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Gliotoxins are epipolythiodioxopiperazine toxins produced by the filamentous fungi, which show great potential in the treatment of liver and lung cancer because of its cytotoxicity. In this study, three novel genes related to gliotoxin biosynthesis, gliT, gliM and gliK encoding thioredoxin reductase, O-methyltransferase and gamma-glutamyl cyclotransferase, respectively, from the deep-sea-derived fungus Geosmithia pallida were cloned from G. pallida and expressed in Escherichia coli. The recombinant GliT, GliM and GliK proteins were expressed and purified by Ni affinity column, which was demonstrated by SDS-PAGE and Western blot analysis. The inclusion bodies of GliT were renatured and the corresponding enzymatic properties of the two enzymes were further investigated. Using DTNB as a substrate, GliT showed the highest enzymatic activity of 11041 mU/L at pH 7.0, and the optimal reaction temperature was 40 °C. Using EGCG as a substrate, GliM showed the highest enzymatic activity of 239.19 mU/mg at pH 7.0, the optimum temperature was 35 °C. GliK from G. pallida was firstly reported to show bi-function of glutymal cyclotransferase and acetyltransfearse actvity with highest enzymatic activity of 615.5 U/mg in this study. The results suggested the important enzymatic function of GliT, GliM and GliK in the gliotoxin biosynthesis in G. pallida, which would lay a foundation for the mechanism elucidation of the gliotoxin biosynthesis in G. pallida and the exploitation of novel gliotoxin derivaties.

Increasing evidence suggests the significant contribution of high levels of thioredoxin reductase (TrxR) in various stages of tumorigenesis and resistance to tumor chemotherapy. Thus, inhibition of TrxR with small molecules is an attractive strategy for cancer therapy. Eriocalyxin B (EriB), a naturally occurring diterpenoid extracted from Isodon eriocalyx, has reflected potential anticancer activities through numerous pathways. Here, we describe that EriB covalently modifies GSH and selectively inhibits TrxR activity by targeting the Sec residue of the enzyme. Pharmacological inhibition of TrxR by EriB results in elevated ROS levels, reduced total GSH and thiols content, which ultimately induced potent RKO cell apoptosis mediated by oxidative stress. Importantly, EriB indicates potent synthetic lethality with GSH inhibitors, BSO, in RKO cells. In summary, our results highlight that targeting TrxR by EriB explores a novel mechanism for the biological action of EriB. This opened up a new therapeutic indication for using EriB to combat cancers.

Chicken hepatocytes were cultured in vitro and 240 specific pathogen-free (SPF) white leghorns chickens (7 days old) were obtained. The hepatocytes and chickens were randomly allocated to one of six treatment groups: control group; chitosan (COS) group; sodium selenite (Na2SeO3) group; selenide chitosan (COS-Se) group; chitosan sulfate (LS-COS) group; and selenide chitosan sulfate (LS-COS-Se) group. Our results showed that LS-COS-Se increased (P

Thioredoxin which is induced by thioredoxin reductase causes the proliferation of cancerous cells and metastasis due to its effects on cell growth, besides its regulatory effects on the amount of reactive oxygen species. One of the procedures recently used in cancer treatment is thioredoxin reductase inhibition. Different types of bioactivities of NHC and metal-NHC complexes have been studied and anti-cancer is one of these activities. In addition to in-vitro anticancer activity, molecular docking methods are also one of the important methods used in drug design. This method achieves foresight about future studies and the mechanisms that are difficult to analyze experimentally. In this study, previously synthesized and characterized [1-(2-methyl-2-propenyl)-3-(4-methylbenzyl) benzimidazolium]+ (1a) and [1-(2-methyl-2-propenyl)-3-(4-isopropylbenzyl) benzimidazolium]+ (1b) molecules and their Ag(I)-NHC complexes (2a and 2b) were investigated using molecular docking method for thioredoxin reductase. In addition, the interaction of these molecules with DNA was evaluated. 2b has the best binding energy of -8.95 kcal/mol with the region that comprised Ile10, Phe254, Ala38, Val41 of thioredoxin reductase. Also, ligands interacted with Cyt11, Gua10, Cyt9, and Thy8 while complexes interacted with Ade5, Ade6, Thy7, and Thy8 part of DNA.

A series of (NHC)Au(I)Cl monocarbene complexes and their gold(III) analogues (NHC)Au(III)Cl3 were prepared and investigated as antibacterial agents and inhibitors of bacterial TrxR. The complexes showed stronger antibacterial effects against the Gram-positive MRSA and E. faecium strains than against several Gram-negative bacteria. All complexes were efficient inhibitors of bacterial thioredoxin reductase, indicating that inhibition of this enzyme might be involved in their mechanism of action. The efficacy of gold(I) and gold(III) analogues was comparable in most of the assays. The cytotoxicity of the gold NHC compounds against cancer and human cells was overall weaker than the activity against the Gram-positive bacteria, suggesting that their optimization as antibacterials warrants further investigation.

The selenoprotein thioredoxin reductase 1 (TrxR1; TXNRD1) participates in multiple cellular processes and is regarded as a cellular target in anti-tumor drug discovery and development. TrxR1 has been reported to reduce menadione to menadiol and to produce superoxide anion radicals. However, the details of TrxR1-mediated menadione reduction have rarely been studied. In this study, we found that wild-type TrxR1 could reduce menadione in a less efficient way, but the U498C mutant variant supported high-efficiency menadione reduction in a Sec-independent manner. Meanwhile, the site-directed mutagenesis results showed that Cys64 mutant increased the Km values and decreased the catalytic efficiency, which was associated with a charge-transfer complex between FAD-Cys64. Mass spectrometry (MS) revealed that in NADPH pre-reduced TrxR1 but not oxidized TrxR1, the highly active Sec498 of wild-type TrxR1 was arylated by menadione and strongly impaired the DTNB reducing activity in a dose-dependent manner. TrxR1 reduced menadione more efficiently than glutathione reductase (GR), and interestingly menadione did not inhibit the GSSG reducing activity of GR. In summary, our results demonstrate that TrxR1 catalyzes the reduction of menadione in a Sec-independent manner, which highly depend on Cys498 instead of N-terminal redox motif, and the Sec498 of TrxR1 is the primary target of menadione. The interaction between menadione and TrxR1 revealed in this study may provide a valuable reference for the development of anticancer drugs targeting selenoprotein TrxR1.

A series of novel piperine derivatives were synthesized with high yield and were evaluated for its antifilarial potential against the bovine filarial parasite Setaria cervi. Among 21 (3a-3u) compounds screened, three of them (3k, 3l, 3s) showed significant potential against all the developmental stages (oocytes, microfilariae and adult) of the filarial worm in time and dose dependent manner. 3l showed the highest efficacy among the selected three compounds. These three compounds were further evaluated for both in vitro and in vivo toxicity analyses which further fortified the benign nature of the selected compounds. The antifilarial activities they exhibited were clearly fuelled through disparity of the internal redox homeostasis as evidenced from the alterations in the enzymatic and non-enzymatic antioxidants level which ultimately shifted towards activation of pro-apoptotic signaling cascade eventually leading to the death of the parasites. The ability of the compound 3l to bind thioredoxin reductase and CED-3 protein are the key findings of this study. The present study supported with several biological experiments is therefore a maiden report on the antifilarial effectiveness of these novel piperine derivatives.

