Real-time measurements of ATP dynamics via ATeams in Plasmodium falciparum reveal drug-class-specific response patterns.

Malaria tropica, caused by the parasite Plasmodium falciparum (P. falciparum), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of P. falciparum is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP2- levels in a ratiometric manner, thereby facilitating in cellulo measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in P. falciparum to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein in vitro and stably integrated the sensors into the genome of the P. falciparum NF54attB cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs' mode of action, with possible implications for target identification and drug development.

Structural Analysis of Plasmodium falciparum Hexokinase Provides Novel Information about Catalysis Due to a Plasmodium-Specific Insertion.

The protozoan parasite Plasmodium falciparum is the causative pathogen of the most severe form of malaria, for which novel strategies for treatment are urgently required. The primary energy supply for intraerythrocytic stages of Plasmodium is the production of ATP via glycolysis. Due to the parasite's strong dependence on this pathway and the significant structural differences of its glycolytic enzymes compared to its human counterpart, glycolysis is considered a potential drug target. In this study, we provide the first three-dimensional protein structure of P. falciparum hexokinase (PfHK) containing novel information about the mechanisms of PfHK. We identified for the first time a Plasmodium-specific insertion that lines the active site. Moreover, we propose that this insertion plays a role in ATP binding. Residues of the insertion further seem to affect the tetrameric interface and therefore suggest a special way of communication among the different monomers. In addition, we confirmed that PfHK is targeted and affected by oxidative posttranslational modifications (oxPTMs). Both S-glutathionylation and S-nitrosation revealed an inhibitory effect on the enzymatic activity of PfHK.

The C-terminus of Sudan ebolavirus VP40 contains a functionally important CXnC motif, a target for redox modifications.

The Ebola virus matrix protein VP40 mediates viral budding and negatively regulates viral RNA synthesis. The mechanisms by which these two functions are exerted and regulated are unknown. Using a high-resolution crystal structure of Sudan ebolavirus (SUDV) VP40, we show here that two cysteines in the flexible C-terminal arm of VP40 form a stabilizing disulfide bridge. Notably, the two cysteines are targets of posttranslational redox modifications and interact directly with the host`s thioredoxin system. Mutation of the cysteines impaired the budding function of VP40 and relaxed its inhibitory role for viral RNA synthesis. In line with these results, the growth of recombinant Ebola viruses carrying cysteine mutations was impaired and the released viral particles were elongated. Our results revealed the exact positions of the cysteines in the C-terminal arm of SUDV VP40. The cysteines and/or their redox status are critically involved in the differential regulation of viral budding and viral RNA synthesis.

Review of neurodevelopmental disorders in patients with HNF1B gene variations.

This review investigates the association between neurodevelopmental disorders (NDD) and variations of the gene HNF1B. Heterozygous intragenetic mutations or heterozygous gene deletions (17q12 microdeletion syndrome) of HNF1B are the cause of a multi-system developmental disorder, termed renal cysts and diabetes syndrome (RCAD). Several studies suggest that in general, patients with genetic variation of HNF1B have an elevated risk for additional neurodevelopmental disorders, especially autism spectrum disorder (ASD) but a comprehensive assessment is yet missing. This review provides an overview including all available studies of patients with HNF1B mutation or deletion with comorbid NDD with respect to the prevalence of NDDs and in how they differ between patients with an intragenic mutation or 17q12 microdeletion. A total of 31 studies was identified, comprising 695 patients with variations in HNF1B, (17q12 microdeletion N = 416, mutation N = 279). Main results include that NDDs are present in both groups (17q12 microdeletion 25.2% vs. mutation 6.8%, respectively) but that patients with 17q12 microdeletions presented more frequently with any NDDs and especially with learning difficulties compared to patients with a mutation of HNF1B. The observed prevalence of NDDs in patients with HNF1B variations seems to be higher than in the general population, but the validity of the estimated prevalence must be deemed insufficient. This review shows that systematical research of NDDs in patients with HNF1B mutations or deletions is lacking. Further studies regarding neuropsychological characteristics of both groups are needed. NDDs might be a concomitant of HFN1B-related disease and should be considered in clinical routine and scientific reports.

Low level of antioxidant capacity biomarkers but not target overexpression predicts vulnerability to ROS-inducing drugs.

