Therapeutic targeting of thioredoxin reductase 1 causes ferroptosis while potentiating anti-PD-1 efficacy in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) faces low response rates to anti-PD-1 immunotherapies, highlighting the need for enhanced treatment strategies. Auranofin, which inhibits thioredoxin reductase (TrxR) through its gold-based composition, has shown potential in cancer treatment. It targets the TrxR system, essential for safeguarding cells from oxidative stress. The overproduction of TrxR in cancerous cells supports their proliferation. However, auranofin's interference with this system can upset the cellular redox equilibrium, boost levels of reactive oxygen species, and trigger the death of cancer cells. This study is the first to highlight TXNRD1 as a crucial factor contributing to resistance to anti-PD-1 treatment in HNSCC. In this study, we identified targetable regulators of resistance to immunotherapy-induced ferroptosis in HNSCC. We observed a link of thioredoxin reductase 1 (TXNRD1) with tumoral PD-L1 expression and ferroptosis suppression in HNSCC. Moreover, HNSCC tumors with aberrant TXNRD1 expression exhibited a lack of PD-1 response, NRF2 overexpression, and PD-L1 upregulation. TXNRD1 inhibition promoted ferroptosis in HNSCC cells with NRF2 activation and in organoid tumors derived from patients lacking a PD-1 response. Mechanistically, TXNRD1 regulated PD-L1 transcription and maintained the redox balance by binding to ribonucleotide reductase regulatory subunit M2 (RRM2). TXNRD1 expression disruption sensitized HNSCC cells to anti-PD-1-mediated Jurkat T-cell activation, promoting tumor killing through ferroptosis. Moreover, TXNRD1 inhibition through auranofin cotreatment synergized with anti-PD-1 therapy to potentiate immunotherapy-mediated ferroptosis by mediating CD8+ T-cell infiltration and downregulating PD-L1 expression. Our findings indicate that targeting TXNRD1 is a promising therapeutic strategy for improving immunotherapy outcomes in patients with HNSCC.

Synergistic targeting of TrxR1 and ATM/AKT pathway in human colon cancer cells.

Thioredoxin reductase 1 (TrxR1) has emerged as a promising target for cancer therapy. In our previous research, we discovered several new TrxR1 inhibitors and found that they all have excellent anti-tumor activity. At the same time, we found these TrxR1 inhibitors all lead to an increase in AKT phosphorylation in cancer cells, but the detailed role of AKT phosphorylation in TrxR1 inhibitor-mediated cell death remains unclear. In this study, we identified the combination of AKT and TrxR1 inhibitor displayed a strong synergistic effect in colon cancer cells. Furthermore, we demonstrated that the synergistic effect of auranofin (TrxR1 inhibitor) and MK-2206 (AKT inhibitor) was caused by ROS accumulation. Importantly, we found that ATM inhibitor KU-55933 can block the increase of AKT phosphorylation caused by auranofin, and exhibited a synergistic effect with auranofin. Taken together, our study demonstrated that the activation of ATM/AKT pathway is a compensatory mechanism to cope with ROS accumulation induced by TrxR1 inhibitor, and synergistic targeting of TrxR1 and ATM/AKT pathway is a promising strategy for treating colon cancer.

Discovery of novel hydroxyamidine based indoleamine 2,3-dioxygenase 1 (IDO1) and thioredoxin reductase 1 (TrxR1) dual inhibitors.

In order to take advantage of both immunotherapeutic and metabolic antitumor agents, novel dual indoleamine 2,3- dioxygenase 1 (IDO1) and thioredoxin reductase 1 (TrxR1) inhibitors were designed. Thioredoxin reductase 1 (TrxR1) is a main ROS modulator within CRC cells. Indoleamine 2,3-dioxygenase (IDO1) is crucial controller for tryptophan (Trp) metabolism that is also important for CRC immunotherapy. Herein, ten compounds 12a-j containing hydroxyamidine scaffold were designed, synthesized and evaluated for inhibitory activities against IDO1/TrxR1 enzyme and CRC cells. Among these compounds, the most active compound 12d (ZC0109) showed excellent and balanced activity against both IDO1 (IC50 = 0.05 µM) and TrxR1 (IC50 = 3.00 ± 0.25 µM) were selected for further evaluation. Compound ZC0109 exhibited good dual inhibition against IDO1 and TrxR1 both in vitro and in vivo. Further mechanistic studies reveal that, through IDO1 and TrxR1 inhibition by ZC0109 treatment, accumulated ROS effectively induced apoptosis and G1/S cell cycle arrest in cancer cells. In vivo evaluation demonstrated excellent anti-tumor effect of ZC0109 with the notable ability of promoting ROS-induced apoptosis, reducing kynurenine level in plasma and restoring anti-tumor immune response. Thus, ZC0109 represents a potential CRC therapy agent for further development.

