Author Correction: Arylmethylamino steroids as antiparasitic agents.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: characterization of the Plasmodium vivax enzyme and inhibitor studies.

BACKGROUND: Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. METHODS: Plasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium. RESULTS: Here the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. CONCLUSION: The characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites' redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.

Redox status of patients before cardiac surgery.

OBJECTIVES: Redox regulation plays a crucial role in balancing the cardiovascular system. In this prospective study we aimed to identify currently unknown correlations valuable to cardiovascular research and patient management. METHODS: Blood samples from 500 patients were collected directly before cardiosurgical interventions (Ethics Committee reference number 85/11). Four central redox parameters were determined together with about 30 clinical, anthropometric, and metabolic parameters. RESULTS: Creatinine levels and pulmonary hypertension were significant predictors of the total antioxidant status (TAOS) in the patients; total glutathione levels were linked to C-peptide, and creatinine, gender, and ventricular arrhythmia influenced nitrate/nitrite levels. Notably, significant interactions were found between medication and redox parameters. Calcium channel blockers (CCBs) were positive predictors of total glutathione levels, whereas angiotensin-converting enzyme inhibitors and CCBs were negative predictors of NOx levels. Age showed the highest correlation with the duration of the intensive care stay, followed by NOx levels, creatinine, TAOS, and C-reactive protein. DISCUSSION: In this prospective study we determined multiple correlations between redox markers and parameters linked to cardiovascular diseases. The data point towards so far unknown interdependencies, particularly between antihypertensive drugs and redox metabolism. A thorough follow-up to these data has the potential to improve patient management. ABBREVIATIONS: A: absorption; ΔA: absorption difference; ABTS: 2,2'-azino-di(3-ethylbenzothiazoline sulfonate); ACE: angiotensin-converting enzyme; AO: antioxidant; ARB: angiotensin receptor blocker; BMI: body mass index; CAD: coronary artery disease; CCB: calcium channel blocker; CDC: coronary heart diseases; COPD: chronic obstructive pulmonary disease; CRP: C-reactive protein; CVD: cardiovascular diseases; Cu-OOH: cumene hydroperoxide; D: dilution factor; DAN: 2,3-diaminonaphtalene; DMSO: dimethylsulfoxide; DNA: deoxyribonucleic acid; DTNB: 5,5-dithiobis(2-nitrobenzoate); ε: extinction coefficient; EDRF: endothelium-derived relaxing factor; fc: final concentration; GPx: glutathione peroxidases; (h)GR: (human) glutathione reductase; GSH: (reduced) glutathione; GSSG: glutathione disulfide; GST: glutathione-S-transferase; Hb: hemoglobin; HDL: high-density lipoprotein; Hk: hematocrit; H2O2: hydrogen peroxide; ICS: intensive care stay; LDH: lactate dehydrogenase; LDL: low-density lipoprotein; MI: myocardial infarction; NED: N-(1-naphthyl)-ethylendiamine-dihydrochloride; NOS: nitric oxide synthase; NOx: nitrate/nitrite; NR: nitrate reductase; PBS: phosphate buffered saline; PCA: principle component analysis; PH: pulmonary hypertension; ROS: reactive oxygen species; RNS: reactive nitrogen species; RT: room temperature (25°C); SA: sulfanilamide; SOD: superoxide dismutase; SSA: sulfosalicylic acid; TAC: total antioxidant capacity; TAOS: total antioxidant status; TEAC: trolox equivalent antioxidative capacity; TG: triglycerides; tGSH: total glutathione; TNB-: 2-nitro-5-thiobenzoate; U: unit; UV: ultraviolet; VA: volume activity; Wc: working concentration; WHR: waist-hip ratio.

H2O2 dynamics in the malaria parasite Plasmodium falciparum.

