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Thioredoxin Reductase (TrxR) functions to recycle thioredoxin (Trx) during hydroperoxide metabolism mediated by peroxiredoxins and is currently being targeted using the FDA-approved anti-rheumatic drug, auranofin (AF), to selectively sensitize cancer cells to therapy. AF treatment decreased TrxR activity and clonogenic survival in small cell lung cancer (SCLC) cell lines (DMS273 and DMS53) as well as the H727 atypical lung carcinoid cell line. AF treatment also significantly sensitized DMS273 and H727 cell lines in vitro to sorafenib, an FDA-approved multi-kinase inhibitor that depleted intracellular glutathione (GSH). The pharmacokinetic, pharmacodynamic, and safety profile of AF was examined in nude mice with DMS273 xenografts administered AF intraperitoneally at 2 mg/kg or 4 mg/kg (IP) once (QD) or twice daily (BID) for 1-5 d. Plasma levels of AF were 10-20 μM (determined by mass spectrometry of gold), and the optimal inhibition of TrxR activity was obtained at 4 mg/kg once daily, with no effect on glutathione peroxidase 1 activity. This AF treatment extended for 14 d, inhibited TrxR (>75%), and resulted in a significant prolongation of median overall survival from 19 to 23 d ( p  = .04, N  = 30 controls, 28 AF). In this experiment, there were no observed changes in animal bodyweight, complete blood counts (CBCs), bone marrow toxicity, blood urea nitrogen, or creatinine. These results support the hypothesis that AF effectively inhibits TrxR both in vitro and in vivo in SCLC, sensitizes NETs and SCLC to sorafenib, and could be repurposed as an adjuvant therapy with targeted agents that induce disruptions in thiol metabolism.

Thioredoxin reductase 1 (TXNRD1) has been identified as one of the promising chemotherapeutic targets in cancer cells. Therefore, a novel TXNRD1 inhibitor could accelerate chemotherapy in clinical anticancer research. In this study, glaucocalyxin A (GlauA), a natural diterpene extracted from Rabdosia japonica var. glaucocalyx, was identified as a novel inhibitor of TXNRD1. We found that GlauA effectively inhibited recombinant TXNRD1 and reduced its activity in gastric cancer cells without affecting the enzyme's expression level. Mechanistically, the selenocysteine residue (U498) of TXNRD1 was irreversibly modified by GlauA through a Michael addition. Additionally, GlauA formed a covalent adduct with glutathione (GSH) and disrupted cellular redox balance by depleting cellular GSH. The inhibition of TXNRD1 and depletion of GSH by GlauA conferred its cytotoxic effects in spheroid culture and Transwell assays in AGS cells. The disulfide stress induced cytotoxicity of GlauA could be mitigated by adding reducing agents, such as DTT and β-ME. Furthermore, the FDA-approval drug auranofin, a TXNRD1 inhibitor, triggered oligomerization of the cytoskeletal protein Talin-1 in AGS cells, indicating that inhibiting TXNRD1 triggered disulfide stress. In conclusion, this study uncovered GlauA as an efficient inhibitor of TXNRD1 and demonstrated the potential of TXNRD1 inhibition as an effective anticancer strategy by disrupting redox homeostasis and inducing disulfide stress., (© 2024 Federation of European Biochemical Societies.)

Canine mammary gland tumour (CMT) is the most common spontaneous tumour in intact female dogs and often exhibits metastases. Auranofin (AF) is a gold complex used for treating rheumatism. The excellent anti-tumour ability of AF has been demonstrated in various types of human and canine tumours. In this study, five CMT cell lines (CIPp, CMT-7364, CHMp, CIPm and CTBp) and three CMT primary cells (G7894, L1883 and L6783) were used to explore the anti-tumour effect of AF on CMT. Two CMT cell lines (CIPp and CMT-7364) were used to search the underlying mechanism of the effect of AF on CMT. The results showed that AF inhibited the growth, migration, invasion, and colony formation abilities of CMT cells. Additionally, the growth of CMT in a 3D cell culture model was effectively suppressed by AF. Furthermore, AF induced cell apoptosis of CMT cells via the PI3K/AKT pathway. In conclusion, AF effectively induces CMT apoptosis by regulating the PI3K/AKT pathway, indicating that AF should be explored as a potential CMT treatment in future studies., (© 2024 John Wiley & Sons Ltd.)

Alzheimer's disease remains an unsolved neurological puzzle with no cure. Current therapies offer only symptomatic relief, hindered by limited uptake through the blood-brain barrier. Auranofin, an FDA-approved compound, exhibits potent antioxidative and anti-inflammatory properties targeting brain disorders. Yet, its oral bioavailability challenge prompts the exploration of nanoformulation-based solutions enhancing blood-brain barrier penetrability. The study aimed to investigate the neuroprotective potential of auranofin nanoparticles in streptozotocin-induced AD rats. Auranofin-containing polylactic-co-glycolic acid nanoparticles were formulated by the multiple emulsion solvent evaporation method. Characterization was done by determining entrapment efficiency, particle size distribution, surface charge, and morphology. An in vivo study was performed by administering streptozotocin (3 mg/kg/i.c.v., days 1 and 3), auranofin (5 and 10 mg/kg), auranofin nanoparticles (2.5 and 5 mg/kg), and donepezil (2 mg/kg) for 14 days orally. Behavioral deficits were evaluated using the open field test, Morris water maze, objective recognition test, change in oxidative stress levels, and AD markers in the brain. Following the decapitation of the rats, the brains were excised to isolate the hippocampus. Subsequent analyses included the quantification of biochemical and neuroinflammatory markers, as well as the assessment of neurotransmitter levels. The characterization of auranofin nanoparticles showed an entrapment efficiency of 98%, an average particle size of 101.5 ± 10.3 nm, a surface charge of 27.5 ± 5.10 mV, and a polydispersity index of 0.438 ± 0.12. In vivo, administration of auranofin and auranofin nanoparticles significantly reversed streptozotocin-induced cognitive deficits, biochemical alteration, neuroinflammatory markers, and neurotransmitter levels. The present finding suggests that auranofin nanoparticles have more significant neuroprotective potential than auranofin alone. The therapeutic efficacy may be attributed to its antioxidant and anti-inflammatory properties, as well as its positive neuromodulatory effects. Therefore, our findings suggest that it could be a promising candidate for Alzheimer's disease therapy., Competing Interests: Declarations. Ethical approval: The experimental protocol received approval from the Institutional Animal Ethics Committee (IAEC) under approval number CPCSEA/LIPH/2019/10. All experimental procedures were conducted according to the guidelines stipulated by CCSEA. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

While highly effective at killing Gram-positive bacteria, auranofin lacks significant activity against Gram-negative species for reasons that largely remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the low susceptibility of the Gram-negative model organism Escherichia coli to auranofin when compared to the Gram-positive model organism Bacillus subtilis . The proteome response of E. coli exposed to auranofin suggests a combination of inactivation of thiol-containing enzymes and the induction of systemic oxidative stress. Susceptibility tests in E. coli mutants lacking proteins upregulated upon auranofin treatment suggested that none of them are directly involved in E. coli 's high tolerance to auranofin. E. coli cells lacking the efflux pump component TolC were more sensitive to auranofin treatment, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested whether E. coli 's thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from B. subtilis , which was not the case. However, E. coli strains lacking the low-molecular-weight thiol glutathione, but not glutathione reductase, showed a high susceptibility to auranofin. Bacterial cells expressing the genetically encoded redox probe roGFP2 allowed us to observe the oxidation of cellular protein thiols in situ . Based on our findings, we hypothesize that auranofin leads to a global disturbance in the cellular thiol redox homeostasis in bacteria, but Gram-negative bacteria are inherently more resistant due to the presence of drug export systems and high cellular concentrations of glutathione.IMPORTANCEAuranofin is an FDA-approved drug for the treatment of rheumatoid arthritis. However, it has also high antibacterial activity, in particular against Gram-positive organisms. In the current antibiotics crisis, this would make it an ideal candidate for drug repurposing. However, its much lower activity against Gram-negative organisms prevents its broad-spectrum application. Here we show that, on the level of the presumed target, there is no difference in susceptibility between Gram-negative and Gram-positive species: thioredoxin reductases from both Escherichia coli and Bacillus subtilis are equally inhibited by auranofin. In both species, auranofin treatment leads to oxidative protein modification on a systemic level, as monitored by proteomics and the genetically encoded redox probe roGFP2. The single largest contributor to E. coli 's relative resistance to auranofin seems to be the low-molecular-weight thiol glutathione, which is absent in B. subtilis and other Gram-positive species., Competing Interests: The authors declare no conflict of interest.

