This method enables the identification of test substances that inhibit DIO1 activity and may thus interfere with thyroid hormone homeostasis. The protocol presented describes a non-radioactive, colorimetric method for assessing the effects of test substances on the enzymatic activity of Deiodinase 1 (DIO1) in human liver microsomes. The assay is based on the Sandell-Kolthoff (SK) reaction, which quantifies iodide released during the deiodination of reverse triiodothyronine (rT3) to diiodothyronine (T2). The yellow cerium (IV) is reduced to colorless cerium (III) in the presence of iodide, allowing photometric detection at 415 nm. In this standardized setup, the cerium (IV) concentration is set to 40 mM to improve signal stability and robustness under the described conditions. To ensure assay reliability and reproducibility, a comprehensive control setup is included: 6-propyl-2-thiouracil (reference item), aurothioglucose (positive control), 1-thio-β-D-glucose sodium salt (negative control), and 1% dimethyl sulfoxide (solvent control). The protocol includes a range-finding assay to determine appropriate test concentrations, standardization of microsome batch-specific iodide release activity and structured chemical interference testing in the absence of microsomes, with an optional follow-up assessment without dithiothreitol (DTT) to identify DTT-dependent artifacts. The DIO1-SK assay is suitable for medium- to high-throughput screening and mechanistic toxicology studies. Its robustness, scalability, and applicability to human microsomes make it a valuable tool for investigating endocrine disruption. This protocol supports regulatory validation efforts.

Praziquantel (PZQ) is currently the only agent for treating schistosomiasis, but it is plagued by suboptimal efficacy to juvenile parasites, looming drug resistance, and inability to prevent reinfection. Thioredoxin glutathione reductase (TGR) is regarded as a promising therapeutic target due to its essential role in maintaining schistosome redox homeostasis. Herein, the crystal structures of Schistosoma japonicum TGR (SjTGR) in multiple redox states and in complex with NADPH, GSH, and the anti-helminthic agent Auranofin were elucidated. Structural analyses identified the hook-shaped conformation at the C-terminal redox center, which DTNB assays further confirmed enhances electron transfer efficiency. Structural and ITC data indicated that R317 was critical for NADPH binding via hydrogen-bond interactions. The analysis also indicated that the structure basis of Auranofin's potency was its tripartite interaction at the redox-active sites. In addition, we investigated the substrate specificity of SjTrx1i and SjTRP14, downstream proteins regulated by SjTGR, and elucidated the structural basis for this specificity by determining their oxidized/reduced structures. Furthermore, in vivo RNAi indicated knockdown of SjTGR or SjTRP14 blocked the survival and oviposition of schistosomes, thus ameliorating egg-induced granulomatous pathology in mice. This work provided a framework for knowledge-based design of novel anti-schistosomals targeting parasite-specific redox vulnerabilities.

Gold-based drugs possess a long history of clinical treatment of rheumatoid arthritis with a favorable safety profile. Their unique mechanism of action involving redox regulation holds promise for overcoming the common resistance and toxicity issues associated with platinum-based drugs. However, the further development of gold drugs still faces several key challenges, primarily including off-target binding, immunosuppressive toxicity, and insufficient selectivity for thioredoxin reductase (TrxR) isoforms. This review summarizes the unique strategies from the group of Zou to address these issues, involving stimulus-responsive gold prodrugs to overcome albumin-mediated off-target binding; a subcellular photocatalysis strategy for selective inhibition of TrxR1; and a dynamic ligand exchange strategy enabling the therapeutic repurposing of the approved drug auranofin at clinically relevant doses for anticancer treatment. Furthermore, we have designed and developed metal-based drugs with immunogenic cell death (ICD) activity and conducted an in-depth exploration of their targets and related mechanisms. Future perspectives highlight critical research pathways required for realizing the full therapeutic potential of these metal complexes in actual clinical settings.