Hydrogen peroxide is an important antimicrobial agent but is also crucially involved in redox signaling and pathogen-host cell interactions. As a basis for systematically investigating intracellular H2O2 dynamics and regulation in living malaria parasites, we established the genetically encoded fluorescent H2O2 sensors roGFP2-Orp1 and HyPer-3 in Plasmodium falciparum. Both ratiometric redox probes as well as the pH control SypHer were expressed in the cytosol of blood-stage parasites. Both redox sensors showed reproducible sensitivity towards H2O2 in the lower micromolar range in vitro and in the parasites. Due to the pH sensitivity of HyPer-3, we used parasites expressing roGFP2-Orp1 for evaluation of short-, medium-, and long-term effects of antimalarial drugs on H2O2 levels and detoxification in Plasmodium. None of the quinolines or artemisinins tested had detectable direct effects on the H2O2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H2O2. The systematic evaluation and comparison of the two genetically encoded cytosolic H2O2 probes in malaria parasites provides a basis for studying parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity.

Arylmethylamino steroids as antiparasitic agents.

In search of antiparasitic agents, we here identify arylmethylamino steroids as potent compounds and characterize more than 60 derivatives. The lead compound 1o is fast acting and highly active against intraerythrocytic stages of chloroquine-sensitive and resistant Plasmodium falciparum parasites (IC50 1-5 nM) as well as against gametocytes. In P. berghei-infected mice, oral administration of 1o drastically reduces parasitaemia and cures the animals. Furthermore, 1o efficiently blocks parasite transmission from mice to mosquitoes. The steroid compounds show low cytotoxicity in mammalian cells and do not induce acute toxicity symptoms in mice. Moreover, 1o has a remarkable activity against the blood-feeding trematode parasite Schistosoma mansoni. The steroid and the hydroxyarylmethylamino moieties are essential for antimalarial activity supporting a chelate-based quinone methide mechanism involving metal or haem bioactivation. This study identifies chemical scaffolds that are rapidly internalized into blood-feeding parasites.

Structural and Functional Characterization of Plasmodium falciparum Nicotinic Acid Mononucleotide Adenylyltransferase.

Nicotinic acid mononucleotide adenylyltransferase (NaMNAT) is an indispensable enzyme for the synthesis of NAD and NAD phosphate. It catalyzes the adenylylation of nicotinic acid mononucleotide (NaMN) to yield nicotinic acid adenine dinucleotide (NaAD). Since NAD(H) and NAD phosphate(H) are essentially involved in metabolic and redox regulatory reactions, NaMNAT is an attractive drug target in the fight against bacterial and parasitic infections. Notably, NaMNAT of the malaria parasite Plasmodium falciparum possesses only 20% sequence identity with the homologous human enzyme. Here, we present for the first time the two X-ray structures of P. falciparum NaMNAT (PfNaMNAT)-in the product-bound state with NaAD and complexed with an α,ß-non-hydrolizable ATP analog-the structures were determined to a resolution of 2.2Å and 2.5Å, respectively. The overall architecture of PfNaMNAT was found to be more similar to its bacterial homologs than its human counterparts although the PPHK motif conserved in bacteria is missing. Furthermore, PfNaMNAT possesses two cysteine residues within the active site that have not been described for any other NaMNATase so far and are likely to be involved in redox regulation of PfNaMNAT activity. Enzymatic studies and surface plasmon resonance data reveal that PfNaMNAT is capable of utilizing NaMN and nicotinamide mononucleotide with a slight preference for NaMN. Surprisingly, a comparison with the active site of Escherichia coli NaMNAT showed very similar architectures, despite different substrate preferences.

Characterization and redox regulation of Plasmodium falciparum methionine adenosyltransferase.