In mature leaves, cell-to-cell transport via plasmodesmata between mesophyll cells links the production of assimilates by photosynthesis with their export to sink organs. This study addresses the question of how signals derived from chloroplasts and photosynthesis influence plasmodesmata permeability. Cell-to-cell transport was analyzed in leaves of the Arabidopsis chlorophyll b-less ch1-3 mutant, the same mutant complemented with a cyanobacterial CAO gene (PhCAO) overaccumulating chlorophyll b, the trxm3 mutant lacking plastidial thioredoxin m3, and the ntrc mutant lacking functional NADPH:thioredoxin reductase C. The regulation of plasmodesmata permeability in these lines could not be traced back to the reduction state of the thioredoxin system or the types and levels of reactive oxygen species produced in chloroplasts; however, it could be related to chloroplast ATP and NADPH production. The results suggest that light enables plasmodesmata closure via an increase in the ATP and NADPH levels produced in photosynthesis, providing a control mechanism for assimilate export based on the rate of photosynthate production in the Calvin–Benson cycle. The level of chlorophyll b influences plasmodesmata permeability via as-yet-unidentified signals. The data also suggest a role of thioredoxin m3 in the regulation of cyclic electron flow around photosystem I.

A series of gold(I) complexes with the general formula [Au(L2)(L′)] (L2=4‐phenyl‐N‐(prop‐2‐yn‐1‐yl)quinazoline‐2‐carboxamide, L′=PPh3 (triphenylphosphine), 1; TPA (1,3,5‐triaza‐7‐phosphaadamantane), 2, and Me2‐imy (1,3‐dimethylimidazol‐2‐ylidene), 3) were synthesized and fully characterized by spectroscopic methods. The alkynyl ligand L2 belongs to the quinazoline carboxamide class of ligands that are known to bind to the translocator protein (TSPO) at the outer mitochondrial membrane. 1 and 2 exert cytotoxic effects in bladder cancer cells with IC50 values in the low micromolar range. Further mechanistic analysis indicated that the two complexes both act by inducing reactive oxygen species and caspase‐mediated apoptosis. The complexes inhibit thioredoxin reductase, an established target of anticancer gold(I) complexes. Docking studies confirmed that after ligand exchange the free ligand L2 can interact with the TSPO binding site., Alkynyl gold(I) complexes with phosphane and NHC co‐ligands have been prepared that are potent inhibitors of thioredoxin reductase. The complexes containing phosphane co‐ligands are also potent cytotoxins that reduce cell viability by inducing reactive oxygen species and caspase‐dependent apoptotic cell death.

α-Diazo homophotalimides were reacted with various propiolic acids on Rh 2 (esp) 2 catalysis. The resulting propiolate esters were transformed into novel, heterocyclic Δ α,β -spirobutenolides in good to excellent product yields. The approach represents a fundamentally novel entry into natural-like Δ α,β -spirobutenolides present in many biologically active natural products as well as fully synthetic compounds endowed with diverse biological activities. The Δ α,β -spirobutenolides thus obtained were shown to inhibit thioredoxin reductase, a selenocysteine enzyme target for cancer. Moreover, for the best compound in the series (TrxR IC 50 1.49 ± 0.08 μM), it was shown, using MALDI-TOF mass-spectrometry, that it selectively binds selenocysteine in the presence of a 10-fold excess of cysteine. This validates the new compound as a promising lead for anticancer therapy development.

Three [1,3‐diethyl‐4‐(p‐methoxyphenyl)‐5‐(3,4,5‐trimethoxyphenyl)imidazol‐2‐ylidene](L)gold(I) complexes, 4 a (L=Cl), 5 a (L=PPh3), and 6 a (L=same N‐heterocyclic carbene (NHC)), and their fluorescent [4‐(anthracen‐9‐yl)‐1,3‐diethyl‐5‐phenylimidazol‐2‐ylidene](L)gold(I) analogues, 4 b, 5 b, and 6 b, respectively, were studied for their localisation and effects in cancer cells. Despite their identical NHC ligands, the last three accumulated in different compartments of melanoma cells, namely, the nucleus (4 b), mitochondria (5 b), or lysosomes (6 b). Ligand L was also more decisive for the site of accumulation than the NHC ligand because the couples 4 a/4 b, 5 a/5 b, and 6 a/6 b, carrying different NHC ligands, afforded similar results in cytotoxicity tests, and tests on targets typically found at their sites of accumulation, such as DNA in nuclei, reactive oxygen species and thioredoxin reductase in mitochondria, and lysosomal membranes. Regardless of the site of accumulation, cancer cell apoptosis was eventually induced. The concept of guiding a bioactive complex fragment to a particular subcellular target by secondary ligand L could reduce unwanted side effects., Striking gold: Through the choice of their secondary ligand L, a series of [4,5‐diarylimidazol‐2‐ylidene](L)gold(I) complexes targeted different cancer cell organelles (nuclei, mitochondria or lysosomes) where they interfered beneficially with molecular targets, such as DNA, thioredoxin reductase, or lysosomal membranes, to elicit p53‐independent apoptosis. Such guided metallodrugs will help to minimise unwanted side effects by addressing limited arrays of targets.

The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative parasite and can cause serious infections in humans. The annotated genome of A. castellanii (strain Neff) suggests the presence of two different thioredoxin reductases (TrxR), of which one is of the small bacterial type and the other of the large vertebrate type. This combination is highly unusual. Similar to vertebrate TrxRases, the gene coding for the large TrxR in A. castellanii contains a UGA stop codon at the C-terminal active site, suggesting the presence of selenocysteine. We characterized the thioredoxin system in A. castellanii in conjunction with glutathione reductase (GR), to obtain a more complete understanding of the redox system in A. castellanii and the roles of its components in the response to oxidative stress. Both TrxRases localize to the cytoplasm, whereas GR localizes to the cytoplasm and the large organelle fraction. We could only identify one thioredoxin (Trx-1) to be indeed reduced by one of the TrxRases, i.e., by the small TrxR. This thioredoxin, in turn, could reduce one of the two peroxiredoxins tested and also methionine sulfoxide reductase A (MsrA). Upon exposure to hydrogen peroxide and diamide, only the small TrxR was upregulated in expression at the mRNA and protein levels, but not the large TrxR. Our results show that the small TrxR is involved in the A. castellanii’s response to oxidative stress. The role of the large TrxR, however, remains elusive.