In this study, we demonstrate that ferroptosis is a component of the cell death mechanism induced by auranofin in HT-1080 cells, in contrast to the gold(III) compounds [Au(phen)Cl 2 ]PF 6 and [Au(bnpy)Cl 2 ]. Additionally, we identify a potential role of Prdx6 in modulating the sensitivity of A-375 cells to auranofin treatment, whereas the gold(III) compounds evaluated here exhibit Prdx6-independent cytotoxicity. Finally, using mass spectrometry, we show that auranofin binds selectively to the catalytic Cys47 residue of Prdx6 in vitro under acidic conditions. No binding was observed with the C47S mutant or at neutral pH., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Auranofin is an approved anti-rheumatic drug that has a broad-range inhibitory action against several microorganisms, including human pathogenic fungi. The auranofin activity against Histoplasma capsulatum, the dimorphic fungus that causes histoplasmosis, has not been properly addressed. Since there are few therapeutic options for this life-threatening systemic mycosis, this study evaluated the effects of auranofin on H. capsulatum growth and expression of virulence factors., Methodology/principal Findings: Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) of auranofin against 15 H. capsulatum strains with distinct genetic backgrounds were determined using the yeast form of the fungus and a microdilution protocol. Auranofin activity was also assessed on a macrophage model of infection and on a Tenebrio molitor invertebrate animal model. Expression of virulence-related genes was compared between auranofin treated and untreated H. capsulatum yeast cells using a quantitative PCR assay. Auranofin affected the growth of different strains of H. capsulatum, with MIC and MFC values ranging from 1.25 to 5.0 μM and from 2.5 to >10 μM, respectively. Auranofin was able to kill intracellular H. capsulatum yeast cells and conferred protection against the fungus in the experimental animal model of infection. Moreover, the expression of catalase A, HSP70, superoxide dismutase, thioredoxin reductase, serine proteinase, cytochrome C peroxidase, histone 2B, formamidase, metallopeptidase, Y20 and YPS3 proteins were reduced after six hours of auranofin treatment. CONCLUSIONS/SIGNIFICANCE: Auranofin is fungicidal against H. capsulatum and reduces the expression of several virulence-related genes, which makes this anti-rheumatic drug a good candidate for new medicines against histoplasmosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 de Abreu Almeida et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Protozoal diarrhea caused by Tritrichomonas foetus (blagburni) is a prevalent, lifelong, and globally distributed burden in domestic cats. Treatment is limited to the use of 5-nitroimidazoles and treatment failure is common. The repurposed gold salt compound auranofin has killing activity against diverse protozoa in vitro but evidence of efficacy in naturally occurring protozoal infections is lacking. This exploratory study investigated the efficacy and safety of auranofin for treatment of cats with naturally occurring, 5-nitroimidazole-resistant, T. foetus infection. The minimum lethal concentration (MLC) of auranofin against 5 isolates of feline T. foetus was determined under aerobic conditions in vitro. Healthy cats and cats with T. foetus infection were treated with immediate release auranofin (range, 0.5-3 mg/cat for 7 days) or guar gum-coated auranofin capsules (0.5 or 3 mg/cat for 7 days). Adverse effects were monitored by clinical signs and clinicopathologic testing. Efficacy was determined by fecal consistency score, bowel movement frequency, and single-tube nested PCR of feces for T. foetus rDNA. Fecal samples were assayed for concentrations of auranofin, known and predicted metabolites of auranofin, gold containing molecules, and total gold content using HPLC, LC-MS, ion mobility-MS, and ICP-MS, respectively. Auranofin was effective at killing isolates of feline T. foetus at MLC ≥ 1 μg/ml. Treatment of cats with T. foetus infection with either immediate release auranofin or a colon-targeted guar gum-coated tablet of auranofin did not eradicate infection. Treatment failure occurred despite fecal concentrations of gold that met or exceeded the equivalent MLC of auranofin. Neither auranofin, known or predicted metabolites of auranofin, nor any gold-containing molecules >100 Da could be detected in fecal samples of treated cats. Adverse effects associated with auranofin treatment were common but minor. These studies identify that in vitro susceptibility test results of auranofin may not translate to treatment effectiveness in vivo even when achieving gold concentrations equivalent to the MLC of auranofin in the target environment. These studies further establish the absence of any predicted or unpredicted gold containing metabolites in feces after oral administration of auranofin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC), the predominant type of liver cancer, is an aggressive malignancy with limited therapeutic options. In this study, we assess a collection of newly designed gold(I) phosphine complexes. Remarkably, the compound GC002 exhibits the greatest toxicity to HCC cells and outperforms established medications, such as sorafenib and auranofin, in terms of antitumor efficacy. GC002 triggers irreversible necroptosis in HCC cells by increasing the intracellular accumulation of reactive oxygen species (ROS). Mechanistically, GC002 significantly suppresses the activity of thioredoxin reductase (TrxR), which plays a crucial role in regulating redox homeostasis and is often overexpressed in HCC by binding directly to the enzyme. Our in vivo xenograft study confirms that GC002 possesses remarkable antitumor activity against HCC without severe side effects. These findings not only highlight the novel mechanism of controlling necroptosis via TrxR and ROS but also identify GC002 as a promising candidate for the further development of antitumor agents targeting HCC.

Objective: Cytotoxicity of the antirheumatic drug auranofin (Aur) and the non-steroidal anti-inflammatory drug meclofenamic acid (MA) on several cancer cell lines and isolated mitochondria was examined to assess whether these drugs behave as oxidative phosphorylation inhibitors., Methods: The effect of Aur or MA for 24 h was assayed on metastatic cancer and non-cancer cell proliferation, energy metabolism, mitophagy and metastasis; as well as on oxygen consumption rates of cancer and non-cancer mitochondria., Results: Aur doses in the low micromolar range were required to decrease proliferation of metastatic HeLa and MDA-MB-231 cells, whereas one or two orders of magnitude higher levels were required to affect proliferation of non-cancer cells. MA doses required to affect cancer cell growth were one order of magnitude higher than those of Aur. At the same doses, Aur impaired oxidative phosphorylation in isolated mitochondria and intact cells through mitophagy induction, as well as glycolysis. Consequently, cell migration and invasiveness were severely affected. The combination of Aur with very low cisplatin concentrations promoted that the effects on cellular functions were potentiated., Conclusion: Aur surges as a highly promising anticancer drug, suggesting that efforts to establish this drug in the clinical treatment protocols are warranted and worthy to undertake., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rodríguez-Enríquez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Auranofin (AF), a former rheumatoid polyarthritis treatment, gained renewed interest for its use as an antimicrobial. AF is an inhibitor of thioredoxin reductase (TrxB), a thiol and protein repair enzyme, with an antibacterial activity against several bacteria including C. difficile, an enteropathogen causing post-antibiotic diarrhea. Several studies demonstrated the effect of AF on C. difficile physiology, but the crucial questions of resistance mechanisms and impact on microbiota remain unaddressed. We explored potential resistance mechanisms by studying the impact of TrxB multiplicity and by generating and characterizing adaptive mutations. We showed that if mutants inactivated for trxB genes have a lower MIC of AF, the number of TrxBs naturally present in clinical strains does not impact the MIC. All stable mutations isolated after AF long-term exposure were in the anti-sigma factor of σ B and strongly affect physiology. Finally, we showed that AF has less impact on human gut microbiota than vancomycin., (© 2024. The Author(s).)

