Ancient Loss of Catalytic Selenocysteine Spurred Convergent Adaptation in a Mammalian Oxidoreductase.

Selenocysteine, the 21st amino acid specified by the genetic code, is a rare selenium-containing residue found in the catalytic site of selenoprotein oxidoreductases. Selenocysteine is analogous to the common cysteine amino acid, but its selenium atom offers physical-chemical properties not provided by the corresponding sulfur atom in cysteine. Catalytic sites with selenocysteine in selenoproteins of vertebrates are under strong purifying selection, but one enzyme, glutathione peroxidase 6 (GPX6), independently exchanged selenocysteine for cysteine <100 million years ago in several mammalian lineages. We reconstructed and assayed these ancient enzymes before and after selenocysteine was lost and up to today and found them to have lost their classic ability to reduce hydroperoxides using glutathione. This loss of function, however, was accompanied by additional amino acid changes in the catalytic domain, with protein sites concertedly changing under positive selection across distant lineages abandoning selenocysteine in glutathione peroxidase 6. This demonstrates a narrow evolutionary range in maintaining fitness when sulfur in cysteine impairs the catalytic activity of this protein, with pleiotropy and epistasis likely driving the observed convergent evolution. We propose that the mutations shared across distinct lineages may trigger enzymatic properties beyond those in classic glutathione peroxidases, rather than simply recovering catalytic rate. These findings are an unusual example of adaptive convergence across mammalian selenoproteins, with the evolutionary signatures possibly representing the evolution of novel oxidoreductase functions.

Piperazine-Fused Cyclic Disulfides Unlock High-Performance Bioreductive Probes of Thioredoxins and Bifunctional Reagents for Thiol Redox Biology.

We report piperazine-fused six-membered-cyclic disulfides as redox substrates that unlock best-in-class bioreduction probes for live cell biology, since their self-immolation after reduction is unprecedentedly rapid. We develop scalable, diastereomerically pure, six-step syntheses that access four key cis- and trans-piperazine-fused cyclic dichalcogenides without chromatography. Fluorogenic redox probes using the disulfide piperazines are activated >100-fold faster than the prior art monoamines, allowing us to deconvolute reduction and cyclization rates during activation. The cis- and trans-fused diastereomers have remarkably different reductant specificities, which we trace back to piperazine boat/chair conformation effects: the cis-fused disulfide C-DiThia is activated only by strong vicinal dithiol reductants, but the trans-disulfide T-DiThia is activated even by moderate concentrations of monothiols such as GSH. Thus, in cellular applications, cis-disulfide probes selectively report on the reductive activity of the powerful thioredoxin proteins, while trans-disulfides are rapidly but promiscuously reactive. Finally, we showcase late-stage diversifications of the piperazine-disulfides, promising their broad applicability as redox-cleavable cores for probes and prodrugs that interface powerfully with cellular thiol/disulfide redox biology, for solid phase synthesis and purification, and for stimulus-responsive linkers in bifunctional reagents and antibody-drug conjugates - in addition to their dithiols' potential as high-performance reducing agents.

Arsenic exposure and increased C-reactive protein are independently associated with lower erythrocyte glutathione peroxidase activity in Bangladeshi children.

Toxic metal contaminants present in food and water have widespread effects on health and disease. Chalcophiles, such as arsenic, cadmium, and mercury, show a high affinity to selenium and exposure to these metals could have a modulating effect on enzymes dependent on selenocysteine in their active sites. The aim of this study was to assess the influence of these metals on the activity of the selenoprotein glutathione peroxidase 1 (GPX1) in erythrocytes of 100 children residing in rural Bangladesh, where drinking water often contains arsenic. GPX1 expression, as measured using high-throughput immunoblotting, showed little correlation with GPX activity (rs = 0.02, p = 0.87) in blood samples. Toxic metals and selenium measured in erythrocytes using inductively coupled plasma mass spectrometry (ICP-MS) and C-reactive protein (CRP) measured in plasma, were all considered as effectors of this divergence in GPX enzymatic activity. Arsenic concentrations in erythrocytes were most influential for GPX1 activity (rs = -0.395, p < 0.0001), and CRP levels also negatively impacted GPX1 activity (rs = -0.443, p < 0.0001). These effects appear independent of each other as arsenic concentrations and CRP showed no correlation (rs = 0.124, p = 0.2204). Erythrocyte selenium, cadmium, and mercury did not show any correlation with GPX1 activity, nor with CRP or arsenic. Our findings suggest that childhood exposure to inorganic arsenic, as well as inflammation triggering the release of CRP, may negatively affect GPX1 activity in erythrocytes.

Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H2O2, and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells.

Long-term treatment of schizophrenia with clozapine (CLZ), an atypical antipsychotic drug, is associated with an increased incidence of metabolic disorders mediated by poorly understood mechanisms. We herein report that CLZ, while slowing down the morphological changes and lipid accumulation occurring during SW872 cell adipogenesis, also causes an early (day 3) inhibition of the expression/nuclear translocation of CAAT/enhancer-binding protein ß and peroxisome proliferator-activated receptor γ. Under the same conditions, CLZ blunts NADPH oxidase-derived reactive oxygen species (ROS) by a dual mechanism involving enzyme inhibition and ROS scavenging. These effects were accompanied by hampered activation of the nuclear factor (erythroid-derived2)-like 2 (Nrf2)-dependent antioxidant responses compared to controls, and by an aggravated formation of mitochondrial superoxide. CLZ failed to exert ROS scavenging activities in the mitochondrial compartment but appeared to actively scavenge cytosolic H2O2 derived from mitochondrial superoxide. The early formation of mitochondrial ROS promoted by CLZ was also associated with signs of mitochondrial dysfunction. Some of the above findings were recapitulated using mouse embryonic fibroblasts. We conclude that the NADPH oxidase inhibitory and cytosolic ROS scavenging activities of CLZ slow down SW872 cell adipogenesis and suppress their Nrf2 activation, an event apparently connected with increased mitochondrial ROS formation, which is associated with insulin resistance and metabolic syndrome. Thus, the cellular events characterised herein may help to shed light on the more detailed molecular mechanisms explaining some of the adverse metabolic effects of CLZ.

TXNL1 has dual functions as a redox active thioredoxin-like protein as well as an ATP- and redox-independent chaperone.

TXNL1 (also named TRP32, for thioredoxin related protein of 32 kDa) is a cytosolic thioredoxin-fold protein expressed in all cell types and conserved from yeast to mammals, but with yet poorly known function. Here, we expressed and purified human TXNL1 together with several Cys-to-Ser variants, characterizing their enzymatic properties. TXNL1 could reduce disulfides in insulin, cystine and glutathione disulfide (GSSG) in reactions coupled to thioredoxin reductase (TXNRD1, TrxR1) using NADPH, similarly to thioredoxin (TXN, Trx1), but with lower catalytic efficacy due to at least one order of magnitude higher Km of TrxR1 for TXNL1 compared to Trx1. However, in sharp contrast to Trx1, we found that TXNL1 also had efficient chaperone activity that did not require ATP. TXNL1 made non-covalent complexes with reduced insulin, thereby keeping it in solution, and TXNL1 provided chaperone function towards whole cell lysate proteins by preventing their aggregation during heating. The chaperone activities of TXNL1 did not require its redox activity or any dithiol-disulfide exchange reactions, as revealed using Cys-to-Ser substituted variants, as well as a maintained chaperone activity of TXNL1 also in the absence of TrxR1 and NADPH. These results reveal that TXNL1 has dual functions, supporting TrxR1-driven redox activities in disulfide reduction reactions, as well as being an ATP-independent chaperone that does not require involvement of its redox activity.

Cardiolipin drives the catalytic activity of GPX4 on membranes: Insights from the R152H mutant.

