Inhibition of Trypanosoma brucei glucose-6-phosphate dehydrogenase by human steroids and their effects on the viability of cultured parasites

Dehydroepiandrosterone ( DHEA) is known as an intermediate in the synthesis of mammalian steroids and a potent uncompetitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH), but not the enzyme from plants and lower eukaryotes. G6PDH catalyzes the first step of the pentose-phosphate pathway supplying cells with ribose 5-phosphate, a precursor of nucleic acid synthesis, and NADPH for biosynthetic processes and protection against oxidative stress. In this paper we demonstrate that also G6PDH of the protozoan parasite Trypanosoma brucei is uncompetitively inhibited by DHEA and epiandrosterone (EA), with K(i) values in the lower micromolar range. A viability assay confirmed the toxic effect of both steroids on cultured T. brucei bloodstream form cells. Additionally, RNAi mediated reduction of the G6PDH level in T. brucei bloodstream forms validated this enzyme as a drug target against Human African Trypanosomiasis. Together these findings show that inhibition of G6PDH by DHEA derivatives may lead to the development of a new class of anti-trypanosomatid compounds. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

Anacardic acid derivatives as inhibitors of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Chagas` disease, a parasitic infection caused by the flagellate protozoan Trypanosoma cruzi, is a major public health problem affecting millions of individuals in Latin America. On the basis of the essential role in the life cycle of T. cruzi, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been considered an attractive target for the development of novel antitrypanosomatid agents. In the present work, we describe the inhibitory effects of a small library of natural and synthetic anacardic acid derivatives against the target enzyme. The most potent inhibitors, 6-n-pentadecyl-(1) and 6-n-dodecylsalicilic acids (10e), have IC(50) values of 28 and 55 mu M, respectively. The inhibition was not reversed or prevented by the addition of Triton X-100, indicating that aggregate-based inhibition did not occur. In addition, detailed mechanistic characterization of the effects of these compounds on the T. cruzi GAPDH-catalyzed reaction showed clear noncompetitive inhibition with respect to both substrate and cofactor. (C) 2008 Elsevier Ltd. All rights reserved.

Classical and hologram QSAR studies on a series of inhibitors of trypanosomatid Glyceraldehyde-3-Phosphate Dehydrogenase

Leishmaniasis and trypanosomiasis are major causes of morbidity and mortality in both tropical and subtropical regions of the world. The current available drugs are limited, ineffective, and require long treatment regimens. Due to the high dependence of trypanosomatids on glycolysis as a source of energy, some glycolytic enzymes have been identified as attractive targets for drug design. In the present work, classical Two-Dimensional Quantitative Structure -Activity Relationships (2D QSAR) and Hologram QSAR (HQSAR) studies were performed on a series of adenosine derivatives as inhibitors of Leishmania mexicana Glyceraldehyde-3-Phosphate Dehydrogenase (LmGAPDH). Significant correlation coefficients (classical QSAR, r(2)=0.83 and q(2) =0.81; HQSAR, r(2)=0.91 and q(2) =0.86) were obtained for the 56 training set compounds, indicating the potential of the models for untested compounds. The models were then externally validated using a test set of 14 structurally related compounds and the predicted values were in good agreement with the experimental results (classical QSAR, r(pred)(2) = 0.94; HQSAR, r(pred)(2) = 0.92).

Structural basis for selective inhibition of trypanosomatid glyceraldehyde-3-phosphate dehydrogenase: Molecular docking and 3D QSAR studies

The glycolytic enzyme glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) is as an attractive target for the development of novel antitrypanosomatid agents. In the present work, comparative molecular field analysis and comparative molecular similarity index analysis were conducted on a large series of selective inhibitors of trypanosomatid GAPDH. Four statistically significant models were obtained (r(2) > 0.90 and q(2) > 0.70), indicating their predictive ability for untested compounds. The models were then used to predict the potency of an external test set, and the predicted values were in good agreement with the experimental results. Molecular modeling studies provided further insight into the structural basis for selective inhibition of trypanosomatid GAPDH.

