Thioredoxin reductase is inhibited by the carbamoylating activity of the anticancer sulfonylhydrazine drug laromustine

The thioredoxin system facilitates proliferative processes in cells and is upregulated in many cancers. The activities of both thioredoxin (Trx) and its reductase (TrxR) are mediated by oxidation/reduction reactions among cysteine residues. A common target in preclinical anticancer research, TrxR is reported here to be significantly inhibited by the anticancer agent laromustine. This agent, which has been in clinical trials for acute myelogenous leukemia and glioblastoma multiforme, is understood to be cytotoxic principally via interstrand DNA crosslinking that originates from a 2-chloroethylating species generated upon activation in situ. The spontaneous decomposition of laromustine also yields methyl isocyanate, which readily carbamoylates thiols and primary amines. Purified rat liver TrxR was inhibited by laromustine with a clinically relevant IC₅₀ value of 4.65 μM. A derivative of laromustine that lacks carbamoylating activity did not appreciably inhibit TrxR while another derivative, lacking only the 2-chloroethylating activity, retained its inhibitory potency. Furthermore, in assays measuring TrxR activity in murine cell lysates, a similar pattern of inhibition among these compounds was observed. These data contrast with previous studies demonstrating that glutathione reductase, another enzyme that relies on cysteine-mediated redox chemistry, was not inhibited by methylcarbamoylating agents when measured in cell lysates. Mass spectrometry of laromustine-treated enzyme revealed significant carbamoylation of TrxR, albeit not on known catalytically active residues. However, there was no evidence of 2-chloroethylation anywhere on the protein. The inhibition of TrxR is likely to contribute to the cytotoxic, anticancer mechanism of action for laromustine.     

Inhibition of Both Thioredoxin Reductase and Glutathione Reductase may Contribute to the Anticancer Mechanism of TH-302

Selenium-containing thioredoxin reductase (TrxR) is an important target of cancer therapy. Many useful anticancer agents including bis-alkylating agents, cisplatin, and arsenic trioxide are known to interact with the selenocysteine dipeptide in the carboxy terminal region of thioredoxin reductase and inactivate its ability to reduce thioredoxin. Some investigators have postulated that the inactivation of TrxR may add to the cytotoxic potential of these anticancer agents. TH-302 is a newly developed antineoplastic drug which represents a potential new class of tumor selective hypoxia-activated prodrugs (HAPs). TH-302 is an inactive prodrug created by the covalent conjugation of 2-nitroimidazole as an oxygen sensor to bromo-isophosphoramide (Br-IPM). In the presence of severe hypoxia and near anoxia, the two imidazole sensor moiety undergoes reduction and the Br-IPM is released in situ. Bromo-IPM is a more potential analog of Chloro-IPM, the active alkylating moiety that is derived by activation of ifosfamide (IFO). We previously demonstrated that IFO could inhibit tumor TrxR activity and chloro-IPM is known to bind covalently to the seleno-cysteine dipeptide in thioredoxin reductase. The present study assessed the ability of TH-302 to activate in the tumors of mice-bearing hepatoma 22 (H22) and inactivate the tumor TrxR. In mice-bearing hepatoma 22 (H22) solid tumors, intraperitoneal (i.p.) injection with TH-302 at the dose of 200 mg/kg administered twice, a regimen which was well tolerated by the mice, significantly inhibited tumor growth. Also in this mice model, i.p. TH-302 at the dose of 300 mg/kg, which would be the maximum single i.p. administration dose tolerated by mice, and which induced only 2% body weight loss, significantly inhibited both TrxR and glutathione reductase (GR) activities by 46% (P?<?0.001) and 60% (P?<?0.001) as compared with the controls, respectively, at 3 h after the injection. Since TrxR is a key player in thioredoxin system and GR is the major reductase for the reduction of oxidized glutathione in glutathione system, the present results imply the anticancer effect of TH-302 is associated concurrently with modulation of TrxR and GR. These findings suggest that the anticancer activity of TH-302 in this model system may associate with both DNA alkylation and the modulation of TrxR and GR. In addition, they suggest that, by inhibition of these two critical reductases, with less glutathione available to intercept the reactive intermediates involved in DNA alkylation, the antitumor effects of the chemotherapy would be enhanced.     

