vol 315 no 7110pp 730ndash732 1997

[24] H J Vial C Ben Mamoun and I W Sherman ldquoPlasmodiumlipids metabolism and functionrdquo in Molecular Approaches toMalaria pp 327ndash352 ASM Press Washington DC USA 2005

[25] G G Holz Jr ldquoLipids and the malarial parasiterdquo Bulletin of theWorld Health Organization vol 55 no 2-3 pp 237ndash248 1977

[26] G Pessi and C Ben Mamoun ldquoPathways for phosphatidyl-choline biosynthesis targets and strategies for antimalarialdrugsrdquo Future Medi Future Lipido vol 1 no 2 pp 173ndash1802006

[27] C S De Macedo M L Uhrig E A Kimura and A M KatzinldquoCharacterization of the isoprenoid chain of coenzyme Q inPlasmodium falciparumrdquo FEMS Microbiology Letters vol 207no 1 pp 13ndash20 2002

[28] P J Proteau ldquo1-Deoxy-D-xylulose 5-phosphate reductoiso-merase an overviewrdquo Bioorganic Chemistry vol 32 no 6 pp483ndash493 2004

[29] S Steinbacher J KaiserW Eisenreich R Huber A Bacher andF Rohdich ldquoStructural basis of fosmidomycin action revealedby the complex with 2-C-methyl-D-erythritol 4-phosphate syn-thase (IspC) implications for the catalytic mechanism and anti-malaria drug developmentrdquo Journal of Biological Chemistry vol278 no 20 pp 18401ndash18407 2003

[30] H R Goulart E A Kimura V J Peres A S Couto F A ADuarte and A M Katzin ldquoTerpenes arrest parasite develop-ment and inhibit biosynthesis of isoprenoids in Plasmodium

falciparumrdquo Antimicrobial Agents and Chemotherapy vol 48no 7 pp 2502ndash2509 2004

[31] S Olepu P K Suryadevara K Rivas et al ldquo2-Oxo-tetrahydro-18-naphthyridines as selective inhibitors ofmalarial protein far-nesyltransferase and as anti-malarialsrdquo Bioorganic and Medici-nal Chemistry Letters vol 18 no 2 pp 494ndash497 2008

[32] R Howe M Kelly J Jimah D Hodge and A R Odom ldquoIso-prenoid biosynthesis inhibition disrupts Rab5 localization andfood vacuolar integrity in Plasmodium falciparumrdquo EukaryoticCell vol 12 no 2 pp 215ndash223 2013

[33] S Fletcher C G Cummings K Rivas et al ldquoPotent plasmo-dium-selective farnesyltransferase inhibitors that arrest thegrowth of malaria parasites structure-activity relationshipsof ethylenediamine-analogue scaffolds and homology modelvalidationrdquo Journal of Medicinal Chemistry vol 51 no 17 pp5176ndash5197 2008

[34] S Lethu D Bosc E Mouray P Grellier and J Dubois ldquoNewprotein farnesyltransferase inhibitors in the 3-arylthiophene 2-carboxylic acid series diversification of the arylmoiety by solid-phase synthesisrdquo Journal of Enzyme Inhibition and MedicinalChemistry vol 28 no 1 pp 163ndash171 2013

[35] J Ohkanda J W Lockman K Yokoyama et al ldquoPep-tidomimetic inhibitors of protein farnesyltransferase showpotent antimalarial activityrdquo Bioorganic and Medicinal Chem-istry Letters vol 11 no 6 pp 761ndash764 2001

[36] I C Moura G Wunderlich M L Uhrig et al ldquoLimonenearrests parasite development and inhibits isoprenylation ofproteins in Plasmodium falciparumrdquo Antimicrobial Agents andChemotherapy vol 45 no 9 pp 2553ndash2558 2001

[37] C T Behrendt A Kunfermann V Illarionova et al ldquoReversefosmidomycin derivatives against the antimalarial drug targetIspC (Dxr)rdquo Journal of Medicinal Chemistry vol 54 no 19 pp6796ndash6802 2011

[38] T Umeda N Tanaka Y KusakabeMNakanishi Y Kitade andK T Nakamura ldquoMolecular basis of fosmidomycinrsquos action onthe human malaria parasite Plasmodium falciparumrdquo ScientificReports vol 1 article no 9 2011

