Identification of novel coenzyme Q10 biosynthetic proteins Coq11 and Coq12 in Schizosaccharomyces pombe.

Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10 has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of the p-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10 synthesis, we investigated CoQ10 production in 400 Schizosaccharomyces pombe gene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion of coq11 (an S. cerevisiae COQ11 homolog) and a novel gene designated coq12 lowered CoQ levels to ∼4% of that of the WT strain. Addition of PHB or p-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of the Δcoq12 strain, but these compounds did not affect the Δcoq11 strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+ reductase domain. We determined that purified Coq12 protein from S. pombe displayed NAD+ reductase activity when incubated with ethanol-extracted substrate of S. pombe. Because purified Coq12 from Escherichia coli did not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC-MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.

Anti-oxidative and anti-hyperglycemic properties of Agastache foeniculum essential oil and oily fraction in hyperglycemia-stimulated and lipopolysaccharide-stimulated macrophage cells: In vitro and in silico studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperglycemia (HG) and lipopolysaccharide (LPS) often promote superoxide accumulation, which may increase oxidative stress. Reducing superoxide production in hyperglycemia and the inflammatory condition is an emerging way to reduce protein and lipid oxidation and diabetes complication. AIM OF STUDY: To examine the effect of Agastache foeniculum essential oil (AFEO) and oil fraction (AFoil) on HG- and LPS-stimulated oxidative stress, the pathogenicity of AFEO and AFoil on oxidative stress was assessed. METHODS: The stimulatory effects of AFEO and AFoil on the activity and expression of NADH oxide (NOX), catalase (CAT), superoxide dismutase (SOD), and the expression of nuclear respiratory factor 2 (NRF2) and nuclear factor-kappa B (NF-kB) in the stimulated macrophage cell line, J774.A1, was studied. The interaction patterns of AFEO and AFoil components with NOX, SOD, CAT, NRF2, and NF-kB proteins were also deduced using molecular docking. RESULTS: Estragole was the main ingredient in AFEO (97%). Linolenic acid (32.10%), estragole (16.22%), palmitic acid (12.62%), linoleic acid (12.04%), and oleic acid (8.73%) were the major chemical components of the AFoil. NOX activation was stimulated in macrophage cells by HG and LPS. At 20 µg/mL, AFEO and AFoil decreased NOX activity while increased SOD and CAT activities in stimulated macrophages. AFoil with estragole and omega-3 fatty acids was better than AFEO with estragole in anti-hyperglycemic and anti-oxidative activity. According to molecular docking research, estragole, linoleic acid, and linolenic acid bind to different hydrophobic pockets of NOX, SOD, CAT, NFR2, and NF-kB using hydrogen bonds, van der Waals bonds, pi-alkyl, and pi-anion interactions, with different binding energies. CONCLUSION: AFEO and AFoil showed antioxidant and anti-diabetic activity. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes and up-regulating superoxide-removing enzymes at gene and protein levels. The AFoil emulsion can be used to reduce the detrimental impacts of hyperglycemia and oxidative stress.

Bioassay-based Corchorus capsularis L. leaf-derived ß-sitosterol exerts antileishmanial effects against Leishmania donovani by targeting trypanothione reductase.

Leishmaniasis, a major neglected tropical disease, affects millions of individuals worldwide. Among the various clinical forms, visceral leishmaniasis (VL) is the deadliest. Current antileishmanial drugs exhibit toxicity- and resistance-related issues. Therefore, advanced chemotherapeutic alternatives are in demand, and currently, plant sources are considered preferable choices. Our previous report has shown that the chloroform extract of Corchorus capsularis L. leaves exhibits a significant effect against Leishmania donovani promastigotes. In the current study, bioassay-guided fractionation results for Corchorus capsularis L. leaf-derived ß-sitosterol (ß-sitosterolCCL) were observed by spectroscopic analysis (FTIR, 1H NMR, 13C NMR and GC-MS). The inhibitory efficacy of this ß-sitosterolCCL against L. donovani promastigotes was measured (IC50 = 17.7 ± 0.43 µg/ml). ß-SitosterolCCL significantly disrupts the redox balance via intracellular ROS production, which triggers various apoptotic events, such as structural alteration, increased storage of lipid bodies, mitochondrial membrane depolarization, externalization of phosphatidylserine and non-protein thiol depletion, in promastigotes. Additionally, the antileishmanial activity of ß-sitosterolCCL was validated by enzyme inhibition and an in silico study in which ß-sitosterolCCL was found to inhibit Leishmania donovani trypanothione reductase (LdTryR). Overall, ß-sitosterolCCL appears to be a novel inhibitor of LdTryR and might represent a successful approach for treatment of VL in the future.

Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme.

Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.

Identification and binding mode of a novel Leishmania Trypanothione reductase inhibitor from high throughput screening.

Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against Leishmaniasis. This enzyme is fundamental for parasite survival in the host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize hydrogen peroxide produced by host macrophages during infection. In order to identify new lead compounds against Leishmania we developed and validated a new luminescence-based high-throughput screening (HTS) assay that allowed us to screen a library of 120,000 compounds. We identified a novel chemical class of TR inhibitors, able to kill parasites with an IC50 in the low micromolar range. The X-ray crystal structure of TR in complex with a compound from this class (compound 3) allowed the identification of its binding site in a pocket at the entrance of the NADPH binding site. Since the binding site of compound 3 identified by the X-ray structure is unique, and is not present in human homologs such as glutathione reductase (hGR), it represents a new target for drug discovery efforts.

A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily.

The short chain dehydrogenase/reductase superfamily (SDR) is a large family of NAD(P)H-dependent enzymes found in all kingdoms of life. SDRs are particularly well-represented in plants, playing diverse roles in both primary and secondary metabolism. In addition, some plant SDRs are also able to catalyse a reductive cyclisation reaction critical for the biosynthesis of the iridoid backbone that contains a fused 5 and 6-membered ring scaffold. Mining the EST database of Plantago major, a medicinal plant that makes iridoids, we identified a putative 5ß-progesterone reductase gene, PmMOR (P. major multisubstrate oxido-reductase), that is 60% identical to the iridoid synthase gene from Catharanthus roseus. The PmMOR protein was recombinantly expressed and its enzymatic activity assayed against three putative substrates, 8-oxogeranial, citral and progesterone. The enzyme demonstrated promiscuous enzymatic activity and was able to not only reduce progesterone and citral, but also to catalyse the reductive cyclisation of 8-oxogeranial. The crystal structures of PmMOR wild type and PmMOR mutants in complex with NADP+ or NAD+ and either 8-oxogeranial, citral or progesterone help to reveal the substrate specificity determinants and catalytic machinery of the protein. Site-directed mutagenesis studies were performed and provide a foundation for understanding the promiscuous activity of the enzyme.

Structural insights into natural compounds as inhibitors of Fasciola gigantica thioredoxin glutathione reductase.

Fascioliasis is caused by the helminth parasites of genus Fasciola. Thioredoxin glutathione reductase (TGR) is an important enzyme in parasitic helminths and plays an indispensable role in its redox biology. In the present study, we conducted a structure-based virtual screening of natural compounds against the Fasciola gigantica TGR (FgTGR). The compounds were docked against FgTGR in four sequential docking modes. The screened ligands were further assessed for Lipinski and ADMET prediction so as to evaluate drug proficiency and likeness property. After refinement, three potential inhibitors were identified that were subjected to 50 ns molecular dynamics simulation and free energy binding analyses to evaluate the dynamics of protein-ligand interaction and the stability of the complexes. Key residues involved in the interaction of the selected ligands were also determined. The results suggested that three top hits had a negative binding energy greater than GSSG (-91.479 KJ · mol-1 ), having -152.657, -141.219, and -92.931 kJ · mol-1 for compounds with IDs ZINC85878789, ZINC85879991, and ZINC36369921, respectively. Further analysis showed that the compound ZINC85878789 and ZINC85879991 displayed substantial pharmacological and structural properties to be a drug candidate. Thus, the present study might prove useful for the future design of new derivatives with higher potency and specificity.