The aim of this study was to examine, in biochemical detail, the functional role of the Arg152 residue in the selenoprotein Glutathione Peroxidase 4 (GPX4), whose mutation to His is involved in Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD). Wild-type and mutated recombinant enzymes with selenopcysteine (Sec) at the active site, were purified and structurally characterized to investigate the impact of the R152H mutation on enzymatic function. The mutation did not affect the peroxidase reaction's catalytic mechanism, and the kinetic parameters were qualitatively similar between the wild-type enzyme and the mutant when mixed micelles and monolamellar liposomes containing phosphatidylcholine and its hydroperoxide derivatives were used as substrate. However, in monolamellar liposomes also containing cardiolipin, which binds to a cationic area near the active site of GPX4, including residue R152, the wild-type enzyme showed a non-canonical dependency of the reaction rate on the concentration of both enzyme and membrane cardiolipin. To explain this oddity, a minimal model was developed encompassing the kinetics of both the enzyme interaction with the membrane and the catalytic peroxidase reaction. Computational fitting of experimental activity recordings showed that the wild-type enzyme was surface-sensing and prone to "positive feedback" in the presence of cardiolipin, indicating a positive cooperativity. This feature was minimal, if any, in the mutant. These findings suggest that GPX4 physiology in cardiolipin containing mitochondria is unique, and emerges as a likely target of the pathological dysfunction in SSMD.

Novel gold(III)-dithiocarbamate complex targeting bacterial thioredoxin reductase: antimicrobial activity, synergy, toxicity, and mechanistic insights.

Introduction: Antimicrobial resistance is a pressing global concern that has led to the search for new antibacterial agents with novel targets or non-traditional approaches. Recently, organogold compounds have emerged as a promising class of antibacterial agents. In this study, we present and characterize a (C^S)-cyclometallated Au(III) dithiocarbamate complex as a potential drug candidate. Methods and results: The Au(III) complex was found to be stable in the presence of effective biological reductants, and showed potent antibacterial and antibiofilm activity against a wide range of multidrug-resistant strains, particularly gram-positive strains, and gram-negative strains when used in combination with a permeabilizing antibiotic. No resistant mutants were detected after exposing bacterial cultures to strong selective pressure, indicating that the complex may have a low propensity for resistance development. Mechanistic studies indicate that the Au(III) complex exerts its antibacterial activity through a multimodal mechanism of action. Ultrastructural membrane damage and rapid bacterial uptake suggest direct interactions with the bacterial membrane, while transcriptomic analysis identified altered pathways related to energy metabolism and membrane stability including enzymes of the TCA cycle and fatty acid biosynthesis. Enzymatic studies further revealed a strong reversible inhibition of the bacterial thioredoxin reductase. Importantly, the Au(III) complex demonstrated low cytotoxicity at therapeutic concentrations in mammalian cell lines, and showed no acute in vivo toxicity in mice at the doses tested, with no signs of organ toxicity. Discussion: Overall, these findings highlight the potential of the Au(III)-dithiocarbamate scaffold as a basis for developing novel antimicrobial agents, given its potent antibacterial activity, synergy, redox stability, inability to produce resistant mutants, low toxicity to mammalian cells both in vitro and in vivo, and non-conventional mechanism of action.

Development of an assay pipeline for the discovery of novel small molecule inhibitors of human glutathione peroxidases GPX1 and GPX4.