Since the survival of lymphoma patients who experience disease progression or relapse remains very poor, new therapeutic approaches and effective drugs are urgently needed. Here we show that auranofin (AF), an anti-rheumatoid drug thought to inhibit thioredoxin reductases (TXNRDs) as its mechanism of action, exhibited potent activity against multiple cancer types, especially effective against B cell lymphoma. Surprisingly, a knockdown of TXNRD1 and TXNRD2 did not cause significant cytotoxicity, suggesting that abrogation of TXNRD enzyme per se was insufficient to cause cancer cell death. Further mechanistic study showed that the interaction of AF with TXNRD could convert this antioxidant enzyme to a ROS-generating molecule via disrupting its electron transport, leading to a leak of electrons that interact with molecular oxygen to form superoxide. AF also suppressed energy metabolism by inhibiting both mitochondria complex II and the glycolytic enzyme GAPDH, leading to a significant depletion of ATP and inhibition of cancer growth in vitro and in vivo. Importantly, we found that the AF-mediated ROS stress could induce PD-L1 expression, revealing an unwanted effect of AF in causing immune suppression. We further showed that a combination of AF with anti-PD-1 antibody could enhance the anticancer activity in a syngeneic immune-competent mouse B-cell lymphoma model. Our study suggests that AF could be a potential drug for lymphoma treatment, and its combination with immune checkpoint inhibitors would be a logical strategy to increase the therapeutic activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Sepsis is a common complication of infections that significantly impacts the survival of critically patients. Currently, effective pharmacological treatment strategies are lacking. Auranofin, known as an inhibitor of Thioredoxin reductase (TrxR), exhibits anti-inflammatory activity, but its role in sepsis is not well understood. Here, we demonstrate the significant inhibitory effect of Auranofin on sepsis in a cecal ligation and puncture (CLP) mouse model. In vitro, Auranofin inhibits pyroptosis triggered by Caspase-11 activation. Further investigations reveal that inhibiting TrxR1 suppresses macrophage pyroptosis induced by E. coli, while TrxR2 does not exhibit this effect. TrxR1, functioning as a reductase, regulates the oxidative-reductive status of Thioredoxin-1 (Trx-1). Mechanistically, the modulation of Trx-1's reductive activity by TrxR1 may be involved in Caspase-11 activation-induced pyroptosis. Additionally, inhibiting TrxR1 maintains Trx-1 in its oxidized state. The oxidized form of Trx-1 interacts with Caveolin-1 (CAV1), regulating outer membrane vesicle (OMV) internalization. In summary, our study suggests that inhibiting TrxR1 suppresses OMV internalization by maintaining the oxidized form of Trx-1, thereby restricting Caspase-11 activation and alleviating sepsis., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The prevalence of carbapenem-resistant Escherichia coli (CREC) is increasing worldwide, and infections caused by CREC are associated with substantial morbidity and mortality rates. It is within this context that combination therapy has been reported as an effective strategy for treating resistant bacteria. Auranofin was approved by the FDA for treating rheumatoid arthritis. We confirmed that auranofin restored the susceptibility of ertapenem to CREC through synergy checkerboard and time-kill analyses. We also demonstrated that sub-MIC levels of auranofin significantly inhibited the expression of carbapenemase (bla KPC ) and efflux pump (acrA, acrD, and tolC) genes. The combination of auranofin and ertapenem suppressed the expression levels of motility (motA and flhD) genes, decreasing motility, which is a known pathogenic factor in CREC. Taken together, our results indicate that auranofin exerted a synergistic effect with ertapenem by suppressing the expression of carbapenemase and efflux pump genes and reducing the motility and virulence factors against CREC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mass spectrometry-based omics technologies are increasingly used in perturbation studies to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by fold change and variation of each molecular parameter, but also by multiple testing corrections. While the fold change is largely determined by the biological system, the variation is determined by experimental workflows. Here, it is shown that memory effects of prior subculture can influence the variation of perturbation profiles using the two colon carcinoma cell lines SW480 and HCT116. These memory effects are largely driven by differences in growth states that persist into the perturbation experiment. In SW480 cells, memory effects combined with moderate treatment effects amplify the variation in multiple omics levels, including eicosadomics, proteomics, and phosphoproteomics. With stronger treatment effects, the memory effect was less pronounced, as demonstrated in HCT116 cells. Subculture homogeneity was controlled by real-time monitoring of cell growth. Controlled homogeneous subculture resulted in a perturbation network of 321 causal conjectures based on combined proteomic and phosphoproteomic data, compared to only 58 causal conjectures without controlling subculture homogeneity in SW480 cells. Some cellular responses and regulatory events were identified that extend the mode of action of arsenic trioxide (ATO) only when accounting for these memory effects. Controlled prior subculture led to the finding of a synergistic combination treatment of ATO with the thioredoxin reductase 1 inhibitor auranofin, which may prove useful in the management of NRF2-mediated resistance mechanisms., Competing Interests: Competing interests statement:P.P. is the founder and CEO of PHIO scientific GmbH. All other authors declare no competing financial interests.

Introduction: Auranofin (AF) is a well-established, FDA-approved, antiarthritic gold drug that is currently being reevaluated for a variety of therapeutic indications through drug repurposing. AF has shown great promise as a potential anticancer agent and has been approved for a few clinical trials in cancer. The renewed interest in AF has led to extensive research into the design, preparation and biological evaluation of auranofin analogs, which may have an even better pharmacological profile than the parent drug., Areas Covered: This article reviews the strategies for chemical modification of the AF scaffold. Several auranofin analogs have been prepared and characterized for medical application in the field of cancer treatment over the last 20 years. Some emerging structure-function relationships are proposed and discussed., Expert Opinion: The chemical modification of the AF scaffold has been the subject of intense activity in recent years and this strategy has led to the preparation and evaluation of several AF analogs. The case of iodauranofin is a particularly promising example. The availability of homogeneous biological data for a group of AF derivatives allows some initial structure-function relationships to be proposed, which may inspire the design and synthesis of new and better AF analogs for cancer treatment.