Molecular cloning, in-silico characterization and functional validation of monodehydroascorbate reductase gene in Eleusine coracana.

Ascorbic acid is a ubiquitous water soluble antioxidant that plays a critical role in plant growth and environmental stress tolerance. It acts as a free radical scavenger as well as a source of reducing power for several cellular processes. Because of its pivotal role in regulating plant growth under optimal as well as sub-optimal conditions, it becomes obligatory for plants to maintain a pool of reduced ascorbic acid. Several cellular processes help in maintaining the reduced ascorbic acid pool, by regulating its synthesis and regeneration processes. Current study demonstrates that monodehydroascorbate reductase is an important enzyme responsible for maintaining the reduced ascorbate pool, by optimizing the recycling of oxidized ascorbate. Cloning and functional characterization of this important stress inducible gene is of great significance for its imperative use in plant stress management. Therefore, we have cloned and functionally validated the role of monodehydroascorbate reductase gene (mdar) from a drought tolerant variety of Eleusine coracana. The cloned Ecmdar gene comprises of 1437bp CDS, encoding a 478 amino acid long polypeptide. The active site analysis showed presence of conserved Tyr348 residue, facilitating the catalytic activity in electron transfer mechanism. qPCR expression profiling of Ecmdar under stress indicated that it is an early responsive gene. The analysis of Ecmdar overexpressing Arabidopsis transgenic lines suggests that monodehydroascorbate reductase acts as a key stress regulator by modulating the activity of antioxidant enzymes to strengthen the ROS scavenging ability and maintains ROS homeostasis. Thus, it is evident that Ecmdar is an important gene for cellular homeostasis and its over-expression could be successfully used to strengthen stress tolerance in crop plants.

Biosurfactant production from Pseudomonas taiwanensis L1011 and its application in accelerating the chemical and biological decolorization of azo dyes.

Dye dispersion and the interaction efficiency between azoreductases and dye molecules are rate-limiting steps for the decolorization of azo dyes. In this study, a biosurfactant-producing strain, Pseudomonas taiwanensis L1011, was isolated from crude oil. To increase the yield of the biosurfactant BS-L1011 from P. taiwanensis L1011, culture conditions were optimized including temperature, initial pH, carbon source, nitrogen source and C/N ratio. A maximum yield of 1.12g/L of BS-L1011 was obtained using D-mannitol as carbon source and yeast extract/urea as compound nitrogen source with C/N ratio of 10/4, pH 7.0 and 28°C. BS-L1011 exhibited a low critical micelle concentration (CMC) of 10.5mg/L and was able to reduce the surface tension of water to 25.8±0.1 mN/m. BS-L1011 was stable over a wide range of temperatures, pH values and salt concentrations. The biosurfactant is reported for the first time to accelerate chemical decolorization of Congo red by sodium hypochlorite, and biological decolorization of Amaranth by Bacillus circulans BWL1061, thus showing a potential in the treatment of dyeing wastewater.

Identification of potential trypanothione reductase inhibitors among commercially available ß-carboline derivatives using chemical space, lead-like and drug-like filters, pharmacophore models and molecular docking.

American trypanosomiasis or Chagas disease caused by the protozoan Trypanosoma cruzi (T. cruzi) is an important endemic trypanosomiasis in Central and South America. This disease was considered to be a priority in the global plan to combat neglected tropical diseases, 2008-2015, which indicates that there is an urgent need to develop more effective drugs. The development of new chemotherapeutic agents against Chagas disease can be related to an important biochemical feature of T. cruzi: its redox defense system. This system is based on trypanothione ([Formula: see text],[Formula: see text]-bis(glutathyonil)spermidine) and trypanothione reductase (TR), which are rather unique to trypanosomes and completely absent in mammalian cells. In this regard, tricyclic compounds have been studied extensively due to their ability to inhibit the T. cruzi TR. However, synthetic derivatives of natural products, such as [Formula: see text]-carboline derivatives ([Formula: see text]-CDs), as potential TR inhibitors, has received little attention. This study presents an analysis of the structural and physicochemical properties of commercially available [Formula: see text]-CDs in relation to compounds tested against T. cruzi in previously reported enzymatic assays and shows that [Formula: see text]-CDs cover chemical space that has not been considered for the design of TR inhibitors. Moreover, this study presents a ligand-based approach to discover potential TR inhibitors among commercially available [Formula: see text]-CDs, which could lead to the generation of promising [Formula: see text]-CD candidates.