Despite a strong rationale for why cancer cells are susceptible to redox-targeting drugs, such drugs often face tumor resistance or dose-limiting toxicity in preclinical and clinical studies. An important reason is the lack of specific biomarkers to better select susceptible cancer entities and stratify patients. Using a large panel of lung cancer cell lines, we identified a set of "antioxidant-capacity" biomarkers (ACB), which were tightly repressed, partly by STAT3 and STAT5A/B in sensitive cells, rendering them susceptible to multiple redox-targeting and ferroptosis-inducing drugs. Contrary to expectation, constitutively low ACB expression was not associated with an increased steady state level of reactive oxygen species (ROS) but a high level of nitric oxide, which is required to sustain high replication rates. Using ACBs, we identified cancer entities with a high percentage of patients with favorable ACB expression pattern, making it likely that more responders to ROS-inducing drugs could be stratified for clinical trials.

Prominent role of cysteine residues C49 and C343 in regulating Plasmodium falciparum pyruvate kinase activity.

The protozoan parasite Plasmodium falciparum causes the most severe form of malaria and is highly dependent on glycolysis. Glycolytic enzymes were shown to be massively redox regulated, inter alia via oxidative post-translational modifications (oxPTMs) of their cysteine residues. In this study, we identified P. falciparum pyruvate kinase (PfPK) C49 and C343 as amino acid residues essentially involved in maintaining structural and functional integrity of the enzyme. The mutation of these cysteines resulted in an altered substrate affinity, lower enzymatic activities, and, as studied by X-ray crystallography, conformational changes within the A-domain where the substrate binding site is located. Although the loss of a cysteine evoked an impaired catalysis in both mutants, the effects observed for mutant C49A were more severe: multiple conformational changes, caused by the loss of two hydrogen bonds, impeded proper substrate binding and thus the transfer of phosphate upon catalysis.

Characterization of a small molecule inhibitor of disulfide reductases that induces oxidative stress and lethality in lung cancer cells.

Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.

Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant.

Aims: Genetically encoded green fluorescent protein (GFP)-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last few years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular environments, which should be investigated. In this context, the unicellular intraerythrocytic parasite Plasmodium, the causative agent of malaria, represents a challenge, as the small size of the organism results in weak fluorescence signals that complicate precise measurements, especially for cell compartment-specific observations. To address this, we have functionally and structurally characterized an enhanced redox biosensor superfolder roGFP2 (sfroGFP2). Results: SfroGFP2 retains roGFP2-like behavior, yet with improved fluorescence intensity (FI) in cellulo. SfroGFP2-based redox biosensors are pH insensitive in a physiological pH range and show midpoint potentials comparable with roGFP2-based redox biosensors. Using crystallography and rigidity theory, we identified the superfolding mutations as being responsible for improved structural stability of the biosensor in a redox-sensitive environment, thus explaining the improved FI in cellulo. Innovation: This work provides insight into the structure and function of GFP-based redox biosensors. It describes an improved redox biosensor (sfroGFP2) suitable for measuring oxidizing effects within small cells where applicability of other redox sensor variants is limited. Conclusion: Improved structural stability of sfroGFP2 gives rise to increased FI in cellulo. Fusion to hGrx1 (human glutaredoxin-1) provides the hitherto most suitable biosensor for measuring oxidizing effects in Plasmodium. This sensor is of major interest for studying glutathione redox changes in small cells, as well as subcellular compartments in general. Antioxid. Redox Signal. 37, 1-18.

Characterization of Plasmodium falciparum macrophage migration inhibitory factor homologue and its cysteine deficient mutants.

Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) is a homologue of the multifunctional human host cytokine MIF (HsMIF). Upon schizont rupture it is released into the human blood stream where it acts as a virulence factor, modulating the host immune system. Whereas for HsMIF a tautomerase, an oxidoreductase, and a nuclease activity have been identified, the latter has not yet been studied for PfMIF. Furthermore, previous studies identified PfMIF as a target for several redox post-translational modifications. Therefore, we analysed the impact of S-glutathionylation and S-nitrosation on the protein's functions. To determine the impact of the four cysteines of PfMIF we produced His-tagged cysteine to alanine mutants of PfMIF via site-directed mutagenesis. Recombinant proteins were analysed via mass spectrometry, and enzymatic assays. Here we show for the first time that PfMIF acts as a DNase of human genomic DNA and that this activity is greater than that shown by HsMIF. Moreover, we observed a significant decrease in the maximum velocity of the DCME tautomerase activity of PfMIF upon alanine replacement of Cys3, and Cys3/Cys4 double mutant. Lastly, using a yeast reporter system, we were able to verify binding of PfMIF to the human chemokine receptors CXCR4, and demonstrate a so-far overlooked binding to CXCR2, both of which function as non-cognate receptors for HsMIF. While S-glutathionylation and S-nitrosation of PfMIF did not impair the tautomerase activity of PfMIF, activation of these receptors was significantly decreased.