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.

Ethaselen synergizes with oxaliplatin in tumor growth inhibition by inducing ROS production and inhibiting TrxR1 activity in gastric cancer.

BACKGROUND: Oxaliplatin is one of the most commonly used chemotherapeutic agent for the treatment of various cancers, including gastric cancer. It has, however, a narrow therapeutic index due to its toxicity and the occurrence of drug resistance. Hence, it is of great significance to develop novel therapies to potentiate the anti-tumor effect and reduce the toxicity of oxaliplatin. In our previous study, we demonstrated that ethaselen (BBSKE), an inhibitor of thioredoxin reductase, effectively inhibited the growth of gastric cancer cells and promoted apoptosis in vitro. In the present study, we investigated whether BBSKE can potentiate the anti-tumor effect of oxaliplatin in gastric cancer in vivo and vitro. METHODS: Cellular apoptosis and ROS levels were analyzed by flow cytometry. Thioredoxin reductase 1 (TrxR1) activity in gastric cancer cells, organoid and tumor tissues was determined by using the endpoint insulin reduction assay. Western blot was used to analyze the expressions of the indicated proteins. Nude mice xenograft models were used to test the effects of BBSKE and oxaliplatin combinations on gastric cancer cell growth in vivo. In addition, we also used the combined treatment of BBSKE and oxaliplatin in three cases of gastric cancer Patient-Derived organoid (GC-PDO) to detect the anti-tumor effect. RESULTS: We found that BBSKE significantly enhanced oxaliplatin-induced growth inhibition in gastric cancer cells by inhibiting TrxR1 activity. Because of the inhibition of TrxR1 activity, BBSKE synergized with oxaliplatin to enhance the production of ROS and activate p38 and JNK signaling pathways which eventually induced apoptosis of gastric cancer cells. In vivo, we also found that BBSKE synergized with oxaliplatin to suppress the gastric cancer tumor growth in xenograft nude mice model, accompanied by the reduced TrxR1 activity. Remarkably, we found that BBSKE attenuated body weight loss evoked by oxaliplatin treatment. We also used three cases of GC-PDO and found that the combined treatment of BBSKE and oxaliplatin dramatically inhibited the growth and viability of GC-PDO with increased ROS level, decreased TrxR1 activity and enhanced apoptosis. CONCLUSIONS: This study elucidates the underlying mechanisms of synergistic effect of BBSKE and oxaliplatin, and suggests that the combined treatment has potential value in gastric cancer therapy.

Jolkinolide B targets thioredoxin and glutathione systems to induce ROS-mediated paraptosis and apoptosis in bladder cancer cells.

Bladder cancer is a clinically heterogeneous disease with a poor prognosis. In the current study, anti-proliferation assay of a Euphorbiaceae diterpenoid library led to the identification of an anti-bladder cancer agent Jolkinolide B (JB). JB showed significant cytotoxicity against a panel of bladder cancer cell lines and suppressed the growth of cisplatin (CDDP)-resistant bladder cancer xenografts in single or combination treatments. Mechanistic study revealed that, besides inducing mitogen-activated protein kinase (MAPK)-related apoptosis, JB could trigger the paraptosis via activation of reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress and extracellular signal-regulated kinase (ERK) pathway. The excessive production of ROS could be induced by JB via inhibition of thioredoxin reductase 1 (TrxR1) and depletion of glutathione (GSH). Collectively, JB that targets thioredoxin and GSH systems to induce two distinct cell death modes may serve as a promising candidate in future anti-bladder cancer drug development.