In the aim to estimate the protective role of calcium (Ca) and ethylene glycol tetraacetic acid (EGTA) against cadmium (Cd)-induced damage, chickpea (Cicer arietinum L.) seeds were exposed to 200 μM Cd stress for 6 days or 3 days then subjected to co-treatment of the metal with either 100 mM CaCl2 or 100 μM EGTA for 3 additional days. The addition of Ca and EGTA improved seedling growth. This protecting effect was correlated to the alleviation of the metal-induced oxidative stress, exemplified by the reduction of hydrogen peroxide (H2O2) contents. Besides, Ca and EGTA stimulated thioredoxin (Trx) and thioredoxin reductase (NTR) activities (2.75- and 1.75-fold increase when compared to Cd-stressed, respectively) protecting, thereby, protein -SH groups from the Cd-mediated oxidation, and modulated ferredoxin (Fdx) activity to a control level. Moreover, Ca and EGTA reinstated the glutathione redox steady state, mainly via preserving a high level of glutathione reduced form (GSH). This effect coincided with the maintaining of the Cd-stimulated glutathione reductase (GR) activity and the decline of glutathione peroxidase (GPX, 43% lower than Cd-stressed shoots) activity. Ca and EGTA counteracted the inhibitory effect of Cd on the activity and gene expression of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) isoenzyme and modulated the activities of catalase (CAT) and ascorbate peroxidase (APX). Overall, our results provided evidence that Ca and EGTA supplement could be a promising approach in the remediation of Cd-contaminated environment.

Thioredoxin reductase (TrxR), which plays a key role in cellular regulations including cell growth, redox signaling pathways and differentiation, belongs to the selenocysteine-containing protein family. Numerous human diseases are associated with thioredoxin reductase/selenocysteine deficiency or overdose. Thus, there is an urgent need to develop new tools for thioredoxin reductase detection. Small molecule fluorescent probes, which have been studied for decades, are powerful chemical tools for biomolecule detection and diagnosis within a bio-system. However, there still remains a challenge in finding a specific target site when designing a new probe. In this work, we presented a Tag-Sec fluorescent probe that could detect both thioredoxin reductase and selenocysteine with high sensitivity, selectivity, broad pH tolerance and good water solubility. We hope that the concept described in this work could serve as a useful guide to design probes for detecting Sec-containing proteins and clinical diagnosis.

Glutaredoxins (Grxs) and thioredoxins (Trxs) play a critical role in resistance to oxidative conditions. However, physiological and biochemical roles of Mycoredoxin 3 (Mrx3) that shared a high amino acid sequence similarity to Grxs remain unknown in Corynebacterium glutamicum. Here we showed that mrx3 deletion strains of C. glutamicum was involved in the protection against oxidative stress. Recombinant Mrx3 not only catalytically reduced the disulfide bonds in ribonucleotide reductase (RNR), insulin and 5,5'-dithiobis-(2-nitro-benzoicacid) (DTNB), but also reduced the mixed disulphides between mycothiol (MSH) and substrate, which was exclusively linked to the thioredoxin reductase (TrxR) electron transfer pathway by a dithiol mechanism. Site-directed mutagenesis confirmed that the conserved Cys17 and Cys20 in Mrx3 were necessary to maintain its activity. The mrx3 deletion mutant showed decreased resistance to various stress, and these sensitive phenotypes were almost fully restored in the complementary strain. The physiological roles of Mrx3 in resistance to various stress were further supported by the induced expression of mrx3 under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. Thus, we presented the first evidence that Mrx3 protected against various oxidative stresses by acting as a disulfide oxidoreductase behaving like Trx.

A series of bis[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes (2a-f) containing methyl, fluoro or methoxy substituents at various positions in the 4-aryl ring was synthesized and evaluated for their anti-cancer properties in A2780 (wild-type and Cisplatin-resistant) ovarian carcinoma as well as LAMA 84 (imatinib-sensitive and -resistant) and HL-60 leukemia cell lines. The bis-NHC gold(i) complexes were more active compared to their related mono-NHC gold(i) analogues and reduced proliferation and metabolic activity in a low micromolar range. With the exception of 2d (3-F), the compounds displayed higher potency than the established drugs Auranofin and Cisplatin. The lack of effects against non-cancerous lung fibroblast SV-80 cells indicated a high selectivity towards tumor cells. All tested complexes generated reactive oxygen species in A2780cis cells; however, the induction of apoptosis was very low. Furthermore, thioredoxin reductase is not the main target of these complexes, because its inhibition pattern did not correlate with their biological activity.

The thiol-based glutathione reductase (GR) and thioredoxin reductase (TrxR) are the major antioxidant enzymes present in various organisms that maintain the internal redox homeostasis. The thioredoxin system has attracted the attention of researchers from diverse investigation fields of biological sciences. Apart from redox regulation, this system is thought to be the major regulator of various biological processes including transcription, apoptosis, etc. Identification and physicobiochemical characterization of the reductase enzyme i.e. Thioredoxin reductase (TrxR) revealed the potency of it to become a promising target. Novel therapeutic interventions by selective targeting of TrxR in parasitic organisms as well as in the cancer cells have now become a usual treatment approach. However, different isoforms and their variation in the penultimate amino acid (Selenocysteine or cysteine) present in the catalytic site of the enzyme have made this enzyme to respond differently towards various drugs and synthetic and/or natural compounds. Therefore, the present article seeks to highlight the importance and the detailed molecular mechanism, functional perspective underlying the TrxR inhibition in various parasitic protozoans, helminthes as well as in cancer cells for devising suitable anti-TrxR candidates.

Significance: Selenenyl sulfides (RSeSRs) and thioseleninic acids (RSeSHs) are the monoselenium (Se) analogs of disulfides and persulfides that contain Se-S bonds. These bonds are found in several antioxidant-regenerating enzymes as derivatives of selenocysteine, making them an important player in redox biology as it pertains to sulfur redox regulation. Recent Advances: Mechanistic studies of redox-regulating selenoenzymes such as thioredoxin reductase and glutathione peroxidase suggest crucial Se-S bonds in the active sites. Peptide models and small-molecule mimics of these active sites have been prepared to study their fundamental chemistry. These advances help pave the road to better understand the functions of the Se-S bond in the body. Critical Issues: The Se-S bond is unstable at atmospheric temperatures and pressures. Therefore, studying their properties proposes a major challenge. Currently, there are no trapping reagents specific to RSeSRs or RSeSHs, making their presence, identity, and fates in biological environments difficult to track. Future Directions: Further understanding of the fundamental chemistry/biochemistry of RSeSRs and RSeSHs is needed to understand what their intracellular targets are and to what extent they impact signaling. Besides antioxidant regeneration and peroxide radical reduction, the roles of RSeSR and RSeSHs in other systems need to be further explored.

The use of readily available 1-aryl-3-arylidenepyrrolidine-2,5-diones in high yielding direct diazo-transfer reactions and subsequent involvement of the resulting diazo compounds in RhII-catalyzed O–H, S–H, and N–H insertion reactions delivered 4-substituted 1-aryl-3-arylidenepyrrolidine-2,5-diones of defined regiochemistry and geometrical configuration. These products are intended to be studied as Michael acceptors capable of inhibiting thioredoxin reductase, a promising cancer target.