Biochemical characterization of the recombinant schistosome tegumental protein SmALDH_312 produced in E. coli and baculovirus expression vector system

BACKGROUND The heterologous expression of parasitic proteins is challenging because the sequence composition often differs significantly from host preferences. However, the production of such proteins is important because they are potential drug targets and can be screened for interactions with new lead compounds. Here we compared two expression systems for the production of an active recombinant aldehyde dehydrogenase (SmALDH_312) from Schistosoma mansoni, which causes the neglected tropical disease schistosomiasis. RESULTS We produced SmALDH_312 successfully in the bacterium Escherichia coli and in the baculovirus expression vector system (BEVS). Both versions of the recombinant protein were found to be active in vitro, but the BEVS-derived enzyme showed 3.7-fold higher specific activity and was selected for further characterization. We investigated the influence of Mg2+, Ca2+ and Mn2+, and found out that the specific activity of the enzyme increased 1.5-fold in the presence of 0.5 mM Mg2+. Finally, we characterized the kinetic properties of the enzyme using a design-of-experiment approach, revealing optimal activity at pH 7.6 and 41C. CONCLUSIONS Although, E. coli has many advantages, such as rapid expression, high yields and low costs, this system was outperformed by BEVS for the production of a schistosome ALDH. BEVS therefore rovides an opportunity for the expression and subsequent evaluation of schistosome enzymes as drug targets

Disentangling symptoms of externalizing disorders in children using multiple measures and informants.

The trait impulsivity theory suggests that a single, highly heritable externalizing liability factor, expressed as temperamental trait impulsivity, represents the core vulnerability for externalizing disorders. The present study sought to test the application of latent factor models derived from this theory to a clinical sample of children. Participants were 474 German children (age 6-12 years, 81% male) with symptoms of attention-deficit/hyperactivity disorder and externalizing behavior problems participating in an ongoing multicenter intervention study. Using confirmatory factor analyses (CFA) and exploratory structural equation modeling (ESEM), we evaluated several factor models of externalizing spectrum disorders (unidimensional; first-order correlated factors; higher-order factor; fully symmetrical bifactor; bifactor S-1 model). Furthermore, we assessed our prevailing factor models for measurement invariance across raters (clinicians, parents, teachers) and assessment modes (interview, questionnaires). While both CFA and ESEM approaches provided valuable insights into the multidimensionality, ESEM solutions were generally superior since they showed a substantially better model fit and less biased factor loadings. Among the models tested, the bifactor S-1 CFA/ESEM models, with a general hyperactivity-impulsivity reference factor, displayed a statistically sound factor structure and allowed for straightforward interpretability. Furthermore, these models showed the same organization of factors and loading patterns, but not equivalent item thresholds across raters and assessment modes, highlighting cross-situational variability in child behavior. Our findings are consistent with the assumption of the trait impulsivity theory that a common trait, presented as hyperactivity-impulsivity symptoms, underlies all externalizing disorders. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Targeting spectrin redox switches to regulate the mechanoproperties of red blood cells.

The mechanical properties of red blood cells (RBCs) are fundamental for their physiological role as gas transporters. RBC flexibility and elasticity allow them to survive the hemodynamic changes in the different regions of the vascular tree, to dynamically contribute to the flow thereby decreasing vascular resistance, and to deform during the passage through narrower vessels. RBC mechanoproperties are conferred mainly by the structural characteristics of their cytoskeleton, which consists predominantly of a spectrin scaffold connected to the membrane via nodes of actin, ankyrin and adducin. Changes in redox state and treatment with thiol-targeting molecules decrease the deformability of RBCs and affect the structure and stability of the spectrin cytoskeleton, indicating that the spectrin cytoskeleton may contain redox switches. In this perspective review, we revise current knowledge about the structural and functional characterization of spectrin cysteine redox switches and discuss the current lines of research aiming to understand the role of redox regulation on RBC mechanical properties. These studies may provide novel functional targets to modulate RBC function, blood viscosity and flow, and tissue perfusion in disease conditions.

Identification of sulfenylation patterns in trophozoite stage Plasmodium falciparum using a non-dimedone based probe.

