Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence pathways in P. aeruginosa, including quorum sensing (QS) and Type IV pili (TFP). We show that auranofin acts via multiple targets, one of which being Vfr. Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.

The emergence and rapid spread of the mobile colistin resistance gene mcr-1 among bacterial species and hosts significantly challenge the efficacy of "last-line" antibiotic colistin. Previously, we reported silver nitrate and auranofin serve as colistin adjuvants for combating mcr-1-positive bacteria. Herein, we uncovered more gold-based drugs and nanoparticles, and found that they exhibited varying degree of synergisms with colistin on killing mcr-1-positive bacteria. However, pre-activation of the drugs by either glutathione or N-acetyl cysteine, thus releasing and accumulating gold ions, is perquisite for their abilities to substitute zinc cofactor from MCR-1 enzyme. X-ray crystallography and biophysical studies further supported the proposed mechanism. This study not only provides basis for combining gold-based drugs and colistin for combating mcr-1-positive bacterial infections, but also undoubtedly opens a new horizon for metabolism details of gold-based drugs in overcoming antimicrobial resistance.

Auranofin (AF), a gold (I)-containing phosphine compound, is being investigated for oncological application as a repurposed drug. We show here that 4~5 µM AF induces paraptosis, a non-apoptotic cell death mode characterized by dilation of the endoplasmic reticulum (ER) and mitochondria, in breast cancer cells. Although the covalent inhibition of thioredoxin reductase (TrxR), an enzyme that critically controls intracellular redox homeostasis, is considered the primary mechanism of AF's anticancer activity, knockdown of TrxR1 did not induce paraptosis. Instead, both TrxR1 knockdown plus the proteasome inhibitor (PI), bortezomib (Bz), and 2 μM AF plus Bz induced paraptosis, thereby mimicking the effect of 5 μM AF. These results suggest that the paraptosis induced by 5 μM AF requires the inhibition of both TrxR1 and proteasome. We found that TrxR1 knockdown/Bz or subtoxic doses of AF and Bz induced paraptosis selectively in breast cancer cells, sparing non-transformed MCF10A cells, whereas 4~5 μM AF killed both cancer and MCF10A cells. GSH depletion was found to be more critical than ROS generation for the paraptosis induced by dual TrxR1/proteasome inhibition. In this process, the ATF4/CHAC1 (glutathione-specific gamma-glutamylcyclotransferase 1) axis leads to GSH degradation, contributing to proteotoxic stress possibly due to the accumulation of misfolded thiol-containing proteins. These results suggest that the paraptosis-inducing strategy of AF plus a PI may provide an effective therapeutic strategy against pro-apoptotic therapy-resistant cancers and reduce the potential side effects associated with high-dose AF.

Macrophages secrete a variety of pro-inflammatory cytokines in response to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) but abnormal release of cytokines unfortunately promotes cytokine storms. Dimethyl fumarate (DMF), an FDA-approved drug for multiple sclerosis (MS) treatment, has been found as an effective therapeutic agent for resolution. In this study, the anti-inflammatory effect of DMF was found to correlate to selenoprotein thioredoxin reductase 1 (TXNRD1). DMF irreversibly modified the Sec498 residue and C-terminal catalytic cysteine residues of TXNRD1 in a time- and dose-dependent manner. In LPS-stimulated RAW 264.7 cells, cellular TXNRD activity was increased through up-regulation of the protein level and DMF inhibited TXNRD activity and the nitric oxide (NO) production of RAW 264.7 cells. Meanwhile, the inhibition of TXNRD1 by DMF would contribute to the redox regulation of inflammation and promote the nuclear factor erythroid 2-related factor 2 (NRF2) activation. Notably, inhibition of cellular TXNRD1 by auranofin or TRi-1 showed anti-inflammatory effect in RAW 264.7 cells. This finding demonstrated that targeting TXNRD1 is a potential mechanism of using immunometabolites for dousing inflammation in response to pathogens and highlights the potential of TXNRD1 inhibitors in immune regulation.

