The folate metabolic network of Falciparum malaria

ARTICLE IN PRESS

G Model MOLBIO 10739 1–13

Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Molecular & Biochemical Parasitology

1

Review

2

The folate metabolic network of Falciparum malaria

3 4

Q1

J. Enrique Salcedo-Sora ∗ , Steve A. Ward Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK

5

6

a r t i c l e

i n f o

a b s t r a c t

7 8 9 10 11

Article history: Received 29 May 2012 Received in revised form 4 February 2013 Accepted 11 February 2013 Available online xxx

12 13 14 15 16 17 18 19 20

21

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Keywords: Folate pABA Plasmodium One-carbon metabolism Folate transport Malaria Q2 Plasmodium cell cycle

The targeting of key enzymes in the folate pathway continues to be an effective chemotherapeutic approach that has earned antifolate drugs a valuable position in the medical pharmacopoeia. The successful therapeutic use of antifolates as antimalarials has been a catalyst for ongoing research into the biochemistry of folate and pterin biosynthesis in malaria parasites. However, our understanding of the parasites folate metabolism remains partial and patchy, especially in relation to the shikimate pathway, the folate cycle, and folate salvage. A sizeable number of potential folate targets remain to be characterised. Recent reports on the parasite specific transport of folate precursors that would normally be present in the human host awaken previous hypotheses on the salvage of folate precursors or by-products. As the parasite progresses through its life-cycle it encounters very contrasting host cell environments that present radically different metabolic milieus and biochemical challenges. It would seem probable that as the parasite encounters differing environments it would need to modify its biochemistry. This would be reflected in the folate homeostasis in Plasmodium. Recent drug screening efforts and insights into folate membrane transport substantiate the argument that folate metabolism may still offer unexplored opportunities for therapeutic attack. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . para-Amino benzoic acid (pABA) biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . An outline of the folate cycle and one-carbon metabolism in Plasmodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential folate salvage opportunities during the Plasmodium life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The Plasmodium-infected hepatocyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The circulating Plasmodium-infected red blood cell and the pABA salvage hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Plasmodium gametocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. The vector Anopheles spp. and the sexual cycle of Plasmodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Folate membrane transport in Plasmodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Flux-balance and standard thermodynamics of the Falciparum folate network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Antifolate targets and gene discovery strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Mutifunctional and chokepoint targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. The antifolates as probes of folate biochemistry and membrane transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Targeting folate transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

39

∗ Corresponding author at: Molecular and Biochemical Parasitology Group, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. Tel.: +44 0151 7053278; fax: +44 0151 7053371. E-mail address: [email protected] (J.E. Salcedo-Sora).

1. Introduction

40

Approximate half of the world’s population live in areas with active malaria transmission and in 2008 there were 190–311 million cases of malaria worldwide: 85% in Africa, 10% in South-East Asia and 4% in Eastern Mediterranean regions. With an estimated 0.71 million deaths in the same year attributed to malaria it is clear this disease continues to be a major global health problem [1]. The

41

0166-6851/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

42 43 44 45 46

ARTICLE IN PRESS

G Model MOLBIO 10739 1–13

J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

2

O

OH

O

O HN 3

4

R

N

N

CH 2

5

A

B

N

N H

C

H 2N 2

1

O

NH

10

NH

9

6

O OH

7

O

8

Pterin

pABA

OH

n

Glu

Fig. 1. Tetrahydropteroic poly-gamma-glutamic acid. The heterocyclic pterin ring is reduced at positions 5,6,7,and 8. The substituent (R) is found mainly as HCO , CH2 , or CH3 linked to positions N5 and/or N10 as 10-formyltetrahydrofolate (for formyl-methionyl-tRNA synthesis), 5,10-methylenetetrahydrofolate (for dTMP synthesis), and 5-mTHF (for methionine synthesis), respectively. Intermediate reactants are NHCH as formininotetrahydrofolate, and CH+ as 5,10-methenyltetrahydrofolate. Folates contain at least one glutamate residue and are sequestered intracellularly as polyglutamated derivatives with usually 3–8 glutamate residues (n = 3–8).

71

deadliest form of human malaria caused by Plasmodium falciparum is endemic in 87 countries from which 451 million cases were estimated for 2007 [2], with the majority of the mortality occurring in young children (<5 years old). Antifolates as chemotherapeutic agents that restrain cell proliferation have been deployed widely in the clinical management over the last 70 years. Current antifolate therapies are established treatments for a number of cancers [3], chronic inflammatory pathologies (e.g. rheumatoid arthritis, psoriasis) [4,5], bacterial infections [6,7], and parasitic infections (e.g. Toxoplasmosis) [8]. The latter category includes malaria, although it is notable that the selective pressure imposed on antifolate target genes has selected for mutations that have resulted in widespread clinical resistance to these drugs [9,10]. Currently antifolates as part of the treatment of uncomplicated malaria are only recommended in combination with artemisinin or amodiaquine [11] and even this utility is being eroded through resistance. However, the historical evidence confirms that the folate pathway is a proven drug target with huge clinical potential and we argue that there is a strong case to continue trying to exploit novel aspects of this essential parasite pathway. Antifolate, antimalarials and antifolate resistance have been extensively reviewed several times over the past decade (e.g. [12–14] and references therein) and as a consequence this important area will not be revisited in this review.

72

2. para-Amino benzoic acid (pABA) biosynthesis

73

The B9 family of vitamins or folates are essential cofactors that carry a one-carbon unit that at different levels of oxidation is utilised in the biosynthesis of thymidylate, methionine, and mitochondrial N-formyl-methionyl-t-RNA. They are found in nature as conjugated pterins: a heterocyclic pterin ring conjugated with paraaminobenzoic acid (pABA) and at least one glutamate residue that can be polymerised through covalent gamma-linked peptide bonds to produce tetrahydropteroylpoly-gamma glutamate (H4 PteGlun ) [15](Fig. 1 and Table 1). They are synthesised de novo by some prokaryotes, lower eukaryotes (e.g. Apicomplexa parasites such as Toxoplasma and Plasmodium), and plants [16]. Organisms that do not synthesise folates de novo have to salvage them from exogenous sources. At key stages in its life cycle the malaria parasite has the metabolic needs of a rapidly proliferating cell. There are at least four distinct genomic replication events in the life cycle of these parasites [17,18] where folate products are essential cofactors

47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70

74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

supporting the conversion of available nutrients into biomass. Consequently, Plasmodium species have evolved to meet their folate needs by preserving the de novo folate pathway (dispensed with by most eukaryotes) and developing mechanisms to salvage folate from external sources [19]. Although it has been established that malaria parasites express the required enzymes for the synthesis of folates from GTP, pABA and glutamate (x–xvi) (Table 2, Fig. 2) [20–25], far less biochemical information is available defining the upstream pathways such as the biosynthesis of pABA. There is pharmacological evidence that pABA synthesis via the shikimate pathway is functional in Plasmodium given that pABA supplementation can rescue the growth inhibitory effects of the herbicide glyphosate [26,27]. Glyphosate is an inhibitor of shikimate kinase/5-enolpyruvyl-shikimate-3-phosphate synthase (v–vi) [28]. A number of shikimate analogues have also been shown to have an inhibitory effect on P. falciparum growth in vitro, an effect that is synergistic with other known antimalarials [29,30]. The shikimate pathway links central carbon metabolism with the synthesis of chorismic acid that is then utilised in the synthesis of a variety of aromatic compounds (aromatic amino acids, hydroxybenzoate for ubiquinone synthesis, and pABA) [31]. It is generally accepted that seven enzymatic steps catalyse the production of chorismic acid (i–vii) (Table 2, Fig. 2). Although six out of seven shikimate biosynthesis enzyme activities have been detected in crude extracts of P. falciparum or Toxoplasma gondii [32,33], only the gene product for chorismate synthase has been characterised. This enzyme was found to be cytoplasmic in contrasts to the plant homologue which localises to the plastid [34]. Found only in microorganisms and plants the shikimate biosynthetic pathway is part of the parasite-specific enzymatic machinery that could offer specific chemotherapeutic targets. pABA is synthesised from chorismic acid in two steps [35]. First, aminodeoxychorismate (ADC) synthase transfers the amide nitrogen of glutamine to chorismic acid (viii) (Table 2, Fig. 2), forming 4-amino-4-deoxychorismate. ADC lyase then removes pyruvate from ADC and aromatises the ring to generate pABA (ix) (Table 2, Fig. 2). While ADC synthase activity is generated from a multisubunit PabA–PabB protein complex in Escherichia coli [35], in eukaryotes (i.e. yeast and plants) it is present as a single protein with fused domains homologous to PabA and PabB [36,37]. The P. falciparum protein encoded by PFI1100w (PlasmoDB 8.0) [38] has previously been identified as a potential pABA synthetase or PabA–PabB homologue [39]. These hybrid proteins in eukaryotes have been commonly named PABA synthase reflecting the assumption that they would catalyse the different steps for pABA synthesis

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134

G Model MOLBIO 10739 1–13

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

3

Table 1 Folates nomenclature [144]. Folates are a group of heterocyclic compounds based on the 4-[(pteridin-6-ylmethyl) amino] benzoic acid skeleton (Fig. 1) conjugated with one or more l-glutamic acid units [145]. The term folates is commonly used to refer to any of the folate products listed in this table. De novo biosynthesis of folates involves the conjugation with an l-glutamate. Any further conjugation with this amino acid is named polyglutamation and abbreviated as Glun , where n is equal to or higher than two. NA: no abbreviation.

135 136 137 138 139 140 141

Common name

IUPAC

Symbol

Abbreviation

DHF THF 5-Formyl tetrahydrofolate 10-Formyl tetrahydrofolate 5,10-Methenyl tetrahydrofolate 5,10-Methylene tetrahydrofolate 5-Methyl tetrahydrofolate Folic acid

7,8-Dihydropteroylglutamate 5,6,7,8-Tetrahydropteroylglutamate 5-Formyl tetrahydropteroylglutamate 10-Formyl tetrahydropteroylglutamate 5,10-Methenyl tetrahydropteroylglutamate 5,10-Methylene tetrahydropteroylglutamate 5-Methyl tetrahydropteroylglutamate Pteroylglutamate

H2 PteGlu H4 PteGlu 5-CHO-H4 PteGlu 10-CHO-H4 PteGlu 5,10-CH+ -H4 PteGlu 5,10-CH2 -H4 PteGlu 5-CH3 -H4 PteGlu PteGlu

DHF THF 5-fTHF 10-fTHF 5,10-meTHF 5,10-myTHF 5-mTHF NA

from chorismate. That view was modified when the first eukaryotic proteins with separate ADC lyase – PabC activity – were characterised [40,41]. ADC lyase is a member of the fold-type IV of pyridoxal 5phosphate dependent enzymes. This activity is carried out by PabC in E. coli [42] and eukaryotes [36,41]. Given the relatively recent discovery of PabC proteins in eukaryotes, there is a possibility

that Plasmodium parasites also encode an equivalent enzyme that had not been found before. A candidate for a PabC homologue in P. falciparum, the gene product of PF14 0557 (Salcedo-Sora et al., unpublished) shows primary structural similarities to ADC lyases (20–32% identity and 44–49% similarity) functionally characterised from E. coli [42], S. cerevisiae (Abz2p) [41], and Streptomyces sp [43]. Although neither gene product PFI1100w or PF14 0557 has been

Fig. 2. Folate metabolic network in P. falciparum. Nomenclature follows the recommendations of the IUPAC [145]. Abbreviations as used in the Kyoto Encyclopedia of Genes and Genomes (KEGG http://www.genome.jp/kegg/). Dotted lines represent reactions present in model organisms but are speculative or presumed present in Plasmodium. Exogenous salvage in the form of pABA and 5-methyltetrahydrofolate is represented by the white boxes. Directionality of the reactions follows their ır G o (2). Four reactions (ix, x, xvii, xxi) with the highest ?rG’o have thicker arrows. Reactions for 10-formyl-THF synthetase and 10-formyl-THF dehydrogenase [153] follow the dashed green and yellow arrows, respectively.

