Localized Sustained Release of Copper Enhances Antitumor Effects of Disulfiram in Head and Neck Cancer.

Drug repurposing uses approved drugs as candidate anticancer therapeutics, harnesses previous research and development efforts, and benefits from available clinically suitable formulations and evidence of patient tolerability. In this work, the drug used clinically to treat chronic alcoholism, disulfiram (DSF), was studied for its antitumor efficacy in a copper-dependent manner. The combination of DSF and copper could achieve a tumor cell growth inhibition effect comparable to those of 5-fluorouracil and taxol on head and neck cancer cells. Both bulk dendrimer hydrogel and microsized dendrimer hydrogel particles were utilized for the localized sustained release of copper in the tumor site. The localized sustained release of copper facilitated the tumor inhibition effect following intratumoral injection in a mouse's head and neck cancer model.

Tumor specific in situ synthesis of therapeutic agent for precision cancer therapy.

BACKGROUND: Traditional chemotherapeutic agents suffer from a lack of selectivity, poor targeting ability, and drug resistance. Developing tumor-specific therapies is crucial for precisely eliminating tumors while circumventing toxicity to normal tissues. Disulfiram (DSF), an FDA-approved drug for treating alcohol dependence, exhibits antitumor effect by forming complexes with copper ions (Cu(DDC)2). Here, we developed a Cu-doped polydopamine-based nanosystem (DSF@CuPDA-PEGM) to achieve in situ generation of toxic Cu(DDC)2. RESULTS: In cancer cells with elevated H2O2 contents, CuPDA responsively degrades to release Cu ions and DSF, allowing on-site synthesis of Cu(DDC)2 with potent antitumor activity. DSF@CuPDA-PEGM exhibits excellent therapeutic efficacy against both drug-sensitive and drug-resistant cancer cells while minimizing toxicity to noncancerous cells. Moreover, DSF@CuPDA-PEGM promotes the immune response by inducing cancer cell immunogenic death, thereby augmenting anti-PD-1-based immune checkpoint blockade therapy. CONCLUSION: A tumor-specifically degradable Cu-doped polydopamine-based nanosystem is developed to achieve in situ synthesis of antitumor compounds, providing a promising approach to precisely eliminate tumors and heighten chemo-immunotherapy.

Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.

Disulfiram/Copper Suppresses Cancer Stem Cell Activity in Differentiated Thyroid Cancer Cells by Inhibiting BMI1 Expression.

Differentiated thyroid carcinomas (DTCs), which have papillary and follicular types, are common endocrine malignancies worldwide. Cancer stem cells (CSCs) are a particular type of cancer cells within bulk tumors involved in cancer initiation, drug resistance, and metastasis. Cells with high intracellular aldehyde hydrogenase (ALDH) activity are a population of CSCs in DTCs. Disulfiram (DSF), an ALDH inhibitor used for the treatment of alcoholism, reportedly targets CSCs in various cancers when combined with copper. This study reported for the first time that DSF/copper can inhibit the proliferation of papillary and follicular DTC lines. DSF/copper suppressed thyrosphere formation, indicating the inhibition of CSC activity. Molecular mechanisms of DSF/copper involved downregulating the expression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and cell cycle-related proteins, including cyclin B2, cyclin-dependent kinase (CDK) 2, and CDK4, in a dose-dependent manner. BMI1 overexpression diminished the inhibitory effect of DSF/copper in the thyrosphere formation of DTC cells. BMI1 knockdown by RNA interference in DTC cells also suppressed the self-renewal capability. DSF/copper could inhibit the nuclear localization and transcriptional activity of c-Myc and the binding of E2F1 to the BMI1 promoter. Overexpression of c-Myc or E2F1 further abolished the inhibitory effect of DSF/copper on BMI1 expression, suggesting that the suppression of c-Myc and E2F1 by DSF/copper was involved in the downregulation of BMI1 expression. In conclusion, DSF/copper targets CSCs in DTCs by inhibiting c-Myc- or E2F1-mediated BMI1 expression. Therefore, DSF is a potential therapeutic agent for future therapy in DTCs.