Fungal infection is a serious global health issue, causing approximately 1.5 million mortalities annually. However, clinically available anti-fungal drugs are limited, especially for multidrug-resistant fungal infections. Therefore, new antifungal drugs are urgently needed to address this clinical challenge. In this study, we proposed two non-antifungal drugs, auranofin and pentamidine, in combination to fight against multidrug-resistant C. albicans. The insufficient antifungal activity of anti-rheumatic drug auranofin is partially due to fungal membrane barrier preventing the drug uptake, and anti-protozoal drug pentamidine was used here to improve the permeability of membrane. The auranofin/pentamidine combination displayed synergistic inhibitory effect against both drug-susceptible and drug-resistant C. albicans, as well as biofilm, and significantly reduced the minimum inhibitory concentration of each drug. At non-antifungal concentration, pentamidine can disrupt the membrane integrity and increase membrane permeability, leading to enhanced cellular uptake of auranofin in C. albicans. This repurposing strategy using the combination of non-antifungal drugs with complementary antifungal mechanism may provide a novel approach for discovery of antifungal drugs to fight against multidrug-resistant fungal infections.

Chronic obstructive pulmonary disease (COPD), a small airway disease, is regarded as a metabolic disorder. To further uncover the metabolic profile of COPD patients, it is necessary to identify metabolism-related differential genes in small airway epithelium (SAE) of COPD. Metabolism-related differential genes in SAE between COPD patients and nonsmokers were screened from GSE128708 and GSE20257 datasets. KEGG, GO, and PPI analyses were performed to evaluate the pathway enrichment, term enrichment, and protein interaction of candidate metabolism-related differential genes, respectively. RT-PCR was used to verify the mRNA expression of the top ten differential genes. Western blotting was used to evaluate the protein expression of TXNRD1. TXNRD1 inhibitor auranofin (AUR) was used to assess the impact of TXNRD1 on oxidative stress and inflammation induced by cigarette smoke extraction (CSE). Twenty-four metabolism-related differential genes were selected. ALDH3A1, AKR1C3, CYP1A1, AKC1C1, CPY1B1, and TXNRD1 in the top ten genes were significantly upregulated after CSE simulation for 24 h in human bronchial epithelial (16HBE) cells. Among them, CYP1A1 and TXNRD1 also have a significant upregulation in primary SAE after simulation of CSE for 24 h. The overexpression of protein TXNRD1 has also been detected in 16HBE cells, primary SAE stimulated with CSE, and mouse lung exposed to cigarette smoke (CS). Additionally, inhibition of TXNRD1 with 0.1 μM AUR alleviated the expression of IL-6 and reactive oxygen species (ROS) induced by CSE by activating the Nrf2/HO-1 pathway in 16HBE cells. This study identified twenty-four metabolism-related differential genes associated with COPD. TXNRD1 might participate in the oxidative stress and inflammation induced by CS by regulating the activation of the Nrf2/HO-1 pathway.

Pro-oxidative shift in redox balance, usually termed as "oxidative stress", can lead to different cell responses depending on its intensity. Excessive accumulation of reactive oxygen species ("oxidative distress") can cause DNA, lipid and protein damage. Physiological oxidative stimulus ("oxidative eustress"), in turn, can favor cell proliferation and differentiation - the processes of paramount importance primarily for stem cells. Functions of antioxidant enzymes in cells is currently a focus of intense research, however the role of different antioxidant pathways in pluripotent cell responses to oxidative distress/eustress is still under investigation. In this study, we assessed the contribution of the thioredoxin reductase (TrxR)-dependent pathways to maintaining the redox homeostasis in human induced pluripotent stem cells and their differentiated progeny cells under basal conditions and under conditions of oxidative stress of varying intensity. Employing the genetically encoded H2O2 biosensor cyto-HyPer and two inhibitors of thioredoxin reductase (auranofin and Tri-1), we show that the reduced activity of TrxR-dependent enzymatic systems leads to the non-cytotoxic disruption of thiol-disulfide metabolism in the cytoplasm of both pluripotent and differentiated cells under basal conditions. Quantifying the cytoplasmic concentrations of peroxide establishing in H2O2-stressed cells, we demonstrate that TrxR-dependent pathways contribute to the antioxidant activity in the cell cytoplasm under conditions of mild but not severe oxidative stress in both cell lines tested. The observed effects may testify about a conservative role of the TrxR-controlled enzymatic systems manifested as a response to physiological redox stimuli rather than a protection against the severe oxidative stress.

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.