16-Bromoepiandrosterone, an activator of the mammalian immune system, inhibits glucose 6-phosphate dehydrogenase from Trypanosoma cruzi and is toxic to these parasites grown in culture

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the pentose-phosphate pathway which supplies cells with ribose 5-phosphate (R5P) and NADPH. R5P is the precursor for the biosynthesis of nucleotides while NADPH is the cofactor of several dehydrogenases acting in a broad range of biosynthetic processes and in the maintenance of the cellular redox state. RNA interference-mediated reduction of G6PDH levels in bloodstream-form Trypanosoma brucei validated this enzyme as a drug target against Human African Trypanosomiasis. Dehydroepiandrosterone (DHEA), a human steroidal pro-hormone and its derivative 16 alpha-bromoepiandrosterone (16BrEA) are uncompetitive inhibitors of mammalian G6PDH. Such steroids are also known to enhance the immune response in a broad range of animal infection models. It is noteworthy that the administration of DHEA to rats infected by Trypanosoma cruzi, the causative agent of Human American Trypanosomiasis (also known as Chagas` disease), reduces blood parasite levels at both acute and chronic infection stages. In the present work, we investigated the in vitro effect of DHEA derivatives on the proliferation of T. cruzi epimastigotes and their inhibitory effect on a recombinant form of the parasite`s G6PDH (TcG6PDH). Our results show that DHEA and its derivative epiandrosterone (EA) are uncompetitive inhibitors of TcG6PDH, with K(i) values of 21.5 +/- 0.5 and 4.8 +/- 0.3 mu M, respectively. Results from quantitative inhibition assays indicate 16BrEA as a potent inhibitor of TcG6PDH with an IC(50) of 86 +/- 8 nM and those from in vitro cell viability assays confirm its toxicity for T. cruzi epimastigotes, with a LD(50) of 12 +/- 8 mu M. In summary, we demonstrated that, in addition to host immune response enhancement, 16BrEA has a direct effect on parasite viability, most likely as a consequence of TcG6PDH inhibition. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta)

The enzyme nitrate reductase (NR) responsible for the conversion of nitrate to nitrite is considered to be the rate-limiting step in nitrogen assimilation. The economically important marine macroalga Gracilaria tenuistipitata presents a circadian oscillation in NR protein content and activity. In order to identify if the regulation of NR in G. tenuistipitata happens at transcriptional levels, the NR cDNA and gene were sequenced and the NR mRNA expression was studied. Analysis of the sequenced gene revealed absence of introns which is unusual for NR genes. The transcriptional profiling revealed a circadian rhythm for NR; furthermore, a rhythm was observed in constant light condition, suggesting a possible regulation by the biological clock at the mRNA levels for NR in G. tenuistipitata.

Three-Dimensional Quantitative Structure-Activity Relationship Study of Antitumor 2-Formylpyridine Thiosemicarbazones Derivatives as Inhibitors of Ribonucleotide Reductase

In order to extend previous SAR and QSAR studies, 3D-QSAR analysis has been performed using CoMFA and CoMSIA approaches applied to a set of 39 alpha-(N)-heterocyclic carboxaldehydes thiosemicarbazones with their inhibitory activity values (IC(50)) evaluated against ribonucleotide reductase (RNR) of H.Ep.-2 cells (human epidermoid carcinoma), taken from selected literature. Both rigid and field alignment methods, taking the unsubstituted 2-formylpyridine thiosemicarbazone in its syn conformation as template, have been used to generate multiple predictive CoMFA and CoMSIA models derived from training sets and validated with the corresponding test sets. Acceptable predictive correlation coefficients (Q(cv)(2) from 0.360 to 0.609 for CoMFA and Q(cv)(2) from 0.394 to 0.580 for CoMSIA models) with high fitted correlation coefficients (r` from 0.881 to 0.981 for CoMFA and r(2) from 0.938 to 0.993 for CoMSIA models) and low standard errors (s from 0.135 to 0.383 for CoMFA and s from 0.098 to 0.240 for CoMSIA models) were obtained. More precise CoMFA and CoMSIA models have been derived considering the subset of thiosemicarbazones (TSC) substituted only at 5-position of the pyridine ring (n=22). Reasonable predictive correlation coefficients (Q(cv)(2) from 0.486 to 0.683 for CoMFA and Q(cv)(2) from 0.565 to 0.791 for CoMSIA models) with high fitted correlation coefficients (r(2) from 0.896 to 0.997 for CoMFA and r(2) from 0.991 to 0.998 for CoMSIA models) and very low standard errors (s from 0.040 to 0.179 for CoMFA and s from 0.029 to 0.068 for CoMSIA models) were obtained. The stability of each CoMFA and CoMSIA models was further assessed by performing bootstrapping analysis. For the two sets the generated CoMSIA models showed, in general, better statistics than the corresponding CoMFA models. The analysis of CoMFA and CoMSIA contour maps suggest that a hydrogen bond acceptor near the nitrogen of the pyridine ring can enhance inhibitory activity values. This observation agrees with literature data, which suggests that the nitrogen pyridine lone pairs can complex with the iron ion leading to species that inhibits RNR. The derived CoMFA and CoMSIA models contribute to understand the structural features of this class of TSC as antitumor agents in terms of steric, electrostatic, hydrophobic and hydrogen bond donor and hydrogen bond acceptor fields as well as to the rational design of this key enzyme inhibitors.

