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Human thioredoxin reductase (TrxR) is a selenoprotein with a central role in cellular redox homeostasis, utilizing a highly reactive and solvent-exposed selenocysteine (Sec) residue in its active site. Pharmacological modulation of TrxR can be obtained with several classes of small compounds showing different mechanisms of action, but most often dependent upon interactions with its Sec residue. The clinical implications of TrxR modulation as mediated by small compounds have been studied in diverse diseases, from rheumatoid arthritis and ischemia to cancer and parasitic infections. The possible involvement of TrxR in these diseases was in some cases serendipitously discovered, by finding that existing clinically used drugs are also TrxR inhibitors. Inhibiting isoforms of human TrxR is, however, not the only strategy for human disease treatment, as some pathogenic parasites also depend upon Sec-containing TrxR variants, including S. mansoni, B. malayi or O. volvulus. Inhibiting parasite TrxR has been shown to selectively kill parasites and can thus become a promising treatment strategy, especially in the context of quickly emerging resistance towards other drugs. Here we have summarized the basis for the targeting of selenoprotein TrxR variants with small molecules for therapeutic purposes in different human disease contexts. We discuss how Sec engagement appears to be an indispensable part of treatment efficacy and how some therapeutically promising compounds have been evaluated in preclinical or clinical studies. Several research questions remain before a wider application of selenoprotein TrxR inhibition as a first-line treatment strategy might be developed. These include further mechanistic studies of downstream effects that may mediate treatment efficacy, identification of isoform-specific enzyme inhibition patterns for some given therapeutic compounds, and the further elucidation of cell-specific effects in disease contexts such as in the tumor microenvironment or in host-parasite interactions, and which of these effects may be dependent upon the specific targeting of Sec in distinct TrxR isoforms.

Cellular redox networks power a multitude of cellular processes, and are often dysregulated in pathologies including cancer and inflammatory diseases. Quantifying the turnover of the key players in redox homeostasis is crucial for understanding their physiological dynamics and for targeting them in pathologies. However, suitably selective probes for assessing specific redox enzyme activities in cells are lacking. We rationally developed the first chemical probes targeting the mammalian selenoprotein thioredoxin reductase (TrxR) while fully resisting other cellular thiols and oxidoreductases. We used a cyclic selenenylsulfide as a thermodynamically stable and kinetically reversible trigger, oriented to harness the chemistry of TrxR's unique selenolthiol active site, and integrated it into modular probes releasing arbitrary cargos upon reduction. The probes showed remarkable selenocysteine-dependent sensitivity to cytosolic TrxR1, against a panel of oxidoreductases. Lead probe RX1 also had excellent TrxR1-selective performance in cells, as cross-validated by TrxR1 knockout, selenium starvation, TrxR1 knock-in, and TrxRselective chemical inhibitors. Its background-free fluorogenicity enabled us to perform the first quantitative high-throughput live cell screen for TrxR1 inhibitors. This indicated that tempered SNAr electrophiles may be a more favorable drug class than classically-used electrophiles. The RX1 design is thus a robust, cellularly validated, high-performance modular system for mammalian TrxR1. This sets the stage for in vivo imaging TrxR1 activity in health and disease, and can also drive and reorient TrxR1-inhibitor drug design. The thermodynamic and kinetic considerations behind RX1's selectivity also outline paths towards rationally-designed probes for other key players in redox biology.

Background Extremely rare progressive diseases like Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) can be neonatally lethal and therefore go undiagnosed or are difficult to treat. Recent sequencing efforts have linked this disease to mutations in GPX4, with consequences in the resulting enzyme, glutathione peroxidase 4. This offers potential diagnostic and therapeutic avenues for those suffering from this disease, though the steps toward these treatments is often convoluted, expensive, and time-consuming. Main body The CureGPX4 organization was developed to promote awareness of GPX4-related diseases like SSMD, as well as support research that could lead to essential therapeutics for patients. We provide an overview of the 21 published SSMD cases and have compiled additional sequencing data for four previously unpublished individuals to illustrate the genetic component of SSMD, and the role of sequencing data in diagnosis. We outline in detail the steps CureGPX4 has taken to reach milestones of team creation, disease understanding, drug repurposing, and design of future studies. Conclusion The primary aim of this review is to provide a roadmap for therapy development for rare, ultra-rare, and difficult to diagnose diseases, as well as increase awareness of the genetic component of SSMD. This work will offer a better understanding of GPx4-related diseases, and help guide researchers, clinicians, and patients interested in other rare diseases find a path towards treatments.

