Heparanase 1 (HPSE1) is the only mammalian endoglycosidase that cleaves heparan sulfate (HS), a glycosaminoglycan (GAG), and is frequently upregulated in cancers, thereby promoting tumor progression. Despite extensive efforts to develop inhibitors of its HS-degrading activity, its non-enzymatic functions limit therapeutic efficacy and pose a major challenge for therapeutic development. Thus, inhibiting HPSE1 expression is critical for controlling its enzymatic and non-enzymatic functions; however, no FDA-approved inhibitors are currently available. Here, we identify auranofin (AUF), an oral gold-containing drug used to treat rheumatoid arthritis, as a potent inhibitor of HPSE1 promoter activity. High-throughput screening revealed that an atypical protein kinase C (aPKC)-NF-κB signaling axis is a key regulator of HPSE1 expression. Notably, AUF treatment reduced HPSE1 expression and significantly suppressed the invasive capacity of MDA-MB-231 cells in a Transwell migration assay. We then investigated the role of HPSE1 in the invasive activity of MDA-MB-231 cells, which produce higher levels of hyaluronan (HA) and HS than non-invasive cells. Neither HS degradation, HA supplementation in Matrigel during Transwell migration, nor HPSE1 overexpression alone was sufficient to drive invasion, suggesting that invasive capacity depends on mesenchymal features and coordinated induction of HPSE1 and GAGs rather than HS degradation. Collectively, our findings demonstrate that AUF-mediated inhibition of aPKC suppresses HPSE1 expression, thereby inhibiting both its enzymatic and non-enzymatic functions and limiting cancer progression, metastasis, and angiogenesis. These results highlight the therapeutic potential of AUF for targeting HPSE1-driven tumor progression and support its repurposing for cancer treatment.

Ovarian cancer is characterized by early metastatic dissemination, frequent relapses, and limited therapeutic options following the onset of platinum resistance. To address these challenges, a dual-action gold-(I) prodrug, RDL-15, was designed to concurrently interfere with intracellular redox regulation and extracellular matrix remodeling. RDL-15 combines the gold-(I) pharmacophore derived from Auranofin with the scaffold of LP-158, a carboxylate-based inhibitor of gelatinases MMP-2 and MMP-9 previously described. The complex retains micromolar cytotoxic activity in ovarian cancer cell lines A2780 and SKOV-3, including the cisplatin and AF-resistant A2780/R variant, and induces an early reduction of thioredoxin reductase activity. In SKOV-3 cells, characterized by high migratory and invasive capacity, RDL-15 significantly suppresses migration and invasion, whereas Et3PAuCl alone shows no intrinsic anti-invasive effect. Computational analyses further support a structure-function relationship linking gold-ligand bonding features to the observed biological profile.

Thioredoxin reductase from Burkholderia cenocepacia (Bc-TrxR) is a recognized intracellular target of antibacterial gold(I) compounds, yet the molecular basis of enzyme inhibition remains unclear. Here, we report an integrated structural, biophysical, and biochemical investigation of recombinant Bc-TrxR and its interaction with three prototypical gold(I) agents: auranofin (AF) and two trimethylphosphine-thiolate derivatives (Au1 and Au2), previously identified as potent enzyme inhibitors. The enzyme was expressed, purified, and its crystal structure solved at 2.52 Å resolution, revealing a homodimeric architecture closely resembling that of the Escherichia coli enzyme. Each subunit contains FAD- and NADPH-binding domains and a catalytic Cys-Cys motif representing a plausible coordination site for gold fragments. High-resolution ESI-MS provided direct molecular evidence for the formation of defined gold-protein adducts, with up to two Au(I) centres bound per subunit. The observed mass shifts are consistent with selective coordination of gold fragments to catalytically relevant cysteine residues, effectively blocking redox turnover. Together, crystallographic and mass spectrometric results disclose the molecular mechanism of Bc-TrxR inhibition by phosphine-thiolate gold(I) complexes and establish a structural framework for the rational design of next-generation antibacterial metallodrugs.