As a methyl group donor for biochemical reactions, S-adenosylmethionine plays a central metabolic role in most organisms. Depletion of S-adenosylmethionine has downstream effects on polyamine metabolism and methylation reactions, and is an effective way to combat pathogenic microorganisms such as malaria parasites. Inhibition of both the methylation cycle and polyamine synthesis strongly affects Plasmodium falciparum growth. Despite its central position in the methylation cycle, not much is currently known about P. falciparum methionine adenosyltransferase (PfalMAT). Notably, however, PfalMAT has been discussed as a target of different redox regulatory modifications. Modulating the redox state of critical cysteine residues is a way to regulate enzyme activity in different pathways in response to changes in the cellular redox state. In the present study, we optimized an assay for detailed characterization of enzymatic activity and redox regulation of PfalMAT. While the presence of reduced thioredoxin increases the activity of the enzyme, it was found to be inhibited upon S-glutathionylation and S-nitrosylation. A homology model and site-directed mutagenesis studies revealed a contribution of the residues Cys52, Cys113 and Cys187 to redox regulation of PfalMAT by influencing its structure and activity. This phenomenon connects cellular S-adenosylmethionine synthesis to the redox state of PfalMAT and therefore to the cellular redox homeostasis.

Benzo[b]quinolizinium Derivatives Have a Strong Antimalarial Activity and Inhibit Indoleamine Dioxygenase.

The heme-containing enzymes indoleamine 2,3-dioxygenase-1 (IDO-1) and IDO-2 catalyze the conversion of the essential amino acid tryptophan into kynurenine. Metabolites of the kynurenine pathway and IDO itself are involved in immunity and the pathology of several diseases, having either immunoregulatory or antimicrobial effects. IDO-1 plays a central role in the pathogenesis of cerebral malaria, which is the most severe and often fatal neurological complication of infection with Plasmodium falciparum. Mouse models are usually used to study the underlying pathophysiology. In this study, we screened a natural compound library against mouse IDO-1 and identified 8-aminobenzo[b]quinolizinium (compound 2c) to be an inhibitor of IDO-1 with potency at nanomolar concentrations (50% inhibitory concentration, 164 nM). Twenty-one structurally modified derivatives of compound 2c were synthesized for structure-activity relationship analyses. The compounds were found to be selective for IDO-1 over IDO-2. We therefore compared the roles of prominent amino acids in the catalytic mechanisms of the two isoenzymes via homology modeling, site-directed mutagenesis, and kinetic analyses. Notably, methionine 385 of IDO-2 was identified to interfere with the entrance of l-tryptophan to the active site of the enzyme, which explains the selectivity of the inhibitors. Most interestingly, several benzo[b]quinolizinium derivatives (6 compounds with 50% effective concentration values between 2.1 and 6.7 nM) were found to be highly effective against P. falciparum 3D7 blood stages in cell culture with a mechanism independent of IDO-1 inhibition. We believe that the class of compounds presented here has unique characteristics; it combines the inhibition of mammalian IDO-1 with strong antiparasitic activity, two features that offer potential for drug development.

Comparison of methods probing the intracellular redox milieu in Plasmodium falciparum.

Glutathione plays a crucial role in the redox regulation of the malaria parasite Plasmodium falciparum and is linked to drug resistance mechanisms, especially in resistance against the antimalarial drug chloroquine (CQ). The determination of the glutathione-dependent redox potential was recently established in living parasites using a cytosolically expressed biosensor comprising redox-sensitive green fluorescent protein coupled to human glutaredoxin 1 (hGrx1-roGFP2). In order to further elucidate redox changes induced by antimalarial drugs and to consolidate the application spectrum of the ratiometric biosensor we systematically compared it to other methods probing thiol and redox metabolism. Among these methods were cell disruptive and non-disruptive approaches including spectrophotometric assays with Ellman's reagent and naphthalene dicarboxyaldehyde as well as molecular probes such as ThiolTracker™ Violet and the dichlorofluorescein-based probe CM-H2DCFDA. To directly compare the methods, blood stages of the CQ-sensitive P. falciparum 3D7 strain were challenged with the oxidative agent diamide and the antimalarial drugs artemisinin and CQ for 1h, 4h, and 24h. For all conditions, dose-dependent changes in the different redox parameters could be monitored which are compared and discussed. We furthermore detected slight differences in thiol status of parasites transiently transfected with hGrx1-roGFP2 in comparison with control 3D7 cells. In conclusion, ThiolTracker™ Violet and, even more so, the hGrx1-roGFP2 probe reacted reliably and sensitively to drug induced changes in intracellular redox metabolism. These results were substantiated by classical cell disruptive methods.