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

142 143 144 145 146 147 148

ARTICLE IN PRESS

G Model MOLBIO 10739 1–13

J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

4

Table 2 Enzymes of the folate metabolic network in P. falciparum. Protein or enzyme activities have been assigned to seventeen reactions that serve the folate biosynthesis and usage pathways in P. falciparum. These are eleven genes, of which six (CS, PTPS, DHPS-PPPK, DHFS-FPGS, DHFR-TS, SHMT) have been functional characterised and eight still at a putative status (SK-EPSP, pabA/pabB, pabC, GTPCH-I, GCV/GCS, MTFMT, MTHFD/MTHFC, FTHFS). Additionally, three cell purified enzyme activities have also been reported (pABA synthase, GTPCH-I, MTHFR, MS). PlasmoDB (Plasmo DB v8.0). Between parenthesis in the first column are the reactions as in Fig. 2. (*) ır G o values from [115,149,150] as defined in [114]. NA: not available. (#) Native protein defined as cell protein extracts that are not denaturated and presumed functional. Name

ır G o (kJ/mol)*

PlasmoDB

EC

Functional characterisation (#)

Shikimate kinase (SK) (v) 3-Phosphoshikimate 1-carboxyvinyl transferase/3enolpyruvylshikimate 5-phosphate synthase (EPSP) (vi) Chorismate synthase (CS) (vii)

−8.4 −8.0

PFB0280w PFB0280w

2.7.1.71 2.5.1.19

Putative bifunctional SK-EPSP

−60.5

PFF1105c

4.2.3.5

[34]

Aminodeoxychorismate synthase (pabA/pabB) (viii) Aminodeoxychorismate lyase (pabC) (ix) GTP cyclohydrolase I (GTPCH-I) (x) Dihydroneopterin aldolase (DHNA)/6pyruvoyltetrahydro-pterin synthase (PTPS) (xi, xxvi)

−33.2

PFI1100w

2.6.1.85

Purified recombinant protein and cytolocalisation in cytosol Putative

−161.0

PF14 0557

4.1.3.38

Putative

This paper

−122.8

PFL1155w

3.5.4.16

Kinetics from native protein

[20,21]

16.6/NA

PFF1360w

4.1.2.25/ 4.2.3.12

[22]

Hydromethyldihydropteridine pyrophosphokinase (PPPK) (xii) Dihydropteroate synthase (DHPS) (xiii) Dihydrofolate synthase (DHFS) (xiv) Folylpolyglutamate synthase (FPGS) (xvi) Dihydrofolate reductase (DHFR) (xv)

−16.1

PF08 0095

2.7.6.3

Recombinant protein renders an atypical eukaryotic PTPS that delivers two alternative products. One of them a pterin that is functional as a substrate for the downstream reaction Recombinant bifunctional PPPK-DHPS protein

−28.6

PF08 0095

2.5.1.15

−25.9

PF13 0140

6.3.2.12

−25.9

PF13 0140

6.3.2.17

−26.8

PFD0830w

1.5.1.3

Thymidylate synthase (TS) (xxi) Serine hydroxymethyltransferase (SHMT) (xvii) T-protein

−52.7

PFD0830w

2.1.1.45

−6.6

PFL1720w

10.5 (GCV) (xviii)

PF13 0345

P-protein H-protein Dihydrolipoamide dehydrogenase (LPD1) 5,10-Methylene-THF reductase (MTHFR) (xix) Methionine synthase (MS) (xx) 5,10-MTHF dehydrogenase/5,10methenyl cyclohydrolase/10-formylTHF synthetase (MTHFD/MTHFC/FTHFS) (xxii/xxiii/xxiv) 10-Formyl-THFdehydrogenase (xxv) Methionyl-tRNA formyltransferase (MTFMT) (xxvi)

References

[39]

[145]

Bifunctional DHFS-FPGS complements homologue yeast gene knockouts

[24,122]

Bifunctional DHFR-TS. Purified recombinant protein for DHFR and DFHR-TS. Purified native and recombinant for TS alone

[146–148]

2.1.2.1

Purified recombinant protein

[45,47]

2.1.2.10

Glycine cleavage system (xviii) (GCV/GCS): Tand H-protein mRNA amplified and H-protein GFP-tagged protein localised to mitochondria. P-protein unidentified and functionality of whole complex still hypothetical

[48,50]

1.5.1.20

Native protein

[79,149,150]

2.1.1.13 1.5.1.5/3.5.4.9/6.3.4.3

Native protein Putative multifunctional homologue of the human MTHFD1 or the isoforms (MTHFD1L, MTHFD2, MTHFD2L)

[151] [123,152]

1.4.4.2 PF11 0339 PFL1550w −39.8 −57.3 4.0/−6.7/−14.8

PFF1490w

1.8.1.4

−27.7

NA

Putative

MAL13P1.67

2.1.2.9

Putative

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

G Model MOLBIO 10739 1–13

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

149 150 151

152 153

154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187

functionally characterised, they are candidates for the enzymes that carry out pABA synthesis previously detected in extracts of P. falciparum-infected cells [32]. 3. An outline of the folate cycle and one-carbon metabolism in Plasmodium The one-carbon unit that folates contribute to metabolic pathways originates from serine or glycine in reactions catalysed by serine hydroxymethyltransferase (SHMT/PFL1720w) (xvii) [44–47] and possibly via the mitochondrial glycine cleavage system (GCV) [48] (xviii) (Table 2, Fig. 2). A protein similar to SHMT initially described as a potential mitochondrial isoform (PF14 0534) of this enzyme [48] and observed by immunofluorescence to localise in this organelle [49] has been found to lack enzyme activity as a SHMT [47]. The canonical GCV system on the other hand, assembles a tetrahydrofolate amino methyltransferase or glycine synthase (T protein), a nonenzymatic nucleating protein (H protein, carrier for lipoic acid as prosthetic group), a glycine decarboxylate (P protein), and a dihydrolipoamide dehydrogenase (L protein). However, a homologue of the P protein is yet to be found in apicomplexan and although the expression and localisation have been verified for P. falciparum H protein [50], the functionality of these proteins as a GCV system in Plasmodium is yet to be established. As in other eukaryotes the bulk of the intracellular folate in Plasmodium parasites is polyglutamated (the majority carry five to eight residues) which renders them highly charged metabolites that exist largely bound to proteins. In P. falciparum the pentaglutamated 5mTHF (Table 1) has been reported as the main folate form found intracellularly [51], and the enzyme that catalyses its polyglutamation (folylpolyglutamate synthase) (xvi) has been characterised as a bifunctional enzyme which also carries a dihydrofolate synthase activity [24]. By comparison with folate pathways in other eukaryotes there is evidence in Plasmodia for at least two distinct folate biosynthetic reactions: thymidylate synthesis using 5,10methylenetetrahydrofolate (xxi), and methionine synthesis via l-homocysteine methylation using 5-mTHF (xx). These and other enzymes, characterised through cell extract activities or characterised as gene products for the folate cycle in Plasmodium are listed in Table 2 with their reaction numbers depicted in Fig. 2.

189

4. Potential folate salvage opportunities during the Plasmodium life cycle

190

4.1. The Plasmodium-infected hepatocyte

191

Antifolates are effective inhibitors of the liver stages of Plasmodium spp, which represents a highly desirable characteristic since the pathology caused by blood stages is avoided [52]. Hepatocytes are also the primary site of folate metabolism in humans with the liver estimated to represent about 50% of the visceral folate pool [53]. Thus, this folate-rich tissue (25–35 ␮M of intracellular folate) could be a very abundant folate source for Plasmodium sporozoites on hepatocyte invason. However, polyglutamated folates as they exist in intracellular pools, are poorly transported through cell membranes [54] and thus the release of 5-mTHF to potentially serve the intracellular parasite in infected hepatocytes will need the hydrolysis of the glutamic acid tail. In hepatocytes this hydrolysis is carried out by the lysosomal gamma-pteroylpolyglutamate hydrolase [55]. Its capacity to cleave both terminal and internal gamma-glutamate peptide bonds ensures the availability of transportable folates. The availability and usage of hepatic folate pools by the intracellular malaria sporozoite and growing/multiplying exoerythrocytic shizont remains unexplored. Several studies have shown that

188

192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208

5

hepatic stages are highly sensitive to the antifolates as a result of their very active DNA replication required to generate thousands of daughter cells in a relatively short time [56,52].