Design, synthesis and biological evaluations of diverse Michael acceptor-based phenazine hybrid molecules as TrxR1 inhibitors.

A series of novel phenazine derivatives (1~27) containing the Michael acceptor scaffolds were designed and synthesized in this study. Some compounds exhibited selective cytotoxicity against Bel-7402 cancer cell line in vitro, in which compound 26 were found to have the best antiproliferative activity. Meanwhile, compound 26 showed no obvious cell toxicity against human normal liver epithelial L02 cells, which means this compound possessed a better safety potential. In the following research, compound 26 was verified to inhibit TrxR1 enzyme activity, ultimately resulting in cellular molecular mechanism events of apoptosis including growth of intracellular ROS level, depletion of reduced Trx1, liberation of ASK1 and up-regulation of p38, respectively. Together, all these evidences implicated that compound 26 acted as the TrxR1 inhibitor against Bel-7402 cells, and could activate apoptosis through the ROS-Trx-ASK1-p38 pathway.

Berberine accelerated wound healing by restoring TrxR1/JNK in diabetes.

The high disability, mortality and morbidity of diabetic ulcers make it urgent to explore effective strategies for diabetic wound repair. TrxR1 plays a vital role in regulating redox homeostasis in various pathologies. In the present study, the effect of berberine (BBR) on diabetic wounds was investigated in streptozotocin (STZ)-induced diabetic rats and a high glucose (HG)-induced cell model, and the mechanism of BBR on TrxR1 was elucidated. BBR treatment remarkably accelerated wound healing and enhanced extracellular matrix (ECM) synthesis and significantly inhibited HG-induced HaCaT cell damage. Further analysis indicated that BBR activated TrxR1, suppressed its downstream JNK signaling, thereby inhibiting oxidative stress and apoptosis, promoted cell proliferation, down-regulated matrix metalloproteinase (MMP) 9 (MMP9) and up-regulated transforming growth factor-ß1 (TGF-ß1) and tissue inhibitors of MMP 1 (TIMP1), resulting in accelerated wound healing. Importantly, the enhancement of BBR on wound repair was further abolished by TrxR1 inhibitor. Moreover, in diabetic wounds induced by a combination of STZ injection and high-fat diet, BBR significantly increased wound closure rate and TrxR1 expression, and this was reversed by TrxR1 inhibitor. These data indicated that topical BBR treatment accelerated diabetic wound healing by activating TrxR1. Targeting TrxR1 may be a novel, effective strategy for restoring redox homeostasis and promoting diabetic wound healing.

Piperlonguminine and Piperine Analogues as TrxR Inhibitors that Promote ROS and Autophagy and Regulate p38 and Akt/mTOR Signaling.

The natural products piperlongumine and piperine have been shown to inhibit cancer cell proliferation through elevation of reactive oxidative species (ROS) and eventually cell death, but only have modest cytotoxic potencies. A series of 14 novel phenylallylidenecyclohexenone analogues based on piperlongumine and piperine therefore were designed and synthesized, and their pharmacological properties were evaluated. Most of the compounds produced antiproliferative activities against five human cancer cells with IC50 values lower than those of piperlongumine and piperine. Among these, compound 9m exerted the most potent antiproliferative activity against drug-resistant Bel-7402/5-FU human liver cancer 5-FU resistant cells (IC50 = 0.8 µM), which was approximately 10-fold lower than piperlongumine (IC50 = 8.4 µM). Further, 9m showed considerably lower cytotoxicity against LO2 human normal liver epithelial cells compared to Bel-7402/5-FU. Mechanistically, compound 9m inhibited thioredoxin reductase (TrxR) activity, increased ROS levels, reduced mitochondrial transmembrane potential (MTP), and induced autophagy in Bel-7402/5-FU cells via regulation of autophagy-related proteins LC3, p62, and beclin-1. Finally, 9m activated significantly the p38 signaling pathways and suppressed the Akt/mTOR signaling pathways. In conclusion, 9m could be a promising candidate for the treatment of drug-resistant cancer cells and, as such, warrants further investigation.