The antineoplastic activity of the thioredoxin reductase 1 (TrxR) inhibitor, auranofin (AF), has already been investigated in various cancer mouse models as a single drug, or in combination with other molecules. However, there are inconsistencies in the literature on the solvent, dose and administration route of AF treatment in vivo. Therefore, we investigated the solvent and administration route of AF in a syngeneic SB28 glioblastoma (GBM) C57BL/6J and a 344SQ non-small cell lung cancer 129S2/SvPasCrl (129) mouse model. Compared to daily intraperitoneal injections and subcutaneous delivery of AF via osmotic minipumps, oral gavage for 14 days was the most suitable administration route for high doses of AF (10-15 mg/kg) in both mouse models, showing no measurable weight loss or signs of toxicity. A solvent comprising 50% DMSO, 40% PEG300 and 10% ethanol improved the solubility of AF for oral administration in mice. In addition, we confirmed that AF was a potent TrxR inhibitor in SB28 GBM tumors at high doses. Taken together, our results and results in the literature indicate the therapeutic value of AF in several in vivo cancer models, and provide relevant information about AF's optimal administration route and solvent in two syngeneic cancer mouse models.

Glioblastoma (GBM) is the most aggressive primary brain tumor. Recently, agents increasing the level of oxidative stress have been proposed as anticancer drugs. However, their efficacy may be lowered by the cytoprotective activity of antioxidant enzymes, often upregulated in neoplastic cells. Here, we assessed the mRNA and protein expression of thioredoxin reductase 1 (TrxR1), a master regulator of cellular redox homeostasis, in GBM and non-tumor brain tissues. Next, we examined the influence of an inhibitor of TrxR1, auranofin (AF), alone or in combination with a prooxidant menadione (MEN), on growth of GBM cell lines, patient-derived GBM cells and normal human astrocytes. We detected considerable amount of TrxR1 in the majority of GBM tissues. Treatment with AF decreased viability of GBM cells and their potential to form colonies and neurospheres. Moreover, it increased the intracellular level of reactive oxygen species (ROS). Pre-treatment with ROS scavenger prevented the AF-induced cell death, pointing to the important role of ROS in the reduction of cell viability. The cytotoxic effect of AF was potentiated by treatment with MEN. In conclusion, our results identify TrxR1 as an attractive drug target and highlights AF as an off-patent drug candidate in GBM therapy.

IRF3 (interferon regulatory factor 3) is a critical component of the antiviral innate immune response. IRF3 deficiency causes detrimental effects to the host during virus infection. Dysregulation of IRF3 functions is associated with viral, inflammatory, and hepatic diseases. Both transcriptional and pro-apoptotic activities of IRF3 are involved in the exacerbated inflammation and apoptosis in liver injury induced by ethanol and high-fat diets. Therefore, regulation of IRF3 activities has consequences, and it is a potential therapeutic target for infectious and inflammatory diseases. We recently revealed that IRF3 is degraded by a small molecule, auranofin, by activating the cellular macroautophagy/autophagy pathway. Autophagy is a catabolic pathway that contributes to cellular homeostasis and antiviral host defense. Degradation of IRF3 by autophagy may be a novel strategy used by the viruses to their benefit. In addition, IRF3 functions are harmful in other diseases, including liver injury and bacterial infection. A better understanding of the role of autophagy in regulating IRF3 functions has significant implications in developing therapeutic strategies. Therefore, autophagy provides checks and balances in the innate immune response.

Auranofin is a thioredoxin reductase-1 inhibitor originally approved for the treatment of rheumatoid arthritis. Recently, auranofin has been repurposed as an anticancer drug, with pharmacological activity reported in multiple cancer types. In this study, we characterized transcriptional and genetic alterations associated with auranofin response in cancer. By integrating data from an auranofin cytotoxicity screen with transcriptome profiling of lung cancer cell lines, we identified an auranofin resistance signature comprising 29 genes, most of which are classical targets of the transcription factor NRF2, such as genes involved in glutathione metabolism (GCLC, GSR, SLC7A11) and thioredoxin system (TXN, TXNRD1). Pan-cancer analysis revealed that mutations in NRF2 pathway genes, namely KEAP1 and NFE2L2, are strongly associated with overexpression of the auranofin resistance gene set. By clustering cancer types based on auranofin resistance signature expression, hepatocellular carcinoma, and a subset of non-small cell lung cancer, head-neck squamous cell carcinoma, and esophageal cancer carrying NFE2L2/KEAP1 mutations were predicted resistant, whereas leukemia, lymphoma, and multiple myeloma were predicted sensitive to auranofin. Cell viability assays in a panel of 20 cancer cell lines confirmed the augmented sensitivity of hematological cancers to auranofin; an effect associated with dependence upon glutathione and decreased expression of NRF2 target genes involved in GSH synthesis and recycling (GCLC, GCLM and GSR) in these cancer types. In summary, the omics-based identification of sensitive/resistant cancers and genetic alterations associated with these phenotypes may guide an appropriate repurposing of auranofin in cancer therapy.