209

4.2. The circulating Plasmodium-infected red blood cell and the pABA salvage hypothesis

212

The mature human erythrocyte does not have known requirements for folate but contains a sequestered vestigial polyglutamated pool of 5-mTHF (1.6–2.0 ␮M) [57] accumulated during erythropoiesis [58]. This perennial presence of sequestered polyglutamated 5-mTHF in mature human erythrocytes is due to the lack of a functional gamma-polyglutamate hydrolase. Consequently, the bulk of this highly charged polyglutamated folate is bound to haemoglobin (present in excess of 5 mM) at physiological oxygen pressures [59,60]. The idea that this abundant source of 5-mTHF in human erythrocytes might be available to the intracellular malaria parasite would be appealing [61]. However, the extensive mass and charge added by the glutamate tail demands an absolute requirement for its hydrolysis down to the monoglutated folate before membrane transport is possible. In the absence of a gammapolyglutamate hydrolase activity [62] or alternative routes for folate salvage from the erythrocytes haemoglobin-attached folate pool it must be assumed that this source of folate is unavailable to the intra-erythrocytic parasite. The only known folate precursor of relevance to human malaria circulating in the host’s plasma is pABA, although at very variable concentrations (0.146–6.56 ␮M) in adults [63]. While an excess of pABA (>7 ␮M) seems to inhibit in vitro parasite growth [64], low levels of pABA have long been reported as being detrimental to Plasmodium growth in vitro and in vivo in a number of malaria models [65–74], including P. falciparum growing in Aotus monkeys [61], and the in vitro growth of mosquito stages of P. berghei [75]. At low concentrations (10 nM) pABA antagonises the antimalarial activity of pyrimethamine and sulfadoxine [76]. pABA has been shown to be more effectively metabolised into polyglutamated folate endproducts by P. falciparum in comparison to the synthetic folate analogues folic and folinic acid [77]. More recently, an in vitro metabolomics study confirmed the active usage of pABA by P. falciparum resulting in the significant depletion of extracellular pABA from standard culture medium, particularly noticeable when the parasite entered mitotic division [78]. The main folate end-product in human plasma 5-mTHF (10–40 nM) can be used (provided at supra-physiological levels, 37 mM) by P. falciparum to synthesise methionine [79]. In contrast 5-mTHF, when used at physiological concentrations (23 nM), failed to antagonise the inhibitory effects of a number of antifolate compounds [80,54]. Folate analogues such as folic acid or 5-formyltetrahydrofolic acid (folinic acid or Leucovorin) are used in diet supplementation. These folate analogues are mostly metabolised in the liver and incorporated into the visceral polyglutamated pool of 5-mTHF [81]. These syhthetic folate analogues have been extensively used in in vitro studies of folate salvage and antifolate mechanism of action studies [19]. It has been shown that folic acid antagonises (at 100 nM or higher) the effect of antifolates such as pyrimethamine, and sulfadoxine [82,76,64]. In metabolic labelling experiments both folic acid and folinic acid have been shown to be incorporated into polyglutamated products by P. falciparum, although not as efficiently as is the case for pABA [77]. Folate metabolites are also present in plasma as folate catabolites in the form of glutamated pABA (estimated 160 nM) [53] and acetamidobenzoylglutamate, together with modified pterin aldehydes. The latter have been measured in urine predominantly as erythroneopterin and biopterin [83]. Glutamated pABA can replace pABA in P. berghei growth assays and is a substrate of dihydropteroate synthase (xiii), although with an apparent Km for the

214

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

210 211

213

215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272

G Model MOLBIO 10739 1–13

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

6

286

glutamated form about 100 times higher than that for pABA [61]. The possible salvage of folate catabolic by-products such as modified pterins has not been explored in Plasmodium. In addition to spontaneous oxidation other protein-regulated mechanisms seem to contribute to active folate catabolism. Ferritin, the major ironstorage protein, has been shown to catalyse in vitro folate cleavage and cellular folate catabolism is nearly always proportionally to the intracellular concentration of ferritin [84]. Pathologies with high levels of ferritin are general inflammatory conditions, including infectious diseases such as malaria [85,86]. Folate catabolism is responsive and directly proportional to folate intake. If folate turnover is increased (i.e. pregnancy and neonatal growth, oral contraceptive use, anticonvulsant drugs use, alcoholism, high levels of thyroid hormone, and cancer) [87] so is folate catabolism.

287

4.3. Plasmodium gametocytes

288

304

There is pharmacological evidence of folate synthesis in gametocytes since pyrimethamine inhibits the survival of early-stage gametocytes of P. falciparum [88,89]. This reflects the parallels in early gametocyte metabolism and asexual P. falciparum metabolism. Interestingly the selective pressure of chemotherapeutic treatment of malaria (particularly with antifolates) has consistently been shown to increase the prevalence of circulating gametocytes [90–93]. The link between gametocyte carriage and infectivity is unclear. In studies where increased gametocytaemia was observed after sulfadoxine–pyrimethamine treatment there was a correlation with higher gametocyte infectivity measured as development of oocysts in the mosquito vector [93]. However, direct feeding of mosquitoes on human subjects, instead of artificial membrane feeding, showed that only 0.5% of mosquitoes fed on sulfadoxine–pyrimethamine treated gametocyte carriers developed oocysts in contrast to 11% of mosquitoes fed on chloroquine-treated gametocyte carriers [94,95].

305

4.4. The vector Anopheles spp. and the sexual cycle of Plasmodium

306

Multicellular organisms excluding plants are folate auxothrops. Despite this, research on folate metabolism in diptera seems to have generated confusing results that have led to claims of a folate biosynthesis capability in mosquitoes. The most likely explanations for these claims include the presence of microbial symbionts that many species of insects carry, dietary impurities in feeding media, or carry-over of maternal vitamins in eggs [96,97]. Pterins, of which folates are a conjugated derivative, have multiple roles in colouration in insects but not in the biosynthesis of folates [98]. Pterins are intermediates in the biosynthesis of pigments first discovered in the wings of butterflies and named from the Greek “pteron” (wing). Other than the cases where symbiotic bacteria provide folate, insects have to meet their folate requirements from their diet. In the case of malaria vectors this probably includes the blood meal procured (2–4 ␮l) by the females of Anopheles spp. This argument is supported by data reporting the over-expression of genes homologous for different classes of folate membrane transporters (Section 9) after the blood meal in Anopheles gambiae [99]. In the Plasmodium-infected blood meal the differentiation of mature gametocytes to gametes (female macrogametes and male microgametes) occurs rapidly after ingestion in the mosquito midgut and is triggered by the changes in pH and temperature, and the presence of xanthurenic acid [100]. The zygote resulting from the fertilisation of the macrogamete within the first hour will be the only parasite form to survive in the aggressive environment of the mosquito gut. In the following 24–30 h the P. falciparum zygote develops into a tetraploid motile ookinete. The successful ookinete exits the peritrophic matrix and crosses the midgut epithelium to reach the midgut basal lamina. The meiotic division of the ookinete

273 274 275 276 277 278 279 280 281 282 283 284 285

289 290 291 292 293 294 295 296 297 298 299 300 301 302 303

307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334

produces oocysts that in 10 or more days divide into sporozoites (the infective stage), of which approximately a quarter successfully reach the salivary glands [101]. The multiplication into oocysts and thereafter into sporozoites are extracellular processes that will have to salvage nutrients from the mosquito’s haemolymph. For instance, the concentration of certain amino acids have been shown to change as a consequence of oocyst infections and the uptake of 3 H-Leucine has been demonstrated [101]. However, in the absence of records regarding the vitamin content in the haemolymph of diptera, the malaria parasites might need to depend on their de novo folate biosynthesis while circulating in the mosquito vector. Antifolates have the capacity to interfere with both vector and parasite. Folates are essential for both mosquitoes oogenesis [102] and Plasmodium sexual reproduction and oocyst multiplication [92,94]. Among the antimetabolites particularly effective at causing sterility in insects, especially females, are analogues of folic acid. Antifolates have also been documented to hinder the development of oocysts and the formation of sporozoites. Thus, antifolates have both insecticidal as well as parasiticidal properties that merit further investigation to pursue inhibitors with improved efficiency at targeting both organisms.

335

5. Folate membrane transport in Plasmodium

356

P. falciparum can take up exogenous folic acid, folinic acid, and pABA and convert it to polyglutamated folate end products [51,77]. A critically important aspect of folate salvage is the mechanism by which folate derivatives are transported into the intracellular parasite. In infected erythrocytes exogenous folates and precursors must pass through at least three membranes: the host erythrocyte’s membrane, the parasitophorous vacuole membrane and the parasite plasma membrane, in addition to the inner membrane traffic of the folates exchanged with the mitochondrion. In P. falciparum iRBCs folates are preferentially transported via the new permeability pathways (NPPs) present in the erythrocytes membrane [103]. The parasitophorous vacuole membrane is a mesh like structure with a size limit of 1.4 kDa and an effective channel pore diameter ˚ that is not expected to be a rate limiting barrier for low of 23 A[104] molecular mass compounds. High affinity folate transport activity has been described in the apicomplexa parasites T. gondii [105] and P. falciparum [103]. The uptake of folates across the parasite plasma membrane is saturable and energy-dependent with the properties of a carrier-mediated membrane transport [103]. Two membrane proteins, PfFT1 and PfFT2, have been identified as homologues of the BT1 family of folate transporters in P. falciparum [54]. The high affinity folate-biopterin transporters – FBT or BT1 family – (TC 2.A.71) [106] were initially identified in lower eukaryotes (e.g. the kinetoplastid Leishmania) that are unable to perform de novo biosynthesis of folates and are heavily dependent on exogenous folates or pterins [107]. These are high affinity transporters for folates and methotrexate (Km 0.25–0.7 ␮M) and their expression is regulated through cellular growth with maximum activity observed in the logarithmic phase [108,109]. Accordingly, the expression of both PfFT1 and PfFT2 seem to be highly regulated in the P. falciparum asexual cycle with the highest levels observed in replicating (mature trophozoites and shizonts) intraerythrocytic forms and gametocytes [110]. P. falciparum PfFT1 and PfFT2 appeared to be sorted predominantly to the plasma membrane in intraerythrocytic stages of P. falciparum where they are presumed to facilitate a symport uptake of folate and protons in the direction of their concentration gradients. PfFT1 and PfFT2 exhibit broad substrate specificity in transporting folate products (e.g. folic and folinic acid as well as 5-mTHF, pteroic acid, and dihydropteroic acid), as well as folate precursors pABA and glutamated pABA [54]. Homologues of BT1 family are not found in humans and the

357

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355

358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397

G Model MOLBIO 10739 1–13

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

398 399 400

401 402

403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460

7

primary structure of the regions predicted to be involved in substrate binding is very dissimilar to the human folate transporters PCFT and RFC [54].