QSAR-Driven Discovery of Novel Chemical Scaffolds Active against Schistosoma mansoni.

Schistosomiasis is a neglected tropical disease that affects millions of people worldwide. Thioredoxin glutathione reductase of Schistosoma mansoni (SmTGR) is a validated drug target that plays a crucial role in the redox homeostasis of the parasite. We report the discovery of new chemical scaffolds against S. mansoni using a combi-QSAR approach followed by virtual screening of a commercial database and confirmation of top ranking compounds by in vitro experimental evaluation with automated imaging of schistosomula and adult worms. We constructed 2D and 3D quantitative structure-activity relationship (QSAR) models using a series of oxadiazoles-2-oxides reported in the literature as SmTGR inhibitors and combined the best models in a consensus QSAR model. This model was used for a virtual screening of Hit2Lead set of ChemBridge database and allowed the identification of ten new potential SmTGR inhibitors. Further experimental testing on both shistosomula and adult worms showed that 4-nitro-3,5-bis(1-nitro-1H-pyrazol-4-yl)-1H-pyrazole (LabMol-17) and 3-nitro-4-{[(4-nitro-1,2,5-oxadiazol-3-yl)oxy]methyl}-1,2,5-oxadiazole (LabMol-19), two compounds representing new chemical scaffolds, have high activity in both systems. These compounds will be the subjects for additional testing and, if necessary, modification to serve as new schistosomicidal agents.

Computational and Investigative Study of Flavonoids Active Against Typanosoma cruzi and Leishmania spp.

Flavonoid compounds active against Trypanosoma cruzi and Leishmania species were submitted to several methodologies in silico: docking with the enzymes cruzain and trypanothione reductase (from T. cruzi), and N-myristoyltransferase, dihydroorotate dehydrogenase, and trypanothiona reductase (from Leishmania spp). Molecular maps of the complexes and the ligands were calculated. In order to compare and evaluate the antioxidant activity of the flavonoids with their antiprotozoal activity, quantum parameters were calculated. Considering the energies, interactions, and hydrophobic surfaces calculated, the flavonoids chrysin dimethyl ether against T. cruzi, and ladanein against Leishmania sp. presented the best results. The antioxidant activity did not show any correlation with anti-parasitic activity; only chrysin and its dimethyl ether showed favorable anti-parasitic results. This study hopes to contribute to existing research on these natural products against these tropical parasites.

Trypanothione reductase: a target protein for a combined in vitro and in silico screening approach.

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC50 values of <1 µM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC50 values down to 2 µM.

Molecular cloning, computational and expression analysis of anthocyanidin reductase in tea (Camellia sinensis).

Tea [Camellia sinensis (L.) O. Kuntze] is one of the most popular non-alcoholic beverages rich in phenolic compounds, which includes epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) and catechin (C). Anthocyanidin reductase (ANR) is responsible for catechin biosynthesis in plants, and analysis of its protein sequences and structures will be valuable for further research in the field. We have screened our dormant bud-specific complementary DNA (cDNA) library and reported 1,322-bp cDNA encoding CsANR. Analysis of the sequence revealed the presence of 1,011-bp open reading frame with coding capacity for a polypeptide of 337 amino acids, flanked by 1,123- and 196-bp 5' and 3' untranslated regions, respectively. Theoretical molecular weight (MW) and isoelectric point (pI) of the deduced ANR protein were predicted (using ProtParam) to be 36.4 kDa and 6.54. For the first time, we have reported 3D model of ANR from C. sinensis. Quality of the predicted model was analysed with PROCHECK analysis. Molecular docking of modelled ANR revealed similar binding pockets for both substrates and products. Expression analyses of CsANR and accumulation pattern of catechins were observed to be varied with developmental age of tissue and seasonal condition. Variation in accumulation pattern of catechins and its fractions was found to be correlated with expression pattern of ANR.