The Nrf2 transcription factor is a key regulator of redox reactions and considered the main target for the multiple sclerosis (MS) drug dimethyl fumarate (DMF). However, exploration of additional Nrf2-activating compounds is motivated, since DMF displays significant off-target effects and has a relatively poor penetrance to the central nervous system (CNS). We de novo synthesized eight vinyl sulfone and sulfoximine compounds (CH-1–CH-8) and evaluated their capacity to activate the transcription factors Nrf2, NFκB, and HIF1 in comparison with DMF using the pTRAF platform. The novel sulfoximine CH-3 was the most promising candidate and selected for further comparison in vivo and later an experimental model for traumatic brain injury (TBI). CH-3 and DMF displayed comparable capacity to activate Nrf2 and downstream transcripts in vitro, but with less off-target effects on HIF1 from CH-3. This was verified in cultured microglia and oligodendrocytes (OLs) and subsequently in vivo in rats. Following TBI, DMF lowered the number of leukocytes in blood and also decreased axonal degeneration. CH-3 preserved or increased the number of pre-myelinating OL. While both CH-3 and DMF activated Nrf2, CH-3 showed less off-target effects and displayed more selective OL associated effects. Further studies with Nrf2-acting compounds are promising candidates to explore potential myelin protective or regenerative effects in demyelinating disorders. Electronic supplementary material The online version of this article (10.1007/s13311-020-00855-0) contains supplementary material, which is available to authorized users.

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.

Chronic autoimmune diseases are associated with mutations in PTPN22, a modifier of T cell receptor signaling. As with all protein tyrosine phosphatases the activity of PTPN22 is redox regulated, but if or how such regulation can modulate inflammatory pathways in vivo is not known. To determine this, we created a mouse with a cysteine-to-serine mutation at position 129 in PTPN22 (C129S), a residue proposed to alter the redox regulatory properties of PTPN22 by forming a disulfide with the catalytic C227 residue. The C129S mutant mouse showed a stronger T cell-dependent inflammatory response and development of T cell dependent autoimmune arthritis due to enhanced TCR signaling and activation of T cells, an effect neutralized by a mutation in Ncf1, a component of the NOX2 complex. Activity assays with purified proteins suggest that the functional results can be explained by an increased sensitivity to oxidation of the C129S mutated PTPN22 protein. We also observed that the disulfide of native PTPN22 can be directly reduced by the thioredoxin system, while the C129S mutant lacking this disulfide was less amenable to reductive reactivation. In conclusion, we show that PTPN22 functionally interacts with Ncf1 and is regulated by oxidation via the non-catalytic C129 residue and oxidation-prone PTPN22 leads to increased severity in the development of T cell-dependent autoimmunity.Significance statementA hitherto unstudied aspect of PTPN22 biology is its regulation by cell redox states. Here we created a mouse model where PTPN22 was mutated to respond differentially to redox levels in vivo and found that PTPN22 function is regulated by reactive oxygen species and that redox regulation of PTPN22 impacts T-cell-dependent autoimmune inflammation.

