Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.

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.

Restrained management of copper level enhances the antineoplastic activity of imatinib in vitro and in vivo.

The present study was designed to investigate if elevated copper level can be targeted to enhance the efficacy of a significant anticancer drug, imatinib (ITB). The antineoplastic activity of this drug was assessed in the HepG2, HEK-293, MCF-7 and MDA-MD-231 cells targeting elevated copper level as their common drug target. The cell lines were treated with the different doses of copper chloride (Cu II) and disulfiram (DSF) alone as well as in their combinations with the drug for 24 h in standard culture medium and conditions. The treated cells were subjected to various assays including MTT, PARP, p-53, caspase-7, caspase-3, LDH and single cell electrophoresis. The study shows that DSF and Cu (II) synergizes the anticancer activity of ITB to a significant extent in a dose-specific way as evidenced by the combinations treated groups. Furthermore, the same treatment strategy was employed in cancer-induced rats in which the combinations of ITB-DSF and ITB-Cu II showed enhanced antineoplastic activity as compared to ITB alone. However, DSF was more effective than Cu (II) as an adjuvant to the drug. Hence, restrained manipulation of copper level in tumor cells can orchestrate the redox and molecular dispositions inside the cells favoring the induction of apoptosis.

Subacute alcohol and/or disulfiram intake affects bioelements and redox status in rat testes.

The aim of the study was to investigate if alcohol and disulfiram (DSF) individually and in combination affect bioelements' and red-ox homeostasis in testes of the exposed rats. The animals were divided into groups according to the duration of treatments (21 and/or 42 days): C21/C42 groups (controls); OL21 and OL22-42 groups (0.5 mL olive oil intake); A1-21 groups (3 mL 20% ethanol intake); DSF1-21 groups (178.5 mg DSF/kg/day intake); and A21+DSF22-42 groups (the DSF ingestion followed previous 21 days' treatment with alcohol). The measured parameters in testes included metals: zinc (Zn), copper (Cu), iron (Fe), magnesium (Mg) and selenium (Se); as well as oxidative stress (OS) parameters: superoxide anion radical (O2•-), glutathione reduced (GSH) and oxidized (GSSG), malondialdehyde (MDA), hydrogen peroxide (H2O2) decomposition and activities of total superoxide dismutase (tSOD), glutathione-S-transferase (GST) and glutathione reductase (GR). Metal status was changed in all experimental groups (Fe rose, Zn fell, while Cu increased in A21+DSF24-32 groups). Development of OS was demonstrated in A1-21 groups, but not in DSF1-21 groups. In A21+DSF22-42 groups, OS was partially reduced compared to A groups (A1-21>MDA>C; A1-21

Inhibitory potential of three zinc chelating agents against the proteolytic, hemorrhagic, and myotoxic activities of Echis carinatus venom.

Viperbites undeniably cause local manifestations such as hemorrhage and myotoxicity involving substantial degradation of extracellular matrix (ECM) at the site of envenomation and lead to progressive tissue damage and necrosis. The principle toxin responsible is attributed to snake venom metalloproteases (SVMPs). Treatment of such progressive tissue damage induced by SVMPs has become a challenging task for researchers and medical practitioners who are in quest of SVMPs inhibitors. In this study, we have evaluated the inhibitory potential of three specific zinc (Zn(2+)) chelating agents; N,N,N',N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD) on Echis carinatus venom (ECV) induced hemorrhage and myotoxicity. Amongst them, TPEN has high affinity for Zn(2+) and revealed potent inhibition of ECV metalloproteases (ECVMPs) in vitro (IC50: 6.7 µM) compared to DTPA and TTD. The specificity of TPEN towards Zn(2+) was confirmed by spectral and docking studies. Further, TPEN, DTPA, and TTD completely blocked the hemorrhagic and myotoxic activities of ECV in a dose dependent manner upon co-injection; whereas, only TPEN successfully neutralized hemorrhage and myotoxicity following independent injection. Histological examinations revealed that TPEN effectively prevents degradation of dermis and basement membrane surrounding the blood vessels in mouse skin sections. TPEN also prevents muscle necrosis and accumulation of inflammatory cells at the site of ECV injections. In conclusion, a high degree of structural and functional homology between mammalian MMPs and SVMPs suggests that specific Zn(2+) chelators currently in clinical practice could be potent first aid therapeutic agents in snakebite management, particularly for local tissue damage.

Inactivation of respiratory syncytial virus by zinc finger reactive compounds.

BACKGROUND: Infectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (Pneumoviridae), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins. RESULTS: Three compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats S.hispidus and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease. CONCLUSIONS: This work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease.

Carbamoylation of brain glutamate receptors by a disulfiram metabolite.

S-Methyl-N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO), a metabolite of the drug disulfiram, is a selective carbamoylating agent for sulfhydryl groups. Treatment of glutamate receptors isolated from mouse brain with DETC-MeSO blocks glutamate binding. In vivo, carbamoylated glutathione, administered directly to mice or formed by reaction of DETC-MeSO with glutathione in the blood, also blocks brain glutamate receptors. Carbamoyl groups appear to be delivered to brain glutamate receptors or to liver aldehyde dehydrogenase in vivo by a novel glutathione-mediated mechanism. Seizures caused by the glutamate analogs N-methyl-D-aspartate and methionine sulfoximine, or by hyperbaric oxygen, are prevented by DETC-MeSO, indicating that carbamoylation of glutamate receptors gives an antagonist effect. These observations offer an explanation for some of the previously reported neurological effects of disulfiram, such as its ability to prevent O2-induced seizures. Furthermore, some of the physiology of the disulfiram-ethanol reaction, that could not be accounted for based on the known inhibition of aldehyde dehydrogenase alone, may be explained by disulfiram's effect on glutamate receptors.

Diethyldithiocarbamate depresses the acoustic startle response in rats.

The relationship between norepinephrine (NE) content in cortex and spinal cord and acoustic startle amplitude was investigated in two experiments. Administration of diethyldithiocarbamate (DDC) depressed startle amplitude at the same time and dose that it most severely depleted NE content. These results support the conclusion that NE facilitates the normal elaboration of the acoustic startle reflex and also support evidence that NE activity in the spinal cord may be of particular importance in the maintenance of normal startle amplitude.