Plasmodium falciparum glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase is a potential drug target.

The malarial parasite Plasmodium falciparum is exposed to substantial redox challenges during its complex life cycle. In intraerythrocytic parasites, haemoglobin breakdown is a major source of reactive oxygen species. Deficiencies in human glucose-6-phosphate dehydrogenase, the initial enzyme in the pentose phosphate pathway (PPP), lead to a disturbed redox equilibrium in infected erythrocytes and partial protection against severe malaria. In P. falciparum, the first two reactions of the PPP are catalysed by the bifunctional enzyme glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase (PfGluPho). This enzyme differs structurally from its human counterparts and represents a potential target for drugs. In the present study we used epitope tagging of endogenous PfGluPho to verify that the enzyme localises to the parasite cytosol. Furthermore, attempted double crossover disruption of the PfGluPho gene indicates that the enzyme is essential for the growth of blood stage parasites. As a further step towards targeting PfGluPho pharmacologically, ellagic acid was characterised as a potent PfGluPho inhibitor with an IC50 of 76 nM. Interestingly, pro-oxidative drugs or treatment of the parasites with H2O2 only slightly altered PfGluPho expression or activity under the conditions tested. Furthermore, metabolic profiling suggested that pro-oxidative drugs do not significantly perturb the abundance of PPP intermediates. These data indicate that PfGluPho is essential in asexual parasites, but that the oxidative arm of the PPP is not strongly regulated in response to oxidative challenge.

Detection of thiol-based redox switch processes in parasites - facts and future.

Malaria and African trypanosomiasis are tropical diseases caused by the protozoa Plasmodium and Trypanosoma, respectively. The parasites undergo complex life cycles in the mammalian host and insect vector, during which they are exposed to oxidative and nitrosative challenges induced by the host immune system and endogenous processes. Attacking the parasite's redox metabolism is a target mechanism of several known antiparasitic drugs and a promising approach to novel drug development. Apart from this aspect, oxidation of cysteine residues plays a key role in protein-protein interaction, metabolic responses to redox events, and signaling. Understanding the role and dynamics of reactive oxygen species and thiol switches in regulating cellular redox homeostasis is crucial for both basic and applied biomedical approaches. Numerous techniques have therefore been established to detect redox changes in parasites including biochemical methods, fluorescent dyes, and genetically encoded probes. In this review, we aim to give an insight into the characteristics of redox networks in the pathogens Plasmodium and Trypanosoma, including a comprehensive overview of the consequences of specific deletions of redox-associated genes. Furthermore, we summarize mechanisms and detection methods of thiol switches in both parasites and discuss their specificity and sensitivity.

Antimalarial NADPH-Consuming Redox-Cyclers As Superior Glucose-6-Phosphate Dehydrogenase Deficiency Copycats.

AIMS: Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum were shown to protect G6PD-deficient populations from severe malaria. Here, we investigated the mechanism of a novel antimalarial series, namely 3-[substituted-benzyl]-menadiones, to understand whether these NADPH-consuming redox-cyclers, which induce oxidative stress, mimic the natural protection of G6PD deficiency. RESULTS: We demonstrated that the key benzoylmenadione metabolite of the lead compound acts as an efficient redox-cycler in NADPH-dependent methaemoglobin reduction, leading to the continuous formation of reactive oxygen species, ferrylhaemoglobin, and subsequent haemichrome precipitation. Structure-activity relationships evidenced that both drug metabolites and haemoglobin catabolites contribute to potentiate drug effects and inhibit parasite development. Disruption of redox homeostasis by the lead benzylmenadione was specifically induced in Plasmodium falciparum parasitized erythrocytes and not in non-infected cells, and was visualized via changes in the glutathione redox potential of living parasite cytosols. Furthermore, the redox-cycler shows additive and synergistic effects in combination with compounds affecting the NADPH flux in vivo. INNOVATION: The lead benzylmenadione 1c is the first example of a novel redox-active agent that mimics the behavior of a falciparum parasite developing inside a G6PD-deficient red blood cell (RBC) giving rise to malaria protection, and it exerts specific additive effects that are inhibitory to parasite development, without harm for non-infected G6PD-sufficient or -deficient RBCs. CONCLUSION: This strategy offers an innovative perspective for the development of future antimalarial drugs for G6PD-sufficient and -deficient populations.