Background: Alanine aminotransferase (ALT) is the most commonly used serum biomarker for chronic liver diseases (CLDs) but may not accurately reflect hepatic disorders and easily underestimates hepatic fibrosis. The previously revised upper limit of normal (ULN) of ALT (19 U/L for women and 30 U/L for men) increases its sensitivity but yields higher numbers of false-positives. Moreover, CLDs patients with ALT lower than the revised ULN may nonetheless have progression of disease. Therefore there is a need of novel biomarkers to complement the use of ALT. Here we have evaluated measurements of serum dithiothreitol-oxidizing capacity (DOC) in cohorts of chronic hepatitis B patients with different stages of disease as an exploratory pilot study for this purpose. Methods: Serum samples obtained from healthy persons and from chronic hepatitis B patients with normal ALT values were used for sensitivity evaluation. The hepatitis B patients encompassed end-stage liver diseases (ELD), chronic hepatitis B (CHB), CHB with persistently normal ALT (CHB-P) and inactive carriers (ICs). Sensitivity was also evaluated with samples from patients with other diseases. The study period was March 2018 to December 2020. Findings: DOC was found to be a robust biomarker that may become complementary to ALT measurements, especially in patients displaying low ALT levels. ROC analyses indicated that the AUC values of DOC reached 0.983 and 0.956 in ELD and CHB patients exhibiting normal ALT levels, respectively. Importantly, the AUC values of DOC reached 0.852 and 0.844 in CHB-P patients and ICs, respectively. Such AUC values permit screening and continued monitoring, corresponding to over 30% and 50% sensitivity with 99% and 95% specificity for CHB-P and ICs, respectively. DOC was also significantly correlated with indicators for fibrosis, assessing both APRI (Pearson r = 0.4905, P < 0.0001) and FIB-4 (Pearson r = 0.4421, P < 0.0001). Surprisingly, the AUC values of DOC in the hepatitis B patients with ALT levels lower than the revised ULN were not compromised. In examined non-liver diseases, DOC was low and normal, including in patients with acute myocardial infection displaying increased ALT levels. Interpretations: The results suggest that DOC can be promising as a complementary biomarker used in addition to ALT for monitoring of disease in chronic hepatitis B patients, especially when ALT levels are normal. DOC should be further evaluated for possible clinical use as biomarker also in other CLDs. Funding: This study was funded by the National Natural Science Foundation of China (Grant numbers: 31771971 and 32001013).

The cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1), and to some extent mitochondrial TrxR2 (TXNRD2), can be inhibited by a wide range of electrophilic compounds. Many such compounds also yield cytotoxicity toward cancer cells in culture or in mouse models, and most compounds are likely to irreversibly modify the easily accessible selenocysteine residue in TrxR1, thereby inhibiting its normal activity to reduce cytosolic thioredoxin (Trx1, TXN) and other substrates of the enzyme. This leads to an oxidative challenge. In some cases, the inhibited forms of TrxR1 are not catalytically inert and are instead converted to prooxidant NADPH oxidases, named SecTRAPs, thus further aggravating the oxidative stress, particularly in cells expressing higher levels of the enzyme. In this review, the possible molecular and cellular consequences of these effects are discussed in relation to cancer therapy, with a focus on outstanding questions that should be addressed if targeted TrxR1 inhibition is to be further developed for therapeutic use.

Dynamically driven cellular redox networks power a broad range of physiological cellular processes, and additionally are often dysregulated in various pathologies including cancer and inflammatory diseases. Therefore it is vital to be able to image and to respond to the turnover of the key players in redox homeostasis, to understand their physiological dynamics and to target pathological conditions. However, selective modular probes for assessing specific redox enzyme activities in cells are lacking. Here we report the development of cargo-releasing chemical probes that target the mammalian selenoprotein thioredoxin reductase (TrxR) while being fully resistant to thiol reductants in cells, such as the monothiol glutathione (GSH). We used a rationally oriented cyclic selenenylsulfide as a thermodynamically stable and kinetically reversible trigger that matches the chemistry of the unique TrxR active site, and integrated this reducible trigger into modular probes that release arbitrary cargos upon reduction. The probes' redox biochemistry was evaluated over a panel of thiol-type oxidoreductases, particularly showing remarkable, selenocysteine-dependent sensitivity of the "RX1" probe design to cytosolic TrxR1, with little response to mitochondrial TrxR2. The probe was cross-validated in cells by TrxR1 knockout, selenium starvation, TrxR1 knock-in, and use of TrxR-selective chemical inhibitors, showing excellent TrxR1-dependent cellular performance. The RX1 design is therefore a robust, cellularly-validated, modular probe system for mammalian TrxR1. This sets the stage for in vivo imaging of TrxR1 activity in health and disease; and the thermodynamic and kinetic considerations behind its selectivity mechanism represent a significant advance towards rationally-designed probes for other key players in redox biology.