Novel Disulfiram Derivatives as ALDH1a1-Selective Inhibitors.

Aldehyde dehydrogenase-1a1 (ALDH1a1), the enzyme responsible for the oxidation of retinal into retinoic acid, represents a key therapeutic target for the treatment of debilitating disorders such as cancer, obesity, and inflammation. Drugs that can inhibit ALDH1a1 include disulfiram, an FDA-approved drug to treat chronic alcoholism. Disulfiram, by carbamylation of the catalytic cysteines, irreversibly inhibits ALDH1a1 and ALDH2. The latter is the isozyme responsible for important physiological processes such as the second stage of alcohol metabolism. Given the fact that ALDH1a1 has a larger substrate tunnel than that in ALDH2, replacing disulfiram ethyl groups with larger motifs will yield selective ALDH1a1 inhibitors. We report herein the synthesis of new inhibitors of ALDH1a1 where (hetero)aromatic rings were introduced into the structure of disulfiram. Most of the developed compounds retained the anti-ALDH1a1 activity of disulfiram; however, they were completely devoid of inhibitory activity against ALDH2.

Mechanism of zinc ejection by disulfiram in nonstructural protein 5A.

Hepatitis C virus (HCV) is a notorious member of the Flaviviridae family of enveloped, positive-strand RNA viruses. Non-structural protein 5A (NS5A) plays a key role in HCV replication and assembly. NS5A is a multi-domain protein which includes an N-terminal amphipathic membrane anchoring alpha helix, a highly structured domain-1, and two intrinsically disordered domains 2-3. The highly structured domain-1 contains a zinc finger (Zf)-site, and binding of zinc stabilizes the overall structure, while ejection of this zinc from the Zf-site destabilizes the overall structure. Therefore, NS5A is an attractive target for anti-HCV therapy by disulfiram, through ejection of zinc from the Zf-site. However, the zinc ejection mechanism is poorly understood. To disclose this mechanism based on three different states, A-state (NS5A protein), B-state (NS5A + Zn), and C-state (NS5A + Zn + disulfiram), we have performed molecular dynamics (MD) simulation in tandem with DFT calculations in the current study. The MD results indicate that disulfiram triggers Zn ejection from the Zf-site predominantly through altering the overall conformation ensemble. On the other hand, the DFT assessment demonstrates that the Zn adopts a tetrahedral configuration at the Zf-site with four Cys residues, which indicates a stable protein structure morphology. Disulfiram binding induces major conformational changes at the Zf-site, introduces new interactions of Cys39 with disulfiram, and further weakens the interaction of this residue with Zn, causing ejection of zinc from the Zf-site. The proposed mechanism elucidates the therapeutic potential of disulfiram and offers theoretical guidance for the advancement of drug candidates.

Disulfiram Chelated with Copper Promotes Apoptosis in Osteosarcoma via ROS/Mitochondria Pathway.

Disulfiram (DSF) chelated with copper has been confirmed to have a strong anti-tumor ability. In this study, we determined that DSF-Cu induced mitochondria-dependent apoptosis in osteosarcoma (OS), reflecting in DSF-Cu induces mitochondrial membrane potential decline, the production of reactive oxygen species (ROS), and inhibiting cells migration and invasion along with decreasing the concentration of intracellular glutathione (GSH) and facilitating the opening of mitochondrial permeability transition pore (PT) in osteosarcoma cells. These anti-tumor activities can be reversed by Cyclosporine A (CsA, PT inhibitors) and N-acetyl-L-cysteine (NAC, antioxidants). Our results suggested that DSF-Cu exerts its anti-tumor effects in OS via regulation of the ROS/Mitochondria pathway. Our findings provide the basis for DSF-Cu to treat osteosarcoma, even might develop as a potential therapy for other tumors.

