Metabolic maps and functions of the Plasmodium mitochondrion

The mitochondrion of Plasmodium species is a validated drug target. However, very little is known about the functions of this organelle. In this review, we utilize data available from the Plasmodium falciparum genome sequencing project to piece together putative metabolic pathways that occur in the parasite, comparing this with the existing biochemical and cell biological knowledge. The Plasmodium mitochondrion contains both conserved and unusual features, including an active electron transport chain and many of the necessary enzymes for coenzyme Q and iron-sulphur cluster biosynthesis. It also plays an important role in pyrimidine metabolism. The mitochondrion participates in an unusual hybrid haem biosynthesis pathway, with enzymes localizing in both the mitochondrion and plastid organelles. The function of the tricarboxylic acid cycle in the mitochondrion is unclear. We discuss directions for future research into this fascinating, yet enigmatic, organelle.     

Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase,a potential drug target

Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc₁ complex inhibitor.     

Diversity in mitochondrial metabolic pathways in parasitic protists Plasmodium and Cryptosporidium

Apicomplexans are obligate intracellular parasites and occupy diverse niches. They have remodeled mitochondrial carbon and energy metabolism through reductive evolution. Plasmodium lacks mitochondrial pyruvate dehydrogenase and H⁺-translocating NADH dehydrogenase (Complex I, NDH1). The mitochondorion contains a minimal mtDNA (～ 6 kb) and carries out oxidative phosphorylation in the insect vector stages, by using 2-oxoglutarate as an alternative means of entry into the TCA cycle and a single-subunit flavoprotein as an alternative NADH dehydrogenase (NDH2). In the blood stages of mammalian hosts, mitochondrial enzymes are down-regulated and parasite energy metabolism relies mainly on glycolysis. Mitosomes of Cryptosporidium parvum and Cryptosporidium hominis (human intestine parasites) lack mtDNA, pyruvate dehydrogenase, TCA cycle enzymes except malate-quinone oxidoreductase (MQO), and ATP synthase subunits except α and β. In contrast, mitosomes of Cryptosporidium muris (a rodent gastric parasite) retain all TCA cycle enzymes and functional ATP synthase and carry out oxidative phosphorylation with pyruvate-NADP⁺ oxidoreductase (PNO) and a simple and unique respiratory chain consisting of NDH2 and alternative oxidase (AOX). Cryptosporidium and Perkinsus are early branching groups of chromoalveolates (apicomplexa and dinoflagellates, respectively), and both Cryptosporidium mitosome and Perkinsus mitochondrion use PNO, MQO, and AOX. All apicomplexan parasites and dinoflagellates share MQO, which has been acquired from ε-proteobacteria via lateral gene transfer. By genome data mining on Plasmodium, Cryptosporidium and Perkinsus, here we summarized their mitochondrial metabolic pathways, which are varied largely from those of mammalian hosts. We hope that our findings will help in understanding the apicomplexan metabolism and development of new chemotherapeutics with novel targets.     

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.     

Mitochondrial function in Babesia bovis.

A variety of anti-mitochondrial drugs that had previously been found to inhibit the growth of the malarial parasite Plasmodium falciparum were tested on Babesia bovis in vitro. Several of these drugs were found to be non-toxic towards B. bovis. However, those drugs that were found to inhibit babesial growth included compounds (shown in parentheses) that have the following putative mitochondrial targets in the parasite: ATP synthetase complex (rhodamine 123, oligomycin, Janus Green); ATP-ADP translocase (bongkrekic acid); electron transport (rotenone, n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), antimycin A); ubiquinone (CoQ) function (BW58C, menoctone); protein synthesis (tetracycline); and the proton pump (CCCP). We have also investigated the effects of some of these drugs on pyrimidine biosynthesis de novo by following the incorporation of [14C]bicarbonate into pyrimidine nucleotides and into the pyrimidine moieties of nucleic acids. The ubiquinone analogues BW58C and menoctone inhibited this pathway in the nM-microM range of concentrations. Inhibitors of electron transport (antimycin A and oligomycin) and an uncoupler (CCCP) were also effective inhibitors of pyrimidine biosynthesis de novo. We conclude that B. bovis has a functional mitochondrion that contributes significantly to pyrimidine biosynthesis de novo and to the overall energy metabolism of the parasite.     