Ethaselen: a potent mammalian thioredoxin reductase 1 inhibitor and novel organoselenium anticancer agent

Mammalian thioredoxin reductase 1 (TrxR1) is considered to be an important anticancer drug target and to be involved in both carcinogenesis and cancer progression. Here, we report that ethaselen, a novel organoselenium compound with anticancer activity, specifically binds to the unique selenocysteine-cysteine redox pair in the C-terminal active site of mammalian TrxR1. Ethaselen was found to be a potent inhibitor rather than an efficient substrate of mammalian TrxR1. It effectively inhibits wild-type mammalian TrxR1 at submicromolar concentrations with an initial mixed-type inhibition pattern. By using recombinant human TrxR1 variants and human glutathione reductase, we prove that ethaselen specifically targets the C-terminal but not the N-terminal active site of mammalian TrxR1. In A549 human lung cancer cells, ethaselen significantly suppresses cell viability in parallel with direct inhibition of TrxR1 activity. It does not, however, alter either the disulfide-reduction capability of thioredoxin or the activity of glutathione reductase. As a downstream effect of TrxR1 inactivation, ethaselen causes a dose-dependent thioredoxin oxidation and enhances the levels of cellular reactive oxygen species in A549 cells. Thus, we propose ethaselen as the first selenium-containing inhibitor of mammalian TrxR1 and provide evidence that selenium compounds can act as anticancer agents based on mammalian TrxR1 inhibition.     

Interactions of nitroaromatic compounds with the mammalian selenoprotein thioredoxin reductase and the relation to induction of apoptosis in human cancer cells

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.     

Interactions of quinones with thioredoxin reductase: a challenge to the antioxidant role of the mammalian selenoprotein

Mammalian thioredoxin reductases (TrxR) are important selenium-dependent antioxidant enzymes. Quinones, a wide group of natural substances, human drugs, and environmental pollutants may act either as TrxR substrates or inhibitors. Here we systematically analyzed the interactions of TrxR with different classes of quinone compounds. We found that TrxR catalyzed mixed single- and two-electron reduction of quinones, involving both the selenium-containing motif and a second redox center, presumably FAD. Compared with other related pyridine nucleotide-disulfide oxidoreductases such as glutathione reductase or trypanothione reductase, the k(ca)(t)/K(m) value for quinone reduction by TrxR was about 1 order of magnitude higher, and it was not directly related to the one-electron reduction potential of the quinones. A number of quinones were reduced about as efficiently as the natural substrate thioredoxin. We show that TrxR mainly cycles between the four-electron reduced (EH(4)) and two-electron reduced (EH(2)) states in quinone reduction. The redox potential of the EH(2)/EH(4) couple of TrxR calculated according to the Haldane relationship with NADPH/NADP(+) was -0.294 V at pH 7.0. Antitumor aziridinylbenzoquinones and daunorubicin were poor substrates and almost inactive as reversible TrxR inhibitors. However, phenanthrene quinone was a potent inhibitor (approximate K(i) = 6.3 +/- 1 microm). As with other flavoenzymes, quinones could confer superoxide-producing NADPH oxidase activity to mammalian TrxR. A unique feature of this enzyme was, however, the fact that upon selenocysteine-targeted covalent modification, which inactivates its normal activity, reduction of some quinones was not affected, whereas that of others was severely impaired. We conclude that interactions with TrxR may play a considerable role in the complex mechanisms underlying the diverse biological effects of quinones.     