Plasmodium falciparum causes the deadliest form of malaria. Adequate redox control is crucial for this protozoan parasite to overcome oxidative and nitrosative challenges, thus enabling its survival. Sulfenylation is an oxidative post-translational modification, which acts as a molecular on/off switch, regulating protein activity. To obtain a better understanding of which proteins are redox regulated in malaria parasites, we established an optimized affinity capture protocol coupled with mass spectrometry analysis for identification of in vivo sulfenylated proteins. The non-dimedone based probe BCN-Bio1 shows reaction rates over 100-times that of commonly used dimedone-based probes, allowing for a rapid trapping of sulfenylated proteins. Mass spectrometry analysis of BCN-Bio1 labeled proteins revealed the first insight into the Plasmodium falciparum trophozoite sulfenylome, identifying 102 proteins containing 152 sulfenylation sites. Comparison with Plasmodium proteins modified by S-glutathionylation and S-nitrosation showed a high overlap, suggesting a common core of proteins undergoing redox regulation by multiple mechanisms. Furthermore, parasite proteins which were identified as targets for sulfenylation were also identified as being sulfenylated in other organisms, especially proteins of the glycolytic cycle. This study suggests that a number of Plasmodium proteins are subject to redox regulation and it provides a basis for further investigations into the exact structural and biochemical basis of regulation, and a deeper understanding of cross-talk between post-translational modifications.

The immunomodulatory potential of the arylmethylaminosteroid sc1o.

Developing resistance mechanisms of pathogens against established and frequently used drugs are a growing global health problem. Besides the development of novel drug candidates per se, new approaches to counteract resistance mechanisms are needed. Drug candidates that not only target the pathogens directly but also modify the host immune system might boost anti-parasitic defence and facilitate clearance of pathogens. In this study, we investigated whether the novel anti-parasitic steroid compound 1o (sc1o), effective against the parasites Plasmodium falciparum and Schistosoma mansoni, might exhibit immunomodulatory properties. Our results reveal that 50 µM sc1o amplified the inflammatory potential of M1 macrophages and shifted M2 macrophages in a pro-inflammatory direction. Since M1 macrophages used predominantly glycolysis as an energy source, it is noteworthy that sc1o increased glycolysis and decreased oxidative phosphorylation in M2 macrophages. The effect of sc1o on the differentiation and activation of dendritic cells was ambiguous, since both pro- and anti-inflammatory markers were regulated. In conclusion, sc1o has several immunomodulatory effects that could possibly assist the immune system by counteracting the anti-inflammatory immune escape strategy of the parasite P. falciparum or by increasing pro-inflammatory mechanisms against pathogens, albeit at a higher concentration than that required for the anti-parasitic effect. KEY MESSAGES: • The anti-parasitic steroid compound 1o (sc1o) can modulate human immune cells. • Sc1o amplified the potential of M1 macrophages. • Sc1o shifts M2 macrophages to a M1 phenotype. • Dendritic cell differentiation and activation was ambiguously modulated. • Administration of sc1o could possibly assist the anti-parasitic defence.

In-vitro safety and off-target profile of the anti-parasitic arylmethylaminosteroid 1o.

Parasite-mediated diseases like malaria and schistosomiasis are growing health problems worldwide and novel drug candidates are urgently needed. In this study, the in-vitro safety profile of steroid compound 1o (sc1o), effective against the parasites Plasmodium falciparum and Schistosoma mansoni with an IC50 value of 5 nM, was characterized. We assessed viability/proliferation, apoptosis and cell cycle tests to determine the cytotoxic profile of sc1o in cancer cells. The mutagenic potential was determined with the AMES test. To identify off-target effects we investigated whether sc1o interacts with safety-relevant molecules such as cytochrome P450 (CYP) enzymes, phosphodiesterases (PDE), histone deacteylases (HDAC) and human ether-a-go-go related gene (hERG). Furthermore, to predict the potential bioavailability of sc1o, its effect on Caco-2 cell barrier integrity, by measurement of the transepithelial electrical resistance (TEER), was determined. Sc1o at 25 µM reduced cell viability, probably through cell-cycle arrest, but did not induce apoptosis in cancer cells. No adverse off-target effects nor mutagenic potential of sc1o were observed. Furthermore, sc1o did not disturb the integrity of the cell barrier, but exhibited low membrane permeability, apparently due to cell adherence. In conclusion, sc1o up to 10 µM showed a good in-vitro safety profile.

Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells.