Au(I) complexes are widely investigated as therapeutic agents due to their high affinity for biological nucleophiles and protein targets. Here, the reactivity of an Auranofin (AF) derivative bearing naproxen as a ligand toward hen egg white lysozyme (HEWL) was investigated by a combined crystallographic and computational approach. Notably, the first observation of lysine metalation in HEWL by an Au complex is reported. The results highlight how ligand substitution can significantly affect Au(I) reactivity toward biomacromolecules, enabling noncanonical targeting and potentially impacting biological activity.

Bacterial toxin-mediated severe systemic diseases, such as Shiga toxin-induced hemolytic uremic syndrome (HUS), are associated with an exceptionally high mortality rate due to life-threatening multi-organ failure and a profound inflammatory surge. Despite this severe clinical burden, targeted therapeutic drugs remain unavailable. Here, we investigated the therapeutic potential of Auranofin (AUR), an FDA-approved compound, in mitigating systemic lethality by targeting the Caspase-9/GSDME-mediated cell death axis. Utilizing a high-throughput screening of 2819 FDA-approved drugs, we identified AUR as a potent inhibitor of Stx2-induced cytotoxicity in THP-1 macrophages. In vitro, AUR pre-treatment (2.5 μM) significantly preserved cell viability, stabilized mitochondrial membrane potential, and suppressed the release of pro-inflammatory IL-1β and LDH. Mechanistic analysis revealed that AUR abrogated the activation of Caspase-9 and Caspase-3, effectively blocking GSDME-mediated pyroptosis. In a C57BL/6 mouse model of Stx2-induced systemic injury, AUR administration significantly prolonged survival time and ameliorated renal and intestinal dysfunction. Histological evaluation confirmed that AUR reduced renal tubular necrosis, fibrin deposition, and the infiltration of macrophages and neutrophils. Western blot analysis of kidney tissues further corroborated the inhibition of the Caspase-9/GSDME axis in vivo. Our findings elucidate that AUR represents a promising drug-repurposing strategy for treating severe systemic syndromes by intercepting the crosstalk between apoptosis and pyroptosis, highlighting a novel, translational therapeutic paradigm for acute toxemia.

Drug repurposing can accelerate oncology translation by leveraging approved compounds with known pharmacology; success depends on a plausible mechanism for the new indication. The microtubule cytoskeleton is a validated anticancer target, yet tubulin-directed activity is often overlooked for non-oncology drugs. Using nanoDSF, FDA-approved compounds were recently screened for effects on tubulin polymerisation, identifying multiple previously unrecognised microtubule-targeting agents (MTA) including Auranofin, Ebselen, Riboflavin, Aprepitant, Benzarone and Nifedipine derivatives, etc. Many hits match reported microtubule perturbation or cancer-relevant phenotypes, and several have been considered for oncology repositioning. These observations suggest microtubule targeting may contribute to anticancer effects more often than appreciated and that tubulin remains an underexploited repurposing target. The aim of this review is to place these newly identified MTAs in the context of current knowledge and to discuss their potential for drug repurposing in oncology.

Hepatocellular carcinoma (HCC) remains a highly aggressive malignancy with a dismal prognosis, largely due to the limited effectiveness of current therapeutic interventions. Although Motesanib (MOT) is a clinically studied VEGFR-centered inhibitor, VEGFR-targeted strategies remain limited by innate or acquired resistance. In this study, we explored a novel pharmacological strategy combining MOT with Auranofin (AF) to enhance therapeutic outcomes in HCC. Our data demonstrate that this dual-targeting approach yields robust synergistic anticancer activity across diverse experimental models. Mechanistically, we discovered that TrxR1 inhibition is critical for this sensitization, which is closely associated with autophagy-related reactive oxygen species (ROS) accumulation. Specifically, genetic depletion of Atg5 effectively suppressed ROS accumulation and attenuated cytotoxicity, suggesting that autophagy contributes to the synergistic oxidative damage. This orchestrated stress response subsequently led to sustained endoplasmic reticulum (ER) stress and JNK pathway activation, culminating in DNA damage, impaired proliferation, and reduced cell viability. Taken together, our results identify the combination of MOT and AF as a promising and mechanistically grounded treatment regimen for advanced HCC (Graphical Abstract).