Flavins and flavoproteins: applications in medicine.

The potential of flavoproteins as targets of pharmacological treatments is immense. In this review we present an overview of the current research progress on medical interventions based on flavoproteins with a special emphasis on cancer, infectious diseases, and neurological disorders.

Protein S-nitrosylation in Plasmodium falciparum.

AIMS: Due to its life in different hosts and environments, the human malaria parasite Plasmodium falciparum is exposed to oxidative and nitrosative challenges. Nitric oxide (NO) and NO-derived reactive nitrogen species can constitute nitrosative stress and play a major role in NO-related signaling. However, the mode of action of NO and its targets in P. falciparum have hardly been characterized. Protein S-nitrosylation (SNO), a posttranslational modification of protein cysteine thiols, has emerged as a principal mechanism by which NO exerts diverse biological effects. Despite its potential importance, SNO has hardly been studied in human malaria parasites. Using a biotin-switch approach coupled to mass spectrometry, we systemically studied SNO in P. falciparum cell extracts. RESULTS: We identified 319 potential targets of SNO that are widely distributed throughout various cellular pathways. Glycolysis in the parasite was found to be a major target, with glyceraldehyde-3-phosphate dehydrogenase being strongly inhibited by S-nitrosylation of its active site cysteine. Furthermore, we show that P. falciparum thioredoxin 1 (PfTrx1) can be S-nitrosylated at its nonactive site cysteine (Cys43). Mechanistic studies indicate that PfTrx1 possesses both denitrosylating and transnitrosylating activities mediated by its active site cysteines and Cys43, respectively. INNOVATION: This work provides first insights into the S-nitrosoproteome of P. falciparum and suggests that the malaria parasite employs the thioredoxin system to deal with nitrosative challenges. CONCLUSION: Our results indicate that SNO may influence a variety of metabolic processes in P. falciparum and contribute to our understanding of NO-related signaling processes and cytotoxicity in the parasites.

Characterization of tryptophan aminotransferase 1 of Malassezia furfur, the key enzyme in the production of indolic compounds by M. furfur.

Malassezia yeasts are responsible for the widely distributed skin disease Pityriasis versicolor (PV), which is characterized by a hyper- or hypopigmentation of affected skin areas. For Malassezia furfur, it has been shown that pigment production relies on tryptophan metabolism. A tryptophan aminotransferase was found to catalyse the initial catalytic step in pigment formation in the model organism Ustilago maydis. Here, we describe the sequence determination, recombinant production and biochemical characterization of tryptophan aminotransferase MfTam1 from M. furfur. The enzyme catalyses the transamination from l-tryptophan to keto acids such as α-ketoglutarate with Km values for both substrates in the low millimolar range. Furthermore, MfTam1 presents a temperature optimum at 40°C and a pH optimum at 8.0. MfTam1 activity is highly dependent on pyridoxal phosphate (PLP), whereas compounds interfering with PLP, such as cycloserine (CS) and aminooxyacetate, inhibit the MfTam1 reaction. CS is known to reverse hyperpigmentation in PV. Thus, the results of the present study give a deeper insight into the role of MfTam1 in PV pathogenesis and as potential target for the development of novel PV therapeutics.

Crystal structure of the Plasmodium falciparum thioredoxin reductase-thioredoxin complex.