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352 +/- 21 and 272 +/- 25 mu M, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1 degrees C and pH 8.6. Above 37 degrees C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly. (C) 2010 Elsevier Inc. All rights reserved.

Novel insights for dihydroorotate dehydrogenase class 1A inhibitors discovery

The enzyme dihydroorotate dehydrogenase (DHODH) has been suggested as a promising target for the design of trypanocidal agents. We report here the discovery of novel inhibitors of Trypanosoma cruzi DHODH identified by a combination of virtual screening and ITC methods. Monitoring of the enzymatic reaction in the presence of selected ligands together with structural information obtained from X-ray crystallography analysis have allowed the identification and validation of a novel site of interaction (S2 site). This has provided important structural insights for the rational design of T cruzi and Leishmania major DHODH inhibitors. The most potent compound (1) in the investigated series inhibits TcDHODH enzyme with K(i)(app) value of 19.28 mu M and possesses a ligand efficiency of 0.54 kcal mol(-1) per non-H atom. The compounds described in this work are promising hits for further development. (C) 2010 Elsevier Masson SAS. All rights reserved.

Use of microprojectile bombardment in transient expression assays to analyze protochlorophyllide reductase gene expression in greening maize seedling leaf cells

In young cells of leaf meristems the progenitors of chloroplasts are small organelles known as proplastids, which divide and differentiate into chloroplasts. However, in the absence of light, proplastids undergo a different sequence of development and become etioplasts. When light is supplied to etiolated plants during the "greening" process, etioplasts differentiate into chloroplasts containing chlorophyll. An important light dependent step in chlorophyll biosynthesis is the photoreduction of protochlorophyllide to chlorophyllide by the NADPH:protochlorophyllide reductase (PCR) enzyme. This enzyme is present at high activity only in etiolated tissue and during early stages of light-induced chlorophyll synthesis. The enzyme and its corresponding mRNAs decrease dramatically with prolonged exposure to light. We have investigated the light-dependent transcriptional regulation of a PCR gene in greening maize leaf cells using a transient expression assay based on microprojectile bombardment. The promoter region was isolated and cloned into a ?-glucuronidase (GUS) reporter gene expression plasmid. We have used this chimeric plasmid in tungsten particle bombardment of both etiolated and greening maize seedling leaves to determine whether the cloned promoter region contains regulatory sequences that control light-responsive PCR gene expression.

The Physiological, Enzymatic, and Genetic Characterization of Staphylococcus sp. Chromium (VI) Reductase Function

A strain of Staphylococcus isolated by Dr. Fekete at the Sandia National Laboratory toxic metal dumping site in Sandia, New Mexico. has been found to reduce toxic Cr(VI) to the less toxic Cr(IlI) state. We have ascertained the environmental parameters for optimal bacterial growth and Cr(VI) reduction. This knowledge may be employed in a comprehensive bioremediation scheme designed to accelerate natural reparation of that Sandia ecosystem. In addition we have investigated the genetic and enzymatic basis for this Cr(VI) reducing ability. This information may allow us to create more effective bioremediation schemes based on the comprehensive knowledge of enzyme and gene function. Preliminary investigations have been carried out toward this end which may serve as the basis for a more thorough investigation.

Insights into the Specificity of Thioredoxin Reductase-Thioredoxin Interactions. A Structural and Functional Investigation of the Yeast Thioredoxin System

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro and in situ activity of carboxymethyl cellulase and glutamate dehydrogenase according to supplementation with different nitrogenous compounds