Design, synthesis, and biological evaluation of a novel indoleamine 2,3-dioxigenase 1 (IDO1) and thioredoxin reductase (TrxR) dual inhibitor.

Targeting the Trp-Kyn pathway is an attractive approach for cancer immunotherapy. Thioredoxin reductase (TrxR) enzymes are reactive oxygen species (ROS) modulators that are involved in the tumor cell growth and survival processes. The 4-phenylimidazole scaffold is well-established as useful for indoleamine 2,3-dioxygenase 1 (IDO1) inhibition, while piperlongumine (PL) and its derivatives have been reported to be inhibitors of TrxR. To take advantage of both immunotherapy and TrxR inhibition, we designed a first-generation dual IDO1 and TrxR inhibitor (ZC0101) using the structural combination of 4-phenylimidazole and PL scaffolds. ZC0101 exhibited better dual inhibition against IDO1 and TrxR in vitro and in cell enzyme assays than the uncombined forms of 4-phenylimidazole and PL. It also showed antiproliferative activity in various cancer cell lines, and a selective killing effect between normal and cancer cells. Furthermore, ZC0101 effectively induced apoptosis and ROS accumulation in cancer cells. Knockdown of TrxR1 and IDO1 expression induced cellular enzyme inhibition and ROS accumulation effects during ZC0101 treatment, but only reduced TrxR1 expression was able to improve ZC0101's antiproliferation effect. This proof-of-concept study provides a novel strategy for cancer treatment. ZC0101 represents a promising lead compound for the development of novel antitumor agents that can also be used as a valuable probe to clarify the relationships and mechanisms of cancer immunotherapy and ROS modulators.

Selenium-independent antioxidant and anti-inflammatory effects of thioredoxin reductase inhibition in alveolar macrophages.

AIMS: Interleukin-1ß (IL-1ß) contributes to the development of bronchopulmonary dysplasia (BPD). Thioredoxin reductase-1 (Txnrd1) inhibition activates nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent responses. Txnrd1 activity is selenium (Se) dependent and Se deficiency is common in prematurity. Auranofin (AFN), a Txnrd1 inhibitor, decreases IL-1ß levels and increases Nrf2 activation in lipopolysaccharide (LPS) treated alveolar macrophages. In lung epithelia, AFN-induced Nrf2 activation is Se dependent. We tested the hypothesis that the effects of Txnrd1 inhibition in alveolar macrophages are Se dependent. MAIN METHODS: To establish Se sufficient (Se+) and deficient (Se-) conditions, alveolar (MH-S) macrophages were cultured in 2.5% fetal bovine serum (FBS) ± 25 nM Na2SeO3. Se- (2.5% FBS) and Se+ (2.5% FBS + 25 nM Na2SeO3) cells were cultured in the presence or absence of 0.05 µg/mL LPS and/or 0.5 µM AFN. Nrf2 activation was determined by measuring NADPH quinone oxidoreductase-1 (Nqo1) and glutathione levels. IL-1ß mRNA (Il1b) and protein levels were measured using qRT-PCR and ELISA. Data were analyzed by ANOVA followed by Tukey's post-hoc. KEY FINDINGS: We detected an independent effect of AFN, but not LPS, on Nqo1 expression and GSH levels in Se+ and Se- cells. LPS significantly increased Il1b and IL-1ß levels in both groups. AFN-mediated attenuation of this effect was not impacted by Se status. SIGNIFICANCE: The beneficial effects of Txnrd1 inhibition in alveolar macrophages are Se-independent and therefore unlikely to be diminished by clinical Se deficiency.

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

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.

Inhibition of thioredoxin reductase 1 correlates with platinum-based chemotherapeutic induced tissue injury.

Platinum-containing drugs (PtDs; e.g. cisplatin, carboplatin, and oxaliplatin) have been widely used as anticancer reagents against various cancers. However, treatment with these drugs results in undesirable adverse effects with unknown mechanisms. Herein, we found a strong correlation between the inhibitory effects of PtDs on cytosolic thioredoxin reductase (TXNRD1) and tissue injury. Of the PtDs tested, cisplatin was found to be the most effective inhibitory PtD against TXRND1, causing the severest kidney injury. The initial inhibition of TXNRD1 in the kidney resulted from cisplatin-induced transcriptional activation of Nrf2-regulated genes including Txnrd1. However, the antioxidant responses in the kidney did not reverse the cisplatin-induced oxidation process. Nephrotoxicity was accompanied with an increase of protein glutathionylation and a cellular thiol redox environment oxidation. These results suggest that the changes of the cellular thiol-dependent redox environment regulated by TXNRD1 is a major event in the adverse effects of cisplatin in kidney.