Inhibition of thioredoxin reductase 1 by porphyrins and other small molecules identified by a high-throughput screening assay

The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K_i = 2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k_inact) of (0.73 ± 0.07) × 10^? 3 μM^? 1 min^? 1. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.     

Effect of thioredoxin reductase 1 on glucocorticoid receptor activity in human outer root sheath cells

Alopecia areata (AA) is a common disease of patchy hair loss on the scalp that can progress to cover the entire scalp and eventually the entire body. Intralesional injection of corticosteroids is the first-line therapy for adult patients, however some patients do not respond to glucocorticoid treatment effectively. To delineate the molecular mechanism underlying glucocorticoid insensitivity, we examined the expression of glucocorticoid receptor (GR) and thioredoxin reductase 1 (TrxR1). In some case of glucocorticoid-resistant AA patients, the expression of TrxR1 was decreased in outer root sheath (ORS). We then investigated the effect of TrxR1 on GR activity using recombinant adenoviruses. Overexpression of TrxR1 markedly increased GR activity in ORS cells cultured in vitro. In addition, TrxR1 protected GR activity against H(2)O(2). Finally, TrxR1-enhanced GR activity was significantly inhibited by the overexpression of dominant negative form of Trx (Trx(C32S/C35S)). These results suggest that decreased TrxR1 may be one putative cause for glucocorticoid resistance in AA, through the impact on intracellular redox system.     

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

Shikonin targets cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in human promyelocytic leukemia HL-60 cells

Shikonin, a major active component of the Chinese herbal plant Lithospermum erythrorhizon, has been applied for centuries in traditional Chinese medicine. Although shikonin demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of shikonin have not been fully defined. We report here that shikonin may interact with the cytosolic thioredoxin reductase (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme with a C-terminal -Gly-Cys-Sec-Gly active site, to induce reactive oxygen species (ROS)-mediated apoptosis in human promyelocytic leukemia HL-60 cells. Shikonin primarily targets the Sec residue in TrxR1 to inhibit its physiological function, but further shifts the enzyme to an NADPH oxidase to generate superoxide anions, which leads to accumulation of ROS and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 attenuates the cytotoxicity of shikonin, whereas knockdown of TrxR1 sensitizes cells to shikonin treatment. Targeting TrxR1 with shikonin thus discloses a previously unrecognized mechanism underlying the biological activity of shikonin and provides an in-depth insight into the action of shikonin in the treatment of cancer.     

Inhibition of thioredoxin reductase by mansonone F analogues: Implications for anticancer activity

Mammalian thioredoxin reductase (TrxR), a ubiquitous selenocysteine containing oxidoreductase, catalyzes the NADPH-dependent reduction of oxidized thioredoxin (Trx). TrxR has been suggested as a potential target for anticancer drugs development for its overexpression in human tumors and its diverse functions in intracellular redox control, cell growth and apoptosis. Mansonone F (MF) compounds have been shown to exhibit antiproliferative effects, but their complex mechanisms are unknown. In the present study, we have investigated the effects of some synthesized MF analogues on TrxR and HeLa cells. The studies of the mode of inhibition and the interactions of IG3, one of the most potent MF analogues, with TrxR showed MF compounds could be partly reduced by the C-terminal selenolthiol active site, and possibly by the N-terminal dithiol motif and/or FAD domain of TrxR simultaneously, accompanied by redox cycling with the generation of superoxide anion radicals. In addition, MF analogues exhibited the potential to inhibit the growth of HeLa cells and reduce TrxR activity in cell lysates. The cell cycle was arrested in G2/M phase and apoptosis was induced in a dose-dependent manner. Furthermore, our results showed that IG3-treated HeLa cells induced the change of intracellular ROS. Taken together, the reported results here suggest that inhibition of TrxR by MF analogues provides a possible complex mechanism for explaining the anticancer activity of MF compounds.     