[39] R Ortmann J Wiesner K Silber G Klebe H Jomaa and MSchlitzer ldquoNovel deoxyxylulosephosphate-reductoisomeraseinhibitors fosmidomycin derivatives with spacious acylresiduesrdquo Archiv der Pharmazie vol 340 no 9 pp 483ndash4902007

[40] K Brucher B Illarionov J Held et al ldquo120572-substituted 120573-Oxaisosteres of fosmidomycin synthesis and biological evaluationrdquoJournal of Medicinal Chemistry vol 55 no 14 pp 6566ndash65752012

[41] K Silber P Heidler T Kurz and G Klebe ldquoAFMoC en-hances predictivity of 3D QSAR a case study with DOXP-reductoisomeraserdquo Journal of Medicinal Chemistry vol 48 no10 pp 3547ndash3563 2005

[42] CHerforth JWiesner P Heidler et al ldquoAntimalarial activity ofN6-substituted adenosine derivativesmdashpart 3rdquo Bioorganic andMedicinal Chemistry vol 12 no 4 pp 755ndash762 2004

[43] A A Escalante A A Lal and F J Ayala ldquoGenetic polymor-phism and natural selection in themalaria parasite Plasmodiumfalciparumrdquo Genetics vol 149 no 1 pp 189ndash202 1998

[44] R M Graze L M McIntyre B J Main M L Wayne and S VNuzhdin ldquoRegulatory divergence in Drosophila melanogasterand D simulans a genomewide analysis of allele-specificexpressionrdquo Genetics vol 183 no 2 pp 547ndash561 2009


[45] I Tiroshauth S Reikhav A A Levy and N Barkai ldquoA yeasthybrid provides insight into the evolution of gene expressionregulationrdquo Science vol 324 no 5927 pp 659ndash662 2009

[46] S Sinha TQidwai K Kanchan et al ldquoDistinct cytokine profilesdefine clinical immune response to falciparum malaria inregions of high or low disease transmissionrdquo European CytokineNetwork vol 21 no 4 pp 232ndash240 2010

[47] S Sinha G N Jha P Anand et al ldquoCR1 levels and genepolymorphisms exhibit differential association with falciparummalaria in regions of varying disease endemicityrdquo HumanImmunology vol 70 no 4 pp 244ndash250 2009


[49] F Mi-Ichi K Kita and T Mitamura ldquoIntraerythrocytic Plas-modium falciparum utilize a broad range of serum-derived fattyacids with limited modification for their growthrdquo Parasitologyvol 133 no 4 pp 399ndash410 2006

[50] C Sparr N Purkayastha B Kolesinska et al ldquoImprovedefficacy of fosmidomycin against Plasmodium and Mycobac-terium species by combinationwith the cell-penetrating peptideoctaargininerdquo Antimicrob Agents Chemother vol 57 no 10 pp4689ndash4698 2013

[51] A M Jansson A Więckowska C Bjorkelid et al ldquoDXRinhibition by potent mono- and disubstituted fosmidomycinanaloguesrdquo Journal of Medicinal Chemistry vol 56 no 15 pp6190ndash6199 2013

[52] C D Rodrigues M Hannus M Prudencio et al ldquoHostscavenger receptor SR-BI plays a dual role in the establishmentof Malaria parasite liver infectionrdquo Cell Host and Microbe vol4 no 3 pp 271ndash282 2008

[53] S Yalaoui T Huby J-F Franetich et al ldquoScavenger receptorBI boosts hepatocyte permissiveness to Plasmodium infectionrdquoCell Host and Microbe vol 4 no 3 pp 283ndash292 2008

[54] M Torrentino-Madamet J Desplans C Travaille Y Jammesand D Parzy ldquoMicroaerophilic respiratory metabolism of Plas-modium falciparum mitochondrion as a drug targetrdquo CurrentMolecular Medicine vol 10 no 1 pp 29ndash46 2010