Further exploration of DVD-445 as a lead thioredoxin reductase (TrxR) inhibitor for cancer therapy: Optimization of potency and evaluation of anticancer potential.

A series of analogs of the earlier reported lead compound DVD-445 (thioredoxin reductase inhibitor with anticancer activity) has been synthesized via a modified Ugi reaction and investigated. Seven most potent compounds (with IC50 below 5.00 µM against recombinant rTrxR1 enzyme) were examined for their effect on cell growth and viability, oxidative stress induction and P-glycoprotein (P-gp) inhibition in human glioblastoma cells cell line U87 and its corresponding multidrug resistant (MDR) cell line U87-TxR. Several of these frontrunner compounds were shown to be superior over DVD-445. Besides providing promising candidates for anticancer therapy, our study further validates the small electrophilic Ugi Michael acceptor (UMA) chemotype as efficacious inhibitor of thioredoxin reductase.

Inhibition of TXNRD or SOD1 overcomes NRF2-mediated resistance to ß-lapachone.

Alterations in the NRF2/KEAP1 pathway result in the constitutive activation of NRF2, leading to the aberrant induction of antioxidant and detoxification enzymes, including NQO1. The NQO1 bioactivatable agent ß-lapachone can target cells with high NQO1 expression but relies in the generation of reactive oxygen species (ROS), which are actively scavenged in cells with NRF2/KEAP1 mutations. However, whether NRF2/KEAP1 mutations influence the response to ß-lapachone treatment remains unknown. To address this question, we assessed the cytotoxicity of ß-lapachone in a panel of NSCLC cell lines bearing either wild-type or mutant KEAP1. We found that, despite overexpression of NQO1, KEAP1 mutant cells were resistant to ß-lapachone due to enhanced detoxification of ROS, which prevented DNA damage and cell death. To evaluate whether specific inhibition of the NRF2-regulated antioxidant enzymes could abrogate resistance to ß-lapachone, we systematically inhibited the four major antioxidant cellular systems using genetic and/or pharmacologic approaches. We demonstrated that inhibition of the thioredoxin-dependent system or copper-zinc superoxide dismutase (SOD1) could abrogate NRF2-mediated resistance to ß-lapachone, while depletion of catalase or glutathione was ineffective. Interestingly, inhibition of SOD1 selectively sensitized KEAP1 mutant cells to ß-lapachone exposure. Our results suggest that NRF2/KEAP1 mutational status might serve as a predictive biomarker for response to NQO1-bioactivatable quinones in patients. Further, our results suggest SOD1 inhibition may have potential utility in combination with other ROS inducers in patients with KEAP1/NRF2 mutations.

FoxO3 reverses 5-fluorouracil resistance in human colorectal cancer cells by inhibiting the Nrf2/TR1 signaling pathway.

5-fluorouracil (5-FU) is widely used in chemotherapy for colorectal cancer (CRC), but a high rate of chemoresistance reduces its effectiveness in clinical treatment. We found remarkably decreased expression of forkhead box 3 (FoxO3) protein, a tumor inhibitor, in 5-FU-resistant SW620 and HCT-8 (SW620/5-FU and HCT-8/5-FU) cells. Moreover, FoxO3 overexpression sensitized SW620/5-FU and HCT-8/5-FU cells to 5-FU. Mechanistically, FoxO3 inhibited the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway by directly binding to Keap1 promoter. Thioredoxin reductase 1 (TR1), a pivotal target gene of Nrf2, was observed to promote 5-FU resistance by reducing intracellular ROS levels. Clinical data also revealed that significant upregulation of TR1 was associated with poor outcome in CRC patients. Auranofin (AUR), a FoxO3 agonist and TR1 inhibitor, enhanced the sensitivity of HCT-8/5-FU and SW620/5-FU cells to 5-FU in vitro and in vivo. Taken together, our results suggest that FoxO3 could reverse 5-FU resistance in CRC via inhibiting the Nrf2/TR1 signaling pathway, and increasing the level of intracellular reactive oxygen species. Chemotherapeutic agents targeting FoxO3 and/or TR1, including AUR, might be promising adjuvant sensitizers to reverse chemoresistance in 5-FU-resistant CRC.