Gambogic acid induces apoptosis in hepatocellular carcinoma SMMC-7721 cells by targeting cytosolic thioredoxin reductase

The thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus and TrxR2 in mitochondria, are essential mammalian selenocysteine (Sec)-containing flavoenzymes with a unique C-terminal -Gly-Cys-Sec-Gly active site. TrxRs are often overexpressed in a number of human tumors, and the reduction of their expression in malignant cells reverses tumor growth, making the enzymes attractive targets for anticancer drug development. Gambogic acid (GA), a natural product that has been used in traditional Chinese medicine for centuries, demonstrates potent anticancer activity in numerous types of human cancer cells and has entered phase II clinical trials. We discovered that GA may interact with TrxR1 to elicit oxidative stress and eventually induce apoptosis in human hepatocellular carcinoma SMMC-7721 cells. GA primarily targets the Sec residue in the antioxidant enzyme TrxR1 to inhibit its Trx-reduction activity, leading to accumulation of reactive oxygen species and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 in cells attenuates the cytotoxicity of GA, whereas knockdown of TrxR1 sensitizes cells to GA. Targeting of TrxR1 by GA thus discloses a previously unrecognized mechanism underlying the biological action of GA and provides useful information for further development of GA as a potential agent in the treatment of cancer.     

硒蛋白TrxR1介导甲萘醌还原：催化特性和抑制作用

硫氧还蛋白还原酶 (Thioredoxin reductase,TrxR) 是一类重要的抗氧化硒蛋白,参与调控肿瘤发生发展。研究表明,萘醌类分子可以靶向抑制TrxR1活性并经由TrxR1介导产生活性氧,导致细胞氧化还原失衡,使其成为潜在的肿瘤化疗药物。本文旨在通过生物化学及质谱分析,探究硒蛋白TrxR1与萘醌化合物甲萘醌的相互作用,进一步揭示TrxR1催化萘醌分子还原的机理和萘醌分子抑制TrxR1活性的机制。通过对TrxR1催化残基的定点突变和突变体的重组表达,我们测定TrxR1突变体介导甲萘醌还原稳态动力学参数,并分析甲萘醌对TrxR1活性抑制,最后通过质谱分析鉴定甲萘醌与TrxR1相互作用。结果表明,硒蛋白TrxR1的Sec498催化甲萘醌还原,但是U498C突变使甲萘醌还原更加高效,表明了甲萘醌还原主要呈现非硒依赖性。突变实验发现C端Cys498发挥主要催化还原作用,而N端Cys64对甲萘醌还原的影响稍强于Cys59。LC-MS结果发现TrxR1存在1分子甲萘醌加合物,推测其不可逆修饰硒蛋白C末端高反应活性的硒代半胱氨酸。本研究揭示了TrxR1可以非硒依赖方式催化甲萘醌还原,同时其活性会受到甲萘醌的不可逆抑制,为靶向TrxR1的萘醌类抗癌药物研发提供有益参考。     

Revealing PACMA 31 as a new chemical type TrxR inhibitor to promote cancer cell apoptosis

Thioredoxin reductase (TrxR) is a pivotal regulator of redox homeostasis, while dysregulation of redox homeostasis is a hallmark for cancer cells. Thus, there is considerable potential to inhibit the aberrantly upregulated TrxR in cancer cells to discover selective cancer therapeutic agents. Nevertheless, the structural types of TrxR inhibitors presented currently are still relatively limited. We herein report that PACMA 31, previously reported to inhibit protein disulfide isomerase (PDI), is a potent TrxR inhibitor. PACMA 31 possesses a pharmacophore scaffold that is structurally different from the announced TrxR inhibitors and exhibits effective cytotoxicity against cervical cancer cells. Our results reveal that PACMA 31 selectively inhibits TrxR over the related glutathione reductase (GR) and in the presence of reduced glutathione (GSH). Further studies with mutant enzyme and molecular docking suggest that the propynamide fragment of PACMA 31 interacts covalently with the selenocysteine residue of TrxR. Moreover, PACMA 31 effectively and selectively curbs TrxR activity in cells and further stimulates the production of reactive oxygen species (ROS) at low micromolar concentrations, which in turn triggers the accumulation of oxidized thioredoxin (Trx) and GSSG in cells. Follow-up studies demonstrate that PACMA 31 targets TrxR in cells to induce oxidative stress-mediated cancer cell apoptosis. Our results provide a new structural type of TrxR inhibitor that may serve as a useful probe for investigating the biology of TrxR-implicated pathways, and uncover a new target of PACMA 31 that contributes to it becoming a candidate for cancer treatment.     