Thioredoxin reductases are frequently overexpressed in various solid tumors as a protective mechanism against heightened oxidative stress. Inhibitors of this system, such as Auranofin, are effective in eradicating cancer cells. However, the clinical significance of thioredoxin reductase 1 (TrxR1) in lung cancer, as well as the potential for its antagonist as a treatment option, necessitated further experimental validation. In this study, we observed significant upregulation of TrxR1 specifically in non-small cell lung cancer (NSCLC), rather than small cell lung cancer. Moreover, TrxR1 expression exhibited associations with survival rate, tumor volume, and histological classification. We developed a novel TrxR1 inhibitor named LW-216 and assessed its antitumor efficacy in NSCLC. Our results revealed that LW-216 is effectively bound with intracellular TrxR1 at sites R371 and G442, facilitating TrxR1 ubiquitination and suppressing TrxR1 expression, while not affecting TrxR2 expression. Treatment of LW-216-induced DNA damage and cell apoptosis in NSCLC cells through the generation of reactive oxygen species (ROS). Importantly, supplementation with N-acetylcysteine (NAC) or ectopic TrxR1 expression reversed LW-216-induced apoptosis. Furthermore, LW-216 displayed potent tumor growth inhibition in NSCLC cell-implanted mice, reducing TrxR1 expression in xenografts. Remarkably, LW-216 exhibited superior antitumor activity compared to Auranofin in vivo. Collectively, our research provides compelling evidence supporting the potential of targeting TrxR1 by LW-216 as a promising therapeutic strategy for NSCLC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background/aim: Synovial sarcoma (SS) is a rare malignant tumor with a poor survival rate. We previously reported that a combination of auranofin (AUR), a thioredoxin reductase inhibitor, and celecoxib (CE), an anti-inflammatory drug, significantly impedes the local progression of osteosarcoma (OS). However, the role of redox regulation in SS remains to be elucidated. This study aimed to investigate the efficacy of combined treatment of AUR and CE on the local progression of SS in vivo., Materials and Methods: Nu/nu mice were implanted with the human SS cell line, Aska-SS, and treated with vehicle control, AUR, or a combination of AUR and CE (AUR-CE). Primary tumor size and weight were evaluated for the study duration and upon resection, respectively. Hematoxylin and eosin (H&E) and Ki-67 staining were performed to assess the local progression of SS., Results: A statistically significant reduction in tumor size and weight was observed in the AUR- and AUR-CE-treated groups upon excision compared to that in the vehicle-treated group. The AUR-CE-treated group showed synergistic inhibition of local tumor growth. H&E staining of local SS tumors revealed decreased cell density and nuclear deformation in the AUR- and AUR-CE-treated groups compared to those in the vehicle-treated group. Immunohistochemical staining revealed a statistically significant decrease in Ki-67-positive cells in the AUR-CE-treated group compared to the vehicle-treated group., Conclusion: The combination of AUR and CE showed significant potential for delaying the local progression of SS. These findings support the repurposing of AUR and CE as early treatment options for SS., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Acute leukemia continues to be a major cause of death from disease worldwide and current chemotherapeutic agents are associated with significant morbidity in survivors. While better and safer treatments for acute leukemia are urgently needed, standard drug development pipelines are lengthy and drug repurposing therefore provides a promising approach. Our previous evaluation of FDA-approved drugs for their antileukemic activity identified disulfiram, used for the treatment of alcoholism, as a candidate hit compound. This study assessed the biological effects of disulfiram on leukemia cells and evaluated its potential as a treatment strategy. We found that disulfiram inhibits the viability of a diverse panel of acute lymphoblastic and myeloid leukemia cell lines (n = 16) and patient-derived xenograft cells from patients with poor outcome and treatment-resistant disease (n = 15). The drug induced oxidative stress and apoptosis in leukemia cells within hours of treatment and was able to potentiate the effects of daunorubicin, etoposide, topotecan, cytarabine, and mitoxantrone chemotherapy. Upon combining disulfiram with auranofin, a drug approved for the treatment of rheumatoid arthritis that was previously shown to exert antileukemic effects, strong and consistent synergy was observed across a diverse panel of acute leukemia cell lines, the mechanism of which was based on enhanced ROS induction. Acute leukemia cells were more sensitive to the cytotoxic activity of disulfiram than solid cancer cell lines and non-malignant cells. While disulfiram is currently under investigation in clinical trials for solid cancers, this study provides evidence for the potential of disulfiram for acute leukemia treatment. KEY MESSAGES: Disulfiram induces rapid apoptosis in leukemia cells by boosting oxidative stress. Disulfiram inhibits leukemia cell growth more potently than solid cancer cell growth. Disulfiram can enhance the antileukemic efficacy of chemotherapies. Disulfiram strongly synergises with auranofin in killing acute leukemia cells by ROS induction. We propose testing of disulfiram in clinical trial for patients with acute leukemia., (© 2024. The Author(s).)

Background: This study explores the repurposing of Auranofin (AF), an anti-rheumatic drug, for treating non-small cell lung cancer (NSCLC) adenocarcinoma and pancreatic ductal adenocarcinoma (PDAC). Drug repurposing in oncology offers a cost-effective and time-efficient approach to developing new cancer therapies. Our research focuses on evaluating AF's selective cytotoxicity against cancer cells, identifying RNAseq-based biomarkers to predict AF response, and finding the most effective co-therapeutic agents for combination with AF., Methods: Our investigation employed a comprehensive drug screening of AF in combination with eleven anticancer agents in cancerous PDAC and NSCLC patient-derived organoids (n = 7), and non-cancerous pulmonary organoids (n = 2). Additionally, we conducted RNA sequencing to identify potential biomarkers for AF sensitivity and experimented with various drug combinations to optimize AF's therapeutic efficacy., Results: The results revealed that AF demonstrates a preferential cytotoxic effect on NSCLC and PDAC cancer cells at clinically relevant concentrations below 1 µM, sparing normal epithelial cells. We identified Carbonic Anhydrase 12 (CA12) as a significant RNAseq-based biomarker, closely associated with the NF-κB survival signaling pathway, which is crucial in cancer cell response to oxidative stress. Our findings suggest that cancer cells with low CA12 expression are more susceptible to AF treatment. Furthermore, the combination of AF with the AKT inhibitor MK2206 was found to be particularly effective, exhibiting potent and selective cytotoxic synergy, especially in tumor organoid models classified as intermediate responders to AF, without adverse effects on healthy organoids., Conclusion: Our research offers valuable insights into the use of AF for treating NSCLC and PDAC. It highlights AF's cancer cell selectivity, establishes CA12 as a predictive biomarker for AF sensitivity, and underscores the enhanced efficacy of AF when combined with MK2206 and other therapeutics. These findings pave the way for further exploration of AF in cancer treatment, particularly in identifying patient populations most likely to benefit from its use and in optimizing combination therapies for improved patient outcomes., (© 2024. The Author(s).)

Triple-negative breast cancer (TNBC) remains a clinical challenge due to molecular, metabolic, and genetic heterogeneity as well as the lack of validated drug targets. Thus, therapies or delivery paradigms are needed. Gold-derived compounds including the FDA-approved drug, auranofin have shown promise as effective anticancer agents against several tumors. To improve the solubility and bioavailability of auranofin, we hypothesized that the nanodelivery of auranofin using biodegradable chitosan modified polyethylene glycol (PEG) nanoparticles (NPs) will enhance anticancer activity against TNBC by comparing the best nanoformulation with the free drug. The selection of the nanoformulation was based on synthesis of various chitosan PEG copolymers via formaldehyde-mediated engraftment of PEG onto chitosan to form [chitosan- g -PEG] copolymer. Furthermore, altered physiochemical properties of the copolymer was based on the formaldehyde ratio towards nanoparticles (CP 1-4 NPs). Following the recruitment of PEG onto the chitosan polymer surface, we explored how this process influenced the stiffness of the nanoparticle using atomic force microscopy (AFM), a factor crucial for in vitro and in vivo studies. Our objective was to ensure the full functionality and inherent properties of chitosan as the parent polymer was maintained without allowing PEG to overshadow chitosan's unique cationic properties while improving solubility in neutral pH. Hence, CP 2 NP was chosen. To demonstrate the efficacy of CP 2 NP as a good delivery carrier for auranofin, we administered a dose of 3 mg/kg of auranofin, in contrast to free auranofin, which was given at 5 mg/kg. In vivo studies revealed the potency of encapsulated auranofin against TNBC cells with a severe necrotic effect following treatment superior to that of free auranofin. In conclusion, chitosan- g -PEG nanoparticles have the potential to be an excellent delivery system for auranofin, increasing its effectiveness and potentially reducing its clinical limitations.

Ferroptotic cancer therapy has been extensively investigated since the genesis of the ferroptosis concept. However, the therapeutic efficacy of ferroptosis induction in heterogeneous and plastic melanoma has been compromised, because the melanocytic and transitory cell subpopulation is resistant to iron-dependent oxidative stress. Here, we report a phenotype-altering liposomal nanomedicine to enable the ferroptosis-resistant subtypes of melanoma cells vulnerable to lipid peroxidation via senescence induction. The strategy involves the ratiometric coencapsulation of a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor (palbociclib) and a ferroptosis inducer (auranofin) within cRGD peptide-modified targeted liposomes. The two drugs showed a synergistic anticancer effect in the model B16F10 melanoma cells, as evidenced by the combination index analysis (<1). The liposomes could efficiently deliver both drugs into B16F10 cells in a targeted manner. Afterward, the liposomes potently induced the intracellular redox imbalance and lipid peroxidation. Palbociclib significantly provoked cell cycle arrest at the G 0 /G 1 phase, which sensitized auranofin-caused ferroptosis through senescence induction. Meanwhile, palbociclib depleted intracellular glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), further boosting ferroptosis. The proof-of-concept was also demonstrated in the B16F10 tumor-bearing mice model. The current work offers a promising ferroptosis-targeting strategy for effectively treating heterogeneous melanoma by manipulating the cellular plasticity.