Selenocysteine (Sec), the sulfur-to-selenium substituted variant of cysteine (Cys), is the defining entity of selenoproteins. These are naturally expressed in many diverse organisms and constitute a unique class of proteins. As a result of the physicochemical characteristics of selenium when compared with sulfur, Sec is typically more reactive than Cys while participating in similar reactions, and there are also some qualitative differences in the reactivities between the two amino acids. This minireview discusses the types of modifications of Sec in selenoproteins that have thus far been experimentally validated. These modifications include direct covalent binding through the Se atom of Sec to other chalcogen atoms (S, O and Se) as present in redox active molecular motifs, derivatization of Sec via the direct covalent binding to non-chalcogen elements (Ni, Mb, N, Au and C), and the loss of Se from Sec resulting in formation of dehydroalanine. To understand the nature of these Sec modifications is crucial for an understanding of selenoprotein reactivities in biological, physiological and pathophysiological contexts.

Cellular antioxidant systems control the levels of hydrogen peroxide (H2O2) within cells. Multiple theoretical models exist that predict the diffusion properties of H2O2 depending on the rate of H2O2 generation and amount and reaction rates of antioxidant machinery components. Despite these theoretical predictions, it has remained unknown how antioxidant systems shape intracellular H2O2 gradients. The relative role of thioredoxin (Trx) and glutathione systems in H2O2 pattern formation and maintenance is another disputed question. Here, we visualized cellular antioxidant activity and H2O2 gradients formation by exploiting chemogenetic approaches to generate compartmentalized intracellular H2O2 and using the H2O2 biosensor HyPer to analyze the resulting H2O2 distribution in specific subcellular compartments. Using human HeLa cells as a model system, we propose that the Trx system, but not the glutathione system, regulates intracellular H2O2 gradients. Antioxid. Redox Signal. 31, 664-670.

Protein-tyrosine phosphatases (PTPs) counteract protein tyrosine phosphorylation and cooperate with receptor-tyrosine kinases in the regulation of cell signaling. PTPs need to undergo oxidative inhibition for activation of cellular cascades of protein-tyrosine kinase phosphorylation following growth factor stimulation. It has remained enigmatic how such oxidation can occur in the presence of potent cellular reducing systems. Here, using in vitro biochemical assays with purified, recombinant protein, along with experiments in the adenocarcinoma cell line A431, we discovered that bicarbonate, which reacts with H(2)O(2) to form the more reactive peroxymonocarbonate, potently facilitates H(2)O(2)-mediated PTP1B inactivation in the presence of thioredoxin reductase 1 (TrxR1), thioredoxin 1 (Trx1), and peroxiredoxin 2 (Prx2) together with NADPH. The cellular experiments revealed that intracellular bicarbonate proportionally dictates total protein phosphotyrosine levels obtained after stimulation with epidermal growth factor (EGF) and that bicarbonate levels directly correlate with the extent of PTP1B oxidation. In fact, EGF-induced cellular oxidation of PTP1B was completely dependent on the presence of bicarbonate. These results provide a plausible mechanism for PTP inactivation during cell signaling and explain long-standing observations that growth factor responses and protein phosphorylation cascades are intimately linked to the cellular acid–base balance.

Auranofin is a gold (1)-containing compound used for the treatment of rheumatic arthritis. Auranofin has anticancer activity in animal models and is approved for clinical trials for lung and ovarian carcinomas. Both the cytosolic and mitochondrial forms of the selenoprotein thioredoxin reductase (TrxR) are well documented targets of auranofin. Auranofin was recently reported to also inhibit proteasome activity at the level of the proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14. We here set out to re-examine the molecular mechanism underlying auranofin cytotoxicity towards cultured cancer cells. The effects of auranofin on the proteasome were examined in cells and in vitro, effects on DUB activity were assessed using different substrates. The cellular response to auranofin was compared to that of the 20S proteasome inhibitor bortezomib and the 19S DUB inhibitor b-AP15 using proteomics. Auranofin was found to inhibit mitochondrial activity and to an induce oxidative stress response at IC50 doses. At 2-3-fold higher doses, auranofin inhibits proteasome processing in cells. At such supra-pharmacological concentrations USP14 activity was inhibited. Analysis of protein expression profiles in drug-exposed tumor cells showed that auranofin induces a response distinct from that of the 20S proteasome inhibitor bortezomib and the DUB inhibitor b-AP15, both of which induced similar responses. Our results support the notion that the primary mechanism of action of auranofin is TrxR inhibition and suggest that proteasome DUB inhibition is an off-target effect. Whether proteasome inhibition will contribute to the antineoplastic effect of auranofin in treated patients is unclear but remains a possibility. (C) 2019 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. Funding Agencies|Swedish Cancer Society; Radiumhemmets Forskningsfonder; Vetenskapsradet; Barncancerfonden; Knut and Alice Wallenbergs Foundation

Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr–double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N -diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.