Thioredoxin glutathione reductase as a novel drug target: evidence from Schistosoma japonicum

Background

Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR) enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia.

Methods and Findings

After cloning the S. japonicum TGR (SjTGR) gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR), glutathione reductase (GR) and glutaredoxin (Grx) activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice.

Conclusions

Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.     

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.     

Novel alkylpolyaminoguanidines and alkylpolyaminobiguanides with potent antitrypanosomal activity

A series of polyaminoguanidines and polyaminobiguanides were synthesized and evaluated as potential antitrypanosomal agents. These analogues inhibit trypanothione reductase (TR) with IC50 values as low as 0.95 microM, but do not inhibit the closely related human enzyme glutathione reductase (GR). The most effective analogues, 7a, 7b and 8d, inhibited parasitic growth in vitro with IC50 values of 0.18, 0.09 and 0.18 microM, respectively. These agents represent a promising new class of potential antitrypanosomal agents.     

Purification, characterization, and immunolocalization of a thioredoxin reductase from adult Fasciola hepatica

Thioredoxin reductase (TrxR), an enzyme belonging to the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases, was isolated from the deoxycholate-soluble extract of the common liver fluke, Fasciola hepatica. Purification to homogeneity of the 60-kDa enzyme from the adult worm was achieved by a combination of ammonium sulfate fractionation, anion exchange, and affinity chromatography on 2',5'-adenosine diphosphate-Sepharose. Using the 5,5'-dithiobis(2-nitrobenzoic acid) assay, the purified TrxR showed a specific activity of 7,117 U min(-1) mg(-1). The enzyme activity was completely inhibited by the presence of the gold compound aurothioglucose (IC50 = 120 nm), indicating that F. hepatica TrxR is a selenoenzyme. Also, the enzyme was capable of reducing disulfide bonds in insulin and was activated by the presence of the reduced form of flavin adenine dinucleotide, properties shared with mammalian TrxRs. Furthermore, the isolated enzyme showed very low glutaredoxin (Grx) activity (0.47 U mg(-1)), but no glutathione reductase activity was detected. Affinity-purified IgGs (20 microg ml(-1)) from the antisera produced against the purified TrxR inhibited its activity about 80% with respect to the control. The enzyme was immunolocalized in cells located within the parenchyma and in the testes, but it was not found in the tegument of the adult fluke.     

Discovery and biochemical characterization of Plasmodium thioredoxin reductase inhibitors from an antimalarial set

Plasmodium falciparum is the most prevalent and deadly species of the human malaria parasites, and thioredoxin reductase (TrxR) is an enzyme involved in the redox response to oxidative stress. Essential for P. falciparum survival, the enzyme has been highlighted as a promising target for novel antimalarial drugs. Here we report the discovery and characterization of seven molecules from an antimalarial set of 13533 compounds through single-target TrxR biochemical screens. We have produced high-purity, full-length, recombinant native enzyme from four Plasmodium species, and thioredoxin substrates from P. falciparum and Rattus norvegicus. The enzymes were screened using a unique, high-throughput, in vitro native substrate assay, and we have observed selectivity between the Plasmodium species and the mammalian form of the enzyme. This has indicated differences in their biomolecular profiles and has provided valuable insights into the biochemical mechanisms of action of compounds with proven antimalarial activity.     

The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress

The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.