6. Flux-balance and standard thermodynamics of the Falciparum folate network Flux balance analysis (FBA) is the mathematical analysis of metabolism using the stoichiometric coefficients for each reaction in the system as the set of constraints under the assumption of steady state. The steady state limitation dictates that the concentrations of compounds in the metabolic network are constant. Depending on the aims of the study, a specific phenotype is selected with a relevant quantitative biological parameter to optimise (maximise or minimise). Examples of these parameters are biomass, energy production/consumption, metabolite production/consumption, and nutrient uptake/excretion [111]. Crucially, knowledge on the kinetics of the enzymes involved is not a prerequisite. A common example of the use of FBA is the finding of essential reactions/metabolites for maximised cellular growth under different culture conditions, namely nutrient availability and their concentration. FBA reconstructions have been performed on the metabolic network thought to operate in P. falciparum [112,113]. Using the biomass function the models were validated by corroborating the essentiality of a number of gene products targeted by known effective antimalarials. Among those targets were folate metabolic enzymes such as CS (vii), PPPK-DHPS (xii/xiii), and DHFR-TS (xv/xxi). The model of Plata et al. [112] predicted a number of essential genes that do not have a homologue in humans. Those included mainly enzymes of the shikimate and pABA synthesis pathways (Table 2). This type of analysis agrees with the essentiality of folate biosynthesis and supports the validation of new folate biosynthesis enzymes as targets. A thermodynamic framework of the folate metabolic network in a biosynthetic organism such as the malaria parasites can also identify the driving reactions for the biosynthesis and assimilation of folate. Metabolic models based on thermodynamic data allow assembly of pathways with their probable directionality or their relative distance from equilibrium. Such models have become possible due to the increased availability of thermodynamic data and the generation of thermodynamic genome-scale models. Particularly useful has been data for the transformed standard Gibbs free energy of formation (f G o ) and reaction (r G o ) [114] calculated using group contribution methodologies and compiled in databases with unprecedented coverage [115]. r G o values for all but two of the reactions of a canonical folate biosynthetic network are available as depicted in Fig. 2 (Table 2). Several features become immediately apparent from this standard system (Fig. 3): the biosynthetic branches from the shikimate and pterin synthesis pathways are much more skewed to the right at equilibrium than the C1- or folate usage cycle, the driving reactions for these synthetic routes are the entry points of high-energy carbohydrates (e.g. E4P and PEP), nucleotides (e.g. GTP and ATP), the final step of pABA synthesis and the reactions for the generation of biomass (Met and dTMP) drive the C1-cycle. The latter, implies a low metabolite flux through the alternative formyl derivatives of folate. It has also been proposed that the magnitude of r G o can imply a role in homoeostatic regulation. Reactions with a highly negative r G o have the potential to be a regulatory point because they can either pull upstream reactants at high rates (end-point reactions) or contribute with highly energised reactants (first-point steps). Reactions with low r G o values (close to zero) would have limited potential for regulation due to their susceptibility to minor changes in the concentration of their reactants. ADC lyase and GTPCHI could

Fig. 3. Standard thermodynamics of the folate biosynthesis. The estimated standard free energy values (Y-axis) are represented for the different reactions (X-axis) as numbered in Fig. 2. The shikimate pathway reactions leading to the synthesis of pABA are represented in red and the pterin synthesis pathway in orange. The dotted line explains the convergence of shikimate and pterin pathways in reaction xiii. The one-carbon cycle (Folate cycle) is represented in blue. The E. coli model [116] as described in the main text is represented in green. Reactants that are part of reactions with significant high negative of ır G o from either the biosynthesis pathways (E4P, PEP, GTP, pABA) or the usage one-carbon cycle (5-MTHF, Met, dTMP) are specifically denoted.

then represent points of the final and first regulatory steps respectively, whereas the C1-cycle reactions such as xviii (SHMT) and xxii–xxiv (MTHFD) would represent low flux steps sensitivity to small changes in metabolite concentrations. A genome-scale model of E. coli metabolite activities and r G o of reactions has been proposed based on thermodynamically feasible reactions or pathways correcting the standard system values for ionic strength and uncertainty of the thermodynamic data [116]. The metabolite activity, defined as the concentration range of a metabolite required for every thermodynamically feasible and essential metabolite for optimal growth was elucidated. Comparing these data against the standard model described above indicate that values for the folate network follow a similar pattern with two differences (Fig. 3). The values for reactions ix and x being lower at −102 and −95.2 kJ/mol (standard model −161 and −122.8 kJ/mol), respectively. Three observations can be taken from this general standard thermodynamic depiction to further our understanding of malaria folate metabolism. Firstly, the ADC lyase reaction and ultimately the synthesis of pABA is essentially irreversible (due to pyruvate being a strong leaving group as well as the stability of the product pABA on aromatisation [117,118]. This serves to assert that in the absence of homologues for chorismate mutase or chorismate lyase [33] (in other organisms these enzymes commit chorismic acid to the synthesis of aromatic amino acids or the production of p-hydroxybenzoate for ubiquinone synthesis), the shikimate pathway in Plasmodium could in effect be a pABA biosynthesis pathway (i–ix, Figs. 2 and 3). Secondly, the more effective use of pABA by comparison to folate products (Section 4.2) is a consequence of the ADC lyase reaction properties that together with the highly energised co-substrate from the pterin synthesis pathway (6-hydromethyl7,8-dihydropterin pyrophosphate) favours the forward reaction (xiii). Metabolites with entry points at the C1-cycle (folates), on the other hand, will be salvaged through points with fluxes more sensitive to metabolite concentrations. Thirdly, the possibility that GTPCHI is a regulatory step in folate metabolism in malaria is supported by evidence that this gene has been amplified as an evolutionary adaptation in P. falciparum under selective pressure

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499

G Model MOLBIO 10739 1–13 8 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

from antifolates [119]. Moreover, the amplification of GTPCHI has more recently been directly linked to increase tolerance to antifolate [120]. The mechanisms of a regulatory feedback for bacterial GTPCHI has been elucidated with dUTP stimulating the reaction in vitro while tetrahydropterins have an inhibitory effect [121]. Salvage via membrane transport of folates and pABA will follow the dynamics of secondary membrane transport which are under the influence of the concentration gradient for substrates, the plasma membrane potential and saturability of the system. With a differential pH of about 0.5 units (cytoplasmic pH 7.3 versus parasitophorous vacuole pH 6.8) in P. falciparum and an expected 1:1 symport mechanism (anion:H+ stoichiometry) as shown for other eukaryotes, the transport of folates in Plasmodium is presumed to be non electrogenic and chiefly dependable of the ratio of the intracellular (Ci) over extracellular (Co) concentrations of exchangeable substrates. At ratios below one the uptake of folates will be favourable with actual rG values of less than −5.93 kJ/mol if we apply the same derivation as for transmembrane ion transport [116]. Given that intracellular folates are polyglutamated and protein-bound the proportion of free intracellular folates would tend to be minimal with a continual Ci/Co ratio far below unity throughout the Plasmodium life cycle (Section 4). The bi-modal operation for the Falciparum folate network shows that the supply of folates from either the biosyntheticonly (prototrophy) or salvaging-only (auxotrophy) folate pathways would not meet parasite demand for folate. The preservation of the biologically costly biosynthetic pathway for folates is an indication that exogenous supply is inadequate for the target biomass of Plasmodium. On the other hand, if exogenous sources of folate reactants are available at any stage of the parasite’s life cycle (e.g. possibly pre- and intraerythrocytic stages) the relative cost of their transport across membranes is vastly outweighted by its contribution towards reducing the cost of de novo synthesis. Hence, the preservation of folate specific membrane transporters that folate prototrophic organisms usually do not have. Furthermore, the model suggests the need for molecular signals and effectors that modulate the activities of folate biosynthesis versus salvage during the parasites complex life-cycle. A strong candidate for one of these regulatory steps is the first reaction of the pterin synthesis GTPCHI. Following the same rationale for energy balance for the folate network, the synthesis of pABA must be also regulated to avoid futile reactions in the presence of exogenous sources of folates.

543

7. Antifolate targets and gene discovery strategies

544

7.1. Mutifunctional and chokepoint targets

545

Progress has been made in the characterisation of the pathway deployed to generate folates from GTP and pABA with much of this work being championed by a handful of research groups [122,19,44,45,47]. Reductionist research driven by the characterisation of single enzymes remains a painstaking and an increasingly unattractive process in the omics era. For instance, pharmacological evidence for the presence of the shikimate pathway in apicomplexa was published 15 years ago as a high impact finding [26,29], and yet only one of the potential eight enzyme activities has been functionally characterised to date. Nonetheless, detailed characterisation of individual enzymes is the only way to obtain their intrinsic kinetic and pharmacological properties. Putative proteins awaiting characterisation include the candidate enzymes for reactions v and vi of the shikimate pathway (Fig. 2), ADC synthase and ADC lyase involved in the synthesis of pABA (viii–ix) and the putative multifunctional MTHFD-homologue for steps xxii–xxiv of the folate cycle. Candidate genes for the other four expected catalytic

546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561

reactions in the shikimate pathway and the three reactions for the folate cycle are yet to be identified (Fig. 2). Multifunctional enzymes are very attractive as pharmacological targets and folate metabolism seems to offer several multifunctional enzymes: SK-EPSP, DHFS-FPGS, DHPS-PPPK, DHFR-TS, and MTHFD (Table 2). Coincidently, *****DHPS-PPPK and DHFR-TS proteins contain the drug targets of the currently deployed antifolates. Potential drug leads have been studied for their ability to inhibit DHFS-FPGS. Two phosphinate analogues of 7,8-dihydrofolate that mimic tetrahedral intermediates formed during DHFS- and FPGScatalysed glutamylation were inhibitory against both DHFS and FPGS. The Ki of an aryl phosphinate analogue against DHFS was 140 nM and the Ki for an alkyl phosphinate against FPGS was 91 nM [122]. Potentially exploitable drug targets are also chokepoint reactions: reactions that either uniquely consume a specific reactant or uniquely produce a specific product in a metabolic network [123]. Importantly the reactant or product must not be a biochemical dead-end. The majority (87.5%) of proposed drug targets in P. falciparum have homologues in other organisms in which there is some evidence of them being chokepoints. It is therefore significant that seven reactions (ix, xii, xiii, xiv, xv, xvi, xxi) of the folate metabolism are in this list [123]. Small molecules capable of inhibiting reactions such as pABA synthesis (viii–ix) have been found in a recent screening programme using the Prestwick Chemical Library [124]. Rubreserine inhibited the plant equivalent to ADC synthase (Fig. 2, viii) and inhibited the in vitro growth of T. gondii and Plasmodium falciparum, with IC50 values of 20 ␮M and 1 ␮M, respectively. In Toxoplasma folate content was decreased by 40–50% in the presence of rubreserine. Importantly addition of pABA or 5-formyltetrahydrofolate into the external medium rescued growth inhibition in the presence of this compound. The Prestwick Chemical Library contains 1200 highly diverse small molecules that are all FDA approved drugs, which significantly derisks future drug discovery projects using these molecules as a starting point. That work builds on previous synthetic chemistry that produced inhibitors that target multiple chorismate-utilising enzymes from bacteria (i.e. Salmonella typhimurium and E. coli) as part of a validation process to substantiate the argument for chorismate branching enzymes as chemically approachable targets [125].