Differential Cytotoxicity Mechanisms of Copper Complexed with Disulfiram in Oral Cancer Cells.

Disulfiram (DSF), an irreversible aldehyde dehydrogenase inhibitor, is being used in anticancer therapy, as its effects in humans are known and less adverse than conventional chemotherapy. We explored the potential mechanism behind the cytotoxicity of DSF-Cu+/Cu2+ complexes in oral epidermoid carcinoma meng-1 (OECM-1) and human gingival epithelial Smulow-Glickman (SG) cells. Exposure to CuCl2 or CuCl slightly but concentration-dependently decreased cell viability, while DSF-Cu+/Cu2+ induced cell death in OECM-1 cells, but not SG cells. DSF-Cu+/Cu2+ also increased the subG1 population and decreased the G1, S, and G2/M populations in OECM-1 cells, but not SG cells, and suppressed cell proliferation in both OECM-1 and SG cells. ALDH enzyme activity was inhibited by CuCl and DSF-Cu+/Cu2+ in SG cells, but not OECM-1 cells. ROS levels and cellular senescence were increased in DSF-Cu+/Cu2+-treated OECM-1 cells, whereas they were suppressed in SG cells. DSF-Cu+/Cu2+ induced mitochondrial fission in OECM-1 cells and reduced mitochondrial membrane potential. CuCl2 increased but DSF- Cu2+ impaired oxygen consumption rates and extracellular acidification rates in OECM-1 cells. CuCl2 stabilized HIF-1α expression under normoxia in OECM-1 cells, and complex with DSF enhanced that effect. Levels of c-Myc protein and its phosphorylation at Tyr58 and Ser62 were increased, while levels of the N-terminal truncated form (Myc-nick) were decreased in DSF-Cu+/Cu2-treated OECM-1 cells. These effects were all suppressed by pretreatment with the ROS scavenger NAC. Overexpression of c-Myc failed to induce HIF-1α expression. These findings provide novel insight into the potential application of DSF-CuCl2 complex as a repurposed agent for OSCC cancer therapy.

Nanoscale Copper(II)-Diethyldithiocarbamate Coordination Polymer as a Drug Self-Delivery System for Highly Robust and Specific Cancer Therapy.

Disulfiram (DSF), an old alcohol-aversion drug, has been repurposed for cancer therapy, and mechanistic studies reveal that it needs to be metabolized to diethyldithiocarbamate (DTC) and subsequently coordinates with copper(II) to form the DTC-copper complex (CuET) for anticancer activation. Here, we utilized this mechanism to construct a CuET self-delivery nanosystem based on the metal coordination polymer for highly robust and selective cancer therapy. In our design, the nanoparticles were facilely prepared under mild conditions by virtue of the strong coordination between Cu2+ and DTC, yielding 100% CuET loading capacity and allowing for further hyaluronic acid (HA) modification (CuET@HA NPs). The CuET@HA NPs could selectively deliver into cancer cells and release the active component of CuET in response to both endo/lysosome acidic pH and intracellular abundant GSH, which induces strong cytotoxicity toward cancer cells over normal cells taking advantage of the p97 pathway interference mechanism. Upon intravenous injection, the self-assembled system could passively accumulate into a tumor and elicit potent tumor growth inhibition at a dose of 1 mg/kg without any noticeable side effects. Given the cost-effective and easily scaled-up preparation, our designed nanosystem provides a promising strategy to pave the way for clinical translation of DSF-based cancer chemotherapy.

Silk Fibroin-Modified Disulfiram/Zinc Oxide Nanocomposites for pH Triggered Release of Zn2+ and Synergistic Antitumor Efficacy.