Auranofin is a gold-containing anti-inflammatory drug used for the treatment of rheumatoid arthritis. Recent studies indicate that auranofin targets cellular antioxidants including the thioredoxin system and the ubiquitin-proteasome system, which can result in cellular oxidative stress. In the present studies, we identified a novel mechanistic site of action of auranofin, namely tetrahydrobiopterin (BH4) biosynthesis. BH4 is an essential cofactor required for aromatic amino acid hydroxylases, nitric oxide synthase and alkylglycerol monooxygenase, enzymes that produce mediators important in regulating inflammation. It is synthesized de novo from GTP via the action of GTP cyclohydrolase I, 6-pyruvoyl-tetrahydrobiopterin synthase and sepiapterin reductase (SPR), which leads to the production of dihydrobiopterin (BH2). Further metabolism of BH2 to BH4 is mediated by dihydrofolate reductase (DHFR). We discovered that auranofin causes a rapid depletion of cellular BH4 in several different cell types; PC12 cells were most sensitive to auranofin followed by Jurkat cells, BeWo cells, HaCaT cells, SKNMC cells, RAW246.7 cells and CX-1 cells. This was due to the inhibition of both SPR and DHFR. BH4 is required for tyrosine hydroxylase and tryptophan hydroxylase, enzymes mediating the synthesis of dopamine and serotonin, respectively. In PC12 cells, blocking BH4 biosynthesis with auranofin correlated directly with inhibition of serotonin and dopamine production indicating that the drug targeted aromatic amino acid hydroxylase activity. Our findings provide novel insights into the metabolic consequences of treating inflammatory diseases or cancer cell growth and metastasis with auranofin.

FAD/NAD(P)H-dependent dehydrogenases form a structurally conserved family of redox enzymes that participate in essential metabolic processes across parasites and higher organisms. Among them, trypanothione reductase (TR) is a key component of the redox metabolism of Leishmania species and represents an attractive target for antileishmanial drug development. However, because several flavoproteins share similar folds and cofactor-binding architectures, the selectivity of TR inhibitors remains a critical issue during early drug discovery. To explore this aspect, we investigated the structural landscape of FAD/NAD(P)H-dependent dehydrogenases in Leishmania infantum by integrating sequence analysis, structural modeling, docking simulations, and in vitro biochemical validation. Reciprocal sequence searches revealed 11 parasite flavoproteins structurally related to TR, including a dihydrolipoamide dehydrogenase (DLD)-like protein, a type II NADH dehydrogenase (NDH2)-like protein, and a dienoyl-CoA reductase (deCoAR)-like protein. Comparative docking analyses across parasite and mammalian homologs allowed us to examine potential cross-reactivity patterns among these enzymes. Enzymatic assays performed on recombinant L. infantum TR (LiTR) and Caldalkalibacillus thermarum NDH2 (CtNDH2) confirmed that selected ligands can exert enzyme-dependent effects. In particular, auranofin and, to a lesser extent, nitrofurazone inhibited LiTR, whereas a terpyridine-Pt-derived compound strongly inhibited LiTR while stimulating CtNDH2 activity. Overall, the results illustrate how structurally related flavoproteins may accommodate common ligands while responding with distinct catalytic outcomes. The integrated computational and biochemical workflow presented here provides a practical framework for assessing ligand selectivity within the FAD/NAD(P)H-dependent dehydrogenase family and may support the development of selective modulators targeting parasite redox metabolism.