Nitric oxide (NO) and hydrogen sulfide (H2S) are two key molecules in plant cells that participate, directly or indirectly, as regulators of protein functions through derived post-translational modifications, mainly tyrosine nitration, S-nitrosation, and persulfidation. These post-translational modifications allow the participation of both NO and H2S signal molecules in a wide range of cellular processes either physiological or under stressful circumstances. NADPH participates in cellular redox status and it is a key cofactor necessary for cell growth and development. It is involved in significant biochemical routes such as fatty acid, carotenoid and proline biosynthesis, and the shikimate pathway, as well as in cellular detoxification processes including the ascorbate–glutathione cycle, the NADPH-dependent thioredoxin reductase (NTR), or the superoxide-generating NADPH oxidase. Plant cells have diverse mechanisms to generate NADPH by a group of NADP-dependent oxidoreductases including ferredoxin-NADP reductase (FNR), NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), NADP-dependent malic enzyme (NADP-ME), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and both enzymes of the oxidative pentose phosphate pathway, designated as glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). These enzymes consist of different isozymes located in diverse subcellular compartments (chloroplasts, cytosol, mitochondria, and peroxisomes) which contribute to the NAPDH cellular pool. We provide a comprehensive overview of how post-translational modifications promoted by NO (tyrosine nitration and S-nitrosation), H2S (persulfidation), and glutathione (glutathionylation), affect the cellular redox status through regulation of the NADP-dependent dehydrogenases.

The thioredoxin system plays an important role in cancer cells. Inhibiting thioredoxin reductase (TrxR) has emerged as an effective strategy to selectively target cancer cells. Withangulatin A (WA), a natural product extracted from the whole herb of Physalis angulata L. (Solanaceae), exhibits potent anticancer activity and other diverse pharmacological activities. To improve activity and targeting, we designed and prepared 41 semisynthetic analogues of WA. Biological evaluation indicated that the most promising compound 13a displayed the most significant effect on HT-29 cells (human colon cancer cells) (IC50 = 0.08 μM). A structure-activity relationship study indicated that α,β-unsaturated ketones and ester are necessary groups, allowing 13a to undergo Michael addition reactions with mercaptan and selenol. Liquid chromatography-mass spectrometry (LC-MS) analysis confirmed that 13a modified selenocysteine 498 (U) residues in the redox centers of TrxR, resulting in enzyme inhibition. Therefore, compound 13a acts as a novel TrxR inhibitor and may be a promising candidate for cancer intervention.

Preeclampsia is a dangerous disease of pregnancy with symptoms such as high blood pressure and proteinuria. The nitric oxide synthase (NOS) enzyme produces nitric oxide (NO) as a mediator of vasodilation and need to calcium and zinc ions for activity. The thioredoxin reductase (TrxR) as an enzyme containing selenium has been neutralized the damaging effects of oxidants. In this study, our aim was to evaluate the activity of eNOS and TrxR enzymes and the amounts of calcium, zinc, and selenium elements in serum of women with preeclampsia.Thirty preeclampsia and 30 healthy pregnant women were enrolled in the study after clinical examination and confirmation by Obstetrician-Gynecologist. Venous blood samples were collected and the activity of NOS, TrxR enzymes, and the concentration of zinc, calcium, and selenium elements were measured in serum.The results of NOS and TrxR activities showed significant decreases in preeclampsia compared with control group (This study identified the role of the NOS and TrxR activities in preeclampsia disorder and may be one of the ways to prevent and reduce the risks of preeclampsia in high-risk women using diet control and stress reduction.

Selenocysteine (Sec) is the 21(st) proteogenic amino acid in the genetic code. Incorporation of Sec into proteins is a complex and bioenergetically costly process that evokes the question: “Why did nature choose selenium?” An answer that has emerged over the past decade is that Sec confers resistance to irreversible oxidative inactivation by reactive oxygen species (ROS). Here, we explore the question of whether this concept can be broadened to include resistance to reactive electrophilic species (RES) since oxygen and related compounds are merely a subset of RES. To test this hypothesis we inactivated mammalian thioredoxin reductase (Sec-TrxR), a mutant containing alpha-methylselenocysteine ((αMe)Sec-TrxR), and a cysteine-ortholog TrxR (Cys-TrxR) with various electrophiles including acrolein, 4-hydroxynonenal, and curcumin. Our results show that the acrolein-inactivated Sec-TrxR and the (αMe)Sec-TrxR mutant could regain 25% and 30% activity respectively, when incubated with 2 mM H(2)O(2) and 5 mM imidazole. In contrast, the Cys-TrxR did not regain activity under the same conditions. We posit that Sec-enzymes can undergo a repair process via β-syn selenoxide elimination that ejects the electrophile, leaving the enzyme in the oxidized selenosulfide state. (αMe)Sec-TrxR was created by incorporating the non-natural amino acid ((αMe)Sec into TrxR by semisynthesis and allowed for rigorous testing of our hypothesis. This Sec-derivative enables higher resistance to both oxidative and electrophilic inactivation because it lacks a backbone C(α)-H, which prevents loss of selenium through formation of dehydroalanine. This is the first time this unique amino acid has been incorporated into an enzyme and is example of state-of-the-art protein engineering.

Utilizing chemotherapeutics to induce immunogenic cell death (ICD) is a promising strategy to sensitize tumor cells and induce anticancer immunity. However, the application of traditional ICD inducers, such as chemodrugs, is largely hindered by their low tumor selectivity and severe side effects. Here, a new unitized ICD nanoinducer with high potency and cancer cell specificity is reported to achieve effective cancer immunotherapy. This nanoinducer is composed of disulfide-bond-incorporated organosilica nanoparticles, curcumin (CUR), and iron oxide nanoparticles, which can deplete intracellular glutathione, produce hydroxyl radicals, and induce cancer-cell-specific Ca2+ depletion as well as thioredoxin reductase inhibition. While the components are unable to induce ICD individually, their complementary pharmaceutical activities significantly elevate intracellular oxidative stress and endoplasmic reticulum stress in parallel. Consequently, ICD and systemic antitumor immunity can be elicited. Compared to the conventional ICD inducer doxorubicin, the unitized nanoinducer exhibits significantly improved ICD-inducing activity and cancer cell selectivity.