Auranofin (AF), a gold(I) compound that is currently used for the treatment of rheumatoid arthritis and is in clinical trials for its promising anticancer activity, was encapsulated within the human H-chain and the horse spleen ferritin nanocages using the alkaline disassembly/reassembly protocol. The aim of the work was to highlight possible differences in their drug loading capacity and efficacy. The drug-loaded ferritins were characterized via UV-vis absorption spectroscopy and inductively coupled plasma-atomic emission spectroscopy to assess AF encapsulation and to define the exact amount of gold atoms trapped in the Ft cavity. The crystal structures allowed us to define the nature of AF interaction with both ferritins and to identify the gold binding sites. Moreover, the biological characterization let us to obtain preliminary information on the cytotoxic effect of AF when bound to the human H-chain.

Gold compounds have a long tradition in medicine and offer many opportunities for new therapeutic applications. Herein, we evaluated the lead compound Auranofin and five related gold(I) complexes as possible inhibitors of SARS-CoV-2 Main Protease (SARS-CoV-2 Mpro), a validated drug target for the COVID-19 disease. The investigational panel of gold compounds included Auranofin; three halido analogues, i.e., Au(PEt3)Cl, Au(PEt3)Br, and Au(PEt3)I; and two gold carbene complexes, i.e., Au(NHC)Cl and [Au(NHC)2]PF6. Notably, all these gold compounds, with the only exception of [Au(NHC)2]PF6, turned out to be potent inhibitors of the catalytic activity of SARS-CoV-2 Mpro: the measured Ki values were in the range 2.1-0.4 μM. The reactions of the various gold compounds with SARS-CoV-2 Mpro were subsequently investigated through electrospray ionization (ESI) mass spectrometry (MS) upon a careful optimization of the experimental conditions; the ESI MS spectra provided clear evidence for the formation of tight metallodrug-protein adducts and for the coordination of well defined gold-containing fragments to the SARS-CoV-2 Mpro, again with the only exception of [Au(NHC)2]PF6, The metal-protein stoichiometry was unambiguously determined for the resulting species. The crystal structures of the metallodrug- Mpro adducts were solved in the case of Au(PEt3)Br and Au(NHC)Cl. These crystal structures show that gold coordination occurs at the level of catalytic Cys 145 in the case of Au(NHC)Cl and at the level of both Cys 145 and Cys 156 for Au(PEt3)Br. Tight coordination of gold atoms to functionally relevant cysteine residues is believed to represent the true molecular basis of strong enzyme inhibition.

Programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) interaction exerts a vital role in tumor-associated immune evasion. While strategies disrupting PD-1/PD-L1 axis have shown clinical benefits in various cancers, the limited response rate prompts us to investigate the complex mechanisms underlying the molecular regulation of PD-L1. Here, we identify the RNA binding protein RBMS3 as a crucial PD-L1 regulator in triple-negative breast cancer (TNBC). Correlation analysis shows that Rbms3 significantly correlates with immunosuppressive CD274, Rbms1, NT5E and ENTPD1. RBMS3 protein binds to CD274 mRNA specifically in TNBC cells to increase PD-L1 levels. Mechanistically, RBMS3 stabilizes CD274 mRNA by interacting with its 3'UTR, which represents as an intrinsic cancer cell mechanism for driving PL-D1 upregulation in TNBC. RBMS3 depletion not only destabilizes the mRNA stability and protein expression of PD-L1, but also suppresses the migratory abilities of TNBC MDA-MB-231 cells. Importantly, combination of RBMS3 ablation with auranofin (AUF), an FDA-approved thioredoxin reductase inhibitor, facilitates anti-tumor T-cell immunity in vivo and improves AUF-mediated anti-cancer effect. Taken together, our findings reveal RBMS3 as a key post-transcriptional regulator of PD-L1 and how they contribute to immune escape in TNBC, which could lead to novel combinatorial therapeutic strategies to enhance the efficacy of cancer immunotherapy.

