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Nature Communications Aug 2023UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and...
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.
Topics: Ubiquitin; Ubiquitin-Conjugating Enzymes; Auranofin; Ubiquitination; Ubiquitin-Activating Enzymes
PubMed: 37558718
DOI: 10.1038/s41467-023-40537-x -
Circulation Research Dec 2023Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular...
BACKGROUND
Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH.
METHODS
Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH.
RESULTS
Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling.
CONCLUSIONS
A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.
Topics: Humans; Mice; Animals; Cyclins; Pulmonary Arterial Hypertension; Cysteine; Endothelial Cells; Cell Proliferation; Pulmonary Artery; Phosphorylation; Cell Cycle Checkpoints; Cyclin D; Cells, Cultured; Cyclin-Dependent Kinase 4
PubMed: 37955182
DOI: 10.1161/CIRCRESAHA.122.321836 -
BioRxiv : the Preprint Server For... May 2024Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that...
Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.
PubMed: 38798568
DOI: 10.1101/2024.05.13.593567 -
International Immunopharmacology Sep 2023Interferon-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...
High throughput screening identifies auranofin and pentamidine as potent compounds that lower IFN-γ-induced Nitric Oxide and inflammatory responses in mice: DSS-induced colitis and Salmonella Typhimurium-induced sepsis.
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® 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 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.
Topics: Mice; Animals; Interferon-gamma; Nitric Oxide; Salmonella typhimurium; Auranofin; Pentamidine; High-Throughput Screening Assays; Nitric Oxide Synthase Type II; Colitis; Sepsis
PubMed: 37392571
DOI: 10.1016/j.intimp.2023.110569 -
Expert Opinion on Drug Discovery Jul 2024Auranofin (AF) is a well-established, FDA-approved, antiarthritic gold drug that is currently being reevaluated for a variety of therapeutic indications through drug... (Review)
Review
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.
Topics: Auranofin; Humans; Drug Design; Antineoplastic Agents; Structure-Activity Relationship; Neoplasms; Animals; Drug Repositioning
PubMed: 38803122
DOI: 10.1080/17460441.2024.2355329 -
Journal of Helminthology Dec 2023Schistosomiasis is a serious tropical disease. Despite extensive research into the etiology of liver fibrosis, effective therapeutic options remain limited. This study...
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 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 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 egg-induced hepatic granuloma and consequent fibrosis.
Topics: Male; Animals; Mice; Schistosoma mansoni; Schistosomiasis mansoni; Auranofin; Prospective Studies; Sirtuin 3; Ovum; Liver; Liver Cirrhosis; Granuloma
PubMed: 38053397
DOI: 10.1017/S0022149X23000792 -
Inflammation Research : Official... Mar 2024Ferroptosis is a reactive oxygen species (ROS)- and iron-dependent form of non-apoptotic cell death process. Previous studies have demonstrated that ferroptosis...
OBJECTIVE
Ferroptosis is a reactive oxygen species (ROS)- and iron-dependent form of non-apoptotic cell death process. Previous studies have demonstrated that ferroptosis participates in the development of inflammatory arthritis. However, the role of ferroptosis in rheumatoid arthritis (RA) inflammatory hypoxic joints remains unclear. This study sought to explore the underlying mechanism of ferroptosis on lipopolysaccharide (LPS)-induced RA fibroblast-like synoviocytes (FLSs).
METHODS
FLSs, isolated from patients with RA, were treated with LPS and ferroptosis inducer (erastin and RSL-3), and ferroptosis inhibitor (Fer-1 and DFO), respectively. The cell viability was measured by CCK-8. The cell death was detected by flow cytometer. The proteins level were tested by Western blot. The cytosolic ROS and lipid peroxidation were determined using DCFH-DA and C11-BODIPY581/591 fluorescence probes, respectively. The small interfering RNA (siRNA) was used to knock down related proteins. The levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, inflammatory cytokines (IL6 and IL8), and LDH were analyzed by commercial kits.
RESULTS
Ferroptosis was activated by LPS in RA FLS with increased cellular damage, ROS and lipid peroxidation, intracellular Fe and IL8, which can be further amplified by ferroptosis inducer (erastin and RSL-3) and inhibited by ferroptosis inhibitor (Fer-1 and DFO). Mechanistically, LPS triggered ferroptosis via NCOA4-mediated ferritinophagy in RA FLSs, and knockdown of NCOA4 strikingly prevent the process of ferroptosis. Intriguingly, LPS-induced RA FLSs became insensitive to ferroptosis and NCOA4-mediated ferritinophagy under hypoxia compared with normoxia. Knockdown of HIF-1α reverted ferroptosis and ferritinophagy evoking by LPS-induced RA FLSs inflammation under hypoxia. In addition, low dose of auranofin (AUR) induced re-sensitization of ferroptosis and ferritinophagy through inhibiting the expression of HIF-1α under hypoxia.