Tuberculosis (TB) is a threat to a large population across the globe. Rapanea melanophloeos (L.) Mez, commonly known as the Cape beech, is a plant that is traditionally used in the treatment of tuberculosis and TB-related symptoms. The aim of this study, was to evaluate the pharmacological effects of a leaf ethanolic extract (1:10, w:v) of R. melanophloeos, as well as different polarity partitions, in conjunction with a fluoroquinolone antimycobacterial drug, ciprofloxacin. The bioassays in this study included the antimycobacterial, antibiofilm and synergistic potential against M. smegmatis. Secondly the antiproliferative and hepatoprotective effect on hepatocytes (HepG2), and, lastly, a potential mechanistic investigation on mycothione, glutathione and thioredoxin reductases. The hexane fraction from the ethanolic plant extract showed relatively good antibiofilm activity with a mean IC50 value of 128.25±4.09 μg/mL. The drug-herb interaction activity of the extract was tested against ciprofloxacin, and it was found that the plant extract acted antagonistically towards the standard drug (FIC > 4). Antiproliferative and hepatoprotective tests were conducted on HepG2 hepatocytes to test the effects on cell viability. The plant was not toxic to the cells but showed no significant hepatoprotective activity against drug-induced hepatotoxic injury, in comparison to the positive control silymarin. Furthermore, the plant extract was tested against glutathione, mycothiol and thioredoxin reductase (human and bacterial analogs) for its inhibitory activity. Rapanea melanophloeos showed higher affinity for the glutathione and mycothiol reductase with relatively high inhibitory activity against the enzymes. The findings from this study indicate the potential adverse effects and drug-herb interactions, associated with the use of R. melanophloeos in the treatment of TB. In addition, this study emphasizes the importance of herb indications and drug interactions, and the term “natural” or “herbal” does not coincide with safe.

Tumor cells are characterized by increased reactive oxygen species production in parallel with an enhanced antioxidant system to avoid oxidative damage. The inhibition of antioxidant systems is an effective way to kill cancer cells, and the thioredoxin system or, more specifically, the cytosolic selenocysteine-containing enzyme thioredoxin reductase (TrxR) has become an interesting target for cancer therapy. We show here that the known cytotoxic and apoptosis-inducing osmium carbonyl cluster Os3(CO)10(NCCH3)2 (1) is a nonsubstrate inhibitor of mammalian TrxR, with an IC50 of 5.3 ± 0.9 μM. It inhibits TrxR selectively over the closely related glutathione reductase (GR) and in the presence of excess reduced glutathione (GSH). This inhibition has also been demonstrated in cell lysates, suggesting that TrxR inhibition is a potential apoptotic pathway for 1.

The down-regulation of zeaxanthin (Zx) epoxidation is important for the regulation of Zx accumulation in xanthophyll cycle and for the development of non-photochemical quenching (NPQ). The NPQ development and Zx accumulation kinetics in rice, barley, and spinach leaves under light of different intensities were highly similar among the three plants. When the leaves were pre-treated with an inhibitor of Zx epoxidase (ZE), salicylaldoxime (SA), the two kinetics patterns in the leaves under low and moderately high light intensities became similar to those of high light intensity-treated leaves. Therefore, we propose that reversible down-regulation of Zx epoxidation plays an important role in plants, and this reversible down-regulation mechanism is a general mechanism in plants which occurs at room temperature under various light conditions as well as under different stress conditions in the presence of light. This reversible down-regulation is different from the irreversible down-regulation mechanism of ZE which involves ZE protein degradation together with D1 protein degradation under photooxidative conditions. There will be discussion on the mechanisms for the actual regulation of ZE activities involving phosphorylation/dephosphorylation of still unknown regulator(s) and/or by the redox regulation involving NADPH thioredoxin reductase C and thioredoxin m.

Ebselen (EbSe), an organo-selenium compound with well-characterized toxicology and pharmacology, exhibited potent antibacterial activity against glutathione (GSH)-positive bacteria when combined with silver ions (Ag+). In this paper, the strong bactericidal activity of EbSe–Ag+ against multidrug-resistant (MDR) Acinetobacter baumannii has been confirmed, and its efficacy was mainly based on the inhibition of thioredoxin reductase (TrxR) activity and the depletion of the total GSH amount. Moreover, the therapeutic effect of EbSe–Ag+ on urinary tract infection was assessed in a mouse model induced with A. baumannii 0361# strain. The treatment with EbSe–Ag+ significantly reduced the bacterial load and expression levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in bladder lesions; meanwhile, the pathological experiment showed that A. baumannii-induced changes in EbSe–Ag+ treated mice were much attenuated than that in the control group. Thus, all the results obtained here may lay the foundation for further analysis and development of EbSe–Ag+ as potential antibacterial agents for MDR A. baumannii-induced urinary tract infection treatment.

Primary amebic meningoencephalitis, caused by brain infection with a free-living ameba, Naegleria fowleri, leads to extensive inflammation of the brain and death within 3-7 days after symptoms begin. Treatment of primary amebic meningoencephalitis relies on amphotericin B in combination with other drugs, but use of amphotericin B is associated with severe adverse effects. Despite a fatality rate of over 97%, economic incentive to invest in development of antiamebic drugs by the pharmaceutical industry is lacking. Development of safe and rapidly acting drugs remains a critical unmet need to avert future deaths. Since FDA-approved anti-inflammatory and antiarthritic drug auranofin is a known inhibitor of selenoprotein synthesis and thioredoxin reductase and the genome of N. fowleri encodes genes for both selenocysteine biosynthesis and thioredoxin reductases, we tested the effect of auranofin against N. fowleri strains of different genotypes from the USA, Europe, and Australia. Auranofin was equipotent against all tested strains with an EC50 of 1-2 μM. Our growth inhibition study at different time points demonstrated that auranofin is fast-acting, and ∼90% growth inhibition was achieved within 16 h of drug exposure. A short exposure of N. fowleri to auranofin led to the accumulation of intracellular reactive oxygen species. This is consistent with auranofin's role in inhibiting antioxidant pathways. Further, combination of auranofin and amphotericin B led to 95% of growth inhibition with 2-9-fold dose reduction for amphotericin B and 3-20-fold dose reduction for auranofin. Auranofin has the potential to be repurposed for the treatment of primary amebic meningoencephalitis.

Bioselenols are important substances for the maintenance of physiological balance and offer anticancer properties; however, their causal mechanisms and effectiveness have not been assessed. One way to explore their physiological functions is the in vivo detection of bioselenols at the molecular level, and one of the most efficient ways to do so is to use fluorescent probes. Various types of bioselenol-specific fluorescent probes have been synthesized and optimized using chemical simulations and by improving biothiol fluorescent probes. Here, we review recent advances in bioselenol-specific fluorescent probes for selenocysteine (Sec), thioredoxin reductase (TrxR), and hydrogen selenide (H2Se). In particular, the molecular design principles of different types of bioselenols, their corresponding sensing mechanisms, and imaging applications are summarized.

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic β-cell damage during the pathogenesis of autoimmune diabetes. While β-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in β-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of β-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that β-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 ( Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 ( Prdx2) had no effect. Together, these results suggest that β-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.