Specialised cellular networks of oxidoreductases coordinate the dithiol/disulfide-exchange reactions that control metabolism, protein regulation, and redox homeostasis. For probes to be selective for redox enzymes and effector proteins (nM to µM concentrations), they must also be able to resist nonspecific triggering by the ca. 50 mM background of non-catalytic cellular monothiols. However, no such selective reduction-sensing systems have yet been established. Here, we used rational structural design to independently vary thermodynamic and kinetic aspects of disulfide stability, creating a series of unusual disulfide reduction trigger units designed for stability to monothiols. We integrated the motifs into modular series of fluorogenic probes that release and activate an arbitrary chemical cargo upon reduction, and compared their performance to that of the literature-known disulfides. The probes were comprehensively screened for biological stability and selectivity against a range of redox effector proteins and enzymes. This design process delivered the first disulfide probes with excellent stability to monothiols, yet high selectivity for the key redox-active protein effector, thioredoxin. We anticipate that further applications of these novel disulfide triggers will deliver unique probes targeting cellular thioredoxins. We also anticipate that further tuning following this design paradigm will deliver redox probes for other important dithiol-manifold redox proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous cellular redox systems.

This protocol describes the proteomics technique called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis or SIESTA 1,2. SIESTA can be used for universal discovery of enzyme substrates that shift in thermal stability or solubility upon post-translational modification (PTM). Experimental design, proteomics sample preparation and data analysis are the key stages of this protocol. Data analysis can be performed using our SIESTA package hosted on GitHub 3. When performed with classical thermal proteome profiling (TPP), the protocol will take 5 days for sample preparation and 14 days of sample analysis by mass spectrometry (the current protocol). If our high-throughput version of TPP called Proteome Integral Solubility Alteration assay (PISA) 4 is used instead, the sample analysis time by mass spectrometry is reduced to 1-2 days for the same number of conditions.

The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties.

Despite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery., The global identification of enzyme substrates is still challenging. Here, the authors develop a method based on proteome-wide thermal shift assays to discover enzyme substrates directly from cell lysates, identifying known and novel oxidoreductase, kinase and poly-(ADP-ribose) polymerase substrates.

The cyclic five-membered disulfide 1,2-dithiolane has been used as the key element in numerous chemical biology probes. Contradictory views of this disulfide motif populate the literature: some reports describe it as being nonspecifically reduced, others as a highly specific substrate for thioredoxin reductase (TrxR). We show that 1,2-dithiolanes are nonspecifically reduced by a broad range of thiol reductants and redox-active proteins, and that their cellular performance is barely affected by TrxR inhibition or knockout. We conclude that inhibitor screenings and probe designs treating 1,2-dithiolanes as TrxR-selective substrates should be treated with caution and previous interpretations may need careful re-evaluation. Considering ring-opening polymerisation, and stringently interpreting assays involving the thiophilic gold-based inhibitor auranofin, are critical to assess 1,2-dithiolane’s true behaviour. We present an approach to control against assay misinterpretation with reducible probes, to ensure that future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient redox probe research in the future.

The article by Fan et al. inaccurately states that ProPerDP is inadequate for protein persulfide detection., The recent report by Fan et al. alleged that the ProPerDP method is inadequate for the detection of protein persulfidation. Upon careful evaluation of their work, we conclude that the claim made by Fan et al. is not supported by their data, rather founded in methodological shortcomings. It is understood that the ProPerDP method generates a mixture of cysteine-containing and non–cysteine-containing peptides. Instead, Fan et al. suggested that the detection of non–cysteine-containing peptides indicates nonspecific alkylation at noncysteine residues. However, if true, then such peptides would not be released by reduction and therefore not appear as products in the reported workflow. Moreover, the authors’ biological assessment of ProPerDP using Escherichia coli mutants was based on assumptions that have not been confirmed by other methods. We conclude that Fan et al. did not rigorously assess the method and that ProPerDP remains a reliable approach for analyses of protein per/polysulfidation.