Kinetic modeling can describe in vivo glycolysis in Entamoeba histolytica

Glycolysis in the human parasite Entamoeba histolytica is characterized by the absence of cooperative modulation and the prevalence of pyrophosphate-dependent (over ATP-dependent) enzymes. To determine the flux-control distribution of glycolysis and understand its underlying control mechanisms, a kinetic model of the pathway was constructed by using the software gepasi. The model was based on the kinetic parameters determined in the purified recombinant enzymes, and the enzyme activities, and steady-state fluxes and metabolite concentrations determined in amoebal trophozoites. The model predicted, with a high degree of accuracy, the flux and metabolite concentrations found in trophozoites, but only when the pyrophosphate concentration was held constant; at variable pyrophosphate, the model was not able to completely account for the ATP production/consumption balance, indicating the importance of the pyrophosphate homeostasis for amoebal glycolysis. Control analysis by the model revealed that hexokinase exerted the highest flux control (73%), as a result of its low cellular activity and strong AMP inhibition. 3-Phosphoglycerate mutase also exhibited significant flux control (65%) whereas the other pathway enzymes showed little or no control. The control of the ATP concentration was also mainly exerted by ATP consuming processes and 3-phosphoglycerate mutase and hexokinase (in the producing block). The model also indicated that, in order to diminish the amoebal glycolytic flux by 50%, it was required to decrease hexokinase or 3-phosphoglycerate mutase by 24% and 55%, respectively, or by 18% for both enzymes. By contrast, to attain the same reduction in flux by inhibiting the pyrophosphate-dependent enzymes pyrophosphate-phosphofructokinase and pyruvate phosphate dikinase, they should be decreased > 70%. On the basis of metabolic control analysis, steps whose inhibition would have stronger negative effects on the energy metabolism of this parasite were identified, thus becoming alternative targets for drug design.     

Revisiting the Central Metabolism of the Bloodstream Forms of Trypanosoma brucei: Production of Acetate in the Mitochondrion Is Essential for Parasite Viability

Background

The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.

Methodology/Principal Findings

A comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.

Conclusions/Significance

Acetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.     

Plasmodium falciparum LipB mutants display altered redox and carbon metabolism in asexual stages and cannot complete sporogony in Anopheles mosquitoes

Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs.     

An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins

Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.     

Characterization of coproporphyrinogen III oxidase in Plasmodium falciparum cytosol

A unique hybrid pathway has been proposed for de novo heme biosynthesis in Plasmodium falciparum involving three different compartments of the parasite, namely mitochondrion, apicoplast and cytosol. While parasite mitochondrion and apicoplast have been shown to harbor key enzymes of the pathway, there has been no experimental evidence for the involvement of parasite cytosol in heme biosynthesis. In this study, a recombinant P. falciparum coproporphyrinogen III oxidase (rPfCPO) was produced in E. coli and confirmed to be active under aerobic conditions. rPfCPO behaved as a monomer of 61 kDa molecular mass in gel filtration analysis. Immunofluorescence studies using antibodies to rPfCPO suggested that the enzyme was present in the parasite cytosol. These results were confirmed by detection of enzyme activity only in the parasite soluble fraction. Western blot analysis with anti-rPfCPO antibodies also revealed a 58 kDa protein only in this fraction and not in the membrane fraction. The cytosolic presence of PfCPO provides evidence for a hybrid heme-biosynthetic pathway in the malarial parasite.     

Flux balance analysis of mycolic acid pathway: targets for anti-tubercular drugs

Mycobacterium tuberculosis is the focus of several investigations for design of newer drugs, as tuberculosis remains a major epidemic despite the availability of several drugs and a vaccine. Mycobacteria owe many of their unique qualities to mycolic acids, which are known to be important for their growth, survival, and pathogenicity. Mycolic acid biosynthesis has therefore been the focus of a number of biochemical and genetic studies. It also turns out to be the pathway inhibited by front-line anti-tubercular drugs such as isoniazid and ethionamide. Recent years have seen the emergence of systems-based methodologies that can be used to study microbial metabolism. Here, we seek to apply insights from flux balance analyses of the mycolic acid pathway (MAP) for the identification of anti-tubercular drug targets. We present a comprehensive model of mycolic acid synthesis in the pathogen M. tuberculosis involving 197 metabolites participating in 219 reactions catalysed by 28 proteins. Flux balance analysis (FBA) has been performed on the MAP model, which has provided insights into the metabolic capabilities of the pathway. In silico systematic gene deletions and inhibition of InhA by isoniazid, studied here, provide clues about proteins essential for the pathway and hence lead to a rational identification of possible drug targets. Feasibility studies using sequence analysis of the M. tuberculosis H37Rv and human proteomes indicate that, apart from the known InhA, potential targets for anti-tubercular drug design are AccD3, Fas, FabH, Pks13, DesA1/2, and DesA3. Proteins identified as essential by FBA correlate well with those previously identified experimentally through transposon site hybridisation mutagenesis. This study demonstrates the application of FBA for rational identification of potential anti-tubercular drug targets, which can indeed be a general strategy in drug design. The targets, chosen based on the critical points in the pathway, form a ready shortlist for experimental testing.     