Background/aim: Osteosarcoma (OS) is a rare malignant tumor with a poor survival rate. Our previous study reported that auranofin (AUR), a thioredoxin reductase inhibitor, suppresses OS pulmonary metastases; however, the local progression of OS is not affected, in vivo. Nonetheless, the development of augmentation therapy with AUR to inhibit OS local progression remains challenging. Celecoxib (CE), an anti-inflammatory drug, potently enhances the therapeutic activity of AUR against colon cancer. Consequently, this study investigated the combined effects of AUR and CE on OS local progression and pulmonary metastases, in vivo., Materials and Methods: C3H/HeSlc mice were implanted with the murine OS cell line, LM8. The mice were treated either with a vehicle control, AUR, or combination of AUR and CE (AUR-CE). The primary tumor size and weight were evaluated for the study duration and at resection, respectively. Hematoxylin and eosin and Ki-67 staining were performed to evaluate OS local progression and pulmonary metastases., Results: Mice in the AUR-CE group showed statistically significantly suppressed tumor sizes and weights at the time of excision compared with those in the vehicle. The mice in the AUR group did not show a statistically significant effect. Histopathological analysis of the primary tumor revealed a statistically significant decrease of the Ki-67-positive cells in the AUR-CE group compared with the vehicle group. Histopathological and quantitative analyses demonstrated that the AUR and AUR-CE groups had statistically significant reductions in the development of OS pulmonary metastases compared with the vehicle group., Conclusion: The combination of AUR and CE significantly inhibited OS local progression and pulmonary metastases., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Auranofin, an FDA-approved drug for rheumatoid arthritis, has emerged as a promising antiparasitic medication in recent years. The gold(I) ion in auranofin is postulated to be responsible for its antiparasitic activity. Notably , aurothiomalate and aurothioglucose also contain gold(I), and, like auranofin, they were previously used to treat rheumatoid arthritis. Whether they have antiparasitic activity remains to be elucidated. Herein, we demonstrated that auranofin and similar derivatives, but not aurothiomalate and aurothioglucose, inhibited the growth of Toxoplasma gondii in vitro . We found that auranofin affected the T. gondii biological cycle (lytic cycle) by inhibiting T. gondii's invasion and triggering its egress from the host cell. However, auranofin could not prevent parasite replication once T. gondii resided within the host. Auranofin treatment induced apoptosis in T. gondii parasites , as demonstrated by its reduced size and elevated phosphatidylserine externalization (PS). Notably, the gold from auranofin enters the cytoplasm of T. gondii, as demonstrated by scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDS) and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).IMPORTANCEToxoplasmosis, caused by Toxoplasma gondii , is a devastating disease affecting the brain and the eyes, frequently affecting immunocompromised individuals. Approximately 60 million people in the United States are already infected with T. gondii , representing a population at-risk of developing toxoplasmosis. Recent advances in treating cancer, autoimmune diseases, and organ transplants have contributed to this at-risk population's exponential growth. Paradoxically, treatments for toxoplasmosis have remained the same for more than 60 years, relying on medications well-known for their bone marrow toxicity and allergic reactions. Discovering new therapies is a priority, and repurposing FDA-approved drugs is an alternative approach to speed up drug discovery. Herein, we report the effect of auranofin, an FDA-approved drug, on the biological cycle of T. gondii and how both the phosphine ligand and the gold molecule determine the anti-parasitic activity of auranofin and other gold compounds. Our studies would contribute to the pipeline of candidate anti- T. gondii agents., Competing Interests: The authors declare no conflict of interest.

Colorectal cancer (CRC) is one of the world's most common and deadly cancers. According to GLOBOCAN2020's global incidence rate and mortality estimates, CRC is the third main cause of cancer and the second leading cause of cancer-related deaths worldwide. The US Food and Drug Administration has approved auranofin for the treatment of rheumatoid arthritis. It is a gold-containing chemical that inhibits thioredoxin reductase. Auranofin has a number of biological activities, including anticancer activity, although it has not been researched extensively in CRC, and the mechanism of action on CRC cells is still unknown. The goal of this research was to see how Auranofin affected CRC cells in vivo and in vitro . The two chemical libraries were tested for drugs that make CRC cells more responsive. The CCK-8 technique was used to determine the cell survival rate. The invasion, migration, and proliferation of cells were assessed using a transwell test and a colony cloning experiment. An electron microscope was used to observe autophagosome formation. Western blotting was also used to determine the degree of expression of related proteins in cells. Auranofin's tumor-suppressing properties were further tested in a xenograft tumor model of human SW620 CRC cells. Auranofin dramatically reduced the occurrence of CRC by decreasing the proliferation, migration, and invasion of CRC cells, according to our findings. Through a mTOR-dependent mechanism, auranofin inhibits the epithelial-mesenchymal transition (EMT) and induces autophagy in CRC cells. Finally, in-vivo tests revealed that auranofin suppressed tumor growth in xenograft mice while causing no harm. In summary, auranofin suppresses CRC cell growth, invasion, and migration. Auranofin inhibits the occurrence and progression of CRC by decreasing EMT and inducing autophagy in CRC cells via a mTOR-dependent mechanism. These findings suggest that auranofin could be a potential chemotherapeutic medication for the treatment of human CRC., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Lung cancer is one of the most lethal diseases in the world. Although there has been significant progress in the treatment of lung cancer, there is still a lack of effective strategies for advanced cases. Lenvatinib, a multi-targeted tyrosine kinase inhibitor, has achieved much attention due to its antitumor properties. Nevertheless, the use of lenvatinib is restricted by the characteristics of poor efficacy and drug resistance. In this study, we assessed the effectiveness of lenvatinib combined with thioredoxin reductase 1 (TrxR1) inhibitors in human lung cancer cells. Our results indicate that the combination therapy involving TrxR1 inhibitors and lenvatinib exhibited significant synergistic antitumor effects in human lung cancer cells. Moreover, siTrxR1 also showed significant synergy with lenvatinib in lung cancer cells. Mechanically, we demonstrated that ROS accumulation significantly contributes to the synergism between lenvatinib and TrxR1 inhibitor auranofin. Furthermore, the combination of lenvatinib and auranofin can activate endoplasmic reticulum stress and JNK signaling pathways to achieve the goal of killing lung cancer cells. Importantly, combination therapy with lenvatinib and auranofin exerted a synergistic antitumor effect in vivo . To sum up, the combination therapy involving lenvatinib and auranofin may be a potential strategy for treating lung cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor which acts as a regulator for cellular oxidative stress, and tightly regulated by Kelch-like ECH-associated protein 1 (Keap1). In this study, we found that auranofin and paclitaxel combination treatment increased TUNEL positive apoptotic cells and enhanced the DNA damage marker γ-H2AX in MCF-7 and MDA-MB-231 breast cancer cells. The immunoblotting analysis revealed the combination of auranofin and paclitaxel significantly increased the FOXO3 expression in a concentration dependent manner. Further we observed that auranofin and paclitaxel treatment prevents the translocation of Nrf2 in a concentration dependent manner. The increased FOXO3 expression might be involved in the cytoplasmic degradation of Nrf1-Keap1 complex. Further, the molecular docking results confirm auranofin act as the agonist for Foxo3. Therefore, the present results suggest that auranofin sensitize the breast cancer cells to paclitaxel via regulating FOXO3/Nrf2/Keap1signaling pathway., (© 2023 John Wiley & Sons Ltd.)