K-Ras is the most prominent driver of oncogenesis and no effective K-Ras inhibitors have been established despite decades of intensive research. Identifying new K-Ras-binding proteins and their interaction domains offers the opportunity for defining new approaches in tackling oncogenic K-Ras. We have identified Galectin-8 as a novel, direct binding protein for K-Ras4B by mass spectrometry analyses and protein interaction studies. Galectin-8 is a tandem-repeat Galectin and it is widely expressed in lung and pancreatic carcinoma cells. siRNA-mediated depletion of Galectin-8 resulted in increased K-Ras4B content and ERK1/2 activity in lung and pancreatic carcinoma cells. Moreover, cell migration and cell proliferation were inhibited by the depletion of Galectin-8. The K-Ras4B-Galectin-8 interaction is indispensably associated with the farnesylation of K-Ras4B. The lysine-rich polybasic domain (PBD), a region that is unique for K-Ras4B as compared to H- and N-Ras, stabilizes the interaction and accounts for the specificity. Binding assays with the deletion mutants of Galectin-8, comprising either of the two carbohydrate recognition domains (CRD), revealed that K-Ras4B only interacts with the N-CRD, but not with the C-CRD. Structural modeling uncovers a potential binding pocket for the hydrophobic farnesyl chain of K-Ras4B and a cluster of negatively charged amino acids for interaction with the positively charged lysine residues in the N-CRD. Our results demonstrate that Galectin-8 is a new binding partner for K-Ras4B and it interacts via the N-CRD with the farnesylated PBD of K-Ras, thereby modulating the K-Ras effector pathways as well as cell proliferation and migration.

The interactome of 2-Cys peroxiredoxins in Plasmodium falciparum.

Peroxiredoxins (Prxs) are crucially involved in maintaining intracellular H2O2 homeostasis via their peroxidase activity. However, more recently, this class of proteins was found to also transmit oxidizing equivalents to selected downstream proteins, which suggests an important function of Prxs in the regulation of cellular protein redox relays. Using a pull-down assay based on mixed disulfide fishing, we characterized the thiol-dependent interactome of cytosolic Prx1a and mitochondrial Prx1m from the apicomplexan malaria parasite Plasmodium falciparum (Pf). Here, 127 cytosolic and 20 mitochondrial proteins that are components of essential cellular processes were found to interact with PfPrx1a and PfPrx1m, respectively. Notably, our data obtained with active-site mutants suggests that reducing equivalents might also be transferred from Prxs to target proteins. Initial functional analyses indicated that the interaction with Prx can strongly impact the activity of target proteins. The results provide initial insights into the interactome of Prxs at the level of a eukaryotic whole cell proteome. Furthermore, they contribute to our understanding of redox regulatory principles and thiol-dependent redox relays of Prxs in subcellular compartments.

Biological activity and stability analyses of knipholone anthrone, a phenyl anthraquinone derivative isolated from Kniphofia foliosa Hochst.

Knipholone (1) and knipholone anthrone (2), isolated from the Ethiopian medicinal plant Kniphofia foliosa Hochst. are two phenyl anthraquinone derivatives, a compound class known for biological activity. In the present study, we describe the activity of both 1 and 2 in several biological assays including cytotoxicity against four human cell lines (Jurkat, HEK293, SH-SY5Y and HT-29), antiplasmodial activity against Plasmodium falciparum 3D7 strain, anthelmintic activity against the model organism Caenorhabditis elegans, antibacterial activity against Aliivibrio fischeri and Mycobacterium tuberculosis and anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs) infected with HIV-1c. In parallel, we investigated the stability of knipholone (2) in solution and in culture media. Compound 1 displays strong cytotoxicity against Jurkat, HEK293 and SH-SY5Y cells with growth inhibition ranging from approximately 62-95% when added to cells at 50 µM, whereas KA (2) exhibits weak to strong activity with 26, 48 and 70% inhibition of cell growth, respectively. Both 1 and 2 possess significant antiplasmodial activity against Plasmodium falciparum 3D7 strain with IC50 values of 1.9 and 0.7 µM, respectively. These results complement previously reported data on the cytotoxicity and antiplasmodial activity of 1 and 2. Furthermore, compound 2 showed HIV-1c replication inhibition (growth inhibition higher than 60% at tested concentrations 0.5, 5, 15 and 50 µg/ml and an EC50 value of 4.3 µM) associated with cytotoxicity against uninfected PBMCs. The stability study based on preincubation, HPLC and APCI-MS (atmospheric-pressure chemical ionization mass spectrometry) analysis indicates that compound 2 is unstable in culture media and readily oxidizes to form compound 1. Therefore, the biological activity attributed to 2 might be influenced by its degradation products in media including 1 and other possible dimers. Hence, bioactivity results previously reported from this compound should be taken with caution and checked if they differ from those of its degradation products. To the best of our knowledge, this is the first report on the anti-HIV activity and stability analysis of compound 2.

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.