Dendrimer-assisted immobilization of alcohol dehydrogenase in nanostructured films for biosensing: Ethanol detection using electrical capacitance measurements

The selective determination of alcohol molecules either in aqueous solutions or in vapor phase is of great importance for several technological areas. In the last years, a number of researchers have reported the fabrication of highly sensitive sensors for ethanol detection, based upon specific enzymatic reactions occurring at the surface of enzyme-containing electrodes. In this study, the enzyme alcohol dehydrogenase (ADH) was immobilized in a layer-by-layer fashion onto Au-interdigitated electrodes (IDEs), in conjunction with layers of PAMAM dendrimers. The immobilization process was followed in Teal time using quartz crystal microbalance (QCM), indicating that an average mass of 52.1 ng of ADH was adsorbed at each deposition step. Detection was carried out using a novel strategy entirely based upon electrical capacitance measurements, through which ethanol could be detected at concentrations of 1 part per million by volume (ppmv). (C) 2007 Elsevier B.V. All rights reserved.

Kinetic and Crystallographic Studies on Glyceraldehyde-3-Phosphate Dehydrogenase from Trypanosoma cruzi in Complex with Iodoacetate

Kinetic and crystallographic studies on the formation of the complex between iodoacetate and the enzyme glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi were conducted in order to investigate the mechanistic and structural basis underlying enzyme inactivation. The crystallographic complex reveal important structural features useful for the design of novel inhibitors.

Discovery of novel Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors

Based on its essential role in the life cycle of Trypanosoma cruzi, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been considered a promising target for the development of novel chemotherapeutic agents for the treatment of Chagas` disease. In the course of our research program to discover novel inhibitors of this trypanosomatid enzyme, we have explored a combination of structure and ligand-based virtual screening techniques as a complementary approach to a biochemical screening of natural products using a standard biochemical assay. Seven natural products, including anacardic acids,. avonoid derivatives, and one glucosylxanthone were identified as novel inhibitors of T. cruzi GAPDH. Promiscuous inhibition induced by nonspecific aggregation has been discarded as specific inhibition was not reversed or affected in all cases in the presence of Triton X-100, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. The structural diversity of this series of promising natural products is of special interest in drug design, and should therefore be useful in future medicinal chemistry efforts aimed at the development of new GAPDH inhibitors having increased potency. (C) 2009 Elsevier Ltd. All rights reserved.

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.

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.

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.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

Os autores padronizaram métodos para a avaliação da atividade da glicose-6-fosfato desidrogenase e glutationa redutase. O princípio geral do primeiro método baseou-se na formação de metahemoglobina pelo nitrito de sódio, seguido da estimulação da via das pentoses pelo azul de metileno. Foram estudados 46 indivíduos adultos, sendo 23 do sexo masculino e 23 do feminino, não deficientes em glicose-6-fosfato desidrogenase (G6PD), com idades variando entre 20 e 30 anos. Os resultados revelaram que a redução da metahemoglobina pelo azul de metileno para sangue total, foram de 154.50 e 139.90 mg/min (p<0.05) respectivamente para o sexo masculino e feminino. Para hemácias lavadas os valores foram de 221.10 e 207.85 mg/min (n.s.) respectivamente. Estas observações permitiram concluir que ao se empregar hemácias lavadas e 0.7 g% de concentração de nitrito de sódio, por um lado não houve diferença entre os sexos e por outro, abreviou o tempo de leitura da quantidade residual de metahemoglobina para 90 minutos. A avaliação da atividade da glutationa redutase foi feita baseado no fato de que a cistamina (agente tiol) liga-se aos grupos SH da hemoglobina formando complexos. Estes complexos são revertidos pela ação da glutationa redutase, ocorrendo conjuntamente nesta reação a redução da metahemoglobina. Foram estudados 32 indivíduos adultos, sendo 16 do sexo masculino e 16 do feminino, não deficientes em G6PD, com idades variando entre 20 e 30 anos. Os resultados revelaram valores de redução da metahemoglobina pela cistamina de 81.27 e 91.13 mg/min (p<0.01) respectivamente para o sexo masculino e feminino. Estas observações permitiram concluir que o emprego de hemácias lavadas e 0.1 molar de concentração de cistamina torna possível a leitura da quantidade residual de metahemoglobina aos 180 minutos de incubação. A atividade da glutationa redutase avaliada por meio da redução da metahemoglobina pela cistamina, foi estudada em 14 indivíduos do sexo feminino antes e após o tratamento com 10 mg por dia de riboflavina durante 8 dias. Os resultados foram de 73.69 e 94.26 mg/min (p<0.01) antes e após o tratamento. Estas observações permitiram concluir que a oferta de riboflavina, mesmo para indivíduos normais, aumenta a atividade da glutationa redutase. Foram ainda avaliados 3 indivíduos da raça negra e deficientes em G6PD, sendo 2 do sexo masculino e 1 do feminino. Houve ativação parcial da G6PD e glutationa redutase, sendo estas alterações mais intensas nos indivíduos do sexo masculino. Considerando-se a raça e as características laboratoriais observadas, foi possível sugerir que a deficiência em G6PD verificada é do tipo Africano, bem como, permitiu considerar os indivíduos do sexo feminino coin o sendo heterozigoto para esta deficiência. Por fim, a análise dos resultados em seu conjunto permitiu concluir que os métodos propostos se mostraram eficientes para avaliar a atividade da G6PD e glutationa redutase. Esta última é dependente da via das pentoses, geradora de NADPH e da riboflavina, vitamina precursora de FAD.

Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging

Replicative life span in Saccharomyces cerevisiae is increased by glucose (G1c) limitation [ calorie restriction (CR)] and by augmented NAD(+). Increased survival promoted by CR was attributed previously to the NAD(+)-dependent histone deacetylase activity of sirtuin family protein Sir2p but not to changes in redox state. Here we show that strains defective in NAD(+) synthesis and salvage pathways (pnc1 Delta, npt1 Delta, and bna6 Delta) exhibit decreased oxygen consumption and increased mitochondrial H2O2 release, reversed over time by CR. These null mutant strains also present decreased chronological longevity in a manner rescued by CR. Furthermore, we observed that changes in mitochondrial H2O2 release alter cellular redox state, as attested by measurements of total, oxidized, and reduced glutathione. Surprisingly, our results indicate that matrix-soluble dihydrolipoyl-dehydrogenases are an important source of CR-preventable mitochondrial reactive oxygen species (ROS). Indeed, deletion of the LPD1 gene prevented oxidative stress in npt1 Delta and bna6 Delta mutants. Furthermore, pyruvate and alpha-ketoglutarate, substrates for dihydrolipoyl dehydrogenase-containing enzymes, promoted pronounced reactive oxygen release in permeabilized wild-type mitochondria. Altogether, these results substantiate the concept that mitochondrial ROS can be limited by caloric restriction and play an important role in S. cerevisiae senescence. Furthermore, these findings uncover dihydrolipoyl dehydrogenase as an important and novel source of ROS leading to life span limitation.

Alcohol dehydrogenase 3 genotype as a risk factor for upper aerodigestive tract cancers

Objective: To assess alcohol dehydrogenase 3 (ADH3) polymorphism at position Ile349Val as indicator of risk factor for upper aerodigestive tract (UADT) cancer to verify its association with UADT cancer in nonalcoholic or nonsmoking individuals.Design: Cross-sectional study.Setting: Primary care or referral center.Patients: the study group consisted of 141 consecutive patients with newly diagnosed squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx admitted for surgical treatment. The comparison group consisted of 94 inpatients without cancer from the A. C. Camargo or other São Paulo (Brazil) hospital and 40 healthy individuals.Intervention: All participants were interviewed and data were collected using a structured questionnaire. After written informed consent was obtained, 20 mL of blood was collected in heparinized tubes.Main Outcome Measures: Odds ratio for ADH3 genotypes using logistic regression models.Results: After adjustment for sex, age, tobacco use, and history of cancer in first-degree family relatives, a significantly higher odds ratio for UADT cancer was observed among individuals with AA genotype and low cumulative alcohol consumption (:5 100 kg of ethanol) (odds ratio=3.8 [95% confidence interval, 1.5-9.7]). A 4-fold increase in odds ratio for UADT cancer among individuals with AA genotype and low tobacco consumption (:525 pack-years) was also found in the adjusted model.Conclusions: These results suggest that genotype AA may be a risk factor for UADT cancer, especially in individuals with low alcohol or tobacco consumption. However, further epidemiological case-control or cohort studies, preferably prospective, are needed to establish the exact role of ADH3 polymorphism and its association with the development of UADT cancers.