Chemical proteomics encompasses novel drug target deconvolution methods in which compound modification is not required. Herein we use Thermal Proteome Profiling, Functional Identification of Target by Expression Proteomics and multiplexed redox proteomics for deconvolution of auranofin targets to aid elucidation of its mechanisms of action. Auranofin (Ridaura®) was approved for treatment of rheumatoid arthritis in 1985. Because several clinical trials are currently ongoing to repurpose auranofin for cancer therapy, comprehensive characterization of its targets and effects in cancer cells is important. Together, our chemical proteomics tools confirmed thioredoxin reductase 1 (TXNRD1, EC:1.8.1.9) as a main auranofin target, with perturbation of oxidoreductase pathways as the top mechanism of drug action. Additional indirect targets included NFKB2 and CHORDC1. Our comprehensive data can be used as a proteomic signature resource for further analyses of the effects of auranofin. Here we also assessed the orthogonality and complementarity of different chemical proteomics methods that can furnish invaluable mechanistic information and thus the approach can facilitate drug discovery efforts in general., Graphical abstract Image 1

Thioredoxin system–mediated persulfidation of Cys residues controls protein function and protects them from oxidative stress., Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

Schistosomiasis is a widespread human parasitic disease currently affecting over 200 million people. Chemotherapy for schistosomiasis relies exclusively on praziquantel. Although significant advances have been made in recent years to reduce the incidence and intensity of schistosome infections, these gains will be at risk should drug resistance parasites evolve. Thioredoxin glutathione reductase (TGR) is a selenoprotein of the parasite essential for the survival of schistosomes in the mammalian host. Several high-throughput screening campaigns have identified inhibitors of Schistosoma mansoni TGR. Follow up analyses of select active compounds form the basis of the present study. We identified eight compounds effective against ex vivo worms. Compounds 1 – 5 are active against all major species and development stages. Ability of these compounds to target immature worms is especially critical because praziquantel is poorly active against this stage. Compounds 1 – 5, 7, and 8 displayed schistosomicidal activity even after only one-hour incubation with the worms. Compounds 1 – 4 meet or exceed standards set by the World Health Organization for leads for schistosomiasis therapy activity. The mechanism of TGR inhibition was studied further with wild type and mutant TGR proteins. Compounds 4 – 6 were found to induce an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in TGR, leading to the production of superoxide and hydrogen peroxide. Collectively, this effort has identified several active compound series that may serve as the basis for the development of new schistosomicidal compounds.

Redox regulation of STAT3 is targeted by direct inhibition of TrxR1, illuminating a promising link for therapeutic development., Because of its key role in cancer development and progression, STAT3 has become an attractive target for developing new cancer therapeutics. While several STAT3 inhibitors have progressed to advanced stages of development, their underlying biology and mechanisms of action are often more complex than would be expected from specific binding to STAT3. Here, we have identified and optimized a series of compounds that block STAT3-dependent luciferase expression with nanomolar potency. Unexpectedly, our lead compounds did not bind to cellular STAT3 but to another prominent anticancer drug target, TrxR1. We further identified that TrxR1 inhibition induced Prx2 and STAT3 oxidation, which subsequently blocked STAT3-dependent transcription. Moreover, previously identified inhibitors of STAT3 were also found to inhibit TrxR1, and likewise, established TrxR1 inhibitors block STAT3-dependent transcriptional activity. These results provide new insights into the complexities of STAT3 redox regulation while highlighting a novel mechanism to block aberrant STAT3 signaling in cancer cells.

The predominantly cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, encoded in human by the TXNRD1 gene) is a key enzyme for protection against oxidative stress and support of many reductive pathways in cells. For several decades, the enzyme has also been studied as a potential drug target for cancer therapy. The notion that TrxR1 may represent a promising target for anticancer therapy was further strengthened in recent years, with several clinical trials using inhibitors of the enzyme currently ongoing and with many novel experimental inhibitors being developed. This chapter will discuss the underlying biochemistry, physiological roles, and functional pathways of TrxR1 that can provide a rationale for targeting this enzyme for cancer therapy.