[55] D Chakrabarti T D Silva J Barger et al ldquoProtein farnesyl-transferase and protein prenylation in Plasmodium falciparumrdquoJournal of Biological Chemistry vol 277 no 44 pp 42066ndash42073 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Fosmidomycin is a broad-spectrum antimicrobial agent andhas been used in clinical trials of combination therapiesfor the treatment of malaria [3] In vitro fosmidomycin isknown to inhibit the deoxyxylulose phosphate reductoiso-merase (DXR) enzyme of isoprenoid biosynthesis from mul-tiple pathogenic organisms Isoprenoid metabolism proceedsthrough DXR even in the presence of fosmidomycin but isinhibited at the level of the downstream enzyme methylery-thritol phosphate cytidylyltransferase (IspD)Overexpressionof IspD in E coli conferred fosmidomycin resistance andfosmidomycin was found to inhibit IspD in vitro [3] Underin vivo conditions fosmidomycin may inhibit IspD directlyor may cause other changes within the cell that reduceIspD activity [3]Mycobacterium tuberculosis synthesizes iso-prenoids via the nonmevalonate or DOXP pathway Previouswork had demonstrated that three enzymes namely DxrIspD and IspF are all required for growth in vitro TheDOXP biosynthetic pathway ofM tuberculosis is specific andessential and represents an attractive potential target for thedesign of new antimycobacterial agents The work by Brownet al (2010) demonstrated that three enzymes in the pathway(Dxr IspD and IspF) are all required for in vitro growth ofM tuberculosis [4] Most eubacteria (not all) synthesize theirisoprenoids using themethylerythritol-4-phosphate pathwaywhereas a minority uses the unrelated mevalonate pathwayand only a few have both [5] Isoprenoids are a large andhighly diverse group of natural products withmany functionsand their synthesis is essential for the parasitersquos survival [6]This paper attempts to cover the different enzymes involved inisoprenoid biosynthesis and their importance in P falciparumand antimalarial drug against these enzymes In additionan insight into host-parasite genetic polymorphisms is alsopresented

2 Scope of Apicoplast as AntimalarialDrug Target

The parasites such as Plasmodium spp Babesia spp Tox-oplasma gondii Cryptosporidium spp Isospora belli andCyclospora cayetanensis infect humans [9] With the excep-tion of Cryptosporidium spp these parasites possess api-coplast a nonphotosynthetic plastid-like organelle The api-coplast genome houses only 68 open reading frames (ORFs)for rRNAs tRNAs ribosomes RNA polymerase transla-tional elongation factor (EF-Tu) ClpC chaperone and Fe-Scluster protein (SufB) [1 9] Most of the proteins involved inthe apicoplastmetabolic pathways are encoded in the nucleussynthesized in the cytoplasm and subsequently importedinto the apicoplast [2]

The apicoplast contains metabolic pathways critical forliver-stage and blood-stage development During the bloodstages parasites lacking an apicoplast can grow in thepresence of isopentenyl pyrophosphate demonstrating thatisoprenoids are the only metabolites produced in the api-coplast which are needed outside of the organelle Two ofthe isoprenoid biosynthesis enzymes are predicted to rely oniron-sulfur (FeS) cluster cofactors however little is knownabout FeS cluster synthesis in the parasite or the roles that FeS

cluster proteins play in parasite biology [10] In P falciparumthe protein SufS and its partner SufE were found exclusivelyin the apicoplast and SufS was shown to have cysteinedesulfurase activity in a complementation assay IscS andits effector Isd11 were solely mitochondrial suggesting thatthe Isc pathway cannot contribute to apicoplast FeS clustersynthesis The Suf pathway was disrupted with a dominantnegative mutant resulting in parasites that were only viablewhen supplemented with IPP These parasites lacked theapicoplast organelle and its organellar genomemdasha phenotypenot observed when isoprenoid biosynthesis was specificallyinhibited with fosmidomycinThese results demonstrate thatthe Suf pathway is essential for parasite survival [10]