Selenoprotein glutathione peroxidases (GPX), like ubiquitously expressed GPX1 and the ferroptosis modulator GPX4, enact antioxidant activities by reducing hydroperoxides using glutathione. Overexpression of these enzymes is common in cancer and can be associated with the development of resistance to chemotherapy. GPX1 and GPX4 inhibitors have thus shown promise as anti-cancer agents, and targeting other GPX isoforms may prove equally beneficial. Existing inhibitors are often promiscuous, or modulate GPXs only indirectly, so novel direct inhibitors identified through screening against GPX1 and GPX4 could be valuable. Here, we developed optimized glutathione reductase (GR)-coupled GPX assays for the biochemical high-throughput screen (HTS) of almost 12,000 compounds with proposed mechanisms of action. Initial hits were triaged using a GR counter-screen, assessed for isoform specificity against an additional GPX isoform, GPX2, and were assessed for general selenocysteine-targeting activity using a thioredoxin reductase (TXNRD1) assay. Importantly, 70% of the GPX1 inhibitors identified in the primary screen, including several cephalosporin antibiotics, were found to also inhibit TXNRD1, while auranofin, previously known as a TXNRD1 inhibitor, also inhibited GPX1 (but not GPX4). Additionally, every GPX1 inhibitor identified (including omapatrilat, tenatoprazole, cefoxitin and ceftibuten) showed similar inhibitory activity against GPX2. Some compounds inhibiting GPX4 but not GPX1 or GPX2, also inhibited TXNRD1 (26%). Compounds only inhibiting GPX4 included pranlukast sodium hydrate, lusutrombopag, brilanestrant, simeprevir, grazoprevir (MK-5172), paritaprevir, navitoclax, venetoclax and VU0661013. Two compounds (metamizole sodium and isoniazid sodium methanesulfate) inhibited all three GPXs but not TXNRD1, while 2,3-dimercaptopropanesulfonate, PI4KIII beta inhibitor 3, SCE-2174 and cefotetan sodium inhibited all tested selenoproteins (but not GR). The detected overlaps in chemical space suggest that the counter screens introduced here should be imperative for identification of specific GPX inhibitors. With this approach, we could indeed identify novel GPX1/GPX2- or GPX4-specific inhibitors, thus presenting a validated pipeline for future identification of specific selenoprotein-targeting agents. Our study also identified GPX1/GPX2, GPX4 and/or TXNRD1 as targets for several previously developed pharmacologically active compounds.

The ferroptosis inducing compounds RSL3 and ML162 are not direct inhibitors of GPX4 but of TXNRD1.

Ferroptosis is defined as cell death triggered by iron-dependent lipid peroxidation that is preventable by antioxidant compounds such as ferrostatin-1. Endogenous suppressors of ferroptosis include FSP-1 and the selenoprotein GPX4, the latter of which directly enzymatically reduces lipid hydroperoxides. Small molecules that trigger ferroptosis include RSL3, ML162, and ML210; these compounds are often used in studies of ferroptosis and are generally considered as GPX4 inhibitors. Here, we found that RSL3 and ML162 completely lack capacity of inhibiting the enzymatic activity of recombinant selenoprotein GPX4. Surprisingly, these compounds were instead found to be efficient inhibitors of another selenoprotein, TXNRD1. Other known inhibitors of TXNRD1, including auranofin, TRi-1 and TRi-2, are also efficient inducers of cell death but that cell death could not be suppressed with ferrostatin-1. Our results collectively suggest that prior studies using RSL3 and ML162 may need to be reevaluated in the context of ferroptosis with regards to additional enzyme targets and mechanisms of action that may be involved.

Selective cellular probes for mammalian thioredoxin reductase TrxR1: rational design of RX1, a modular 1,2-thiaselenane redox probe.

Quantifying the activity of key cellular redox players is crucial for understanding physiological homeostasis, and for targeting their perturbed states in pathologies including cancer and inflammatory diseases. However, cellularly-selective probes for oxidoreductase turnover are sorely lacking. We rationally developed the first probes that selectively target the mammalian selenoprotein thioredoxin reductase (TrxR), using a cyclic selenenylsulfide oriented to harness TrxR's unique selenolthiol chemistry while resisting the cellular monothiol background. Lead probe RX1 had excellent TrxR1-selective performance in cells, cross-validated by knockout, selenium starvation, knock-in, and chemical inhibitors. Its background-free fluorogenicity enabled us to perform the first quantitative high-throughput live cell screen for TrxR1 inhibitors, which indicated that tempered SNAr electrophiles may be more selective TrxR drugs than the classical electrophiles used hitherto. The RX1 design thus sets the stage for in vivo imaging of the activity of this key oxidoreductase in health and disease, and can also drive TrxR1-inhibitor drug design.

Thioredoxin reductase selenoproteins from different organisms as potential drug targets for treatment of human diseases.

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.

Targeting of Nrf2 improves antitumoral responses by human NK cells, TIL and CAR T cells during oxidative stress.