The mammalian thioredoxin system is driven by NADPH through the activities of isoforms of the selenoprotein thioredoxin reductase (TXNRD, TrxR), which in turn help to keep thioredoxins (TXN, Trx) and further downstream targets reduced. Due to a wide range of functions in antioxidant defense, cell proliferation, and redox signaling, strong cellular aberrations are seen upon the targeting of TrxR enzymes by inhibitors. However, such inhibition can nonetheless have rather unexpected consequences. Accumulating data suggest that inhibition of TrxR in normal cells typically yields a paradoxical effect of increased antioxidant defense, with metabolic pathway reprogramming, increased cellular proliferation, and altered cellular differentiation patterns. Conversely, inhibition of TrxR in cancer cells can yield excessive levels of reactive oxygen species (ROS) resulting in cell death and thus anticancer efficacy. The observed increases in antioxidant capacity upon inhibition of TrxR in normal cells are in part dependent upon activation of the Nrf2 transcription factor, while exaggerated ROS levels in cancer cells can be explained by a non-oncogene addiction of cancer cells to TrxR1 due to their increased endogenous production of ROS. These separate consequences of TrxR inhibition can be utilized therapeutically. Importantly, however, a thorough knowledge of the molecular mechanisms underlying effects triggered by TrxR inhibition is crucial for the understanding of therapy outcomes after use of such inhibitors. The mammalian thioredoxin system is driven by thioredoxin reductases (TXNRD, TrxR), which keeps thioredoxins (TXN, Trx) and further downstream targets reduced. In normal cells, inhibition of TrxR yields a paradoxical effect of increased antioxidant defense upon activation of the Nrf2 transcription factor. In cancer cells, however, inhibition of TrxR yields excessive reactive oxygen species (ROS) levels resulting in cell death and thus anticancer efficacy, which can be explained by a non-oncogene addiction of cancer cells to TrxR1 due to their increased endogenous production of ROS. These separate consequences of TrxR inhibition can be utilized therapeutically.

Over the past seven decades, research on autotrophic and heterotrophic model organisms has defined how the flow of electrons (“reducing power”) from high-energy inorganic sources, through biological systems, to low-energy inorganic products like water, powers all of Life’s processes. Universally, an initial major biological recipient of these electrons is nicotinamide adenine dinucleotide-phosphate, which thereby transits from an oxidized state (NADP(+)) to a reduced state (NADPH). A portion of this reducing power is then distributed via the cellular NADPH-dependent disulfide reductase systems as sequential reductions of disulfide bonds. Along the disulfide reduction pathways, some enzymes have active sites that use the selenium-containing amino acid, selenocysteine, in place of the common but less reactive sulfur-containing cysteine. In particular, the mammalian/metazoan thioredoxin systems are usually selenium-dependent as, across metazoan phyla, most thioredoxin reductases are selenoproteins. Among the roles of the NADPH-dependent disulfide reductase systems, the most universal is that they provide the reducing power for the production of DNA precursors by ribonucleotide reductase (RNR). Some studies, however, have uncovered examples of NADPH-independent disulfide reductase systems that can also support RNR. These systems are summarized here and their implications are discussed.

Thioredoxin-related protein of 14 kDa (TRP14; also named TXNDC17 for thioredoxin domain-containing protein 17) is a highly conserved and ubiquitously expressed oxidoreductase. It is expressed in parallel with thioredoxin 1 (Trx1, TXN; TXN1), an efficient substrate for the mammalian cytosolic selenoprotein thioredoxin reductase 1 (TrxR1; TXNRD1). However, TRP14, in sharp contrast to Trx1, cannot support the activities of ribonucleotide reductase, peroxiredoxins or methionine sulfoxide reductases, thus is unable to directly support cell proliferation or antioxidant defence through these pathways. However, TRP14 has been shown to efficiently reduce l-cystine, which thereby indirectly supports glutathione synthesis. TRP14 can also suppress NF-κB signalling, is functionally linked to STAT3 signalling, and can directly reactivate oxidized protein-tyrosine phosphatase PTP1B. Furthermore, TRP14 can efficiently reduce persulfidated or nitrosylated cysteine residues in many proteins, thereby having the capacity to modulate signalling through hydrogen sulfide or NO. Additional bioinformatics analyses and observations suggest further roles for TRP14; therefore, further studies of its functions are warranted. Collectively, the results available suggest that TRP14 is a member of the thioredoxin system dedicated to the control of cellular redox signalling pathways. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.