Fatty acid isoprenoid haem biosynthesis Fe-S clustersand DNA transactions are prominent pathways in api-coplast The most important metabolic functions and themevalonate independent 1-deoxy-D-xylulose-5-phosphate(DOXP) pathway of isoprenoid synthesis and the type IIfatty acid synthesis system operate inside the apicoplastClassical antibacterial drugs such as ciprofloxacin tetra-cycline doxycycline clindamycin and spiramycin inhibitthe apicoplast-located gyrase and translation machineryrespectively and are currently used for the treatment ofinfections with apicomplexan parasites Fosmidomycin aninhibitor of isoprenoid synthesis was proven to be effectiveagainst acute falciparum malaria in clinical phase II studiesFosmidomycin alone or in combination with clindamycinwas evaluated for the treatment of acute uncomplicatedfalciparum malaria Monotherapy using fosmidomycin ledto a fast parasite clearance but was inefficient in radicalelimination of the parasites [11] Triclosan an inhibitor offatty acid synthesis was active in a malaria mouse model Invitro antimalarial activity was shown for inhibitors of peptidedeformylase and the import of apicoplast-targeted proteinsIn the drug discovery against P falciparum the presence ofapicoplast has been a milestone [12]

3 Isoprenoid Biosynthesis

It is required for the production of isopentenyl pyrophosphateand dimethylallyl pyrophosphate that plays a role in thebiosynthesis of molecules used in protein prenylation cellmembrane maintenance hormones protein anchoring andN-glycosylation Plants and apicomplexan protozoa such asmalaria parasites have the ability to produce their isoprenoidsby means of an alternative pathway (nonmevalonate path-way) which takes place in their plastids Interestingly mostbacteria including important pathogens such as M tuber-culosis synthesize IPP and DMAPP via the nonmevalonatepathway

31 Nonmevalonate (MEPDOXP Pathway) Plants and api-complexan protozoa such as malaria parasites produce theirisoprenoids by utilizing an additional alternative pathwaycalled the methylerythritol phosphate (MEP) or nonmeval-onate pathway which takes place in their plastids (Figure 1)

Higher plants possess the MEV pathway in the cytosolin addition to the MEP pathway in the plastids In con-trast the MEV pathway has not yet been detected in


Apicoplast

35kb Doxycycline



prenylated proteins

Parasite

Erythrocyte

IPPOPP

Fosmidomycin

Pyruvate+

G3PDOXP MEP

IPPOPP OPP

+

DMAPP









DOXP synthase

CDP MEP synthase










(a)

H3C

H3C

H3C

H2C

H3C H3C

H3C

H3C

O O

COOH+ H

O

O

O OO

O

O

O

O

CMPO

P

P P

P P P

OH

OHOH

OH OH

OH OHOH

OH OH

OH

OH OH

OPO32minus OPO3

2minus

OPO32minus

OPO32minus

CO2

DXS

MEP

CMS

CMP

CMPMCS

CTP

PPi

NADPHH+ NADP+

DXR


Ominus

ADP ATP

CMK

HDS

2 Fdred

2 Fdox

H2OH2O

OH

CH3

CH3

CH3O

O

O

O

O

O

O

O O

O

O

O

Ominus Ominus

Ominus

Ominus OminusOminus

OminusOminus

Ominus

NAD(P)HH+ NAD(P)+

HDR

P

P P

P DMAPP

IPP

CDP-MECDP-MEPMEcPP

HMB-PP

DXPG3PPyr

(b)


























H3N+

H2PO3

PO3H2

OH

Pamidronate

NHO

OH

OHPO

O

O

H

N

OH

OHOH

P

HO

O

NOH

OHOH

P

H

O

O

N HO

HO

OHOH

OH

P

P

O

O

NHO

OH

OHP

O

O

CH3

OHOH

OH

N P

O

OCH3

P OHOHN

HOO O

O

CH3


FR900098


(a) (b)









734 6 2


240 0 0



537 4 0
















6 Discussion

















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Apicoplast

35kb Doxycycline



prenylated proteins

Parasite

Erythrocyte

IPPOPP

Fosmidomycin

Pyruvate+

G3PDOXP MEP

IPPOPP OPP

+

DMAPP









DOXP synthase

CDP MEP synthase










(a)

H3C

H3C

H3C

H2C

H3C H3C

H3C

H3C

O O

COOH+ H

O

O

O OO

O

O

O

O

CMPO

P

P P

P P P

OH

OHOH

OH OH

OH OHOH

OH OH

OH

OH OH

OPO32minus OPO3

2minus

OPO32minus

OPO32minus

CO2

DXS

MEP

CMS

CMP

CMPMCS

CTP

PPi

NADPHH+ NADP+

DXR


Ominus

ADP ATP

CMK

HDS

2 Fdred

2 Fdox

H2OH2O

OH

CH3

CH3

CH3O

O

O

O

O

O

O

O O

O

O

O

Ominus Ominus

Ominus

Ominus OminusOminus

OminusOminus

Ominus

NAD(P)HH+ NAD(P)+

HDR

P

P P

P DMAPP

IPP

CDP-MECDP-MEPMEcPP

HMB-PP

DXPG3PPyr

(b)


























