Showing papers by "Elias S.J. Arnér published in 2020"

Control of protein function through oxidation and reduction of persulfidated states

Abstract: 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.

Irreversible TrxR1 inhibitors block STAT3 activity and induce cancer cell death.

Abstract: 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.

Direct Observation of Methylmercury and Auranofin Binding to Selenocysteine in Thioredoxin Reductase.

Abstract: 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.

Characterization of Lead Compounds Targeting the Selenoprotein Thioredoxin Glutathione Reductase for Treatment of Schistosomiasis.

Abstract: 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-resistant 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. The 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 1 h 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.

Common modifications of selenocysteine in selenoproteins.

Abstract: 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.

Cyclic 5-Membered Disulfides Are Not Selective Substrates of Thioredoxin Reductase, but Are Opened Nonspecifically by Thiols

Abstract: 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.

Inhibition and crosslinking of the selenoprotein thioredoxin reductase-1 by p-benzoquinone.

Abstract: Quinones are common in nature, and often cytotoxic. Their proposed toxicity mechanisms involve redox cycling with radical generation, and/or reactions with nucleophiles, such as protein cysteine (Cys) residues, forming adducts via Michael addition reactions. The selenenyl anion of selenocysteine (Sec) is a stronger nucleophile, more prevalent at physiological pH, and more reactive than the corresponding thiolate anion of Cys. We therefore hypothesized that Sec residues should be readily modified by quinones and with potential consequences for the structure and function of selenoproteins. Here, we report data on the interaction of p-benzoquinone (BQ) with the selenoprotein thioredoxin reductase-1 (TrxR1), which exposes an accessible Sec residue upon physiological reduction by NADPH. Our results reveal that BQ targets NADPH-reduced TrxR1 and inhibits its activity using 5,5′-dithiobis(2-nitrobenzoic acid) or juglone as model substrates, consistent with the targeting of both the Cys and Sec residues of TrxR1. In the absence of NADPH, BQ modified the non-catalytic Cys residues, leading to subunit crosslinking, mainly through disulfides, which also resulted in some loss of activity. This crosslinking was time-dependent and independent of the Sec residue. Addition of NADPH after BQ pre-treatment could resolve the disulfide-linked crosslinking. TrxR activity loss was also observed upon incubation of J774A.1 cells or cell lysates with BQ. These data suggest that BQ readily targets TrxR1, albeit in a rather complex manner, which results in structural changes and loss of enzyme activity. We suggest that TrxR1 targeting can explain some of the cytotoxicity of BQ, and potentially also that of other quinone compounds.

Perspectives of TrxR1-based cancer therapies

Abstract: 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.

To inhibit TrxR1 is to inactivate STAT3-Inhibition of TrxR1 enzymatic function by STAT3 small molecule inhibitors.

Abstract: The transcription factor STAT3 plays a key role in cancer and immunity, being widely explored as a potential drug target for the development of novel immunomodulatory or anticancer therapeutics. The mechanisms of small molecule-derived inhibition of STAT3 appear, however, to be more complex than initially perceived. Our recent discovery, that some novel STAT3 inhibitors were bona fide inhibitors of the cytosolic selenoprotein oxidoreductase TrxR1 (TXNRD1), led us to explore the effects of a wide array of previously described STAT3 inhibitors on TrxR1 function. We found that 17 out of 23 tested STAT3 small molecule inhibitors indeed inhibited purified TrxR1 at the reported concentrations yielding STAT3 inhibition. All tested compounds were electrophilic as shown by direct reactivities with GSH, and several were found to also be redox cycling substrates of TrxR1. Ten compounds previously shown to inhibit STAT3 were here found to irreversibly inhibit cellular TrxR1 activity (Auranofin, Stattic, 5,15-DPP, Galiellalactone, LLL12, Napabucasin, BP1-102, STA-21, S3I-201 and Degrasyn (WP1130)). Our findings suggest that targeting of TrxR1 may be a common feature for many small molecules that inhibit cellular STAT3 function. It is possible that prevention of STAT3 activation in cells by several small molecules classified as STAT3 inhibitors can be a downstream event following TrxR1 inhibition. Therefore, the relationship between TrxR1 and STAT3 should be considered when studying inhibition of either of these promising drug targets.

Identification and targeting of selective vulnerability rendered by tamoxifen resistance.

Abstract: The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments. To identify compounds able to overcome TMX resistance, we used short-term and long-term viability assays in cancer cells in vitro and in patient samples in 3D ex vivo, analysis of gene expression profiles and cell line pharmacology database, shRNA screen, CRISPR-Cas9 genome editing, real-time PCR, immunofluorescent analysis, western blot, measurement of oxidative stress using flow cytometry, and thioredoxin reductase 1 enzymatic activity. Here, for the first time, we provide an ample evidence that a high level of the detoxifying enzyme SULT1A1 confers resistance to TMX therapy in both in vitro and ex vivo models and correlates with TMX resistance in metastatic samples in relapsed patients. Based on the data from different approaches, we identified three anticancer compounds, RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis), aminoflavone (AF), and oncrasin-1 (ONC-1), whose tumor cell inhibition activity is dependent on SULT1A1. We discovered thioredoxin reductase 1 (TrxR1, encoded by TXNRD1) as a target of bio-activated RITA, AF, and ONC-1. SULT1A1 depletion prevented the inhibition of TrxR1, induction of oxidative stress, DNA damage signaling, and apoptosis triggered by the compounds. Notably, RITA efficiently suppressed TMX-unresponsive patient-derived breast cancer cells ex vivo. We have identified a mechanism of resistance to TMX via hyperactivated SULT1A1, which renders selective vulnerability to anticancer compounds RITA, AF, and ONC-1, and provide a rationale for a new combination therapy to overcome TMX resistance in breast cancer patients. Our novel findings may provide a strategy to circumvent TMX resistance and suggest that this approach could be developed further for the benefit of relapsed breast cancer patients.

Effects of Mammalian Thioredoxin Reductase Inhibitors.

Abstract: 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.

System-wide identification and prioritization of enzyme substrates by thermal analysis (SIESTA)

Abstract: Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying and prioritizing substrate proteins which are modulated in the structural and functional levels through modification. Here we describe a high-throughput unbiased proteomic method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins might change their thermal stability. 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 in up to a depth of 7179 proteins. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on signal transduction, and facilitate drug discovery.