Enzalutamide, as a second-generation anti-androgen agent, has been used to treat castration-resistant prostate cancer (CRPC) or metastatic castration-sensitive prostate cancer (mCSPC). However, enzalutamide resistance inevitably developed for most treated CRPC/mCSPC, and limited effective therapies are currently available for these enzalutamide-resistant prostate cancers. In this study, we utilize our established enzalutamide-resistant prostate cancer cell lines to reveal a vulnerability of these cancer cells to GPX4-targeted ferroptosis. Interestingly, the established enzalutamide-resistant prostate cancer cells are mixed populations that predominantly exhibit stem cell-like (SCL) and neuroendocrine-like (NEL) phenotypes and may reflect cellular heterogeneity during the development of enzalutamide resistance in prostate cancer. We further demonstrated that ACSL4, a long-chain fatty acid-CoA ligase, was upregulated by the JAK/STAT pathway in enzalutamide-resistant SCL/NEL cells, thereby facilitating tumor proliferation and metastasis while increasing sensitivity to ferroptosis. To antagonize the ACSL4-conferred ferroptosis risk, SCL/NEL cells upregulated GPX4 through AP-1 transcription complex to suppress ferroptosis and thus promoted the malignant progression of SCL/NEL cells. Notably, we characterized Auranofin, an anti-rheumatoid arthritis drug, as a ferroptosis inducer for these SCL/NEL cells in vitro and in vivo by targeting AP-1 and decreasing GPX4 expression, suggesting a new application for Auranofin in treating enzalutamide-resistant stem cell-like AP-1[High] CRPC.

Auranofin (AF), an FDA-approved drug for rheumatoid arthritis, exhibits strong antibacterial activity against Gram-positive bacteria, while Gram-negative species remain largely tolerant. This study assessed the antimicrobial activity of AF and three analogues against clinically relevant Gram-negative pathogens and explored tolerance mechanisms in Pseudomonas aeruginosa. Broth microdilution assays were performed on reference strains and clinical isolates of Escherichia coli, Klebsiella pneumoniae, and P. aeruginosa. Synergy studies with the most active analogue, PEt3AuCl (AF-Cl), were conducted against P. aeruginosa using polymyxin B (PMB), two efflux-pump inhibitors, and two glutathione (GSH) depletors. Gold compounds showed MICs between 4 and >64 µg/mL, with AF-Cl displaying the highest activity. AF-Cl activity was markedly enhanced by PMB and efflux-pump inhibitors, indicating that outer membrane permeability and efflux contribute to tolerance. Additionally, GSH depletion significantly potentiated AF-Cl, implicating redox homeostasis in resistance. Overall, AF-Cl shows potential against Gram-negative bacteria when combined with agents targeting membrane integrity, efflux systems, or redox balance, supporting combinatorial strategies to overcome resistance in P. aeruginosa and related pathogens.

Multidrug-resistant (MDR) Acinetobacter baumannii is a WHO priority pathogen that endures in hospitals and complicates care in intensive care units. Drug repositioning offers a faster path to therapies by reusing approved agents with known safety. This review compiles in vitro and in vivo evidences for repurposed candidates against MDR A. baumannii. Repurposing of anticancer drugs displayed bacterial load reduction and enhancement of polymyxin B through membrane permeabilization. Additionally, antiparasitics increased colistin efficacy by altering surface charge. Furthermore, central nervous system (CNS) agents contributed synergy with traditional antibiotics. Anti-inflammatory and other agents also showed promise: diclofenac enhanced colistin killing, desloratadine blocked growth and synergized with meropenem, and auranofin paired with pentamidine achieved bactericidal effects. Translating these signals will require attention to dosing, delivery, and safety, and rigorous clinical trials. In total, repurposing, particularly in rational combinations, could supply near-term options for severe MDR A. baumannii infections.