Opportunistic fungal infections have become an increasing health threat, particularly in immunocompromised individuals, due to their high virulence, biofilm-forming capacity, and resistance to conventional antifungals. Given the limited therapeutic arsenal, drug repurposing represents a promising strategy for antifungal discovery. This study evaluated the antifungal activity and mechanisms of auranofin and iodoquinol against filamentous fungi listed as World Health Organization (WHO) priority pathogens, including Aspergillus fumigatus, Fusarium oxysporum, Scedosporium boydii, Lomentospora prolificans, Rhizopus oryzae, Mucor velutinosus, and Cunninghamella sp. Although Aspergillus flavus is not included in the WHO priority list, it was included in this study due to its clinical relevance as a major cause of aspergillosis and fungal rhinosinusitis. Auranofin displayed fungicidal activity against Aspergillus spp., L. prolificans, S. boydii, and R. oryzae, while iodoquinol exhibited potent but mainly fungistatic effects, with minimum inhibitory concentration values ranging from 0.625 to 20 µM. Both compounds inhibited early growth and reduced preformed biofilms by over 50% in biomass and metabolic activity. Stress susceptibility and fluorescence assays indicated that auranofin interferes with lipid homeostasis, cell wall integrity, and oxidative stress responses, whereas iodoquinol alters membrane lipids, mannose, and chitin distribution, suggesting multitarget surface effects. Scanning electron microscopy revealed consistent ultrastructural alterations, including hyphal deformation and increased extracellular matrix deposition in A. fumigatus and F. oxysporum treated with sub-inhibitory drug concentrations, corroborating the observed cellular stress responses. Drug interaction assays demonstrated additive or synergistic effects with voriconazole and amphotericin B, but not with posaconazole. Overall, auranofin and iodoquinol exhibit broad-spectrum antifungal activity, disrupt fungal morphology and physiology, and represent promising repurposed candidates for developing novel combination therapies against refractory mycoses.IMPORTANCEMycoses caused by opportunistic fungi are an increasingly public health problem, particularly in immunocompromised individuals, due to their high virulence and resistance to conventional antifungals. The World Health Organization's priority fungal pathogens list points out a variety of filamentous fungi, such as Aspergillus fumigatus, A. flavus, Fusarium oxysporum, Scedosporium boydii, Lomentospora prolificans, Rhizopus oryzae, Mucor velutinosus, and Cunninghamella sp. Given the limited therapeutic arsenal to combat them, drug repurposing represents a promising strategy for antifungal discovery. The present study evaluated the antifungal activity and some cellular alterations caused by auranofin and iodoquinol, which are already available in clinical settings to treat rheumatoid arthritis and infections by amoeba, respectively. The data presented in the study revealed that both drugs display a wide spectrum of action against different fungal pathogens, as well as contribute to highlight the potential of them as repurposing drugs to be investigated as alternatives for the treatment of mycoses.

Melanoma is a malignant neoplasm that originates from melanocytes. It continues to pose a significant challenge in oncology due to its aggressive nature and limited treatment options. This study investigates the potential additive effects of PHEC-66, a cannabis extract, in combination with conventional chemotherapeutic agents auranofin, docetaxel, and cisplatin on the viability of a range of melanoma cell lines. These combinations were evaluated using the MTT assay on MM418-C1, MM329, C32, and D24 melanoma cells. There was a nuanced response observed when PHEC-66 was combined with docetaxel and auranofin in these cells, suggesting a potential additive effect. Contrastingly, the combination of PHEC-66 with cisplatin elicited an antagonistic effect, wherein the expected cytotoxicity of this drug was compromised. This unexpected interaction may stem from complex interplays between the agents that influence drug uptake, DNA damage response, and cell survival pathways. These findings underscore the importance of careful selection and assessment of drug combinations, as an additive effect and antagonistic interactions can significantly impact therapeutic outcomes. Further studies are warranted to elucidate the molecular mechanisms behind these interactions and to validate these observations using in vivo models.