Background:Central Nervous System (CNS) tumors have a poor survival prognosis due to their invasive and heterogeneous nature, in addition to the resistance to multiple treatments.Objective:In this paper, the main aspects of brain tumor biology and pathogenesis are reviewed both for primary tumors of the brain, (i.e., gliomas) and for metastasis from other malignant tumors, namely lung cancer, breast cancer and malignant melanoma which account for a high percentage of overall malignant brain tumors. We review the role of antioxidant systems, namely the thioredoxin and glutathione systems, in the genesis and/or progression of brain tumors.Methods:Although overexpression of Thioredoxin Reductase (TrxR) and Thioredoxin (Trx) is often linked to increased malignancy rate of brain tumors, and higher expression of Glutathione (GSH) and Glutathione S-Transferases (GST) are associated to resistance to therapy, several knowledge gaps still exist regarding for example, the role of Peroxiredoxins (Prx), and Glutaredoxins (Grx).Conclusion:Due to their central role in redox homeostasis and ROS scavenging, redox systems are potential targets for new antitumorals and examples of innovative therapeutics aiming at improving success rates in brain tumor treatment are discussed.

Many cancer cells critically rely on antioxidant systems for cell survival and are vulnerable to further oxidative impairment triggered by agents generating reactive oxygen species (ROS). Therefore, the classical design and development of inhibitors that target antioxidant defense enzymes such as thioredoxin reductase (TrxR) can be a promising anticancer strategy. Herein, it is shown that a gold(I) complex containing an oleanolic acid derivative (4 b) induces apoptosis of ovarian cancer A2780 cells by activating endoplasmic reticulum stress (ERS). It can inhibit TrxR enzyme activity to elevate ROS, mediate ERS and mitochondrial dysfunction, and finally leads to cell cycle arrest and apoptosis of A2780 cells. Notably, this complex inhibits A2780 xenograft tumor growth accompanied by increased ERS level and decreased TrxR activity in tumor tissues.

Metal complexes provide a versatile platform to develop novel anticancer pharmacophores, and they form stable compounds with N-heterocyclic carbene (NHC) ligands, some of which have been shown to inhibit the cancer-related selenoenzyme thioredoxin reductase (TrxR). To expand a library of isostructural NHC complexes, we report here the preparation of RhIII- and IrIII(Cp*)(NHC)Cl2 (Cp* = η5-pentamethylcyclopentadienyl) compounds and comparison of their properties to the RuII- and OsII(cym) analogues (cym = η6-p-cymene). Like the RuII- and OsII(cym) complexes, the RhIII- and IrIII(Cp*) derivatives exhibit cytotoxic activity with half maximal inhibitory concentration (IC50) values in the low micromolar range against a set of four human cancer cell lines. In studies on the uptake and localization of the compounds in cancer cells by X-ray fluorescence microscopy, the Ru and Os derivatives were shown to accumulate in the cytoplasmic region of treated cells. In an attempt to tie the localization of the compounds to the inhibition of the tentative target TrxR, it was surprisingly found that only the Rh complexes showed significant inhibitory activity at IC50 values of ∼1 μM, independent of the substituents on the NHC ligand. This indicates that, although TrxR may be a potential target for anticancer metal complexes, it is unlikely the main target or the sole target for the Ru, Os, and Ir compounds described here, and other targets should be considered. In contrast, Rh(Cp*)(NHC)Cl2 complexes may be a scaffold for the development of TrxR inhibitors.

Selenoenzymes, containing a selenocysteine (Sec) residue, fulfill important roles in biology. The mammalian thioredoxin reductase selenoenzymes are key regulators of antioxidant defense and redox signaling and are inhibited by methylmercury species and by the gold-containing drug auranofin. It has been proposed that such inhibition is mediated by metal binding to Sec in the enzyme. However, direct structural observations of these classes of inhibitors binding to selenoenzymes have been few to date. Here we therefore have used extended X-ray absorption fine structure as a direct structural probe to investigate binding to the selenium site in recombinant rat thioredoxin reductase 1 (TrxR1). The results demonstrate for the first time the direct and complete binding of the metal atom of the inhibitors to the selenium atom in TrxR1 for both methylmercury and auranofin, indicating that TrxR1 inhibition indeed can be attributed to such direct metal-selenium binding.

Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, doxorubicinol. Identification of the enzymes responsible for doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to doxorubicinol formation were evaluated: carbonyl reductase (Cbr) 1, Cbr3, and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to doxorubicinol formation in mouse liver but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the doxorubicinol-forming activity in mouse liver cytosol but that the majority of the doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT: Earlier studies suggested carbonyl reductase (Cbr) 1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes—Cbr3 and thioredoxin reductase 1—play no role, and that the majority of the activity remains unidentified. Thus, targeting Cbr1 is necessary but not sufficient to eliminate doxorubicinol-associated cardiotoxicity; identification of the additional doxorubicinol-forming activity is an important next challenge.

Background Gastric cancer is one of the leading causes of cancer-related deaths. Allylated monocarbonyl analogs of curcumin (MACs) have been reported to selectively inhibit a broad range of human cancers including gastric cancer. However, the precise molecular mechanisms underlying the inhibitory activities of MACs are not fully known. Methods In this study, we examined the anti-tumor activities of an allylated MAC, CA6, on gastric cancer cells and gastric cancer xenograft mouse model. The potential molecular anti-tumor mechanisms of CA6 were also elucidated. Results Our data show that CA6 exhibited significant cytotoxicity in gastric cancer cells, which was seen as an induction of G2/M cell cycle arrest and apoptosis. These activities were mediated through an elaboration of ROS levels in gastric cancer cells and induction of endoplasmic reticulum stress. CA6 increased ROS levels through directly binding to and inhibiting thioredoxin reductase R1 (TrxR1). Also, CA6-generated ROS inhibited Akt and activated forkhead O3A (FoxO3a), causing cytotoxicity in gastric cancer cells. Finally, CA6 treatment dose-dependently reduced the growth of gastric cancer xenografts in tumor-bearing mice, which was associated with reduced TrxR1 activity and increased ROS in the tumor. Conclusion In summary, our studies demonstrate that CA6 inhibited gastric cancer growth by inhibiting TrxR1 and increasing ROS, which in turn activated FoxO3a through suppressing Akt. CA6 is a potential candidate for the treatment of gastric cancer.

Gastric cancer (GC) is the third leading cause of cancer deaths worldwide. Conventional chemotherapy has been proven useful to only a portion of the patients. Previous developed targeted drugs are more effective and tolerable than conventional drugs. Thus the development of novel drugs targeting markers is an urgent task and the main direction for future research. Ethaselen, an inhibitor of thioredoxin reductase (TrxR), has been considered an important anticancer target drug. Previous studies show that it is effective on treating many kinds of cancers. In this paper, we examined that ethaselen effectively inhibited the growth of gastric cancer cells and promoted apoptosis. Organoids were cultured from patient-derived cells in a three-dimension form which are widely used in cancer research to help us understand cancer cells behavior at the sub-organ level and develop novel drugs. We established a drug testing and screening system using GC-derived organoids by recapitulating tumor microenvironment. We confirmed that the TrxR-targeting ethaselen could be a novel and effective drug for gastric cancer treatment.