H3N+

H2PO3

PO3H2

OH

Pamidronate

NHO

OH

OHPO

O

O

H

N

OH

OHOH

P

HO

O

NOH

OHOH

P

H

O

O

N HO

HO

OHOH

OH

P

P

O

O

NHO

OH

OHP

O

O

CH3

OHOH

OH

N P

O

OCH3

P OHOHN

HOO O

O

CH3


FR900098


(a) (b)









734 6 2


240 0 0



537 4 0
















6 Discussion

















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


DOXP synthase

CDP MEP synthase










(a)

H3C

H3C

H3C

H2C

H3C H3C

H3C

H3C

O O

COOH+ H

O

O

O OO

O

O

O

O

CMPO

P

P P

P P P

OH

OHOH

OH OH

OH OHOH

OH OH

OH

OH OH

OPO32minus OPO3

2minus

OPO32minus

OPO32minus

CO2

DXS

MEP

CMS

CMP

CMPMCS

CTP

PPi

NADPHH+ NADP+

DXR


Ominus

ADP ATP

CMK

HDS

2 Fdred

2 Fdox

H2OH2O

OH

CH3

CH3

CH3O

O

O

O

O

O

O

O O

O

O

O

Ominus Ominus

Ominus

Ominus OminusOminus

OminusOminus

Ominus

NAD(P)HH+ NAD(P)+

HDR

P

P P

P DMAPP

IPP

CDP-MECDP-MEPMEcPP

HMB-PP

DXPG3PPyr

(b)


























H3N+

H2PO3

PO3H2

OH

Pamidronate

NHO

OH

OHPO

O

O

H

N

OH

OHOH

P

HO

O

NOH

OHOH

P

H

O

O

N HO

HO

OHOH

OH

P

P

O

O

NHO

OH

OHP

O

O

CH3

OHOH

OH

N P

O

OCH3

P OHOHN

HOO O

O

CH3


FR900098


(a) (b)









734 6 2


240 0 0



537 4 0
















6 Discussion

















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























H3N+

H2PO3

PO3H2

OH

Pamidronate

NHO

OH

OHPO

O

O

H

N

OH

OHOH

P

HO

O

NOH

OHOH

P

H

O

O

N HO

HO

OHOH

OH

P

P

O

O

NHO

OH

OHP

O

O

CH3

OHOH

OH

N P

O

OCH3

P OHOHN

HOO O

O

CH3


FR900098


(a) (b)









734 6 2


240 0 0



537 4 0
















6 Discussion

















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















H3N+

H2PO3

PO3H2

OH

Pamidronate

NHO

OH

OHPO

O

O

H

N

OH

OHOH

P

HO

O

NOH

OHOH

P

H

O

O

N HO

HO

OHOH

OH

P

P

O

O

NHO

OH

OHP

O

O

CH3

OHOH

OH

N P

O

OCH3

P OHOHN

HOO O

O

CH3


FR900098


(a) (b)









734 6 2


240 0 0



537 4 0
















6 Discussion

















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


H3N+

H2PO3

PO3H2

OH

Pamidronate

NHO

OH

OHPO

O

O

H

N

OH

OHOH

P

HO

O

NOH

OHOH

P

H

O

O

N HO

HO

OHOH

OH

P

P

O

O

NHO

OH

OHP

O

O

CH3

OHOH

OH

N P

O

OCH3

P OHOHN

HOO O

O

CH3


FR900098


(a) (b)









734 6 2


240 0 0



537 4 0
















6 Discussion

















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







734 6 2


240 0 0



537 4 0
















6 Discussion

















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



6 Discussion

















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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Review Article Exploring Drug Targets in Isoprenoid ...downloads.hindawi.com/journals/bri/2014/657189.pdfcomplexan protozoa such as malaria parasites produce their isoprenoids by utilizing