BACKGROUND: Adoptive cell therapy using cytotoxic lymphocytes is an efficient immunotherapy against solid and hematological cancers. However, elevated levels of reactive oxygen species (ROS) in the hostile tumor microenvironment can impair NK cell and T cell function. Auranofin, a gold (I)-containing phosphine compound, is a strong activator of the transcription factor Nrf2. Nrf2 controls a wide range of downstream targets important for the cells to obtain increased resistance to ROS. In this study, we present a strategy using auranofin to render human cytotoxic lymphocytes resistant toward oxidative stress. METHODS: Melanoma patient-derived tumor infiltrating lymphocytes (TIL) and healthy donor-derived NK cells and CD19-directed CAR T cells were pretreated with a low dose of auranofin. Their resistance toward oxidative stress was assessed by measuring antitumoral responses (killing-assay, degranulation/CD107a, cytokine production) and intracellular ROS levels (flow cytometry) in conditions of oxidative stress. To confirm that the effects were Nrf2 dependent, the transcription level of Nrf2-driven target genes was analyzed by qPCR. RESULTS: Pretreatment of human TIL and NK cells ex vivo with a low-dose auranofin significantly lowered their accumulation of intracellular ROS and preserved their antitumoral activity despite high H2O2 levels or monocyte-derived ROS. Furthermore, auranofin pretreatment of CD19 CAR-T cells or TIL increased their elimination of CD19 +tumor cells or autologous tumor spheroids, respectively, especially during ROS exposure. Analysis of Nrf2-driven target genes revealed that the increased resistance against ROS was Nrf2 dependent. CONCLUSION: These novel findings suggest that Nrf2 activation in human cytotoxic lymphocytes could be used to enhance the efficacy of adoptive cell therapy.

Redox regulation of PTPN22 affects the severity of T-cell-dependent autoimmune inflammation.

Chronic autoimmune diseases are associated with mutations in PTPN22, a modifier of T cell receptor (TCR) 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 noncatalytic C129 residue and oxidation-prone PTPN22 leads to increased severity in the development of T-cell-dependent autoimmunity.

Cyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically.

The cyclic five-membered disulfide 1,2-dithiolane has been widely used in chemical biology and in redox probes. Contradictory reports have described it either as nonspecifically reduced in cells, or else as a highly specific substrate for thioredoxin reductase (TrxR). Here we show that 1,2-dithiolane probes, such as "TRFS" probes, are nonspecifically reduced by thiol reductants and redox-active proteins, and their cellular performance is barely affected by TrxR inhibition or knockout. Therefore, results of cellular imaging or inhibitor screening using 1,2-dithiolanes should not be interpreted as reflecting TrxR activity, and previous studies may need re-evaluation. To understand 1,2-dithiolanes' complex behaviour, probe localisation, environment-dependent fluorescence, reduction-independent ring-opening polymerisation, and thiol-dependent cellular uptake must all be considered; particular caution is needed when co-applying thiophilic inhibitors. We present a general approach controlling against assay misinterpretation with reducible probes, to ensure future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research.

Biochemical and structural characterizations of thioredoxin reductase selenoproteins of the parasitic filarial nematodes Brugia malayi and Onchocerca volvulus.

Enzymes in the thiol redox systems of microbial pathogens are promising targets for drug development. In this study we characterized the thioredoxin reductase (TrxR) selenoproteins from Brugia malayi and Onchocerca volvulus, filarial nematode parasites and causative agents of lymphatic filariasis and onchocerciasis, respectively. The two filarial enzymes showed similar turnover numbers and affinities for different thioredoxin (Trx) proteins, but with a clear preference for the autologous Trx. Human TrxR1 (hTrxR1) had a high and similar specific activity versus the human and filarial Trxs, suggesting that, in vivo, hTrxR1 could possibly be the reducing agent of parasite Trxs once they are released into the host. Both filarial TrxRs were efficiently inhibited by auranofin and by a recently described inhibitor of human TrxR1 (TRi-1), but not as efficiently by the alternative compound TRi-2. The enzyme from B. malayi was structurally characterized also in complex with NADPH and auranofin, producing the first crystallographic structure of a nematode TrxR. The protein represents an unusual fusion of a mammalian-type TrxR protein architecture with an N-terminal glutaredoxin-like (Grx) domain lacking typical Grx motifs. Unlike thioredoxin glutathione reductases (TGRs) found in platyhelminths and mammals, which are also Grx-TrxR domain fusion proteins, the TrxRs from the filarial nematodes lacked glutathione disulfide reductase and Grx activities. The structural determinations revealed that the Grx domain of TrxR from B. malayi contains a cysteine (C22), conserved in TrxRs from clade IIIc nematodes, that directly interacts with the C-terminal cysteine-selenocysteine motif of the homo-dimeric subunit. Interestingly, despite this finding we found that altering C22 by mutation to serine did not affect enzyme catalysis. Thus, although the function of the Grx domain in these filarial TrxRs remains to be determined, the results obtained provide insights on key properties of this important family of selenoprotein flavoenzymes that are potential drug targets for treatment of filariasis.