Cyanobacteria are potentially useful photosynthetic microorganisms for bioremediation under oligotrophic environments. Here, the biphenyl degradation pathway genes of β-proteobacterium Acidovorax sp. strain KKS102 were co-expressed in cyanobacterium Synechocystis sp. PCC6803 cells under control of the photo-inducible psbE promoter. In the KKS102 cells, biphenyl is dioxygenated by bphA1 and bphA2 gene products complex using electrons supplied from NADH via bphA4 and bphA3 gene products (BphA4 and BphA3, respectively), and converted to benzoic acid by bphB, bphC and bphD gene products. Unexpectedly, biphenyl was effectively hydroxylated in oligotrophic BG11 medium by co-expressing the bphA3, bphA1 and bphA2 genes without the bphA4 gene, suggesting that endogenous cyanobacteria-derived protein(s) can supply electrons to reduce BphA3 at the start of the biphenyl degradation pathway. Furthermore, biphenyl was converted to benzoic acid by cyanobacterial cells co-expressing bphA3, bphA1, bphA2, bphB, bphC and bphD. Structural gene-screening using recombinant Escherichia coli cells co-expressing bphA3, bphA1, bphA2, bphB and bphC suggested that petH, which encodes long- and short-type NADP-ferredoxin oxidoreductase isomers (FNRL and FNRS, respectively), and slr0600, which is annotated as an NADPH-thioredoxin reductase gene in CyanoBase, were BphA3-reducible proteins. Purified FNRL and FNRS, and the slr0600 gene product showed BphA3 reductase activity dependent on NADPH and the reduced form of glutathione, respectively, potentially shedding light on the physiological roles of the slr0600 gene product in cyanobacterial cells. Collectively, our results demonstrate the utility of Synechocystis sp. PCC6803 cells as a host for bioremediation of biphenyl compounds under oligotrophic environments without an organic carbon source.

New alkynylgold(I) with P(NMe2)3 (HMPT) phosphane complexes, [Au(C≡C-R)(HMPT)] (R= 4-Ph, 4-MePh, 4-OMe, 4-Br, 4-Cl, 2-py, and 3-py) have been synthesized and characterized, including X-ray studies of complexes with R= 4-OMe and 4-Br; additionally, their physicochemical properties and anticancer activity have been tested. Due to the great water solubility of the HMPT phosphane, all the complexes exhibit an optimal balance of hydrophilicity/lipophilicity. Also, all of these complexes are quite stable in physiological conditions and interact well enough with the transport protein BSA. All complexes exhibit a higher anticancer activity against Caco-2 cells than cisplatin, and some of them do not present cytotoxic activity against enterocyte-like differentiated cells. The selective complexes are proapoptotic drugs by the exposure of phosphatidylserine, results that are also confirmed in primary cultures from mouse colon tumors. Complexes with a halogen unit also arrest the cell cycle in G2/M phase. It is thought that maybe these apoptosis processes are promoted by the observed oxidative damage in the membrane lipids, as a consequence of the inhibition of the thioredoxin reductase enzyme. Based on our results, we conclude that five of our complexes are good candidates to be used in chemotherapy.

An organoruthenium(II) complex with pyrithione (2-mercaptopyridine N-oxide) 1 a has previously been identified by our group as a compound with promising anticancer potential without cytotoxicity towards non-cancerous cells. To expand the rather limited research on compounds of this type, an array of novel chlorido and 1,3,5-triaza-7-phosphaadamantane (pta) organoruthenium(II) complexes with methyl-substituted pyrithiones has been prepared. After thorough investigation of the aqueous stability of these complexes, their modes of action have been elucidated at the cellular level. Minor structural alterations in the ruthenium-pyrithionato compounds resulted in fine-tuning of their cytotoxicities. The best performing compounds, 1 b and 2 b, with a chlorido or pta ligand bound to ruthenium, respectively, and a methyl group at the 3-position of the pyrithione scaffold, have been further investigated. Both compounds trigger early apoptosis, induce the generation of reactive oxygen species and G1 arrest in A549 cancer cells, and show no strong interaction with DNA. However, only 1 b also inhibits thioredoxin reductase. Wound healing assays and mitochondrial function evaluation have revealed differences between these two compounds at the cellular level.

The natural products piperlongumine (1) and ligustrazine (2) have been reported to exert antiproliferative effects against various types of cancer cells by up-regulating the level of reactive oxidative species (ROS). However, the moderate activities of 1 and 2 limit their application. To improve their potential antitumor activity, novel piperlongumine/ligustrazine derivatives were designed and prepared, and their potential pharmacological effects were determined in vitro and in vivo. Among the derivatives obtained, 11 exerted more prominent inhibitory activities against proliferation of drug-sensitive/-resistant cancer cells with lower IC50 values than 1. Particularly, the IC50 value of 11 against drug-resistant Bel-7402/5-FU cells was 0.9 μM, which was about 9-fold better than that of 1 (IC50 value of 8.4 μM). Mechanistic studies showed that 11 demonstrated thioredoxin reductase (TrxR) inhibitory activity, increase of ROS levels, decrease of mitochondrial transmembrane potential levels, and occurrence of DNA damage and autophagy, in a dose-dependent manner, via regulation of DNA damage protein H2AX and autophagy-associated proteins LC3, beclin-1, and p62 in drug-resistant Bel-7402/5-FU cells. Finally, compound 11 at 5 mg/kg displayed potent antitumor activity in vivo with tumor suppression of 76% (w/w). Taken together, compound 11 may represent a promising candidate drug for the chemotherapy of drug-resistant hepatocellular carcinoma and warrant more intensive study.

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA–selenium complexes.

Food colorants are widely used by humans in food production and preparation, however, their potential toxicity requires an in-depth analysis. In this study, five out of 15 commercial food colorants, namely, lutein, betanin, caramel, crocin and chlorophyll, significantly inhibited wild type selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1) in vitro. The hyperactive Sec498 residue of TrxR1 was targeted by those five colorants, which was confirmed by the site-directed mutagenesis of TrxR1. Furthermore, two colorants, chlorophyll and betanin, triggered the oligomerization of TrxR1. A chlorophyll-derived compound, chlorophyllin, irreversibly inhibited the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) reducing activity of TrxR1 with Kinact = 6.96 × 10−3 ± 0.49 × 10−3 µM−1 min−1. Moreover, chlorophyllin reduced the cellular TrxR activity, leading to reactive oxygen species (ROS) accumulation and, subsequently, promoting cancer cell death. In conclusion, this study might contribute to understand the food safety of commercial colorants and provide chemotherapeutic compounds by targeting TrxR1.