562

8. The antifolates as probes of folate biochemistry and membrane transport

602

Experimental investigation of phenotypic and genotypic antifolate drug resistance has contributed to our fundamental understanding of folate biochemistry. The investigation of methotrexate (MTX) resistance in kinetoplastids resulted in the discovery of the biopterin transporter (BT1) [126]. Together with the main folate transporter (FT1) [109] BT1 was found in amplicons isolated using functional cloning strategies. This discovery generated the BT1/FBT family of folate transporters which include examples from plants, photosynthesising bacteria, and the malaria homologues PfFT1 and PfFT2. MTX studies also uncovered a missing link in the folate metabolism of S. cerevisiae. Despite its known capability to salvage external folate, the molecular identity of the membrane proteins capable of transporting this vitamin in yeast was unknown. A recent genome-wide screening for drug carriers found the high-affinity nicotinamide riboside and thiamine transporter NRT1 to be a locus that mediate sensitivity to MTX in this very important eukaryote model [127]. Subsequently, NRT1 has been shown to directly transport both MTX and folic acid (SalcedoSora et al., unpublished). Resistance to the next generation antifolate pemetrexed (PMX) has helped in elucidating the role of a folate transporter in the

604

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601

603

605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624

G Model MOLBIO 10739 1–13

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652

genetic basis of a recessive human folate deficiency syndrome [128]. PMX is a pyrrolopyrimidine that was discovered through structure–activity relationship studies based on Lometrexol [3]. The drug is rapidly polyglutamated in human cells and proceeds to inhibit multiple folate targets including TS and DHFR, together with inhibition of purine synthesis enzymes. PMX resistance was selected under drug pressure from a HeLa cell line lacking the better known RFC and therefore already resistant to MTX. Extensive genomic comparisons between the two RFC-deleted cell lines, MTX resistant and MTX plus PMX resistant lines, highlighted the involvement of three loci encoding for membrane proteins in PMX resistance. One of these loci contained the folate transporter with activity at low pH and subsequently named the Proton-Coupled Folate Transporter or PCFT [128,129]. The use for MTX as a pharmacological probe tool has also been very fruitful. It is particularly noteworthy that this anticancer antifolate is also a potent inhibitor of P. falciparum growth in vitro, including multi-drug resistant strains (IC50 <50 nM) [80,130]. This observation has also been reported in eleven different strains of P. vivax, including a spectrum of drug resistant parasites, with a median IC50 of 2.6 nM [131]. It is therefore puzzling why MTX, the best studied antifolate with a very illustrious research and clinical career, continues to be for the most part ignored as a research tool and a starting point for a first line antimalarial candidate. The research with polyglutamatable antifolates that require membrane transport should deliver, with a rather good degree of certainty, new insights into malaria metabolism and therefore help to expand the list of desirable chemotherapeutic targets.

653

9. Targeting folate transport

654

Folate transport in higher eukaryotes can be achieved through at least eight different types of membrane protein [129]: the Reduced Folate Carrier (RFC, solute carrier SLC19A1), proton-coupled folate transporter (PCFT, SLC46A1), folate receptors (Pfam PF03024) [132], high affinity folate-biopterin transporters FBT or BT1 family (Pfam PF03092), the mitochondrial carrier family (Pfam PF00153), multidrug-resistance-associated proteins of ATP-binding-cassette (ABC) pumps, as well as other solute carriers (SLC21 and SLC22). In P. falciparum the two folate transporters identified thus far belong to the BT1 family as described in Section 5 [54]. It is estimated that over half of the drugs currently on the market target integral membrane proteins. However, the majority of those membrane proteins are receptors, among them folate receptors have been widely studied and used in cancer diagnosis and therapy [132]. The vast majority of other membrane transporters: primarily active membrane transporters (pumps), secondarily active transporters, and channels, remain largely unexplored as pharmacological targets. The absence of folate and pABA in the in vitro culture media or the inhibition of their transport by unspecific blockers of organic anion transporters such as probenecid (PBN) has been shown to sensitise Plasmodium to the cytotoxicity of antifolates [54,103]. This effect has been observed in a number of Plasmodium strains and field isolates with different drug-resistance phenotypes. The PBN effect has been reported in clinical studies where time of parasite clearance was significantly shorter, together with lower gametocytaemia, in children treated with pyrimethamine–sulphadoxine and PBN compared to those treated with pyrimethamine–sulphadoxine alone [133,134]. PBN, however, also sensitises parasites to different classes of antimalarials including aminoquinolines (particularly piperaquine) [80,135–137]. The unexpected results of PBN lowering the inhibitory concentration of antimalarials unconnected to the antifolates may reflect the general importance of organic anions transport to parasite viability and homeostasis. Prime

655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687

9

candidates targets of PBN include pantothenate uptake as well as lactate efflux. The latter is an interesting possibility given that it is an abundant catabolite in Plasmodium that is avidly excreted by a proton-symport mechanism [138]. Lactate membrane transport, both efflux and uptake, has been blocked by PBN in mammalians cells [139]. Thus, the effects of PBN on membrane transport in P. falciparum may be broad spectrum, inhibiting a number of organic anion transporters including folate transporters. Surprisingly, PBN did not seem to affect the inhibitory concentrations of MTX and trimetrexate in P. falciparum [80]. This is an unexpected finding as MTX requires uptake to exert activity and the usual uptake route is a PBN inhibitable folate transporter suggesting the need for an as yet unidentified anion transporter that is resistant to PBN. A more likely possibility is that MTX targets enzymes involved in both folate synthesis and salvage, and therefore the blocking of folate transport does not influence the salvage pathway already compromised by MTX inhibitory effects. The latter possibility is supported by evidence that the addition of physiological concentrations of folates [80] or pABA (Salcedo-Sora et al., unpublished) does not significantly increase the IC50 values of MTX for P. falciparum. Taken together the cross-sensitisation of PBN and the atypical effects of MTX in Plasmodium it seems plausible that by screening for compounds that interfere with folate transport and salvage in Plasmodium, molecules that cross-react with other malaria anion transport systems could be found. They could include inhibitors of the uptake of essential nutrients such as pantothenate as well as of the efflux of lactate. Thus, aniontransport inhibitors have great potential to fulfil the expectation of exploiting the specifics of membrane transport for antimalarial chemotherapy.

688

10. Concluding remarks

720

The preceding three decades of malaria research have improved our understanding of the pterin and folate biosynthesis of the parasite and filled in some blank spaces in the folate enzymatic map. However, on reviewing the different pathways that contribute to the network that delivers folates to the malaria parasite, including membrane transport and potential salvage routes, it becomes apparent that unexplored sections of the network provide ample room for further target characterisation and drug development. Plasmodium folate enzymes already considered as newly reported targets are SHMT [140,46] and DHFS-FPGS [122], with ADC synthase in waiting [124]. These examples of novel antifolate drug discovery opportunities should help to reinforce that there is much that can still be done to exploit this proven antimalarial metabolic network. Each of the converging biosynthetic pathways of the folate network have a reaction that has evolved to maximise the metabolic rate and therefore the thermodynamic driving forces for the pathway: ADC lyase for pABA synthesis, GTPCH-I for pterin synthesis and TS/MTHFR for the folate cycle (Table 2, Fig. 2). If prioritising is a pre-requisite in drug discovery we should start where there is already clear light. Host-parasite interactions shape the metabolism of the parasite. Understanding the interconnectivity between the different folate pools of the host:parasite relationship will be crucial in the pursuit for effective strategies aimed at selectively reducing cellular folate. In order to gain that level of understanding requires a systemic and comprehensive quantitative view of the cellular metabolism of folates in malaria parasites obtained through the use of new experimental and conceptual approaches [141–143] touched on in this review.

721

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719

722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749

G Model MOLBIO 10739 1–13 10

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

750

Acknowledgement

751

This work was funded by the Medical Research Council (G0400173-69712).

752

References

753 754

Q3

755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828

Q4

[1] WHO. World malaria report. WHO Press; 2012. [2] Hay SI, Okiro EA, Gething PW, et al. Estimating the global clinical burden of Plasmodium falciparum malaria in 2007. PLoS Medicine 2010;7(6):e1000290. [3] Walling J. From methotrexate to pemetrexate and beyond. A review of the pharmacodynamic and clinical properties of antifolates. Investigational New Drugs 2006;24:37–77. [4] Feely MG, Erickson A, O’Dell JR. Therapeutic options for rheumatoid arthritis. Expert Opinion on Pharmacotherapy 2009;10(13):2095–106. [5] Said S, Jeffes EWB, Weinstein GD. Methotrexate. Clinics in Dermatology 1997;15:781–97. [6] Masters PA, O’Bryan TA, Zurlo J, Miller DQ, Joshi N. Trimethoprimsulfamethoxazole revisited. Archives of Internal Medicine 2003;163(4):402–10. [7] Walker SL, Lockwood DNJ. Leprosy. Clinics in Dermatology 2007;25:165–72. [8] Guerina NG, Hsu H-W, Meissner HC, et al. Neonatal serologic screening and early treatment for congenital Toxoplasma gondii infection. New England Journal of Medicine 1994;330(26):1858–63. [9] Escalante AA, Smith DL, Kim Y. The dynamics of mutations associated with anti-malarial drug resistance in Plasmodium falciparum. Trends in Parasitology 2009;25(12):557–63. [10] Mita T, Tanabe K, Kita K. Spread and evolution of Plasmodium falciparum drug resistance. Parasitology International 2009;58(3):201–9. [11] WHO. Guidelines for the treatment of malaria; 2006. [12] Müller IB, Hyde JE. Antimalarial drugs: modes of action and mechanisms of parasite resistance. Future Microbiology 2010;5(12):1857–73. [13] Nzila A. Inhibitors of de novo folate enzymes in Plasmodium falciparum. Drug Discovery Today 2006;11(1920):939–44, http://dx.doi.org/10.1016/j.drudis.2006.08.003 http://www.sciencedirect. com/science/article/pii/S1359644606003102 [14] Yuthavong YKS, Leartsakulpanich UCP. Folate metabolism as a source of molecular targets for antimalarials. Future Microbiology 2006;1:113–25. [15] Shane B. Folate metabolism. In: Picciano MF, Stokstad ELR, Gregory III JFJ, editors. Folic acid metabolism in health and disease. Wiley-Liss; 1990. p. 65–78. [16] Bermingham A, Derrick JP. The folic acid biosynthesis pathway in bacteria: evaluation of potential for antibacterial drug discovery. BioEssays 2002;24(7):637–48. [17] White JH, Kilbey BJ. DNA replication in the malaria parasite. Parasitology Today 1996;12:151–5. [18] Gerald N, Mahajan B, Kumar S. Mitosis in the human malaria parasite Plasmodium falciparum. Eukaryotic Cell 2011;10(4):474–82, http://dx.doi.org/10.1128/EC. 00314-10 http://ec.asm.org/content/ 10/4/474.abstract [19] Hyde JE. Exploring the folate pathway in Plasmodium falciparum. Acta Tropica 2005;94:191–206. [20] Krungkrai J, Yuthavong Y, Webster HK. Guanosine triphosphate cyclohydrolase in Plasmodium falciparum and other Plasmodium species. Molecular and Biochemical Parasitology 1985;17(3):265–76. [21] Lee CS, Salcedo E, Wang Q, Wang P, Sims PF, Hyde JE. Characterization of three genes encoding enzymes of the folate biosynthetic pathway in Plasmodium falciparum. Parasitology 2001;122(1):1–13. [22] Dittrich S, Mitchell SL, Blagborough AM, et al. An atypical orthologue of 6-pyruvoyltetrahydropterin synthase can provide the missing link in the folate biosynthesis pathway of malaria parasites. Molecular Microbiology 2008;67(3):609–18, http://dx.doi.org/10.1111/j. 1365-2958.2007.06073.x http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2229834&tool= pmcentrez&rendertype=abstract [23] Triglia T. Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum. Proceedings of the National Academy of Sciences 1994;91(15):7149–53, http://dx.doi.org/10.1073/pnas.91.15.7149 http://www.pnas.org/content/91/15/7149.short [24] Salcedo E, Cortese JF, Plowe CV, Sims PF, Hyde JE. A bifunctional dihydrofolate synthetasefolylpolyglutamate synthetase in Plasmodium falciparum identified by functional complementation in yeast and bacteria. Molecular and Biochemical Parasitology 2001;112(2):239–52, http://dx.doi.org/10.1016/S0166-6851(00)00370-4. [25] Hall SJ, Sims PF, Hyde JE. Functional expression of the dihydrofolate reductase and thymidylate synthetase activities of the human malaria parasite Plasmodium falciparum in Escherichia coli. Molecular and Biochemical Parasitology 1991;45(2):317–30. [26] Roberts F, Roberts CW, Johnson JJ, et al. Evidence for the shikimate pathway in apicomplexan parasites. Nature 1998;393(6687):801–5, http://dx.doi.org/10.1038/31723. [27] Roberts CW, Roberts F, Lyons RE, et al. The shikimate pathway and its branches in apicomplexan parasites. Journal of infectious