Pancreatic ductal adenocarcinoma is characterized by increased generation of reactive oxygen species that can cause lethal oxidative stress. Here, we evaluated the combined inhibition of the glutathione and thioredoxin antioxidant systems in preclinical models of pancreatic ductal adenocarcinoma, using buthionine sulfoximine (BSO) that targets glutathione synthesis, and auranofin that targets thioredoxin recycling. BSO potentiated the cytotoxicity of auranofin and induced lethal oxidative stress in primary pancreatic cancer cells. As assessed by the cellular thermal shift assay, auranofin engaged with thioredoxin reductase 1 in primary cells at concentrations known to induce cell death. Moreover, we used imaging mass cytometry to map the biodistribution of atomic gold in patient-derived xenografts treated with auranofin, and the drug was readily detectable throughout the epithelial and stromal compartments after treatment with a clinically relevant dose. In conclusion, combinatorial treatment with BSO and auranofin could serve as a potential therapeutic strategy in pancreatic ductal adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most prevalent form of non-small cell lung cancer (NSCLC) and a leading cause of cancer death. Immune checkpoint inhibitors (ICIs) of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) signaling induce tumor regressions in a subset of LUAD, but many LUAD tumors exhibit resistance to ICI therapy. Here, we identified Prkci as a major determinant of response to ICI in a syngeneic mouse model of oncogenic mutant Kras/Trp53 loss (KP)-driven LUAD. Protein kinase Cι (PKCι)-dependent KP tumors exhibited resistance to anti-PD-1 antibody therapy (α-PD-1), whereas KP tumors in which Prkci was genetically deleted (KPI tumors) were highly responsive. Prkci-dependent resistance to α-PD-1 was characterized by enhanced infiltration of myeloid-derived suppressor cells (MDSCs) and decreased infiltration of CD8+ T cells in response to α-PD-1. Mechanistically, Prkci regulated YAP1-dependent expression of Cxcl5, which served to attract MDSCs to KP tumors. The PKCι inhibitor auranofin inhibited KP tumor growth and sensitized these tumors to α-PD-1, whereas expression of either Prkci or its downstream effector Cxcl5 in KPI tumors induced intratumoral infiltration of MDSCs and resistance to α-PD-1. PRKCI expression in tumors of patients with LUAD correlated with genomic signatures indicative of high YAP1-mediated transcription, elevated MDSC infiltration and low CD8+ T cell infiltration, and with elevated CXCL5/6 expression. Last, PKCι-YAP1 signaling was a biomarker associated with poor response to ICI in patients with LUAD. Our data indicate that immunosuppressive PKCι-YAP1-CXCL5 signaling is a key determinant of response to ICI, and pharmacologic inhibition of PKCι may improve therapeutic response to ICI in patients with LUAD.