Adoptive natural killer (NK) cell therapy (ANKCT) is a promising strategy for hepatocellular carcinoma (HCC); however, its efficacy is hampered by insufficient NK cell homing and the immunosuppressive activity of M2-polarized tumor-associated macrophages (TAMs). Activation of the cyclic guanosine monophosphate (GMP)-adenosine monophosphate synthase (AMP) synthase-stimulator of interferon genes (cGAS-STING) pathway enhances NK-cell recruitment and reprograms TAMs toward a proinflammatory M1 phenotype. Radiation therapy (RT) activates the cGAS-STING pathway by inducing reactive oxygen species (ROS)-mediated DNA damage. Compared with high-dose irradiation, low-dose radiotherapy (LDRT) offers advantages, including reduced toxicity and enhanced antitumor immunity. However, the robust thioredoxin (Trx) and glutathione (GSH) antioxidant systems in HCC inhibit LDRT-induced ROS accumulation, thereby limiting immune activation. These limitations highlight the need for auxiliary strategies to complement LDRT-induced immunogenicity. Here, we developed a biomimetic nanoparticle in which auranofin-loaded MOF-199 is cloaked with tumor cell membranes (A@MMOF). A@MMOF disrupts tumor redox homeostasis by irreversibly inhibiting the GSH and Trx antioxidant systems while inducing GSH-dependent Cu²⁺ release from the MOF framework. The subsequent reduction of Cu²⁺ to Cu⁺ catalyzes Fenton-like reactions, markedly amplifying the intracellular ROS levels. By providing a sustained redox-driven stimulus, A@MMOF compensates for the insufficient oxidative stress induced by LDRT, leading to robust activation of the cGAS-STING pathway. Thus, A@MMOF synergizes with LDRT to remodel the tumor microenvironment, enhance NK cell infiltration and activation, and ultimately improve the efficacy of ANKCT in HCC.

Auranofin, a gold(I)-based compound initially developed for the treatment of rheumatoid arthritis, has emerged as a promising anticancer agent with a multimodal mechanism of action. This review comprehensively examines the therapeutic potential of auranofin in oncology focusing on its ability to synergize with conventional and emerging cancer treatments. Here, we discuss the unique pharmacological properties of auranofin, including thioredoxin reductase inhibition, reactive oxygen species induction, and modulation of key apoptotic pathways. Moreover, this article highlights new recent evidence on its ability to synergize with other cancer treatments such as chemotherapy, immunotherapy, and targeted therapies. Particular emphasis is placed on the role of auranofin in overcoming drug resistance and its potential as an adjuvant in precision medicine. By analyzing both preclinical and clinical data, this review provides critical insights into the repositioning of auranofin as a versatile component in contemporary cancer treatment paradigms, while addressing current challenges and future directions for gold-based therapeutics in oncology.

Cyclometallated gold(III) compounds were evaluated for their chemoselective capability to promote C-Se coupling reactions under biocompatible conditions. Competitive reactions with selenium and sulfur substrates highlighted the preference for selenium, and this selectivity was further confirmed in selenopeptide models mimicking the GPx active site. Given that thioredoxin reductase (TXNRD1) is a canonical target for gold compounds, we confirmed that our complexes also inhibit this enzyme, with the two six-membered metallacycles exhibiting a higher potency than auranofin. Expanding beyond TXNRD1, the compounds were further investigated as inhibitors of other selenoenzymes, specifically glutathione peroxidase isoenzymes (GPx1, GPx4). The metallacycles were potent inhibitors of GPx1, while in vitro GPx4 inhibition was overall less pronounced, with LC/MS studies identifying selenocysteine (Sec51) as the primary arylation site on GPx1. We demonstrated that this chemoselectivity could be translated to an intracellular setting. The selectivity towards Sec over Cys was further explored using A375 GPx4 WT and A375 GPx4 U46C mutant cell lines, where proliferation assays showed a greater effect in the GPx4 WT cells. By integrating structural and functional insights across selenoenzyme families, this study reveals glutathione peroxidases as pivotal molecular targets of cyclometallated gold(III) compounds and lays the groundwork for designing selective Sec-targeting metallodrugs, an approach with untapped potential in anticancer therapy.