[28] [29]

[30]

[31] [32] [33]

[34]

[35]

[36]

[37]

[38]

[39] [40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

diseases 2002;185(Suppl. 1):S25–36, http://dx.doi.org/10.1086/338004 http://jid.oxfordjournals.org/content/185/Supplement 1/S25.full Duke SO, Powles SB. Glyphosate: a once-in-a-century herbicide. Pest Management Science 2008;64(4):319–25. McConkey GA. Targeting the shikimate pathway in the malaria parasite Plasmodium falciparum. Antimicrobial Agents and Chemotherapy 1999;43(1):175–7. McRobert L, Jiang S, Stead A, McConkey GA. Plasmodium falciparum: interaction of shikimate analogues with antimalarial drugs. Experimental Parasitology 2005;111(3):178–81. Herrmann KM, Weaver LM. The shikimate pathway. Annual Review of Plant Physiology and Plant Molecular Biology 1999;50(1):473–503. Dieckmann A, Jung A. Mechanisms of sulfadoxine resistance in Plasmodium falciparum. Molecular and Biochemical Parasitology 1986;19(2):143. McConkey GA, Pinney JW, Westhead DR, et al. Annotating the Plasmodium genome and the enigma of the shikimate pathway. Trends in Parasitology 2004;20(2):60–5. Fitzpatrick T, Ricken S, Lanzer M, Amrhein N, Macheroux P, Kappes B. Subcellular localization and characterization of chorismate synthase in the apicomplexan Plasmodium falciparum. Molecular Microbiology 2001;40(1):65–75. Nichols BP, Seibold AM, Doktor SZ. Para-aminobenzoate synthesis from chorismate occurs in two steps. Journal of Biological Chemistry 1989;264(15):8597–601. Basset GJC, Quinlivan EP, Ravanel S, et al. Folate synthesis in plants: the paminobenzoate branch is initiated by a bifunctional PabA–PabB protein that is targeted to plastids. Proceedings of the National Academy of Sciences of the United States of America 2004;101(6):1496–501. Edman JC, Goldstein AL, Erbe JG. Para-aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctional enzyme. Yeast 1993;9(6):669–75. Aurrecoechea C, Brestelli J, Brunk BP, et al. PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Research 2009;37:D539–43 http://nar.oxfordjournals.org/cgi/content/abstract/gkn814v2 Triglia T, Cowman AF. Plasmodium falciparum: a homologue of paminobenzoic acid synthetase. Experimental Parasitology 1999;92:154–8. Basset GJC, Ravanel S, Quinlivan EP, et al. Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase. Plant Journal: for Cell and Molecular Biology 2004;40(4):453–61, http://dx.doi.org/10.1111/j.1365-313X.2004.02231.x http://www.ncbi.nlm.nih.gov/pubmed/15500462 Botet J, Mateos L, Revuelta JL, Santos MA. A chemogenomic screening of sulfanilamide-hypersensitive Saccharomyces cerevisiae mutants uncovers ABZ2 the gene encoding a fungal aminodeoxychorismate lyase. Eukaryotic Cell 2007;6(11):2102–11. Green JM, Nichols BP. p-Aminobenzoate biosynthesis in Escherichia coli. Purification of aminodeoxychorismate lyase and cloning of pabC. Journal of Biological Chemistry 1991;266(20):12971–5. Zhang Y, Bai L, Deng Z. Functional characterization of the first two actinomycete 4-amino-4-deoxychorismate lyase genes. Microbiology (Reading, England) 2009;155(Pt 7):2450–9, http://dx.doi.org/10.1099/mic.0.026336-0 http://mic.sgmjournals.org/content/155/7/2450.full Alfadhli S, Rathod PK. Gene organization of a Plasmodium falciparum serine hydroxymethyltransferase and its functional expression in Escherichia coli. Molecular and Biochemical Parasitology 2000;110(2):283. Maenpuen S, Sopitthummakhun K, Yuthavong Y, Chaiyen P, Leartsakulpanich U. Characterization of Plasmodium falciparum serine hydroxymethyltransferase – a potential antimalarial target. Molecular and Biochemical Parasitology 2009;168(1):63–73. Pornthanakasem W, Kongkasuriyachai D, Uthaipibull C, Yuthavong Y, Leartsakulpanich U. Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization. Malaria Journal 2012;11(1):387, http://dx.doi.org/10.1186/1475-2875-11-387,. http://www.malariajournal.com/content/11/1/387 Pang CKT, Hunter JH, Gujjar R, et al. Catalytic and ligand-binding characteristics of Plasmodium falciparum serine hydroxymethyltransferase. Molecular and Biochemical Parasitology 2009;168(1):74–83. Salcedo E, Sims PFG, Hyde JE. A glycine-cleavage complex as part of the folate one-carbon metabolism of Plasmodium falciparum. Trends in Parasitology 2005;21(9):406–11, http://dx.doi.org/10.1016/j.pt.2005.07.001. Read M, Müller IB, Mitchell SL, Sims PFG, Hyde JE. Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum. Malaria Journal 2010;9(1):351, http://dx.doi.org/10.1186/1475-2875-9-351 http://www.malariajournal.com/content/9/1/351 Spalding MD, Allary M, Gallagher JR, Prigge ST. Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion. Molecular and Biochemical Parasitology 2010;172(2):156–60, http://dx.doi.org/10.1016/j.molbiopara.2010.04.005 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2875341&tool= pmcentrez&rendertype=abstract Krungkrai J, Webster HK, Yuthavong Y. De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum. Molecular and Biochemical Parasitology 1989;32:25–38.

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913

G Model MOLBIO 10739 1–13

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998

[52] Rodrigues T, Prudêncio M, Moreira R, Mota MM, Lopes F. Targeting the liver stage of malaria parasites: a yet unmet goal. Journal of Medicinal Chemistry 2012;55(3):995–1012, http://dx.doi.org/10.1021/jm201095h. [53] Lin Y, Dueker SR, Follett JR, et al. Quantitation of in vivo human folate metabolism. American Journal of Clinical Nutrition 2004;80:680–91. [54] Salcedo-Sora JE, Ochong E, Beveridge S, et al. The molecular basis of folate salvage in Plasmodium falciparum: characterization of two folate transporters. Journal of Biological Chemistry 2011;286(52):44659–68, http://dx.doi.org/10.1074/jbc.M111.286054 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3247980&tool= pmcentrez&rendertype=abstract [55] Wang TT, Chandler CJ, Halsted CH. Intracellular pteroylpolyglutamate hydrolase from human jejunal mucosa. Isolation and characterization. Journal of Biological Chemistry 1986;261(29):13551–5 http://www.jbc.org/content/261/29/13551.short [56] Eriksson B, Courtier B, Bjorkman A, Miltgen F, Mazier D. Activity of dihydrofolate reductase inhibitors on the hepatic stages of Plasmodium yoelii yoelii in vitro. Transactions of the Royal Society of Tropical Medicine and Hygiene 1991;85:725–6. [57] Bagley PJ, Selhub J. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proceedings of the National Academy of Sciences of the United States of America 1998;95:13217–20. [58] Koury MJ, Ponka P. New insights into erythropoiesis: the roles of folate, vitamin B12 and Iron. Annual Review of Nutrition 2004;24:105–31. [59] Benesch RE, Benesch R, Kwong S, Baugh CM. A pteroylpolyglutamate binds to tetramers in deoxyhemoglobin but to dimers in oxyhemoglobin. Proceedings of the National Academy of Sciences of the United States of America 1983;80(20):6202–5. [60] Benesch R, Waxman S, Benesch R, Baugh C. The binding of folyl polyglutamates by hemoglobin. Biochemical and Biophysical Research Communications 1982;106(4):1359–63. [61] Ferone R. Folate metabolism in malaria. Bulletin of the World Health Organization 1977;55(2–3):291–8. [62] Krungkrai J, Yuthavong Y, Webster HK. High-performance liquid chromatographic assay for pteroylpolyglutamate hydrolase. Journal of Chromatography 1987;417:47–56. [63] Schapira A, Bygbjerg IC, Jepsen S, Flachs H, Bentzon MW. The susceptibility of Plasmodium falciparum to sulfadoxine and pyrimethamine: correlation of in vivo and in vitro results. American Journal of Tropical Medicine and Hygiene 1986;35(2):239–45 http://www.ncbi.nlm.nih.gov/pubmed/3513641 [64] Chulay JD, Watkins WM, Sixsmith DG. Synergistic antimalarial activity of pyrimethamine and sulfadoxine against Plasmodium falciparum in vitro. American Journal of Tropical Medicine and Hygiene 1984;33(3):325–30. [65] Hawking F. Milk diet p-aminobenzoic acid and malaria (P. berghei). British Medical Journal 1953:1201–2. [66] Maegraith BG, Deegan T, Jones ES. Suppression of malaria (P. Berghei) by milk. British Medical Journal 1952;2(4799):1382–4. [67] Ramakrishnan S, Prakash S, Krishnaswami S, Singh L. Studies on P. berghei vincki and lipes, 1948. XIII. Effect of glucose, biotin, PABA and methionine on the course of blood induced infection in starving albino rats. Indian Journal of Malariology 1953;7:225–8. [68] Ramakrishnan SP, Krishnaswami AK, Singh C. Studies on Plasmodium berghei n. sp. Vincke and Lips 1984. IX. Effect of milk diet on the course of bloodinduced infection in albino rats. Indian Journal of Malariology 1953;7(1):61–5 http://www.ncbi.nlm.nih.gov/pubmed/13128815 [69] Jacobs RL. Role of p-aminobenzoic acid in Plasmodium berghei infection in the mouse. Experimental Parasitology 1964;15:213–25. [70] Bray RS, Garnham PC. Effect of milk diet on P. cynomolgi infections in monkeys. British Medical Journal 1953;1(4821):1200–1. [71] Greenberg J, Taylor DJ, Trembley HL. The effect of milk diets on the course of sporozoite-induced Plasmodium gallinaceum infections in chicks. American Journal of Hygiene 1954;60(2):99–105. [72] Keshavarz-Valian H, Alger NE, Boissonneault GA. Effects of p-aminobenzoic acid methionine threonine and protein levels on susceptibility of mice to Plasmodium berghei. Journal of Nutrition 1985;115(12): 1613–20. [73] Ramakrishnan S, Bhatnagar V, Prakash S, Misra B. Effect of milk diet on Plasmodium gallinaceum infection in its vertebrate and invertebrate hosts. Indian Journal of Malariology 1953;7:261–5. [74] Kicska GA, Ting L-M, Schramm VL, Kim K. Effect of dietary p-aminobenzoic acid on murine Plasmodium yoelii infection. Journal of Infectious Diseases 2003;188(11):1776–81. [75] Al-Olayan EM, Beetsma AL, Butcher GA, Sinden RE, Hurd H. Complete development of mosquito phases of the malaria parasite in vitro. Science 2002;295(5555):677–9. [76] Watkins W, Sixsmith D, Chulay J, Spencer H. Antagonism of sulfadoxine and pyrimethamine antimalarial activity in vitro by p-aminobenzoic acid, p-aminobenzoylglutamic acid and folic acid. Molecular and Biochemical Parasitology 1985;14(1):55–61, http://dx.doi.org/10.1016/0166-6851(85)90105-7. [77] Wang P, Nirmalan N, Wang Q, Sims PF, Hyde JE. Genetic and metabolic analysis of folate salvage in the human malaria parasite Plasmodium falciparum. Molecular and Biochemical Parasitology 2004;135(1):77–87, http://dx.doi.org/10.1016/j.molbiopara.2004.01.008.