Alternative antimicrobial therapies are urgently required for the multidrug-resistant bacterial pathogen Neisseria gonorrhoeae, for which currently ceftriaxone is the only remaining recommended first-line therapy. Repurposing of drugs that are approved for other clinical applications offers an efficient approach for development of alternative antimicrobial therapies. Auranofin, cannabidivarin, and tolfenamic acid were recently identified to display antimicrobial activity against N. gonorrhoeae. Here, we investigated their activity against a collection of 575 multidrug-resistant clinical isolates. All three compounds displayed consistent antimicrobial activity against all isolates, including against strains associated with the high-level ceftriaxone-resistant FC428 clone, with both the mode and MIC90 for auranofin of 0.5 mg/L, while both the mode and MIC90 for cannabidivarin and tolfenamic acid were 8 mg/L. Correlations between MICs of ceftriaxone and auranofin, cannabidivarin or tolfenamic acid were low, indicating that development of cross-resistance is unlikely. Furthermore, antimicrobial synergy analysis between ceftriaxone and auranofin, cannabidivarin, or tolfenamic acid by determination of the fractional inhibitory concentration index (FICI) resulted in an interpretation of indifference. Finally, time-kill analyses showed that all three compounds are bactericidal against both the N. gonorrhoeae ATCC 49226 reference strain and an FC428-associated clinical isolate, with particularly cannabidivarin displaying rapid bactericidal activity. Overall, auranofin, cannabidivarin, and tolfenamic acid displayed consistent antimicrobial activity against multidrug-resistant N. gonorrhoeae, warranting further exploration of their suitability as alternative antimicrobials for treatment of gonococcal infections. IMPORTANCE Neisseria gonorrhoeae is a major public health concern because of the high incidence of gonorrhea and the increasingly limited options for antimicrobial therapy. Strains associated with the FC428 clone are a particular concern because they have shown global dissemination and they display high-level resistance against the currently recommended ceftriaxone therapy. Therefore, development of alternative antimicrobial therapies is urgently required to ensure treatment of gonorrhea remains available in the future. Repurposing of clinically approved drugs could be a rapid approach for the development of such alternative antimicrobials. In this study, we showed that repurposing of auranofin, cannabidivarin, and tolfenamic acid for antimicrobial therapy of gonorrhea deserves further clinical explorations because these compounds displayed consistent antimicrobial activity against a large collection of contemporary multidrug-resistant gonococcal isolates that included strains associated with the FC428 clone.

Metal-based drugs, such as cisplatin and auranofin, are used for the treatment of cancer and rheumatoid arthritis, respectively. Auranofin and other gold-derived compounds have been shown to possess anticancer, anti-inflammatory, antimicrobial, and antiparasitic activity in preclinical and clinical trials. Unlike platinum agents which are known to target DNA, the target of gold is not well elucidated. To better understand the targets and effects of gold agents in mammalian cells, we used a targeted CRISPR (ToxCRISPR) screen in K562 cancer cells to identify genes that modulate cellular sensitivity to gold. We synthesized a novel chiral gold(I) compound, JHK-21, with potent anticancer activity. Among the most sensitizing hits were proteins involved in mitochondrial carriers, mitochondrial metabolism, and oxidative phosphorylation. Further analysis revealed that JHK-21 induced inner mitochondria membrane dysfunction and modulated ATP-binding cassette subfamily member C (ABCC1) function in a manner distinct from auranofin. Characterizing the therapeutic effects and toxicities of metallodrugs in mammalian cells is of growing interest to guide future drug discovery, and cellular and preclinical/clinical studies.

Osteoarthritis, a prevalent orthopedic disease, can affect the elderly and causes impairment. The degradation and aberrant homeostasis of cartilage extracellular matrix figure pivotally in the progression of osteoarthritis. Thioredoxin systems plays a role in a wide range of biological processes, including cell proliferation, apoptosis, and oxidative stress. The present study aimed to investigate the unique function and underlying pathophysiological mechanism of TXNRD1 in chondrocytes. An upregulated expression of TXNRD1 was observed in the articular cartilage of osteoarthritis patients compared with normal articular cartilage. Furthermore, in vitro experiments showed that the expression of TXNRD1 was also abnormally increased in IL-1β-induced primary mouse chondrocytes. Silencing TXNRD1 using siRNA in chondrocytes could effectively inhibit the expression of ADAMTS5 and MMP13, and enhance the expression of COL2A1 and SOX9. The same was true for auranofin, an inhibitor of TXNRD1. This phenomenon indicated that inhibition of TXNRD1 attenuated il-1β-induced metabolic imbalance of extracellular matrix (ECM) and the progression of chondrocyte osteoarthritis. Further mechanism analysis revealed that the activation of Nrf2 signaling pathway and the expression of heme oxygenase-1 (HO-1) were increased upon TXNRD1 inhibition. Furthermore, auranofin was found to attenuate DMM-induced osteoarthritis progression in vivo. Therefore, the pharmacological downregulation of TXNRD1 may provide an effective novel therapy for OA.