Disulfiram (DSF) is an FDA-approved anti-alcoholic drug that has recently proven to be effective in cancer treatment. However, the short half-life in the bloodstream and the metal ion-dependent antitumor activity significantly limited the further application of DSF in the clinical field. To this end, we constructed a silk fibroin modified disulfiram/zinc oxide nanocomposites (SF/DSF@ZnO) to solubilize and stabilize DSF, and, more importantly, achieve pH triggered Zn2+ release and subsequent synergistic antitumor activity. The prepared SF/DSF@ZnO nanocomposites were spherical and had a high drug loading. Triggered by the lysosomal pH, SF/DSF@ZnO could induce the rapid release of Zn2+ under the acidic conditions and caused nanoparticulate disassembly along with DSF release. In vitro experiments showed that cytotoxicity of DSF could be enhanced by the presence of Zn2+, and further amplified when encapsulated into SF/DSF@ZnO nanocomposites. It was confirmed that the significantly amplified cytotoxicity of SF/DSF@ZnO was resulted from pH-triggered Zn2+ release, inhibited cell migration, and increased ROS production. In vivo study showed that SF/DSF@ZnO nanocomposites significantly increased the tumor accumulation and prolonged the retention time. In vivo antitumor experiments in the xenograft model showed that SF/DSF@ZnO exerted the highest tumor-inhibition rate among all the drug treatments. Therefore, this exquisite study established silk fibroin-modified disulfiram/zinc oxide nanocomposites, SF/DSF@ZnO, where ZnO not only acted as a delivery carrier but also served as a metal ion reservoir to achieve synergistic antitumor efficacy. The established DSF nanoformulation displayed excellent therapeutic potential in future cancer treatment.

A disulfiram-loaded electrospun poly(vinylidene fluoride) nanofibrous scaffold for cancer treatment.

Disulfiram (DSF), an FDA approved drug for the treatment of alcoholism, has shown its effectiveness against diverse cancer types. Thus, we developed a disulfiram-loaded scaffold using the electrospinning method to enhance the stability of DSF and to facilitate its appropriate distribution to tumor tissues. The drug release profile of the disulfiram-loaded scaffold was examined by high-performance liquid chromatography. We obtained mechanical and morphological characterizations of A549 cells treated with different scaffolds by various techniques to evaluate its antitumor properties. This work revealed that the cells after the treatment with the disulfiram-loaded scaffold exhibited a lower height and a larger elastic modulus compared with the untreated cells and those treated with the neat electrospun fibers. The changes were the indicators of cell apoptosis. Taken collectively, the results indicate that DSF was successfully incorporated into the electrospun fibers, and the disulfiram-loaded scaffold has great potential for inhibiting the regional recurrence of cancer.

Is Alcohol in Hand Sanitizers Absorbed Through the Skin or Lungs? Implications for Disulfiram Treatment.

AIM: In view of the increase in the use of ethanol-containing hand sanitizers throughout the world due to the current COVID-19 pandemic, we wished to review the possible risks to patients treated with disulfiram, following a case report in which an apparent DER (disulfiram-ethanol reaction) was attributed to the cutaneous absorption of alcohol from hand sanitizers as well as by inhalation of vapour. METHOD: Simple experiments to assess the levels of absorption by each route separately. RESULTS: Our results strongly suggest that while amounts of alcohol sufficient to cause a DER may be inhaled when hand sanitizers are used in confined spaces, absorption can be avoided by dispersal of the fumes, and absorption from the skin alone does not occur in pharmacologically significant quantities. CONCLUSION: Warnings about absorption of alcohol through the skin from hand sanitizers and products such as perfumes, deodorants and after-shave (whose use is often warned against when disulfiram is prescribed) should be modified accordingly.

Soybean lecithin stabilizes disulfiram nanosuspensions with a high drug-loading content: remarkably improved antitumor efficacy.