11

[78] Olszewski KL, Morrisey JM, Wilinski D, et al. Host-parasite interactions revealed by Plasmodium falciparum metabolomics. Cell Host & Microbe 2009;5(2):191–9. [79] Asawamahasakda W, Yuthavong Y. The methionine synthesis cycle and salvage of methyltetrahydrofolate from host red cells in the malaria parasite (Plasmodium falciparum). Parasitology 1993;107:1–10. [80] Nduati E, Diriye A, Ommeh S, et al. Effect of folate derivatives on the activity of antifolate drugs used against malaria and cancer. Parasitology Research 2008;102(6):1227–34. [81] Wright AJA, Finglas PM, Dainty JR, et al. Differential kinetic behavior and distribution for pteroylglutamic acid and reduced folates: a revised hypothesis of the primary site of PteGlu metabolism in humans. Journal of nutrition 2005;135(3):619–23 http://www.ncbi.nlm.nih.gov/pubmed/15735104 [82] Wang P, Brobey RK, Horii T, Sims PF, Hyde JE. Utilization of exogenous folate in the human malaria parasite Plasmodium falciparum and its critical role in antifolate drug synergy. Molecular Microbiology 1999;32(6):1254–62 http://www.ncbi.nlm.nih.gov/pubmed/10383765 [83] Krumdieck CL, Fukushima K, Fukushima T, Shiota T, Butterworth JCE. A longterm study of the excretion of folate and pterins in a human subject after ingestion of 14C folic acid with observations on the effect of diphenylhydantoin administration. American Journal of Clinical Nutrition 1978;31(1):88–93. [84] Suh JR, Oppenheim EW, Girgis S, Stover PJ. Purification and properties of a folate-catabolizing enzyme. Journal of Biological Chemistry 2000;275(45):35646–55, http://dx.doi.org/10.1074/jbc.M005864200 http://www.ncbi.nlm.nih.gov/pubmed/10978335 [85] Odunukwe NN, Salako LA, Okany C, Ibrahim MM. Serum ferritin and other haematological measurements in apparently healthy adults with malaria parasitaemia in Lagos. Nigeria Tropical Medicine & International Health 2000;5(8):582–6. [86] Stoltzfus RJ, Chwaya HM, Albonico M, Schulze KJ, Savioli L, Tielsch JM. Serum ferritin erythrocyte protoporphyrin and hemoglobin are valid indicators of iron status of school children in a malariaholoendemic population. Journal of Nutrition 1997;127(2):293–8 http://www.ncbi.nlm.nih.gov/pubmed/9039830 [87] Suh JR, Herbig AK, Stover PJ. New perspectives on folate catabolism. Annual Review of Nutrition 2001;21:255–82, http://dx.doi.org/10.1146/annurev.nutr.21.1.255 http://www.ncbi.nlm.nih. gov/pubmed/11375437 [88] Lang-Unnasch N, Murphy AD. Metabolic changes of the malaria parasite during the transition from the human to the mosquito host. Annual Review of Microbiology 1998;52:562–90. [89] Omar MS, Collins WE, Contacos PG. Gametocytocidal and sporontocidal effects of antimalarial drugs on malaria parasites. Experimental Parasitology 1974;36:167–77. [90] Barnes KI, Little F, Mabuza A, et al. Increased gametocytemia after treatment: an early parasitological indicator of emerging sulfadoxinepyrimethamine resistance in Falciparum malaria. Journal of Infectious Diseases 2008;197(11):1605–13, http://dx.doi.org/10.1086/587645 http://jid.oxfordjournals.org/content/197/11/1605.abstract [91] Bhasin VK, Trager W. Gametocyte-forming and non-gametocyte-forming clones of Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 1984;33(4):534–7. [92] White NJ. The role of anti-malarial drugs in eliminating malaria. Malaria Journal 2008;7(Suppl. 1):S8, http://dx.doi.org/10.1186/1475-2875-7-S1-S8 http://www.malariajournal.com/content/7/S1/S8 [93] Drakeley C, Sutherland C, Bousema JT, Sauerwein RW, Targett GAT. The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion. Trends in Parasitology 2006;22(9):424–30. [94] Beavogui AH, Djimde AA, Gregson A, et al. Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali. International Journal for Parasitology 2010;40(10):1213–20. [95] Kone A, van de Vegte-Bolmer M, Siebelink-Stoter R, et al. Sulfadoxinepyrimethamine impairs Plasmodium falciparum gametocyte infectivity and Anopheles mosquito survival. International Journal for Parasitology 2010;40(10):1221–8, http://dx.doi.org/10.1016/j.ijpara.2010.05.004 http://www.sciencedirect.com/science/article/pii/S0020751910001852 [96] Dadd RH. Insect nutrition: current developments and metabolic implications. Annual Review of Entomology 1973;18: 381–420. [97] Blatch S, Meyer KW, Harrison JF. Effects of dietary folic acid level and symbiotic folate production on fitness and development in the fruit fly Drosophila melanogaster. Fly 2010;4(4):312–9 http://www.landesbioscience.com/journals/fly/article/13258/ [98] Summers K, Howells A, Pyliotis N. Biology of eye pigmentation in insects. In: Treherne J, editor. Advances in insect physiology. Academic Press; 1982. p. 119–66. [99] Baker D, Nolan T, Fischer B, Pinder A, Crisanti A, Russell S. A comprehensive gene expression atlas of sex- and tissuespecificity in the malaria vector Anopheles gambiae. BMC Genomics 2011;12(1):296, http://dx.doi.org/10.1186/1471-2164-12-296 http://www.biomedcentral.com/1471-2164/12/296 [100] Sinden RE. Molecular interactions between Plasmodium and its insect vectors. Cellular Microbiology 2002;4(11):713–24 http://www.ncbi.nlm.nih.gov/pubmed/12427094

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083

G Model MOLBIO 10739 1–13 12 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

[101] Beier JC. Malaria parasite development in mosquitoes. Annual Review of Entomology 1998;43(1):519–43. [102] Perry AS, Miller S. The essential role of folic acid and the effect of antimetabolites on growth and metamorphosis of housefly larvae Musca domestica L. Journal of Insect Physiology 1965;11:1277–87. [103] Wang P, Wang Q, Sims PFG, Hyde JE. Characterisation of exogenous folate transport in Plasmodium falciparum. Molecular and Biochemical Parasitology 2007;154(1):40–51, http://dx.doi.org/10.1016/j.molbiopara.2007.04.002 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1906846&tool= pmcentrez&rendertype=abstract [104] Desai SA, Rosenberg RL. Pore size of the malaria parasite’s nutrient channel. Proceedings of the National Academy of Sciences of the United States of America 1997;94(5):2045–9. [105] Massimine KM, Doan LT, Atreya CA, et al. Toxoplasma gondii is capable of exogenous folate transport: a likely expansion of the BT1 family of transmembrane proteins. Molecular and Biochemical Parasitology 2005;144(1):44–54. [106] Saier MH. A functional-phylogenetic classification system for transmembrane solute transporters. Microbiology and Molecular Biology Reviews MMBR 2000;64(2):354–411 urlhttp://www.pubmedcentral.nih.gov/articlerender. fcgi?artid=98997&tool=pmcentrez&rendertype=abstracthttp://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=98997&tool= pmcentrez&rendertype=abstract. [107] Ouellette M, Drummelsmith J, El Fadili A, Kundig C, Richard D, Roy G. Pterin transport and metabolism in Leishmania and related trypanosomatid parasites. International Journal for Parasitology 2002;32(4):385–98. [108] Richard D, Kündig C, Ouellette M. A new type of high affinity folic acid transporter in the protozoan parasite Leishmania and deletion of its gene in methotrexate-resistant cells. Journal of Biological Chemistry 2002;277(33):29460–7, http://dx.doi.org/10.1074/jbc.M204796200 http://www.ncbi.nlm.nih.gov/pubmed/12023977 [109] Richard D, Leprohon P, Drummelsmith J, Ouellette M. Growth phase regulation of the main folate transporter of Leishmania infantum and its role in methotrexate resistance. Journal of Biological Chemistry 2004;279(52):54494–501, http://dx.doi.org/10.1074/jbc.M409264200 http://www.ncbi.nlm.nih.gov/pubmed/15466466 [110] Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL. The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biology 2003;1(1):e5, http://dx.doi.org/10.1371/journal.pbio.0000005. [111] Gianchandani EP, Chavali AK, Papin JA. The application of flux balance analysis in systems biology. Wiley Interdisciplinary Reviews: Systems Biology and Medicine 2010;2(3):372–82, http://dx.doi.org/10.1002/wsbm.60. [112] Plata G, Hsiao T-L, Olszewski KL, Llinás M, Vitkup D. Reconstruction and fluxbalance analysis of the Plasmodium falciparum metabolic network. Molecular Systems Biology 2010;6:408, http://dx.doi.org/10.1038/msb.2010.60 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2964117&tool= pmcentrez&rendertype=abstract [113] Huthmacher C, Hoppe A, Bulik S, Holzhutter H-G. Antimalarial drug targets in Plasmodium falciparum predicted by stagespecific metabolic network analysis. BMC Systems Biology 2010;4(1):120, http://dx.doi.org/10.1186/1752-0509-4-120 http://www. biomedcentral.com/1752-0509/4/120 [114] Alberty RA, Cornish-Bowden A, Goldberg RN, Hammes GG, Tipton K, Westerhoff HV. Recommendations for terminology and databases for biochemical thermodynamics. Biophysical Chemistry 2011;155(23):89–103 http://www.sciencedirect.com/science/article/pii/S0301462211000640 [115] Noor E, Bar-Even A, Flamholz A, Lubling Y, Davidi D, Milo R. An integrated open framework for thermodynamics of reactions that combines accuracy and coverage. Bioinformatics 2012;28(15):2037–44, http://dx.doi.org/10.1093/bioinformatics/bts317 http://bioinformatics. oxfordjournals.org/content/28/15/2037.abstract [116] Henry CS, Broadbelt LJ, Hatzimanikatis V. Thermodynamics-based metabolic flux analysis. Biophysical Journal 2007;92(5):1792–805, http://dx.doi.org/10.1529/biophysj.106.093138 http://www.sciencedirect. com/science/article/pii/S0006349507709876 [117] Nakai T, Mizutani H, Miyahara I, et al. Three-dimensional structure of 4amino-4-deoxychorismate lyase from Escherichia coli. Journal of Biochemistry 2000;128(1):29–38 http://jb.oxfordjournals.org/content/128/1/29.abstract [118] Spraggon G, Kim C, Nguyen-Huu X, Yee MC, Yanofsky C, Mills SE. The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates chorismate and glutamine and a product glutamate and (ii) its end-product inhibitor l-tryptophan. Proceedings of the National Academy of Sciences of the United States of America 2001;98(11):6021–6, http://dx.doi.org/10.1073/pnas.111150298 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=33415&tool= pmcentrez&rendertype=abstract [119] Nair S, Miller B, Barends M, et al. Adaptive copy number evolution in malaria parasites. PLoS Genetics 2008;4(10):e1000243. [120] Heinberg A, Siu E, Stern C, et al. Direct evidence for the adaptive role of copy number variation on antifolate susceptibility in Plasmodium falciparum. Molecular Microbiology 2013, http://dx.doi.org/10.1111/mmi.12162. [121] Schoedon G, Redweik U, Frank G, Cotton RG, Blau N. Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli. European Journal of Biochemistry/FEBS 1992;210(2):561–8 http://www.ncbi.nlm.nih.gov/pubmed/1459137