Expressing recombinant selenoproteins using redefinition of a single UAG codon in an RF1-depleted E. coli host strain.

Selenoproteins containing the rare amino acid selenocysteine (Sec), typically being enzymes utilizing the selenium atom of Sec for promoted catalysis of redox reactions, are challenging to obtain at high amounts in pure form. The technical challenges limiting selenoprotein supply derive from intricacies in their translation, necessitating the recoding of a UGA stop codon to a sense codon for Sec. This, in turn, involves the interactions of a Sec-dedicated elongation factor, either directly or indirectly, with a structure in the selenoprotein-encoding mRNA called a SECIS element (Selenocysteine Insertion Sequence), a dedicated tRNA species for Sec with an anticodon for the UGA, and several accessory enzymes and proteins involved in the selenoprotein synthesis. Here, we describe an alternative method for recombinant selenoprotein production using UAG as the Sec codon in a specific strain of E. coli lacking other UAG codons and lacking the release factor RF1 that normally terminates translation at UAG. We also describe how such recombinant selenoproteins can be purified and further analyzed for final Sec contents. The methodology can be used for production of natural selenoproteins in recombinant form as well as for production of synthetic selenoproteins that may be designed to use the unique biophysical properties of Sec for diverse biotechnological applications.

Thioredoxin Reductase Inhibition for Cancer Therapy.

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.

Evaluation of dithiothreitol-oxidizing capacity (DOC) as a serum biomarker for chronic hepatitis B in patients exhibiting normal alanine aminotransferase levels: a pilot study towards better monitoring of disease.

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).

Comprehensive chemical proteomics analyses reveal that the new TRi-1 and TRi-2 compounds are more specific thioredoxin reductase 1 inhibitors than auranofin.

Anticancer drugs that target cellular antioxidant systems have recently attracted much attention. Auranofin (AF) is currently evaluated in several clinical trials as an anticancer agent that targets the cytosolic and mitochondrial forms of the selenoprotein thioredoxin reductase, TXNRD1 and TXNRD2. Recently, two novel TXNRD1 inhibitors (TRi-1 and TRi-2) have been developed that showed anticancer efficacy comparable to AF, but with lower mitochondrial toxicity. However, the cellular action mechanisms of these drugs have not yet been thoroughly studied. Here we used several proteomics approaches to determine the effects of AF, TRi-1 and TRi-2 when used at IC50 concentrations with the mouse B16 melanoma and LLC lung adenocarcinoma cells, as these are often used for preclinical mouse models in evaluation of anticancer drugs. The results demonstrate that TRi-1 and TRi-2 are more specific TXNRD1 inhibitors than AF and reveal additional AF-specific effects on the cellular proteome. Interestingly, AF triggered stronger Nrf2-driven antioxidant responses than the other two compounds. Furthermore, AF affected several additional proteins, including GSK3A, GSK3B, MCMBP and EEFSEC, implicating additional effects on glycogen metabolism, cellular differentiation, inflammatory pathways, DNA replication and selenoprotein synthesis processes. Our proteomics data provide a resource for researchers interested in the multidimensional analysis of proteome changes associated with oxidative stress in general, and the effects of TXNRD1 inhibitors and AF protein targets in particular.

Development of therapies for rare genetic disorders of GPX4: roadmap and opportunities.

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.