Disulfiram (DSF) has been considered as "Repurposing drug" in cancer therapy in recent years based on its good antitumor efficacy. DSF is traditionally used as an oral drug in the treatment of alcoholism. To overcome its rapid degradation and instability, DSF nanosuspensions (DSF/SPC-NSps) were prepared using soybean lecithin (SPC) as a stabilizer of high drug-loaded content (44.36 ± 1.09%). Comprehensive characterization of the nanosuspensions was performed, and cell cytotoxicity, in vivo antitumor efficacy and biodistribution were studied. DSF/SPC-NSps, having a spherical appearance with particle size of 155 nm, could remain very stable in different physiological media, and sustained release. The in vitro MTT assay indicated that the cytotoxicity of DSF/SPC-NSps was enhanced remarkably compared to free DSF against the 4T1 cell line. The IC50 value decreased by 11-fold (1.23 vs. 13.93 µg/mL, p < 0.01). DSF/SPC-NSps groups administered via intravenous injections exhibited better antitumor efficacy compared to the commercial paclitaxel injection (PTX injection) and had a dose-dependent effect in vivo. Notably, DSF/SPC-NSps exhibited similar antitumor activity following oral administration as PTX administration via injection into a vein. These results suggest that the prepared nanosuspensions can be used as a stable delivery vehicle for disulfiram, which has potential application in breast cancer chemotherapy.

Evaluation of Bronopol and Disulfiram as Potential Candidatus Liberibacter asiaticus Inosine 5'-Monophosphate Dehydrogenase Inhibitors by Using Molecular Docking and Enzyme Kinetic.

Citrus huanglongbing (HLB) is a destructive disease that causes significant damage to many citrus producing areas worldwide. To date, no strategy against this disease has been established. Inosine 5'-monophosphate dehydrogenase (IMPDH) plays crucial roles in the de novo synthesis of guanine nucleotides. This enzyme is used as a potential target to treat bacterial infection. In this study, the crystal structure of a deletion mutant of CLas IMPDHΔ98-201 in the apo form was determined. Eight known bioactive compounds were used as ligands for molecular docking. The results showed that bronopol and disulfiram bound to CLas IMPDHΔ98-201 with high affinity. These compounds were tested for their inhibition against CLas IMPDHΔ98-201 activity. Bronopol and disulfiram showed high inhibition at nanomolar concentrations, and bronopol was found to be the most potent molecule (Ki = 234 nM). The Ki value of disulfiram was 616 nM. These results suggest that bronopol and disulfiram can be considered potential candidate agents for the development of CLas inhibitors.

An integrative bioinformatics pipeline to demonstrate the alteration of the interaction between the ALDH2*2 allele with NAD+ and Disulfiram.

Alcohol use disorder (AUD) is a multifactorial psychiatric behavior disorder. Disulfiram is the first approved drug by the Food and Drug Administration for alcohol-dependent patients, which targets the ALDH2 enzyme. Several genes are known to be involved in alcohol metabolism; mutations in any of these genes are known to be associated with AUD. The E504K mutation in the ALDH2 of the precursor protein or the E487K of the mature protein (E504K/E487K; ALDH2*2 allele) is carried by approximately 8% of the world population. In this study, we aimed to test the known inactive allele ALDH2*2, to validate the use of our extensive computational pipeline (in silico tools, molecular modeling, and molecular docking) for testing the interaction between the ALDH2*2 allele, NAD+, and Disulfiram. In silico predictions showed that the E504K variant of ALDH2 to be pathogenic and destabilizing with the maximum number of prediction in silico tools. Consequently, we studied the effect of this mutation mainly on the interaction between NAD+ -E504K and Disulfiram-E504K complexes using molecular docking technique, and molecular dynamics (MD) analysis. From the molecular docking analysis with NAD+ , we observed that the interaction affinity of the NAD+ decreases with the impact of E504K variant. On the other hand, the drug Disulfiram showed similar interaction in both the native and mutant ALDH2 proteins. Further, the comprehensive MD analysis predicted that the E504K destabilizes the protein and influences the NAD+ and Disulfiram interactions. Our findings reveal that the interaction of NAD+ to the protein is disturbed by the E504K/E487K variant whereas the drug Disulfiram has a similar effect as both native ALDH2 and ALDH2 bearing E504K/E487K variant. This study provides a platform to understand the effect of E504K/E487K on the molecular interaction with NAD+ and Disulfiram.