Study of bio-degradation and bio-decolourization of azo dye by Enterobacter sp. SXCR.

The objective of this study was to evaluate the decolourization potential of textile dyes by a relatively newly identified bacteria species, Enterobacter sp. SXCR which was isolated from the petroleum polluted soil samples. The bacterial strain was identified by 16S rRNA gene sequence analysis. The effects of operational conditions like initial dye concentration, pH, and temperature were optimized to develop an economically feasible decolourization process. The isolate was able to decolourize sulphonated azo dye (Congo red) over a wide range (0.1-1 gl(-1)), pH 5-9, and temperature 22-40 degrees C in static condition. Anaerobic condition with minimal salt medium supplemented with 2 gl(-1) glucose, pH 7 and 34 degrees C were considered to be the optimum decolourizing condition. The bacterial isolate SXCR showed a strong ability to decolourize dye (0.2 gl(-1)) within 93 h. The biodegradation was monitored by UV-vis, fourier transform infra-red spectroscopy (FTIR) spectroscopy and high performance liquid chromatography (HPLC). Furthermore, the involvement of azoreductase in the decolourization process was identified in this strain. Cells of Enterobacter cloacae were immobilized by entrapment in calcium-alginate beads. Immobilized bacterial cells were able to reduced azo bonds enzymatically and used as a biocatalyst for decolourization of azo dye Congo red. Michaelis-Menten kinetics was used to describe the correlation between the decolourization rate and the dye concentration.

Identification of thioredoxin glutathione reductase inhibitors that kill cestode and trematode parasites.

Parasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR) is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms) and trematoda (flukes), while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites.

The siderophore-interacting protein YqjH acts as a ferric reductase in different iron assimilation pathways of Escherichia coli.

Siderophore-interacting proteins (SIPs), such as YqjH from Escherichia coli, are widespread among bacteria and commonly associated with iron-dependent induction and siderophore utilization. In this study, we show by detailed biochemical and genetic analyses the reaction mechanism by which the YqjH protein is able to catalyze the release of iron from a variety of iron chelators, including ferric triscatecholates and ferric dicitrate, displaying the highest efficiency for the hydrolyzed ferric enterobactin complex ferric (2,3-dihydroxybenzoylserine)(3). Site-directed mutagenesis revealed that residues K55 and R130 of YqjH are crucial for both substrate binding and reductase activity. The NADPH-dependent iron reduction was found to proceed via single-electron transfer in a double-displacement-type reaction through formation of a transient flavosemiquinone. The capacity to reduce substrates with extremely negative redox potentials, though at low catalytic rates, was studied by displacing the native FAD cofactor with 5-deaza-5-carba-FAD, which is restricted to a two-electron transfer. In the presence of the reconstituted noncatalytic protein, the ferric enterobactin midpoint potential increased remarkably and partially overlapped with the effective E(1) redox range. Concurrently, the observed molar ratios of generated Fe(II) versus NADPH were found to be ~1.5-fold higher for hydrolyzed ferric triscatecholates and ferric dicitrate than for ferric enterobactin. Further, combination of a chromosomal yqjH deletion with entC single- and entC fes double-deletion backgrounds showed the impact of yqjH on growth during supplementation with ferric siderophore substrates. Thus, YqjH enhances siderophore utilization in different iron acquisition pathways, including assimilation of low-potential ferric substrates that are not reduced by common cellular cofactors.

Improved inhibitors of trypanothione reductase by combination of motifs: synthesis, inhibitory potency, binding mode, and antiprotozoal activities.