PURPOSE: Tumor cells are dependent on the glutathione and thioredoxin antioxidant pathways to survive oxidative stress. Since the essential amino acid methionine is converted to glutathione, we hypothesized that methionine restriction (MR) would deplete glutathione and render tumors dependent on the thioredoxin pathway and its rate-limiting enzyme thioredoxin reductase (TXNRD). METHODS: Triple (ER/PR/HER2)-negative breast cancer (TNBC) cells were treated with control or MR media and the effects on reactive oxygen species (ROS) and antioxidant signaling were examined. To determine the role of TXNRD in MR-induced cell death, TXNRD1 was inhibited by RNAi or the pan-TXNRD inhibitor auranofin, an antirheumatic agent. Metastatic and PDX TNBC mouse models were utilized to evaluate in vivo antitumor activity. RESULTS: MR rapidly and transiently increased ROS, depleted glutathione and decreased the ratio of reduced glutathione/oxidized glutathione in TNBC cells. TXNRD1 mRNA and protein levels were induced by MR via a ROS-dependent mechanism mediated by the transcriptional regulators NRF2 and ATF4. MR dramatically sensitized TNBC cells to TXNRD1 silencing and the TXNRD inhibitor auranofin, as determined by crystal violet staining and caspase activity; these effects were suppressed by the antioxidant N-acetylcysteine. H-Ras-transformed MCF-10A cells, but not untransformed MCF-10A cells, were highly sensitive to the combination of auranofin and MR. Furthermore, dietary MR induced TXNRD1 expression in mammary tumors and enhanced the antitumor effects of auranofin in metastatic and PDX TNBC murine models. CONCLUSION: MR exposes a vulnerability of TNBC cells to the TXNRD inhibitor auranofin by increasing expression of its molecular target and creating a dependency on the thioredoxin pathway.

Oxidative stress is hypothesized to play a role in pancreatic β-cell damage, potentially contributing to β-cell dysfunction and death in both type 1 and type 2 diabetes. Oxidative stress arises when naturally occurring reactive oxygen species (ROS) are produced at levels that overwhelm the antioxidant capacity of the cell. ROS, including superoxide and hydrogen peroxide, are primarily produced by electron leak during mitochondrial oxidative metabolism. Additionally, peroxynitrite, an oxidant generated by the reaction of superoxide and nitric oxide, may also cause β-cell damage during autoimmune destruction of these cells. β-cells are thought to be susceptible to oxidative damage based on reports that they express low levels of antioxidant enzymes compared to other tissues. Furthermore, markers of oxidative damage are observed in islets from diabetic rodent models and human patients. However, recent studies have demonstrated high expression of various isoforms of peroxiredoxins, thioredoxin, and thioredoxin reductase in β-cells and have provided experimental evidence supporting a role for these enzymes in promoting β-cell function and survival in response to a variety of oxidative stressors. This mini-review will focus on the mechanism by which thioredoxins and peroxiredoxins detoxify ROS and on the protective roles of these enzymes in β-cells. Additionally, we speculate about the role of this antioxidant system in promoting insulin secretion.

Auranofin (AF), a gold compound, has been used to treat rheumatoid arthritis (RA) for more than 40 years; however, its mechanism of action remains unknown. We revealed that AF inhibited the induction of proinflammatory proteins and their mRNAs by the inflammatory stimulants, cyclooxygenase-2 and inducible nitric oxide synthase, and their upstream regulator, NF-κB. AF also activated the proteins peroxyredoxin-1, Kelch-like ECH-associated protein 1, and NF-E2-related factor 2, and inhibited thioredoxin reductase, all of which are involved in oxidative or electrophilic stress under physiological conditions. Although the cell membrane was previously considered to be permeable to AF because of its hydrophobicity, the mechanisms responsible for transporting AF into and out of cells as well as its effects on the uptake and excretion of other drugs have not yet been elucidated. Antibodies for cytokines have recently been employed in the treatment of RA, which has had an impact on the use of AF. Trials to repurpose AF as a risk-controlled agent to treat cancers or infectious diseases, including severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019, are ongoing. Novel gold compounds are also under development as anti-cancer and anti-infection agents.

Lymphatic filariasis and its associated health hazards have taken enormous especially in the tropical and sub-tropical countries round the globe. Our present work contemplates the immunomodulatory role of filarial Thioredoxin reductase (TrxR) for the survival of the parasite inside the human host. For this, the protein TrxR was purified from the filarial parasite Setaria cervi and further substantiated through specific anti-TrxR antibody raised in mice. Both commercially available anti-TrxR antibody and laboratory raised antibody produced a single band with a molecular mass of ~80 kDa on western blot. The protein is optimally active at pH 7.0 and at temperature 37 °C. This protein contains both alpha helix and beta pleated sheet with selenocysteine at its active site. The Km was found to be 2.75 ± 0.49 mM. TrxR was found to downregulate lipopolysaccharide (LPS)-induced inflammation in macrophages due to inhibition of TLR4-NF-κB pathway. The result was further supported by the downregulation of inflammasome pathway and activation of alternatively activated macrophages upon TrxR treatment. Hence this study projects insights into the importance of filarial TrxR in host-parasite interface as well as it illustrates novel therapeutic strategy towards anti-filarial drug development.

Aquatic organisms are frequently exposed to multiple stressors including low dissolved oxygen (O2) and metals such as cadmium (Cd). Reduced O2 concentration and Cd exposure alter cellular function in part by impairing energy metabolism and dysregulating reactive oxygen species (ROS) homeostasis. However, little is known about the role of mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) in ROS homeostasis in fish and its response to environmental stress. In this study, mGPDH activity and the effects of anoxia-reoxygenation (A-RO) and Cd on ROS (as hydrogen peroxide, H2O2) emission in rainbow trout liver mitochondria during oxidation of glycerol 3-phosphate (G3P) were probed. Trout liver mitochondria exhibited low mGPDH activity that supported a low respiratory rate but substantial H2O2 emission rate. Cd evoked a low concentration stimulatory-high concentration inhibitory H2O2 emission pattern that was blunted by A-RO. At specific redox centers, Cd suppressed H2O2 emission from site IQ, but stimulated emission from sites IIIQo and GQ. In contrast, A-RO stimulated H2O2 emission from site IQ following 15 min exposure and augmented Cd-stimulated emission from site IIF after 30 min exposure but did not alter the rate of H2O2 emission from sites IIIQo and GQ. Additionally, Cd neither altered the activities of catalase, glutathione peroxidase, or thioredoxin reductase nor the concentrations of total glutathione, reduced glutathione, or oxidized glutathione. Overall, this study indicates that oxidation of G3P drives ROS production from mGPDH and complexes I, II and III, whereas Cd directly modulates redox sites but not antioxidant defense systems to alter mitochondrial H2O2 emission.

A series of ruthenium(II) complexes with N-heterocyclic carbene (NHC) ligands of the general type (arene)(NHC)Ru(II)X2 (where X = halide) was prepared, characterized, and evaluated as antibacterial agents in comparison to the respective metal free benzimidazolium cations. The ruthenium(II) NHC complexes generally triggered stronger bacterial growth inhibition than the metal free benzimidazolium cations. The effects were much stronger against Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) than against Gram-negative bacteria (Escherichia coli, Acinetobacter&nbsp, baumannii, Pseudomonas aeruginosa), and all complexes were inactive against the fungus Candida albicans. Moderate inhibition of bacterial thioredoxin reductase was confirmed for selected complexes, indicating that inhibition of this enzyme might be a contributing factor to the antibacterial effects.