Systematic chemical screening identifies disulfiram as a repurposed drug that enhances sensitivity to cisplatin in bladder cancer: a summary of preclinical studies.

BACKGROUND: Since the standard gemcitabine and cisplatin (GC) chemotherapy for advanced bladder cancer yields limited therapeutic effect due to chemoresistance, it is a clinical challenge to enhance sensitivity to GC. METHODS: We performed high-throughput screening by using a library of known chemicals and repositionable drugs. A total of 2098 compounds were administered alone or with GC to human bladder cancer cells, and chemicals that enhanced GC effects were screened. RESULTS: Disulfiram (DSF), an anti-alcoholism drug, was identified as a candidate showing synergistic effects with cisplatin but not with gemcitabine in multiple cell lines. Co-administration of DSF with GC affected cellular localisation of a cisplatin efflux transporter ATP7A, increased DNA-platinum adducts and promoted apoptosis. Micellar DSF nanoparticles (DSF-NP) that stabilised DSF in vivo, enhanced the inhibitory effect of cisplatin in patient-derived and cell-based xenograft models without severe adverse effects. A drug susceptibility evaluation system by using cancer tissue-originated spheroid culture showed promise in identifying cases who would benefit from DSF with cisplatin. CONCLUSIONS: The present study highlighted the advantage of drug repurposing to enhance the efficacy of anticancer chemotherapy. Repurposing of DSF to a chemotherapy sensitiser may provide additional efficacy with less expense by using an available drug with a well-characterised safety profile.

Antibacterial activity of disulfiram and its metabolites.

AIMS: Disulfiram (Antabuse™) and its metabolites formed in vivo were evaluated as antibacterial agents against thirty species of Gram-positive and Gram-negative bacteria. The synergistic potential of disulfiram (DSF) and metabolite diethyldithiocarbamate (DDTC) with approved antibiotics were also compared by isobologram (checkerboard) analysis. METHODS AND RESULTS: Standard microdilution susceptibility testing showed that most DSF metabolites did not possess appreciable antibacterial activity except for DDTC in Bacillus anthracis. Checkerboard studies revealed similarities between the combination drug effects of DSF and DDTC with standard antibiotics. CONCLUSIONS: It was concluded from the susceptibility data that the metabolites would not extend the antibacterial spectrum of DSF in vivo. The data also suggest that the DDTC by-product of DSF metabolism potentiates the antibacterial activity of DSF as both a standalone and combination agent. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a greater understanding of the antibacterial effects of Antabuse and its metabolites. This research also demonstrates the potential application of DSF as an antibiotic adjuvant for the treatment of resistant staph infections.

Repurposing existing drugs: identification of irreversible IMPDH inhibitors by high-throughput screening.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for the production of guanine nucleotides. Disruption of IMPDH activity has been explored as a therapeutic strategy for numerous purposes, such as for anticancer, immunosuppression, antiviral, and antimicrobial therapy. In the present study, we established a luciferase-based high-throughput screening system to identify IMPDH inhibitors from our chemical library of known bioactive small molecules. The screening of 1400 compounds resulted in the discovery of three irreversible inhibitors: disulfiram, bronopol, and ebselen. Each compound has a distinct chemical moiety that differs from other reported IMPDH inhibitors. Further evaluation revealed that these compounds are potent inhibitors of IMPDHs with kon values of 0.7 × 104 to 9.3 × 104 M-1·s-1. Both disulfiram and bronopol exerted similar degree of inhibition to protozoan and mammalian IMPDHs. Ebselen showed an intriguing difference in mode of inhibition for different IMPDHs, with reversible and irreversible inhibition to each Cryptosporidium parvum IMPDH and human IMPDH type II, respectively. In the preliminary efficacy experiment against cryptosporidiosis in severe combined immunodeficiency (SCID) mouse, a decrease in the number of oocyst shed was observed upon the oral administration of disulfiram and bronopol, providing an early clinical proof-of-concept for further utilization of these compounds as IMPDH inhibitors.