Ferroptosis is a form of regulated cell death driven by lipid peroxidation, in which the diffusion of lipid peroxidation substrates on the plasma membrane is hindered by lipid rafts, thereby inhibiting the ferroptosis process. Lipid rafts are microdomains composed of cholesterol, sphingolipids, and specific proteins. Notably, cholesterol acyltransferase sterol O-acyltransferase 1 (SOAT1) is highly expressed in various malignant tumors, where it promotes tumor cell growth by enhancing intracellular cholesterol storage. To alleviate the obstruction of ferroptosis progression by lipid rafts, we developed human serum albumin nanoparticles (HSA@Aur&Ava) for codelivery of the SOAT1 inhibitor avasimibe (Ava) and the ferroptosis inducer auranofin (Aur). Ava indirectly disrupts the structure of lipid rafts by blocking cholesterol esterification, while Aur induces ferroptosis by inhibiting thioredoxin reductase. The synergistic effect of these two agents enhances the accumulation of lipid peroxides and the occurrence of ferroptosis. Additionally, tumor cells release damage-associated molecular patterns (DAMPs), which enhance the maturation of dendritic cells (DCs) and further recruit CD8+ T cells, thereby activating the immune response. The nanoparticles significantly inhibit tumor progression through chemotherapy and immunotherapy, offering an insight for highly effective antitumor strategies from the standpoint of cholesterol metabolism.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major therapeutic challenge and poses a significant global health threat. Developing adjuvants to enhance the efficacy of existing antibiotics represents a promising strategy to address this issue. In this study, we evaluated auranofin as an adjuvant to potentiate the activity of linezolid against MRSA. Auranofin significantly increased MRSA susceptibility to linezolid, promoted intracellular linezolid accumulation, and suppressed the emergence of MRSA resistance to linezolid. Mechanistically, auranofin inhibited the Trx/TrxR system, inducing redox imbalance and reactive oxygen species (ROS) accumulation, which triggers DNA damage and transcriptional dysregulation. Auranofin synergized with linezolid to achieve dual inhibition of MRSA protein synthesis. Furthermore, auranofin downregulated the global regulator sarA, impaired SarA DNA-binding activity, and enhanced SarA phosphorylation, thereby attenuating SarA-mediated virulence factors (eg, adhesins and toxins) and biofilm formation. Importantly, auranofin fully restored anti-MRSA activity of linezolid in both Galleria mellonella and murine bacteremia models. Collectively, these findings identify auranofin as a promising adjuvant to linezolid and highlight its potential to improve therapeutic outcomes against invasive MRSA infections.

Genome instability is most commonly caused by replication stress, which also renders cancer cells extremely vulnerable once their response to replication stress is impeded. Topoisomerase II binding protein 1 (TOPBP1), an allosteric activator of ataxia telangiectasia and Rad3-related kinase (ATR), coordinates ATR in replication stress response and has emerged as a potential therapeutic target for tumors. Here, we identify auranofin, the FDA-approved drug for rheumatoid arthritis, as a lead compound capable of binding to the BRCT 7-8 domains and blocking TOPBP1 interaction with PHF8 and FANCJ. The liquid-liquid phase separation of TOPBP1 is also disrupted by auranofin. Through targeting these TOPBP1-nucleated molecular machineries, auranofin leads to an accumulation of replication defects by impairing ATR activation and attenuating replication protein A loading on perturbed replication forks, and it shows significant anti-breast tumor activity in combination with a PARP inhibitor. This study provides mechanistic insights into how auranofin challenges replication integrity and expands the application of this FDA-approved drug in breast tumor intervention.

To investigate the antibacterial activity of ceftazidime/avibactam (CZA) combined with auranofin (AUR) against Carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro and in vivo. The minimum inhibitory concentrations (MICs) of CZA and AUR in 40 non-redundant CRKP isolates were determined by broth microdilution method. Drug resistance genes were detected by polymerase chain reaction (PCR). Checkerboard assays, time-kill assays and Galleria mellonella infection models were employed to assess the effect of CZA plus AUR in vitro and in vivo. Additionally, serial subculture experiments were used to evaluate resistance progression. 75.0% (30/40) of clinical isolates exhibited resistance to CZA. The addition of AUR resulted in a remarkable 16-fold reduction in MIC50 (from 128 to 8 mg/L) and MIC90 > 64-fold (from > 2048 to 32 mg/L). Fractional inhibitory concentration index (FICI) analysis demonstrated synergistic activity in 67.5% (27/40) of isolates, including 80.0% (24/30) CZA-resistant strains.At sub-inhibitory concentrations, CZA monotherapy increased the MIC 256-fold, in contrast to CZA combined with AUR treatment which significantly reduced the level of resistance. Monotherapy with either agent showed limited bactericidal effects in vivo, whereas combination therapy achieved complete bacterial eradication within 72-96 h. AUR synergizes with CZA on killing a broad spectrum of carbapenem or CZA resistant Klebsiella pneumoniae both in vitro and in vivo, and slows down the development of CZA resistance. The combination of CZA and AUR might be a potential therapeutic strategy for combating superbugs CZA resistant CRKP.