Schistosomiasis is a serious tropical disease. Despite extensive research into the etiology of liver fibrosis, effective therapeutic options remain limited. This study aims to assess the effectiveness of auranofin in treating hepatic granuloma and fibrogenesis produced by Schistosoma (S.) mansoni eggs. Auranofin is a gold complex that contains thioglucose tetraacetate and triethylphosphine. Eighty BALB/c male mice were divided into four groups (n=20/group): negative control (GI), positive control (GII), and early (GIII) and late (GIV) treatment groups with oral auranofin according to beginning of treatment 4th week and 6th week post-infection. Mice were infected subcutaneously in a dose of 60±10 cercariae/mouse. Worm counts, egg loads, and oogram patterns were determined. Biochemical, histological, and immunostaining of interleukin-1β (IL-1β), Sirtuin 3 (SIRT3), and smooth muscle actin (SMA) were assessed. GIII showed a significant decrease in the total S. mansoni worm burden and ova/gram in liver tissue (with reduction percent of 63.07% and 78.26%, respectively). Schistosomal oogram patterns, immature and mature ova, also showed a significant decrease. The reduction in granuloma number and size was 40.63% and 48.66%, respectively, in GIII, whereas in GIV, the reduction percent was 76.63% and 67.08%. In addition, the degree of fibrosis was significantly diminished in both treated groups. GIV showed significant reduction in IL-1β and SMA expression and increase in SIRT3 expression. These findings reveal how auranofin suppresses the development of liver fibrosis. Therefore, it is crucial to take another look at auranofin as a prospective medication for the treatment of S. mansoni egg-induced hepatic granuloma and consequent fibrosis.

The intrinsic redox status of cancer cells limits the efficacy of chemotherapeutic drugs. Auranofin, a Food and Drug Administration-approved gold-containing compound, documented with effective pharmacokinetics and safety profiles in humans, has recently been repurposed for anticancer activity. This study examined the paclitaxel-sensitizing effect of auranofin by targeting redox balance in the MDA-MB-231 and MCF-7 breast cancer cell lines. Auranofin treatment depletes the activities of superoxide dismutase, catalase, and glutathione peroxidase and alters the redox ratio in the breast cancer cell lines. Furthermore, it has been noticed that auranofin augmented paclitaxel-mediated cytotoxicity in a concentration-dependent manner in both MDA-MB-231 and MCF-7 cell lines. Moreover, auranofin increased the levels of intracellular reactive oxygen species (observed using 2, 7-diacetyl dichlorofluorescein diacetate staining) and subsequently altered the mitochondrial membrane potential (rhodamine-123 staining) in a concentration-dependent manner. Further, the expression of apoptotic marker p21 was found to be higher in auranofin plus paclitaxel-treated breast cancer cells compared to paclitaxel-alone treatment. Thus, the present results illustrate the chemosensitizing property of auranofin in MDA-MB-231 and MCF-7 breast cancer cell lines via oxidative metabolism. Therefore, auranofin could be considered a chemosensitizing agent during cancer chemotherapy., (© 2023 John Wiley & Sons Ltd.)

Targeting overexpressed thioredoxin reductase (TrxR) in cancer cells to induce oxidative stress has been proved to be an effective strategy for cancer therapy. However, the treatment was hindered by the low efficiency and frequent administration of TrxR inhibitors, and hence more potent TrxR inhibitors were urgently needed. Herein, we designed and synthesized a series of TrxR inhibitors based on arsenicals. Among these, compound 1d inhibited the proliferation of a variety of cancer cells at low micromolar concentrations and exhibited low toxicity to normal cells. Importantly, compound 1d induced the accumulation of reactive oxygen species (ROS) by inhibiting the TrxR activity, further causing the collapse of the redox system, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and DNA damage, followed by oxidative stress-induced cell apoptosis. In vivo data showed that, compared with the clinical TrxR inhibitor auranofin (AUR), compound 1d could more effectively eliminate tumors by 90 % at a dose of 1.5 mg/kg without any obvious side effects. These results indicated that compound 1d was a potent TrxR inhibitor against cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Aims: Studies in the past have shown that inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase sensitizes cancer cells to genotoxic anticancer treatments, however, clinical use of ATR inhibitors in combination with DNA damaging chemotherapy is limited due to toxicity in healthy tissues. In this study, we investigated the synergistic anticancer effect between ATR inhibition and oxidative DNA damage induced by the thioredoxin reductase inhibitor auranofin., Main Methods: Cytotoxicity was evaluated by cell viability assays. Western blot, comet assay, immunostaining and flow cytometry were performed to dissect the underlying mechanisms. In vivo efficacy was examined against tumor xenografts., Key Findings: Nontoxic doses of auranofin alone increased the levels of reactive oxygen species (ROS) in cancer but not noncancerous cells, resulting in oxidative DNA damage and activation of the ATR DNA damage response pathway selectively in cancer cells. Inhibition of ATR in auranofin-treated cancer cells resulted in unscheduled firing of dormant DNA replication origins, abrogation of the S phase cell cycle checkpoint and extensive DNA breakage, leading to replication catastrophe and potent synergistic lethality. Both the antioxidant NAC and the DNA polymerase inhibitor aphidicolin reduced replication stress and synergistic cytotoxicity, implicating replication stress-driven catastrophic cell death resulted from collision between oxidative DNA damage and dysregulated DNA replication. In vivo, auranofin and VE822 coadministration enabled marked regressions of tumor xenografts, while each drug alone had no effect., Significance: As increased generation of ROS is a universal feature of tumors, our findings may open new routes to broaden the therapeutic potential of ATR inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Three obligate intracellular protozoan parasite species, which are responsible for significant morbidity and mortality and settle in macrophage cells, affect more than one-half of the world's population, namely, Trypanosoma cruzi, Leishmania tropica and Toxoplasma gondii, which are causative agents of Chagas disease, leishmaniasis and toxoplasmosis, respectively. In the current study, it was aimed to investigate the in vitro and ex vivo antiprotozoal activity of auranofin on T. cruzi, L. tropica and T. gondii., Methods: The in vitro drug efficacy (IC50) of auranofin was investigated by haemocytometry and the CellTiter-Glo assay methods and the ex vivo drug efficacy (IC50) by light microscopic examination of Giemsa-stained slides. Also, the cytotoxic activity (CC50) of auranofin was examined by the CellTiter-Glo assay. The selectivity index (SI) was calculated for auranofin., Results: According to IC50, CC50 and SI data, auranofin did not exhibit cytotoxic activity on Vero cells, but exhibited antiprotozoal activity on epimastigotes and intracellular amastigotes of T. cruzi, promastigotes and intracellular amastigotes of L. tropica and intracellular tachyzoites of T. gondii (p<0.05)., Conclusions: The detection antiprotozoal activity of auranofin on T. cruzi, L. tropica and T. gondii according to the IC50, CC50 and SI values is considered an important and promising development. This is significant because auranofin may be an effective alternative treatment for Chagas disease, leishmaniasis and toxoplasmosis in the future., (© The Author(s) 2023. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Clostridioides difficile infections (CDIs) are responsible for a significant number of antibiotic-associated diarrheal cases. The standard-of-care antibiotics for C. difficile are limited to fidaxomicin and vancomycin, with the recently obsolete metronidazole recommended if both are unavailable. No new antimicrobials have been approved for CDI since fidaxomicin in 2011, despite varying rates of treatment failure among all standard-of-care drugs. Drug repurposing is a rational strategy to generate new antimicrobials out of existing therapeutics approved for other indications. Auranofin is a gold-containing anti-rheumatic drug with antimicrobial activity against C. difficile and other microbes. In a previous report, our group hypothesized that inhibition of selenoprotein biosynthesis was auranofin's primary mechanism of action against C. difficile. However, in this study, we discovered that C. difficile mutants lacking selenoproteins are still just as sensitive to auranofin as their respective wild-type strains. Moreover, we found that selenite supplementation dampens the activity of auranofin against C. difficile regardless of the presence of selenoproteins, suggesting that selenite's neutralization of auranofin is not because of compensation for a chemically induced selenium deficiency. Our results clarify the findings of our original study and may aid drug repurposing efforts in discovering the compound's true mechanism of action against C. difficile., (© 2023. Springer Nature Limited.)