Two types of lung cells (epithelial cancer lung cells, A-549 and lung fibroblasts MRC-5) were exposed to the clinically established gold drug auranofin at concentrations close to the half-maximal inhibitory drug concentrations (IC50). Collected cells were subjected to speciation analysis using inductively coupled plasma mass spectrometry (ICP-MS). Auranofin showed better affinity toward proteins than DNA, RNA, and hydrophilic small molecular weight compounds. It can bind to proteins that vary in size (~20 kDa, ~75 kDa, and ≥200 kDa) and pI. However, the possibility of dimerization and protein-protein complex formation should also be taken into account. µRPLC/CZE-ESI-MS/MS studies on trypsinized proteins allowed the indication of 76 peptides for which signal intensity was influenced by auranofin presence in cells. Based on it, identity was proposed for 20 proteins. Except for thioredoxin reductase (TrxR), which is directly targeted by gold complex, the proteins were found to be transformed. Five indicated proteins: myosin, plectin, talin, two annexins, and kinase M3K5, are responsible for cell-cell, cell-protein interactions, and cell motility. A wound healing test confirmed their regulation by auranofin as cell migration decreased by 40% while the cell cycle was not interrupted.

Reactive oxygen species (ROS) homeostasis and mitochondrial metabolism are critical for the survival of cancer cells, including cancer stem cells (CSCs), which often cause drug resistance and cancer relapse. Auranofin is a mono-gold anti-rheumatic drug, and it has been repurposed as an anticancer agent working by the induction of both ROS increase and mitochondrial dysfunction. Hypothetically, increasing auranofin's positive charges via incorporating more gold atoms to enhance its mitochondria-targeting capacity could enhance its anti-cancer efficacy. Hence, in this work, both mono-gold and bi-gold compounds were designed and evaluated to test our hypothesis. The results showed that bi-gold compounds generally suppressed cancer cells proliferation better than their mono-gold counterparts. The most potent compound, BGC2a, substantially inhibited the antioxidant enzyme TrxR and increased the cellular ROS. BGC2a induced cell apoptosis, which could not be reversed by the antioxidant agent vitamin C, implying that the ROS induced by TrxR inhibition might not be the decisive cause of cell death. As expected, a significant proportion of BGC2a accumulated within mitochondria, likely contributing to mitochondrial dysfunction, which was further confirmed by measuring oxygen consumption rate, mitochondrial membrane potential, and ATP production. Moreover, BGC2a inhibited colony formation and reduced stem-like side population (SP) cells of A549. Finally, the compound effectively suppressed the tumor growth of both A549 and PANC-1 xenografts. Our study showed that mitochondrial disturbance may be gold-based compounds' major lethal factor in eradicating cancer cells, providing a new approach to developing potent gold-based anti-cancer drugs by increasing mitochondria-targeting capacity.

A new series of seven gold(I) complexes (1-7) containing 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene (IPr) and phosphane ligands (L1-L7) were synthesized and evaluated for antitumor activity in ovarian cancer (OvCa) models. The synthesized complexes were characterized by IR, mass spectrometry and NMR spectroscopy, and complex 6 was characterized by XRD crystallography. The antiproliferative effect of the new complexes (1-7) was found to be higher than cisplatin and auranofin in OvCa cells sensitive and resistant to cisplatin. The anticancer activity of the most active complex 6 was investigated using OvCa in vitro models, including three-dimensional (3D) multicellular tumor spheroids and in vivo tumor xenografts. Both cisplatin and auranofin were used for comparative purposes. Complex 6 induced apoptosis, mitochondrial reactive oxygen species, and DNA damage; caused a G1 phase cell cycle arrest, inhibited proteasome activity, and cell migration; modified actin polymerization; and significantly inhibited OvCa murine xenografts. These promising results suggest further preclinical testing of these complexes for future applications.