Over the last decades, malaria parasites have been rapidly developing resistance against antimalarial drugs, which underlines the need for novel drug targets. Thioredoxin reductase (TrxR) is crucially involved in redox homeostasis and essential for Plasmodium falciparum. Here, we report the first crystal structure of P. falciparum TrxR bound to its substrate thioredoxin 1. Upon complex formation, the flexible C-terminal arm and an insertion loop of PfTrxR are rearranged, suggesting that the C-terminal arm changes its conformation during catalysis similar to human TrxR. Striking differences between P. falciparum and human TrxR are a Plasmodium-specific insertion and the conformation of the C-terminal arm, which lead to considerable differences in thioredoxin binding and disulfide reduction. Moreover, we functionally analyzed amino acid residues involved in substrate binding and in the architecture of the intersubunit cavity, which is a known binding site for disulfide reductase inhibitors. Cell biological experiments indicate that P. falciparum TrxR is indeed targeted in the parasite by specific inhibitors with antimalarial activity. Differences between P. falciparum and human TrxR and details on substrate reduction and inhibitor binding provide the first solid basis for structure-based drug development and lead optimization.

Thioredoxin and glutathione systems in Plasmodium falciparum.

Despite a 50% decrease in malaria infections between 2000 and 2010, malaria is still one of the three leading infectious diseases with an estimated 216 million cases worldwide in 2010. More than 90% of all malaria infections were caused by Plasmodium falciparum, a unicellular eukaryotic parasite that faces oxidative stress challenges while developing in Anopheles mosquitoes and humans. Reactive oxygen and nitrogen species threatening the parasite are either endogenously produced by heme derived from hemoglobin degradation or they are from exogenous sources such as the host immune defense. In order to maintain the intracellular redox balance, P. falciparum employs a complex thioredoxin and glutathione system based on the thioredoxin reductase/thioredoxin and glutathione reductase/glutathione couples. P. falciparum thioredoxin reductase reduces thioredoxin and a range of low molecular weight compounds, while glutathione reductase is highly specific for its substrate glutathione disulfide. Since Plasmodium spp. lack catalase and a classical glutathione peroxidase, their redox balance depends on a complex set of five peroxiredoxins differentially located in the cytosol, apicoplast, mitochondria, and nucleus with partially overlapping substrate preferences. Moreover, P. falciparum employs a set of members belonging to the thioredoxin superfamily such as three thioredoxins, two thioredoxin-like proteins, a dithiol and three monocysteine glutaredoxins, and a redox-active plasmoredoxin with largely redundant functions. This review aims at summarizing our current knowledge on the functional redox networks of the malaria parasite P. falciparum.

Discovery of a Plasmodium falciparum glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase inhibitor (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (ML276) that reduces parasite growth in vitro.

A high-throughput screen of the NIH's MLSMR collection of ∼340000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is important for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In P. falciparum , the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 11 (ML276), is a submicromolar inhibitor of PfG6PD (IC(50) = 889 nM). It is completely selective for the enzyme's human isoform, displays micromolar potency (IC(50) = 2.6 µM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting PfG6PD in vivo are in progress.

Subcellular localization of adenylate kinases in Plasmodium falciparum.

Adenylate kinases (AK) play a key role in nucleotide signaling processes and energy metabolism by catalyzing the reversible conversion of ATP and AMP to 2 ADP. In the malaria parasite Plasmodium falciparum this reaction is mediated by AK1, AK2, and a GTP:AMP phosphotransferase (GAK). Here, we describe two additional adenylate kinase-like proteins: PfAKLP1, which is homologous to human AK6, and PfAKLP2. Using GFP-fusion proteins and life cell imaging, we demonstrate a cytosolic localization for PfAK1, PfAKLP1, and PfAKLP2, whereas PfGAK is located in the mitochondrion. PfAK2 is located at the parasitophorous vacuole membrane, and this localization is driven by N-myristoylation.

Glucose-6-phosphate metabolism in Plasmodium falciparum.

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.