Cystamine and Disulfiram Inhibit Human Transglutaminase 2 via an Oxidative Mechanism.

The catalytic activity of transglutaminase 2 (TG2), a ubiquitously expressed mammalian enzyme, is regulated by multiple post-translational mechanisms. Because elevated activity of TG2 in the extracellular matrix is associated with organ-specific diseases such as celiac disease and renal fibrosis, there is growing therapeutic interest in inhibitors of this enzyme. Cystamine, a symmetric disulfide compound, is one of the earliest reported TG2 inhibitors. Despite its widespread use as a tool compound to block TG2 activity in vitro and in vivo, its mechanism of action has remained unclear. Here, we demonstrate that cystamine irreversibly inhibits human TG2 ( kinh/ Ki = 1.2 mM-1 min-1) via a mechanism fundamentally distinct from those proposed previously. Through mass spectrometric disulfide mapping and site-directed mutagenesis, we show that cystamine promotes the formation of a physiologically relevant disulfide bond between Cys370 and Cys371 that allosterically abrogates the catalytic activity of human TG2. This discovery led us to evaluate clinically useful thiol → disulfide oxidants for TG2 inhibitory activity. It is demonstrated that disulfiram, a relatively safe oral thiuram disulfide, is a fairly potent TG2 inhibitor ( kinh/ Ki = 8.3 mM-1 min-1) and may therefore provide a practical tool for clinically validating this emerging therapeutic target in intestinal disorders such as celiac disease.

Inhibition of Proteasomal Deubiquitinase by Silver Complex Induces Apoptosis in Non-Small Cell Lung Cancer Cells.

BACKGROUND/AIMS: The ubiquitin proteasome system (UPS) is responsible for the degradation of most intracellular proteins, and proteasomal deubiquitinases (DUBs) have recently been highlighted as novel anticancer targets. It is well documented that copper complexes can inhibit UPS function through targeting both 20S proteasome and proteasomal DUBs. The antineoplastic activities of silver complexes have received much attention, but the exact mechanisms are not fully elucidated. In this study, we aim to investigate the effects of a novel silver complex [Ag(S2CN(C2H5)2)]6 (AgDT) on UPS function and its anticancer potential in non-small cell lung cancer (NSCLC). METHODS: Cell viability assay (i.e., the MTS assay) and flow cytometry assay were used to analyze the cell viability and apoptosis. Proteasome inhibition was measured using 20S proteasome activity assay and 19S proteasomal DUBs activity assay. Western blot analysis and immunohistochemistry were performed to detect protein levels. The in vivo antitumor activity of AgDT was assessed with nude xenografts. RESULTS: Silver ions, alone or in combination with disulfiram (DSF), induced UPS inhibition in NSCLC cells mainly through inhibition of proteasomal DUBs activities. Silver complex AgDT triggered intracellular accumulation of ubiquitinated proteins, and prevented the degradation of surrogate substrate GFPu. Mechanistically, AgDT potently inhibited the activities of proteasomal DUBs USP14 and UCHL5, without altering the 20S proteasome peptidases. Moreover, AgDT induced apoptosis in NSCLC cells and significantly inhibited tumor growth in xenografts. CONCLUSION: Our findings suggest that silver complex AgDT is a novel metal-based proteasomal DUBs inhibitor, and pharmacologic inhibition of USP14 and UCHL5 could prove to be an effective therapeutic strategy for NSCLC.