[122] Wang P, Wang Q, Yang Y, et al. Characterisation of the bifunctional dihydrofolate synthase-folylpolyglutamate synthase from Plasmodium falciparum; a potential novel target for antimalarial antifolate inhibition. Molecular and Biochemical Parasitology 2010;172(1):41–51, http://dx.doi.org/10.1016/j.molbiopara.2010.03.012 http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=2877875&tool= pmcentrez&rendertype=abstract [123] Yeh I, Hanekamp T, Tsoka S, Karp PD, Altman RB. Computational analysis of Plasmodium falciparum metabolism: organizing genomic information to facilitate drug discovery. Genome Research 2004;14(5):917–24, http://dx.doi.org/10.1101/gr.2050304 http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=479120&tool= pmcentrez&rendertype=abstract [124] Camara D, Bisanz C, Barette C, et al. Inhibition of p-aminobenzoate and folate syntheses in plants and apicomplexan parasites by natural product rubreserine. Journal of Biological Chemistry 2012;287(26):22367–76, http://dx.doi.org/10.1074/jbc.M112.365833 http://www.jbc.org/content/ 287/26/22367.abstract [125] Ziebart KT, Dixon SM, Avila B, et al. Targeting multiple chorismateutilizing enzymes with a single inhibitor: validation of a three-stage design. Journal of Medicinal Chemistry 2010;53(9):3718–29, http://dx.doi.org/10.1021/jm100158v. [126] Kunding C, Haimeur A, Legare D, Papadopoulou B, Ouellette M. Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan Leishmania tarentolae. EMBO Journal 1999;18: 2342–51. [127] Lanthaler K, Bilsland E, Dobson P, et al. Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast. BMC Biology 2011;9(1):70 http://www.biomedcentral.com/1741-7007/9/70 [128] Qiu A, Jansen M, Sakaris A, et al. Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell 2006;127(5):917–28, http://dx.doi.org/10.1016/j.cell.2006.09.041 http://www.cell.com/fulltext/S0092-8674(06)01347-X [129] Zhao R, Diop-Bove N, Visentin M, Goldman ID. Mechanisms of membrane transport of folates into cells and across epithelia. Annual Review of Nutrition 2011;31:177–201, http://dx.doi.org/10.1146/annurev-nutr-072610-145133 http://www.ncbi.nlm.nih.gov/pubmed/21568705 [130] Dar O, Khan MS, Adagu I. The potential use of methotrexate in the treatment of falciparum malaria: in vitro assays against sensitive and multidrugresistan falciparum strains. Japanese Journal of Infectious Diseases 2008;61: 210–1. [131] Imwong M, Russell B, Suwanarusk R, et al. Methotrexate is highly potent against pyrimethamine-resistant Plasmodium vivax. Journal of Infectious Diseases 2011;203(2):207–10. [132] Antony AC. Folate receptors. Annual Review of Nutrition 1996;16:501–21. [133] Sowunmi A, Adedeji AA, Fateye BA, Fehintola FA. Comparative effects of pyrimethamine-sulfadoxine with and without probenecid, on Plasmodium falciparum gametocytes in children with acute, uncomplicated malaria. Annals of Tropical Medicine and Parasitolhttp://dx.doi.org/10.1179/000349804X3243 ogy 2004;98(8):873–8, http://www.ncbi.nlm.nih.gov/pubmed/15667719 [134] Sowunmi A, Fehintola FA, Adedeji AA, et al. Open randomized study of pyrimethamine-sulphadoxine vs. pyrimethamine-sulphadoxine plus probenecid for the treatment of uncomplicated Plasmodium falciparum malaria in children. Tropical Medicine & International Health: TM & IH 2004;9(5):606–14, http://dx.doi.org/10.1111/j. 1365-3156.2004.01233.x http://www.ncbi.nlm.nih.gov/pubmed/15117306 [135] Masseno V, Muriithi S, Nzila A. In vitro chemosensitization of Plasmodium falciparum to antimalarials by verapamil and probenecid. Antimicrobial Agents and Chemotherapy 2009;53(7):3131–4. [136] Nzila A, Mberu E, Bray P, et al. Chemosensitization of Plasmodium falciparum by probenecid in vitro. Antimicrobial Agents and Chemotherapy 2003;47:2108–12. [137] Tahar R, Basco LK. Molecular epidemiology of malaria in Cameroon. XXVI. Twelve-year in vitro and molecular surveillance of pyrimethamine resistance and experimental studies to modulate pyrimethamine resistance. American journal of Tropical Medicine and Hygiene 2007;77(2):221–7 http://www.ncbi.nlm.nih.gov/pubmed/17690390 [138] Elliott JL, Saliba KJ, Kirk K. Transport of lactate and pyruvate in the intraerythrocytic malaria parasite Plasmodium falciparum. Biochemical Journal 2001;355(Pt 3):733–9 http://www.biochemj.org/bj/355/bj3550733.htm, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed &dopt=Citation&list uids=11311136 [139] Kuhr WG, van den Berg CJ, Korf J. In vivo identification and quantitative evaluation of carrier-mediated transport of lactate at the cellular level in the striatum of conscious, freely moving rats. Journal of Cerebral Blood Flow and Metabolism 1988;8:848–56, http://dx.doi.org/10.1038/jcbfm.1988.142. [140] Sopitthummakhun K, Thongpanchang C, Vilaivan T, Yuthavong Y, Chaiyen P, Leartsakulpanich U. Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay. Malaria Journal 2012;11(1):194, http://dx.doi.org/10.1186/1475-2875-11-194 http://www.malariajournal.com/content/11/1/194

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251

G Model MOLBIO 10739 1–13

ARTICLE IN PRESS J.E. Salcedo-Sora, S.A. Ward / Molecular & Biochemical Parasitology xxx (2013) xxx–xxx

1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270

[141] Bennett BD, Yuan J, Kimball EH, Rabinowitz JD. Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. Nature Protocols 2008;3(8):1299–311. [142] Kwon YK, Lu W, Melamud E, Khanam N, Bognar A, Rabinowitz JD. A domino effect in antifolate drug action in Escherichia coli. Nature Chemical Biology 2008;4(10):602–8. [143] Yuan J, Bennett BD, Rabinowitz JD. Kinetic flux profiling for quantitation of cellular metabolic fluxes. Nature protocols 2008;3(8):1328–40, http://dx.doi.org/10.1038/nprot.2008.131 http://www.pubmedcentral.nih. gov/articlerender.fcgi?artid=2710581&tool=pmcentrez&rendertype=abstract [144] IUPAC-IUB CBN. Nomenclature and symbols for folic acid and related compounds. Pure and Applied Chemistry 1987;59(6):833–6. [145] Kasekarn W, Sirawaraporn R, Chahomchuen T, Cowman AF, Sirawaraporn W. Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum. Molecular and Biochemical Parasitology 2004;137(1): 43–53. [146] Krungkrai J, Yuthavong Y, Webster HK. High-performance liquid chromatographic assay for thymidylate synthase from the human malaria parasite, Plasmodium falciparum. Journal of Chromatography 1989;487:51–9.

13

[147] Sirawaraporn W, Sirawaraporn R, Cowman AF, Yuthavong Y, Santi D. Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum. Biochemistry 1990;29:10779–85. [148] Hekmat-Nejad M, Rathod PK. Kinetics of Plasmodium falciparum thymidylate synthase: interactions with high-affinity metabolites of 5-fluoroorotate and D1694. Antimicrobial Agents and Chemotherapy 1996;40(7):1628–32. [149] Green JM, Ballou DP, Matthews RG. Examination of the role of methylenetetrahydrofolate reductase in incorporation of methyltetrahydrofolate into cellular metabolism. FASEB Journal 1988;2(1):42–7 http://www.fasebj.org/content/2/1/42.abstract [150] Maden BE. Why methanopterin? Comparative bioenergetics of the reactions catalyzed by methylene tetrahydrofolate reductase and methylene tetrahydromethanopterin reductase. Biochemical Society Transactions 1996;24:466S. [151] Krungkrai J, Webster HK, Yuthavong Y. Characterization of cobalamindependent methionine synthase purified from the human malaria parasite, Plasmodium falciparum. Parasitology Research 1989;75:512–7. [152] Tibbetts AS, Appling DR. Compartmentalization of Mammalian folate-mediated one-carbon metabolism. Annual Review of Nutrition 2010;30:57–81, http://dx.doi.org/10.1146/annurev.nutr.012809.104810.

Please cite this article in press as: Salcedo-Sora JE, Ward SA. The folate metabolic network of Falciparum malaria. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.02.003

1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290