Two experiments were carried out to evaluate the effect of supplementation with different nitrogenous compounds on the activities of carboxymethil cellulase (CMCase) and glutamate dehydrogenase (GDH). In the first experiment, four treatments were evaluated in vitro: cellulose, cellulose with casein, cellulose with urea, and cellulose with casamino acids. After 6, 12 and 24 hours of incubation, CMCase and GDH activity, pH, and concentrations of ammonia nitrogen (AN) and microbial protein were measured. In the three incubation periods, the concentration of AN was higher when urea was used as a supplemental source of nitrogen. The activity of CMCase was higher with the addition of urea and casamino acids when compared with the control and the casein treatment. Supplementation with casamino acids provided higher GDH activity when compared with the control at 6 hours of incubation. At 12 hours of incubation, the GHD activity was also stimulated by casein. At 24 hours, there was no difference in GHD activity among treatments. In the second experiment, three rumen-fistulated bulls were used for in situ evaluation. Animals were fed Tifton hay (Cynodon sp.) ad libitum. The treatments consisted of control (no supplementation), supplementation with non-protein nitrogenous compounds (urea and ammonium sulphate, 9:1) and supplementation with protein (albumin). In treatments with nitrogenous compound supplementation, 1 g of crude protein/kg of body weight was supplied. The experiment was conducted in a 3 × 3 Latin square design. The measurements were performed at 6, 12 and 24 hours after supplementation. No difference in GDH activity was observed among treatments. The control treatment showed higher CMCase activity when compared with the treatments containing supplemental sources of nitrogen. However, urea supplementation provided higher CMCase activity compared to albumin.

Recombinant expression of glycerol-3-phosphate dehydrogenase using the Pichia pastoris system

In the present study, the GPD2 gene from Saccharomyces cerevisiae, which codifies for the enzyme glycerol-3-phosphate dehydrogenase (GPDH), was cloned from the pPICZ-alpha expression vector and used with the purpose of inducing the extracellular expression of the glycerol-3-phosphate dehydrogenase under the control of the methanol-regulated AOX promoter. The presence of the GPD2 insert was confirmed by PCR analysis. Pichia pastoris X-33 (Mut(+)) was transformed with linearized plasmids by electroporation and transformants were selected on YPDS plates containing 100 mu g/mL of zeocin. Several clones were selected and the functionality of this enzyme obtained in a culture medium was assayed. Among the mutants tested, one exhibited 3.1 x 10(-2) U/mg of maximal activity. Maximal enzyme activity was achieved at 6 days of growth. Medium composition and pre-induction osmotic stress influenced protein production. Pre-induction osmotic stress (culturing cells in medium with either 0.35 M sodium chloride or 1.0 M sorbitol for 4h prior to induction) led to an increase in cell growth with sorbitol and resulted in a significant increase in GPDH productivity with sodium chloride in 24h of induction approximately fivefold greater than under standard conditions (without pre-induction). (C) 2010 Elsevier B.V. All rights reserved.

Growth hormone and heart failure: Oxidative stress and energetic metabolism in rats

Several evidences point for beneficial effects of growth hormone (GH) in heart failure (HF). Taking into account that HF is related with changes in myocardial oxidative stress and in energy generation from metabolic pathways, it is important to clarify whether GH increase or decrease myocardial oxidative stress and what is its effect on energetic metabolism in HF condition. Thus, this study investigated the effects of two different doses of GH on energetic metabolism and oxidative stress in myocardium of rats with HF. Male Wistar rats (n = 25) were submitted to aortic stenosis (AS). The HF was evidenced by tachypnea and echocardiographic criteria around 28 weeks of AS. The rats were then randomly divided into three groups: (HF) with HF, treated with saline (0.9% NaCl); (HF-GHI), treated with 1 mk/kg/day recombinant human growth hormone (rhGH), and (HF-GH2) treated with 2 mg/kg/day rhGH. GH was injected, subcutaneously, daily for 2 weeks. A control group (sham; n = 12), with the same age of the others rats was evaluated to confirm data for AS. HF had lower IGF-I (insulin-like growth factor-I) than sham-operated rats, and both GH treatments normalized IGF-I level. HF-GH1 animals had lower lipid hydroperoxide (LH), LH/total antioxidant substances (TAS) and glutathione-reductase than HF. Glutathione peroxidase (GSH-Px), hydroxyacyl coenzyme-A dehydrogenase, lactate dehydrogenase(LDH) were higher in HF-GH1 than in HF. HF-GH2 compared with HF, had increased LH/TAS ratio, as well as decreased oxidized glutathione and LDH activity. Comparing the two GH doses, GSH-Px, superoxide dismutase and LDH were lower in HF-GH2 than in HF-GHI. In conclusion, GH effects were dose-dependent and both tested doses did not aggravate the heart dysfunction. The higher GH dose, 2 mg/kg exerted detrimental effects related to energy metabolism and oxidative stress. The lower dose, 1 mg/kg GH exerted beneficial effects enhancing antioxidant defences, reducing oxidative stress and improving energy generation in myocardium of rats with heart failure. (c) 2007 Elsevier Ltd. All rights reserved.