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

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.

ROS-mediated inactivation of the PI3K/AKT pathway is involved in the antigastric cancer effects of thioredoxin reductase-1 inhibitor chaetocin.

Novel drugs are urgently needed for gastric cancer (GC) treatment. The thioredoxin-thioredoxin reductase (TRX-TRXR) system has been found to play a critical role in GC tumorigenesis and progression. Thus, agents that target the TRX-TRXR system may be highly efficacious as GC treatments. In this study, we showed that chaetocin, a natural product isolated from the Chaetomium species of fungi, inhibited proliferation, induced G2/M phase arrest and caspase-dependent apoptosis in both in vitro and in vivo models (cell xenografts and patient-derived xenografts) of GC. Chaetocin inactivated TRXR-1, resulting in the accumulation of reactive oxygen species (ROS) in GC cells; overexpression of TRX-1 as well as cotreatment of GC cells with the ROS scavenger N-acetyl-L-cysteine attenuated chaetocin-induced apoptosis; chaetocin-induced apoptosis was significantly increased when GC cells were cotreated with auranofin. Moreover, chaetocin was shown to inactivate the PI3K/AKT pathway by inducing ROS generation; AKT-1 overexpression also attenuated chaetocin-induced apoptosis. Taken together, these results reveal that chaetocin induces the excessive accumulation of ROS via inhibition of TRXR-1. This is followed by PI3K/AKT pathway inactivation, which ultimately inhibits proliferation and induces caspase-dependent apoptosis in GC cells. Chaetocin therefore may be a potential agent for GC treatment.

Novel electrophilic amides amenable by the Ugi reaction perturb thioredoxin system via thioredoxin reductase 1 (TrxR1) inhibition: Identification of DVD-445 as a new lead compound for anticancer therapy.

A series of peptidomimetic compounds incorporating an electrophilic moiety was synthesized using the Ugi reaction. These compounds (termed the Ugi Michael acceptors or UMAs) were designed to target the selenocysteine catalytic residue of thioredoxin reductase 1 (TrxR1), a promising cancer target. The compounds were assessed for their potential to inhibit TrxR1 using human neuroblastoma (SH-SY5Y) cell lysate. Based on this initial screening, six compounds were selected for testing against recombinant rat TrxR1 and in the insulin assay to reveal low-micromolar to submicromolar potency of these inhibitors. The same frontrunner compounds were evaluated for their ability to exert antiproliferative activity and induce cell death and this activity was compared to the UMA effects on the levels of reactive oxygen and nitrogen species (RONS). Collectively, the UMA compounds class presented itself as a rich source of leads for TrxR1 inhibitor discovery for anticancer application. Compound 7 (DVD-445) was nominated a lead for further optimization.

A fast and specific fluorescent probe for thioredoxin reductase that works via disulphide bond cleavage.

Small molecule probes are indispensable tools to explore diverse cellular events. However, finding a specific probe of a target remains a high challenge. Here we report the discovery of Fast-TRFS, a specific and superfast fluorogenic probe of mammalian thioredoxin reductase, a ubiquitous enzyme involved in regulation of diverse cellular redox signaling pathways. By systematically examining the processes of fluorophore release and reduction of cyclic disulfides/diselenides by the enzyme, structural factors that determine the response rate and specificity of the probe are disclosed. Mechanistic studies reveal that the fluorescence signal is switched on by a simple reduction of the disulfide bond within the probe, which is in stark contrast to the sensing mechanism of published probes. The favorable properties of Fast-TRFS enable development of a high-throughput screening assay to discover inhibitors of thioredoxin reductase by using crude tissue extracts as a source of the enzyme.