Interferon-gamma (IFN-γ) is a type II interferon produced primarily by T cells and natural killer cells. IFN-γ induces the expression of inducible nitric oxide synthase (NOS2) to catalyze Nitric Oxide (NO) production in various immune and non-immune cells. Excessive IFN-γ-activated NO production is implicated in several inflammatory diseases, including peritonitis and inflammatory bowel diseases. In this study, we screened the LOPAC® 1280 library in vitro on the H6 mouse hepatoma cell line to identify novel non-steroidal small molecule inhibitors of IFN-γ-induced NO production. Compounds with the highest inhibitory activity were validated, which led to identifying the lead compounds: pentamidine, azithromycin, rolipram, and auranofin. Auranofin was the most potent compound determined based on IC 50 and goodness of fit analyses. Mechanistic investigations revealed that majority of the lead compounds suppress the IFN-γ-induced transcription of Nos2 without negatively affecting NO-independent processes, such as the IFN-γ-induced transcription of Irf1, Socs1 and MHC class 1 surface expression. However, all four compounds lower IFN-γ-induced reactive oxygen species amounts. In addition, auranofin significantly reduced IFN-γ-mediated NO and IL6 production in resident as well as thioglycolate-elicited peritoneal macrophages (PMs). Finally, in vivo testing of the lead compounds in the pre-clinical DSS-induced ulcerative colitis mice model revealed pentamidine and auranofin to be the most potent and protective lead compounds. Also, pentamidine and auranofin greatly increase the survival of mice in another inflammatory model: Salmonella Typhimurium-induced sepsis. Overall, this study identifies novel anti-inflammatory compounds targeting IFN-γ-induced NO-dependent processes to alleviate two distinct inflammatory models of disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1's ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration., (© 2023. Springer Nature Limited.)

Gold compounds form a new class of promising anticancer agents with innovative modes of action. It is generally believed that anticancer gold compounds, at variance with clinically established platinum drugs, preferentially target proteins rather than nucleic acids. The reactions of several gold compounds with a few model proteins have been systematically explored in recent years through ESI MS measurements to reveal adduct formation and identify the main features of those reactions. Here, we focus our attention on a group of five gold compounds of remarkable medicinal interest, i.e., Auranofin, Au(NHC)Cl, [Au(NHC) 2 ]PF 6 , Aubipyc, and Auoxo6, and on their reactions with four different biomolecular targets, i.e., the proteins HEWL, hCA I, HSA and the C-terminal dodecapeptide of the enzyme thioredoxin reductase. Complete ESI MS data are available for those reactions due to previous experimental work conducted in our laboratory. From the comparative analysis of the ESI MS reaction profiles, some characteristic trends in the metallodrug-protein reactivity may be identified as detailed below. The main features are described and analyzed in this review. Overall, all these observations are broadly consistent with the concept that cytotoxic gold drugs preferentially target cancer cell proteins, with a remarkable selectivity for the cysteine and selenocysteine proteome. These interactions typically result in severe damage to cancer cell metabolism and profound alterations in the redox state, leading to eventual cancer cell death.

Auranofin, a gold(I)-based complex, is under clinical trials for application as an anticancer agent for the treatment of nonsmall-cell lung cancer and ovarian cancer. In the past years, different derivatives have been developed, modifying gold linear ligands in the search for new gold complexes endowed with a better pharmacological profile. Recently, a panel of four gold(I) complexes, inspired by the clinically established compound auranofin, was reported by our research group. As described, all compounds possess an [Au{P(OMe) 3 }] + cationic moiety, in which the triethylphosphine of the parent compound auranofin was replaced with an oxygen-rich trimethylphosphite ligand. The gold(I) linear coordination geometry was complemented by Cl - , Br - , I - , and the auranofin-like thioglucose tetraacetate ligand. As previously reported, despite their close similarity to auranofin, the panel compounds exhibited some peculiar and distinctive features, such as lower log P values which can induce relevant differences in the overall pharmacokinetic profiles. To get better insight into the P-Au strength and stability, an extensive study was carried out for relevant biological models, including three different vasopressin peptide analogues and cysteine, using 31 P NMR and LC-ESI-MS. A DFT computational study was also carried out for a better understanding of the theoretical fundamentals of the disclosed differences with regard to triethylphosphine parent compounds.

Purpose: Cisplatin resistance is the major obstacle in the clinical treatment of ovarian cancer patients. Molecular mechanisms of cisplatin resistance are multifaceted. Gold(I)-compounds, i.e. N-heterocyclic carbene-gold(I)-complexes (NHC-Au(I)) has been regarded as promising cytotoxic drug candidates. However, their potential to overcome cisplatin resistance has hardly been addressed yet. Here we investigated the activity of the gold(I) drug auranofin and the NHC-Au(I)-compound MC3 in W1CR and A2780cis cisplatin-resistant ovarian cancer cells., Methods: Cytotoxicity of auranofin and MC3 was detected by MTT assay, correlated with intracellular gold(I) content, analyzed by AAS, and with flow cytometric detection of the cell cycle. Insight into cellular redox balance was provided by fluorimetric ROS-formation assay and western blotting thioredoxin (Trx) and Nrf2. The role of ERK was elucidated by using the inhibitor SCH772984 and its impact on cytotoxicity upon co-treatment with cisplatin and Au(I)-compounds, respectively., Results: MC3 overcomes cisplatin resistance in A2780cis and W1CR, and auranofin in W1CR cells completely, which is neither reflected by intracellular gold levels nor cell cycle changes. Upregulated redox balance appears as a basis for resistance. W1CR cells possess higher Trx levels, whereas A2780cis cells display strong Nrf2 expression as anti-oxidative protection. Nevertheless, overcoming redox balance appears not primary mode of activity comparing cisplatin and gold(I)-compounds. pERK emerges as a critical component and thus a promising target for overcoming resistance, regulating apoptosis differently in response to either gold(I) or cisplatin in A2780 cells., Conclusion: These data reflect the complexity of cisplatin resistance in cell models and emphasize NHC-Au(I)-complexes as prospective cytotoxic agents for further investigations in that respect., (© 2023. The Author(s).)

(1) Background: Tuberous sclerosis complex (TSC) mutations directly affect mTORC activity and, as a result, protein synthesis. In several cancer types, TSC mutation is part of the driver mutation panel. TSC mutations have been associated with mitochondrial dysfunction, tolerance to reactive oxygen species due to increased thioredoxin reductase (TrxR) enzyme activity, tolerance to endoplasmic reticulum (ER) stress, and apoptosis. The FDA-approved drug rapamycin is frequently used in clinical applications to inhibit protein synthesis in cancers. Recently, TrxR inhibitor auranofin has also been involved in clinical trials to investigate the anticancer efficacy of the combination treatment with rapamycin. We aimed to investigate the molecular background of the efficacy of such drug combinations in treating neoplasia modulated by TSC mutations. (2) Methods: TSC2 mutant and TSC2 wild-type (WT) cell lines were exposed to rapamycin and auranofin in either mono- or combination treatment. Mitochondrial membrane potential, TrxR enzyme activity, stress protein array, mRNA and protein levels were investigated via cell proliferation assay, electron microscopy, etc. (3) Results: Auranofin and rapamycin normalized mitochondrial membrane potential and reduced proliferation capacity of TSC2 mutant cells. Database analysis identified peroxiredoxin 5 (Prdx5) as the joint target of auranofin and rapamycin. The auranofin and the combination of the two drugs reduced Prdx5 levels. The combination treatment increased the expression of heat shock protein 70, a cellular ER stress marker. (4) Conclusions: After extensive analyses, Prdx5 was identified as a shared target of the two drugs. The decreased Prdx5 protein level and the inhibition of both TrxR and mTOR by rapamycin and auranofin in the combination treatment made ER stress-induced cell death possible in TSC2 mutant cells.