Phosphoenolpyruvate-succinate-glyoxylate pathway in the filarial parasiteSetaria digitata

Setaria digitata, a filarial parasite of cattle possesses certain unique characteristics like cyanide insensitivity, and lack of cytochromes. In the present study, we have shown that the parasite has an incomplete tricarboxylic acid cycle with the absence of activities of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthase. However the parasite showed the existence of glyoxylate cycle and phosphoenolpyruvate-succinate pathway. The widely used antifilarial drug diethylcarbamazine caused general inhibition of all enzymes of phosphoenolpyruvate-succinate pathway and glyoxylate cycle except that of fumarase and isocitrate lyase. The results may pave the way for new targets for chemotherapy in the control of filarial parasites.     

Dissecting Leishmania infantum Energy Metabolism - A Systems Perspective

Leishmania infantum, causative agent of visceral leishmaniasis in humans, illustrates a complex lifecycle pertaining to two extreme environments, namely, the gut of the sandfly vector and human macrophages. Leishmania is capable of dynamically adapting and tactically switching between these critically hostile situations. The possible metabolic routes ventured by the parasite to achieve this exceptional adaptation to its varying environments are still poorly understood. In this study, we present an extensively reconstructed energy metabolism network of Leishmania infantum as an attempt to identify certain strategic metabolic routes preferred by the parasite to optimize its survival in such dynamic environments. The reconstructed network consists of 142 genes encoding for enzymes performing 237 reactions distributed across five distinct model compartments. We annotated the subcellular locations of different enzymes and their reactions on the basis of strong literature evidence and sequence-based detection of cellular localization signal within a protein sequence. To explore the diverse features of parasite metabolism the metabolic network was implemented and analyzed as a constraint-based model. Using a systems-based approach, we also put forth an extensive set of lethal reaction knockouts; some of which were validated using published data on Leishmania species. Performing a robustness analysis, the model was rigorously validated and tested for the secretion of overflow metabolites specific to Leishmania under varying extracellular oxygen uptake rate. Further, the fate of important non-essential amino acids in L. infantum metabolism was investigated. Stage-specific scenarios of L. infantum energy metabolism were incorporated in the model and key metabolic differences were outlined. Analysis of the model revealed the essentiality of glucose uptake, succinate fermentation, glutamate biosynthesis and an active TCA cycle as driving forces for parasite energy metabolism and its optimal growth. Finally, through our in silico knockout analysis, we could identify possible therapeutic targets that provide experimentally testable hypotheses.     

Identification of enzymes that have helminth-specific active sites and are required for Rhodoquinone-dependent metabolism as targets for new anthelmintics

Soil transmitted helminths (STHs) are major human pathogens that infect over a billion people. Resistance to current anthelmintics is rising and new drugs are needed. Here we combine multiple approaches to find druggable targets in the anaerobic metabolic pathways STHs need to survive in their mammalian host. These require rhodoquinone (RQ), an electron carrier used by STHs and not their hosts. We identified 25 genes predicted to act in RQ-dependent metabolism including sensing hypoxia and RQ synthesis and found 9 are required. Since all 9 have mammalian orthologues, we used comparative genomics and structural modeling to identify those with active sites that differ between host and parasite. Together, we found 4 genes that are required for RQ-dependent metabolism and have different active sites. Finding these high confidence targets can open up in silico screens to identify species selective inhibitors of these enzymes as new anthelmintics.     

Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii displays some unusual localisations of carbohydrate converting enzymes, which is due to the presence of a vestigial, non-photosynthetic plastid, referred to as the apicoplast. It was recently demonstrated that the single pyruvate dehydrogenase complex (PDH) in T. gondii is exclusively localised inside the apicoplast but absent in the mitochondrion. This raises the question about expression, localisation and function of enzymes for the tricarboxylic acid (TCA)-cycle, which normally depends on PDH generated acetyl-CoA. Based on the expression and localisation of epitope-tagged fusion proteins, we show that all analysed TCA cycle enzymes are localised in the mitochondrion, including both isoforms of malate dehydrogenase. The absence of a cytosolic malate dehydrogenase suggests that a typical malate-aspartate shuttle for transfer of reduction equivalents is missing in T. gondii. We also localised various enzymes which catalyse the irreversible steps in gluconeogenesis to a cellular compartment and examined mRNA expression levels for gluconeogenesis and TCA cycle genes between tachyzoites and in vitro bradyzoites. In order to get functional information on the TCA cycle for the parasite energy metabolism, we created a conditional knock-out mutant for the succinyl-CoA synthetase. Disruption of the sixth step in the TCA cycle should leave the biosynthetic parts of the cycle intact, but prevent FADH2 production. The succinyl-CoA synthetase depletion mutant displayed a 30% reduction in growth rate, which could be restored by supplementation with 2 microM succinate in the tissue culture medium. The mitochondrial membrane potential in these parasites was found to be unaltered. The lack of a more severe phenotype suggests that a functional TCA cycle is not essential for T. gondii replication and for maintenance of the mitochondrial membrane potential.     

The non-mevalonate isoprenoid biosynthesis of plants as a test system for new herbicides and drugs against pathogenic bacteria and the malaria parasite

Higher plants and several photosynthetic algae contain the plastidic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate pathway (DOXP/MEP pathway) for isoprenoid biosynthesis. The first four enzymes and their genes are known of this novel pathway. All of the ca. 10 enzymes of this isoprenoid pathway are potential targets for new classes of herbicides. Since the DOXP/MEP pathway also occurs in several pathogenic bacteria, such as Mycobacterium tuberculosis, and in the malaria parasite Plasmodium falciparum, all inhibitors and potential herbicides of the DOXP/MEP pathway in plants are also potential drugs against pathogenic bacteria and the malaria parasite. Plants with their easily to handle DOXP/MEP-pathway are thus very suitable test-systems also for new drugs against pathogenic bacteria and the malaria parasite as no particular security measures are required. In fact, the antibiotic herbicide fosmidomycin specifically inhibited not only the DOXP reductoisomerase in plants, but also that in bacteria and in the parasite P. falciparum, and cures malaria-infected mice. This is the first successful application of a herbicide of the novel isoprenoid pathway as a possible drug against malaria.     

Elementary Flux Modes Analysis of Functional Domain Networks Allows a Better Metabolic Pathway Interpretation

Metabolic network analysis is an important step for the functional understanding of biological systems. In these networks, enzymes are made of one or more functional domains often involved in different catalytic activities. Elementary flux mode (EFM) analysis is a method of choice for the topological studies of these enzymatic networks. In this article, we propose to use an EFM approach on networks that encompass available knowledge on structure-function. We introduce a new method that allows to represent the metabolic networks as functional domain networks and provides an application of the algorithm for computing elementary flux modes to analyse them. Any EFM that can be represented using the classical representation can be represented using our functional domain network representation but the fine-grained feature of functional domain networks allows to highlight new connections in EFMs. This methodology is applied to the tricarboxylic acid cycle (TCA cycle) of Bacillus subtilis, and compared to the classical analyses. This new method of analysis of the functional domain network reveals that a specific inhibition on the second domain of the lipoamide dehydrogenase (pdhD) component of pyruvate dehydrogenase complex leads to the loss of all fluxes. Such conclusion was not predictable in the classical approach.     

Identification of Selective Inhibitors of the Plasmodium falciparum Hexose Transporter PfHT by Screening Focused Libraries of Anti-Malarial Compounds

Development of resistance against current antimalarial drugs necessitates the search for novel drugs that interact with different targets and have distinct mechanisms of action. Malaria parasites depend upon high levels of glucose uptake followed by inefficient metabolic utilization via the glycolytic pathway, and the Plasmodium falciparum hexose transporter PfHT, which mediates uptake of glucose, has thus been recognized as a promising drug target. This transporter is highly divergent from mammalian hexose transporters, and it appears to be a permease that is essential for parasite viability in intra-erythrocytic, mosquito, and liver stages of the parasite life cycle. An assay was developed that is appropriate for high throughput screening against PfHT based upon heterologous expression of PfHT in Leishmania mexicana parasites that are null mutants for their endogenous hexose transporters. Screening of two focused libraries of antimalarial compounds identified two such compounds that are high potency selective inhibitors of PfHT compared to human GLUT1. Additionally, 7 other compounds were identified that are lower potency and lower specificity PfHT inhibitors but might nonetheless serve as starting points for identification of analogs with more selective properties. These results further support the potential of PfHT as a novel drug target.