Trypanothione reductase (TR) is an essential enzyme in the trypanothione-based redox metabolism of trypanosomatid parasites. This system is absent in humans and, therefore, offers a promising target for the development of selective new drugs against African sleeping sickness and Chagas' disease. Over the past two decades, a variety of nonpeptidic small-molecule ligands of the parasitic enzyme were discovered. A current goal is to decipher the binding mode of these known inhibitors in order to optimize their structures. We analyzed the binding mode of recently reported 1-(1-(benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) analogues using computer modeling methods. This led us to conclude that the analogues occupy a different region of the active site than the diaryl sulfide-based class of inhibitors. A combination of the two motifs significantly increased affinity for the enzyme compared to the respective parent compounds. The newly synthesized conjugates exhibit K(ic) values for TR as low as 0.51±0.1 µM and high selectivity for the parasitic enzyme over the related human glutathione reductase (hGR), as was predicted by our molecular modeling studies. In vitro studies showed IC(50) values in the low micromolar to submicromolar range against Trypanosoma brucei rhodesiense, often in combination with low cytotoxicity against mammalian cells. Interestingly, even stronger activities were found against Plasmodium falciparum.

Gene expression of monodehydroascorbate reductase and dehydroascorbate reductase during fruit ripening and in response to environmental stresses in acerola (Malpighia glabra).

Acerola (Malpighia glabra) is an exotic fruit cultivated primarily for its abundant ascorbic acid (AsA) content. The molecular mechanisms that regulate the metabolism of AsA in acerola have yet to be defined. Monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) are key enzymes of the ascorbate-glutathione cycle that maintain reduced pools of ascorbic acid and serve as important antioxidants. cDNAs encoding MDHAR and DHAR were isolated from acerola using RT-PCR and RACE. Phylogenetic trees associated acerola MDHAR and DHAR with other plant cytosolic MDHARs and DHARs. Expressions of the two genes correlated with their enzymatic activities and were differentially regulated during fruit ripening. Interestingly, MDHAR expression was only detected in overripe fruits, whereas the transcript level of DHAR was highest at the intermediate stage of fruit ripening. Under dark conditions, there was a sharp and significant decline in the total and reduced ascorbate contents, accompanied by a decrease in the level of transcripts and enzyme activities of the two genes in acerola leaves. MDHAR and DHAR transcripts and enzyme activities were significantly up-regulated in the leaves of acerola under cold and salt stress conditions, indicating that expression of both genes are transcriptionally regulated under these stresses.

Inhibition of Schistosoma mansoni thioredoxin-glutathione reductase by auranofin: structural and kinetic aspects.

Schistosomiasis is a parasitic disease affecting over 200 million people currently treated with one drug, praziquantel. A possible drug target is the seleno-protein thioredoxin-glutathione reductase (TGR), a key enzyme in the pathway of the parasite for detoxification of reactive oxygen species. The enzyme is a unique fusion of a glutaredoxin domain with a thioredoxin reductase domain, which contains a selenocysteine (Sec) as the penultimate amino acid. Auranofin (AF), a gold-containing compound already in clinical use as an anti-arthritic drug, has been shown to inhibit TGR and to substantially reduce worm burden in mice. Using x-ray crystallography we solved (at 2.5 A resolution) the structure of wild type TGR incubated with AF. The electron density maps show that the actual inhibitor is gold, released from AF. Gold is bound at three different sites not directly involving the C-terminal Sec residue; however, because the C terminus in the electron density maps is disordered, we cannot exclude the possibility that gold may also bind to Sec. To investigate the possible role of Sec in the inactivation kinetics, we tested the effect of AF on a model enzyme of the same superfamily, i.e. the naturally Sec-lacking glutathione reductase, and on truncated TGR. We demonstrate that the role of selenium in the onset of inhibition by AF is catalytic and can be mimicked by an external source of selenium (benzeneselenol). Therefore, we propose that Sec mediates the transfer of gold from its ligands in AF to the redox-active Cys couples of TGR.