Microbe entry through catheter ports can lead to biofilm accumulation and complications from catheter-related bloodstream infection and ultimately require antimicrobial treatment and catheter replacement. Although strides have been made with microbial prevention by applying standardized antiseptic techniques during catheter implantation, both bacterial and fungal microbes can present health risks to already sick individuals. To reduce microbial adhesion, murine and human catheters were coated with polyurethane and auranofin using a dip coating method and compared to non-coated materials. Upon passage of fluid through the coated material in vitro , flow dynamics were not impacted. The unique antimicrobial properties of the coating material auranofin has shown inhibitory activity against bacteria such as Staphylococcus aureus and fungi such as Candida albicans . Auranofin coating on catheters at 10mg/mL reduced C . albicans accumulation in vitro from 2.0 x 10 8 to 7.8 x 10 5 CFU for mouse catheters and from 1.6 x 10 7 to 2.8 x 10 6 for human catheters, showing an impact to mature biofilms. Assessment of a dual microbe biofilm on auranofin-coated catheters resulted in a 2-log reduction in S . aureus and a 3-log reduction in C . albicans compared to uncoated catheters. In vivo assessment in a murine subcutaneous model demonstrated that catheters coated with 10 mg/mL auranofin reduced independent S . aureus and C . albicans accumulation by 4-log and 1-log, respectively, compared to non-coated catheters. In conclusion, the auranofin-coated catheters demonstrate proficiency at inhibiting multiple pathogens by decreasing S . aureus and C . albicans biofilm accumulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Felix, Whitely, Tharmalingam, Mishra, Vera-Gonzalez, Mylonakis, Shukla and Fuchs.)

Advanced mesothelioma is considered an incurable disease and new treatment strategies are needed. Previous studies have demonstrated that mitochondrial antioxidant defense proteins and the cell cycle may contribute to mesothelioma growth, and that the inhibition of these pathways may be effective against this cancer. We demonstrated that the antioxidant defense inhibitor auranofin and the cyclin-dependent kinase 4/6 inhibitor palbociclib could decrease mesothelioma cell proliferation alone or in combination. In addition, we determined the effects of these compounds on colony growth, cell cycle progression, and the expression of key antioxidant defense and cell cycle proteins. Auranofin and palbociclib were effective in decreasing cell growth and inhibiting the above-described activity across all assays. Further study of this drug combination will elucidate the contribution of these pathways to mesothelioma activity and may reveal a new treatment strategy.

As unicellular parasites are highly dependent on NADPH as a source for reducing equivalents, the main NADPH-producing enzymes glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway are considered promising antitrypanosomatid drug targets. Here we present the biochemical characterization and crystal structure of Leishmania donovani 6PGD ( Ld 6PGD) in complex with NADP(H). Most interestingly, a previously unknown conformation of NADPH is visible in this structure. In addition, we identified auranofin and other gold(I)-containing compounds as efficient Ld 6PGD inhibitors, although it has so far been assumed that trypanothione reductase is the sole target of auranofin in Kinetoplastida . Interestingly, 6PGD from Plasmodium falciparum is also inhibited at lower micromolar concentrations, whereas human 6PGD is not. Mode-of-inhibition studies indicate that auranofin competes with 6PG for its binding site followed by a rapid irreversible inhibition. By analogy with other enzymes, this suggests that the gold moiety is responsible for the observed inhibition. Taken together, we identified gold(I)-containing compounds as an interesting class of inhibitors against 6PGDs from Leishmania and possibly from other protozoan parasites. Together with the three-dimensional crystal structure, this provides a valid basis for further drug discovery approaches.

Background: Infections caused by azole-resistant Aspergillus are a rising public health threat with high mortality rates, high treatment costs and limited available antifungals, indicating an urgent need for new antifungals or strategies. Our aim was to investigate antifungal and antibiofilm activities of auranofin, an FDA-approved anti-antirheumatic drug., Methods: Fungal susceptibility testing for auranofin was carried out by the broth-based microdilution methods. Cell viability treated by auranofin was tested by resazurin dye testing. The synergistic effect of auranofin and antifungal drugs was evaluated using checkboard assay. The inhibitory of biofilms were measured by crystal violet staining. Gene expression level analysis and enzyme activity was investigated with qRT-PCR analysis and DTNB assay. The key amino acid residues in the binding of auranofin with A. fumigatus thioredoxin reductase (AfTrxR) were indicated by structural analyses, site-directed mutagenesis, and microscale thermophoresis (MST) assays., Results: Auranofin has fungicidal activity and in vitro antifungal spectrum including Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Aspergillus niger, even itraconazole (ITC)-resistant A. fumigatus. Additionally, it has antibiofilm activities against ITC-resistant A. fumigatus by reducing the expression level of SomA and MedA. Moreover, we discovered a synergistic effect of auranofin and ITC or amphotericin B against ITC-resistant A. fumigatus. Auranofin downregulated the gene transcription of AfTrxR, and strongly inhibited the enzyme activity of AfTrxR through interacting with residues C145 and C148., Conclusions: Auranofin has fungicidal and antibiofilm activities in Aspergillus spp. and is also a potentiator of ITC or amphotericin B in vitro., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest., (Copyright © 2023 SFMM. Published by Elsevier Masson SAS. All rights reserved.)

Multidrug-resistant (MDR) Gram-negative bacteria have become a global threat to human life and health, and novel antibiotics are urgently needed. The thioredoxin (Trx) system can be used as an antibacterial target to combat MDR bacteria. Here, we found that two active gold(I) selenium N-heterocyclic carbene complexes H7 and H8 show more promising antibacterial effects against MDR bacteria than auranofin. Both H7 and H8 irreversibly inhibit the bacterial TrxR activity via targeting the redox-active motif, abolishing the capacity of TrxR to quench reactive oxygen species (ROS) and finally leading to oxidative stress. The increased cellular superoxide radical levels impact a variety of functions necessary for bacterial survival, such as cellular redox balance, cell membrane integrity, amino acid metabolism, and lipid peroxidation. In vivo data present much better antibacterial activity of H7 and H8 than auranofin, promoting the wound healing and prolonging the survival time of Carbapenem-resistant Acinetobacter baumannii (CRAB) induced peritonitis. Most notably in this study, we revealed the influence of gold(I) complexes on both the Trx system and the cellular metabolic states to better understand their killing mechanism and to support further antibacterial drug design., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Drug repositioning has gained significant attention over the past several years. The anti-rheumatoid arthritis drug auranofin has been investigated for the treatment of other diseases, including liver fibrosis. Because auranofin is rapidly metabolized, it is necessary to identify the active metabolites of auranofin that have detectable levels in the blood and reflect its therapeutic effects. In the present study, we investigated whether aurocyanide as an active metabolite of auranofin, can be used to evaluate the anti-fibrotic effects of auranofin. Incubation of auranofin with liver microsomes showed that auranofin was susceptible to hepatic metabolism. Previously, we found that the anti-fibrotic effects of auranofin are mediated via system x c - -dependent inhibition of the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome. Therefore, we tried to identify active metabolites of auranofin based on their inhibitory effects on system x c - and NLRP3 inflammasome in bone marrow-derived macrophages. Among the seven candidate metabolites, 1-thio-β-D-glycopyrano-sato-S-(triethyl-phosphine)-gold(I) and aurocyanide potently inhibited system x c -  and NLRP3 inflammasome. A pharmacokinetics study on mice detected significant plasma levels of aurocyanide after auranofin administration. Oral administration of aurocyanide significantly prevented thioacetamide-induced liver fibrosis in mice. Moreover, the in vitro anti-fibrotic effects of aurocyanide were assessed in LX-2 cells, where aurocyanide significantly decreased the migratory ability of the cells. In conclusion, aurocyanide is metabolically stable and detectable in plasma, and has inhibitory effects on liver fibrosis, suggesting that it is a potential marker of the therapeutic effects of auranofin., (© 2023. The Pharmaceutical Society of Korea.)

Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)