CONCLUSIONS
NCOA4-mediated ferritinophagy was a key driver of ferroptosis in inflammatory RA FLSs. The suppression of NCOA4-mediated ferritinophagy protected RA FLSs from ferroptosis in LPS-induced inflammation under hypoxia. Targeting HIF-1α/NCOA4 and ferroptosis could be an effective and valuable therapeutic strategy for synovium hyperplasia in the patients with RA.
Topics: Humans; Synoviocytes; Lipopolysaccharides; Reactive Oxygen Species; Ferroptosis; Interleukin-8; Arthritis, Rheumatoid; Inflammation; Hypoxia; Transcription Factors; RNA, Small Interfering; Fibroblasts; Iron; Nuclear Receptor Coactivators
PubMed: 38189810
DOI: 10.1007/s00011-023-01842-9 -
Redox Biology Jul 2023Selenoprotein glutathione peroxidases (GPX), like ubiquitously expressed GPX1 and the ferroptosis modulator GPX4, enact antioxidant activities by reducing hydroperoxides...
Selenoprotein glutathione peroxidases (GPX), like ubiquitously expressed GPX1 and the ferroptosis modulator GPX4, enact antioxidant activities by reducing hydroperoxides using glutathione. Overexpression of these enzymes is common in cancer and can be associated with the development of resistance to chemotherapy. GPX1 and GPX4 inhibitors have thus shown promise as anti-cancer agents, and targeting other GPX isoforms may prove equally beneficial. Existing inhibitors are often promiscuous, or modulate GPXs only indirectly, so novel direct inhibitors identified through screening against GPX1 and GPX4 could be valuable. Here, we developed optimized glutathione reductase (GR)-coupled GPX assays for the biochemical high-throughput screen (HTS) of almost 12,000 compounds with proposed mechanisms of action. Initial hits were triaged using a GR counter-screen, assessed for isoform specificity against an additional GPX isoform, GPX2, and were assessed for general selenocysteine-targeting activity using a thioredoxin reductase (TXNRD1) assay. Importantly, 70% of the GPX1 inhibitors identified in the primary screen, including several cephalosporin antibiotics, were found to also inhibit TXNRD1, while auranofin, previously known as a TXNRD1 inhibitor, also inhibited GPX1 (but not GPX4). Additionally, every GPX1 inhibitor identified (including omapatrilat, tenatoprazole, cefoxitin and ceftibuten) showed similar inhibitory activity against GPX2. Some compounds inhibiting GPX4 but not GPX1 or GPX2, also inhibited TXNRD1 (26%). Compounds only inhibiting GPX4 included pranlukast sodium hydrate, lusutrombopag, brilanestrant, simeprevir, grazoprevir (MK-5172), paritaprevir, navitoclax, venetoclax and VU0661013. Two compounds (metamizole sodium and isoniazid sodium methanesulfate) inhibited all three GPXs but not TXNRD1, while 2,3-dimercaptopropanesulfonate, PI4KIII beta inhibitor 3, SCE-2174 and cefotetan sodium inhibited all tested selenoproteins (but not GR). The detected overlaps in chemical space suggest that the counter screens introduced here should be imperative for identification of specific GPX inhibitors. With this approach, we could indeed identify novel GPX1/GPX2- or GPX4-specific inhibitors, thus presenting a validated pipeline for future identification of specific selenoprotein-targeting agents. Our study also identified GPX1/GPX2, GPX4 and/or TXNRD1 as targets for several previously developed pharmacologically active compounds.
Topics: Humans; Glutathione; Glutathione Peroxidase GPX1; Neoplasms; Selenoproteins
PubMed: 37244126
DOI: 10.1016/j.redox.2023.102719 -
Anticancer Research Jun 2024Synovial sarcoma (SS) is a rare malignant tumor with a poor survival rate. We previously reported that a combination of auranofin (AUR), a thioredoxin reductase...
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.
Topics: Celecoxib; Animals; Sarcoma, Synovial; Auranofin; Humans; Mice; Disease Progression; Cell Line, Tumor; Xenograft Model Antitumor Assays; Mice, Nude; Antineoplastic Combined Chemotherapy Protocols; Cell Proliferation
PubMed: 38821602
DOI: 10.21873/anticanres.17052