New mono- and di-nuclear thio-purine and thio-purine nucleoside gold(I) complexes were synthesized, characterized, and evaluated in vitro for biological activities in comparison to related known purine complexes. By combining known anti-tumoral thio-purines with R3PAu moieties as present in auranofin, complexes with enhanced effects and selectivities were obtained, which not only act as cytostatics, but also disrupt tumor-specific processes. Their IC50 values in cytotoxicity test with tumor cell lines ranged from three-digit nanomolar to single-digit micromolar, revealing a tentative structure-activity relationship (SAR). Both the residues R2 of the phosphane ligand and R1 at C2 of the pyrimidine ring had a significant impact on the cytotoxicity. In most cases, the introduction of a ribo-furanosyl group at N9 of the purine led to a distinctly more cytotoxic complex. Most complexes were more active against multi-drug-resistant tumor cells or such lacking functional p53 when compared to the respective untreated wild type cell lines. Some nucleoside complexes displayed an interesting dose-dependent dual mode of action regarding cell cycle arrest and DNA repair mechanism. Some phosphane(purine-6-thiolato)gold (I) complexes had a stronger inhibitory effect on the thioredoxin reductase (TrxR) and on the reactive oxygen species (ROS) generation in cancer cells than is typical of other gold complexes. They also led to DNA fragmentation and showed anti-angiogenic effects. Their stability under test conditions was demonstrated by 77Se NMR monitoring of an exemplary selenopurine complex.

Low-grade, sustained inflammation in white adipose tissue (WAT) characterizes obesity and coincides with type 2 diabetes mellitus (T2DM). However, pharmacological targeting of inflammation lacks durable therapeutic effects in insulin-resistant conditions. Through a computational screen, we discovered that the FDA-approved rheumatoid arthritis drug auranofin improved insulin sensitivity and normalized obesity-associated abnormalities, including hepatic steatosis and hyperinsulinemia in mouse models of T2DM. We also discovered that auranofin accumulation in WAT depleted inflammatory responses to a high-fat diet without altering body composition in obese wild-type mice. Surprisingly, elevated leptin levels and blunted beta-adrenergic receptor activity achieved by leptin receptor deletion abolished the antidiabetic effects of auranofin. These experiments also revealed that the metabolic benefits of leptin reduction were superior to immune impacts of auranofin in WAT. Our studies uncover important metabolic properties of anti-inflammatory treatments and contribute to the notion that leptin reduction in the periphery can be accomplished to treat obesity and T2DM.

Auranofin (Ridaura®, AUF) is a gold complex originally approved as an antirheumatic agent that has emerged as a potential candidate for multiple repurposed therapies. The best-studied anticancer mechanism of AUF is the inhibition of thioredoxin reductase (TrxR). However, a number of reports indicate a more complex and multifaceted mode of action for AUF that could be cancer cell type- and dose-dependent. In this study, we observed that AUF displayed variable cytotoxicity in five triple-negative breast cancer cell lines. Using representative MDA-MB-231 cells treated with moderate and cytotoxic doses of AUF, we evidenced that an AUF-mediated TrxR inhibition alone may not be sufficient to induce cell death. Cytotoxic doses of AUF elicited rapid and drastic intracellular oxidative stress affecting the mitochondria, cytoplasm and nucleus. A "redoxome" proteomics investigation revealed that a short treatment with a cytotoxic dose AUF altered the redox state of a number of cysteines-containing proteins, pointing out that the cell proliferation/cell division/cell cycle and cell-cell adhesion/cytoskeleton structure were the mostly affected pathways. Experimentally, AUF treatment triggered a dose-dependent S-phase arrest and a rapid disintegration of the actin cytoskeleton structure. Our study shows a new spectrum of AUF-induced early effects and should provide novel insights into the complex redox-based mechanisms of this promising anticancer molecule.

Amyotrophic lateral sclerosis (ALS) is a fatal disease of motor neurons that mainly affects the motor cortex, brainstem, and spinal cord. Under disease conditions, microglia could possess two distinct profiles, M1 (toxic) and M2 (protective), with the M2 profile observed at disease onset. SOD1 (superoxide dismutase 1) gene mutations account for up to 20% of familial ALS cases. Comparative gene expression differences in M2-protective (early) stage SOD1G93A microglia and age-matched SOD1G93A motor neurons are poorly understood. We evaluated the differential gene expression profiles in SOD1G93A microglia and SOD1G93A motor neurons utilizing publicly available transcriptomics data and bioinformatics analyses, constructed biomolecular networks around them, and identified gene clusters as potential drug targets. Following a drug repositioning strategy, 5 small compounds (belinostat, auranofin, BRD-K78930611, AZD-8055, and COT-10b) were repositioned as potential ALS therapeutic candidates that mimic the protective state of microglia and reverse the toxic state of motor neurons. We anticipate that this study will provide new insights into the ALS pathophysiology linking the M2 state of microglia and drug repositioning.