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.

Targeting Cancer Metabolism Plasticity with JX06 Nanoparticles via Inhibiting PDK1 Combined with Metformin for Endometrial Cancer Patients with Diabetes.

Diabetes is closely related to the occurrence of endometrial cancer (EC) and its poor prognosis. However, there is no effective clinical treatment for EC patients with diabetes (patientEC+/dia+ ). To explore new therapeutic targets, Ishikawa is cultured with high glucose (IshikawaHG ) mimicking hyperglycemia in patientEC+/dia+ . Subsequently, it is discovered that IshikawaHG exhibits glucose metabolic reprogramming characterized by increased glycolysis and decreased oxidative phosphorylation. Further, pyruvate dehydrogenase kinase 1 (PDK1) is identified to promote glycolysis of IshikawaHG by proteomics. Most importantly, JX06, a novel PDK1 inhibitor combined metformin (Met) significantly inhibits IshikawaHG proliferation though IshikawaHG is resistant to Met. Furthermore, a reduction-sensitive biodegradable polymer is adopted to encapsulate JX06 to form nanoparticles (JX06-NPs) for drug delivery. It is found that in vitro JX06-NPs have better inhibitory effect on the growth of IshikawaHG as well as patient-derived EC cells (PDC) than JX06. Additionally, it is found that JX06-NPs can accumulate to the tumor of EC-bearing mouse with diabetes (miceEC+/dia+ ) after intravenous injection, and JX06-NPs combined Met can significantly inhibit tumor growth of miceEC+/dia+ . Taken together, the study demonstrates that the combination of JX06-NPs and Met can target the cancer metabolism plasticity, which significantly inhibits the growth of EC, thereby provides a new adjuvant therapy for patientsEC+/dia+ .

The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin.

Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors.

A novel PDK1 inhibitor, JX06, inhibits glycolysis and induces apoptosis in multiple myeloma cells.

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 µM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM.

New Disulfiram Derivatives as MAGL-Selective Inhibitors.

Monoacylglycerol lipase (MAGL) is a key enzyme in the human endocannabinoid system. It is also the main enzyme responsible for the conversion of 2-arachidonoyl glycerol (2-AG) to arachidonic acid (AA), a precursor of prostaglandin synthesis. The inhibition of MAGL activity would be beneficial for the treatment of a wide range of diseases, such as inflammation, neurodegeneration, metabolic disorders and cancer. Here, the author reports the pharmacological evaluation of new disulfiram derivatives as potent inhibitors of MAGL. These analogues displayed high inhibition selectivity over fatty acid amide hydrolase (FAAH), another endocannabinoid-hydrolyzing enzyme. In particular, compound 2i inhibited MAGL in the low micromolar range. However, it did not show any inhibitory activity against FAAH.

Development of new disulfiram analogues as ALDH1a1-selective inhibitors.

Disulfiram is an FDA-approved drug used to treat chronic alcoholism. This drug works by blocking the second step of ethanol metabolism by inhibiting aldehyde dehydrogenase-2 (ALDH2), the enzyme responsible for acetaldehyde oxidation into acetic acid. This leads to the accumulation of acetaldehyde in the blood following alcohol ingestion and to highly unpleasant symptoms known as acetaldehyde syndrome. Disulfiram also inhibits ALDH1a1, another member of the aldehyde dehydrogenases that catalyzes the oxidation of retinal into retinoic acid. ALDH1a1 represents a key therapeutic target for the treatment of important diseases such as cancer and obesity. The substrate tunnel is larger in ALDH1a1 than in ALDH2; therefore. Thus, replacing disulfiram ethyl groups with larger groups will yield selective ALDH1a1 inhibitors. In this work, we successfully synthesized derivative 2b, in which two ethyl groups were replaced by two para fluorobenzyl groups. The 2b derivative showed a comparable activity to disulfiram against ALDH1a1; however, it was completely devoid of inhibitory activity against ALDH2.

Identification of the New Covalent Allosteric Binding Site of Fructose-1,6-bisphosphatase with Disulfiram Derivatives toward Glucose Reduction.

Fructose 1,6-bisphosphatase (FBPase) has attracted substantial interest as a target associated with cancer and type 2 diabetes. Herein, we found that disulfiram and its derivatives can potently inhibit FBPase by covalently binding to a new C128 allosteric site distinct from the original C128 site in APO FBPase. Further identification of the allosteric inhibition mechanism reveals that the covalent binding of a fragment of 214 will result in the movement of C128 and the dissociation of helix H4 (123-128), which in turn allows S123 to more easily form new hydrogen bonds with K71 and D74 in helix H3 (69-72), thereby inhibiting FBPase activity. Notably, both disulfiram and 212 might moderately reduce blood glucose output in vivo. Therefore, our current findings not only identify a new covalent allosteric site of FBPase but also establish a structural foundation and provide a promising way for the design of covalent allosteric drugs for glucose reduction.

Anti-alcohol abuse drug disulfiram inhibits human PHGDH via disruption of its active tetrameric form through a specific cysteine oxidation.

Due to rising costs and the difficulty to identify new targets, drug repurposing appears as a viable strategy for the development of new anti-cancer treatments. Although the interest of disulfiram (DSF), an anti-alcohol drug, to treat cancer was reported for many years, it is only very recently that one anticancer mechanism-of-action was highlighted. This would involve the inhibition of the p97 segregase adaptor NPL4, which is essential for the turnover of proteins involved in multiple regulatory and stress-response intracellular pathways. However, recently DSF was also reported as one of the first phosphoglycerate dehydrogenase (PHGDH) inhibitors, a tetrameric enzyme catalyzing the initial step of the serine synthetic pathway that is highly expressed in numerous cancer types. Here, we investigated the structure-activity relationships (SAR) of PHGDH inhibition by disulfiram analogues as well as the mechanism of action of DSF on PHGDH via enzymatic and cell-based evaluation, mass spectrometric and mutagenesis experiments.

Disulfiram-based disulfides as narrow-spectrum antibacterial agents.

Sixteen disulfides derived from disulfiram (Antabuse™) were evaluated as antibacterial agents. Derivatives with hydrocarbon chains of seven and eight carbons in length exhibited antibacterial activity against Gram-positive Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Listeria spp. A comparison of the cytotoxicity and microsomal stability with disulfiram further revealed that the eight carbon chain analog was of lower toxicity to human hepatocytes and has a longer metabolic half-life. In the final analysis, this investigation concluded that the S-octylthio derivative is a more effective growth inhibitor of Gram-positive bacteria than disulfiram and exhibits more favorable cytotoxic and metabolic parameters over disulfiram.

The selective cytotoxicity of DSF-Cu attributes to the biomechanical properties and cytoskeleton rearrangements in the normal and cancerous nasopharyngeal epithelial cells.

Cancer initiation and progression follow complex changes of cellular architecture and biomechanical property. Cancer cells with more submissive (or "softer") than their healthy counterparts attributed to the reorganization of the complex cytoskeleton structure, may be considered as a potential anti-tumor therapeutic target. In this study, atomic force microscopy (AFM) was carried out to detect the topographical and biophysical changes of nasopharyngeal carcinoma CNE-2Z cells and normal nasopharyngeal epithelial cells NP69-SV40T by treating the Disulfiram chelated with Cu2+ (DSF-Cu). DSF-Cu induced the apoptotic population, ROS production and decreased the NF-κB-p65 expression of CNE-2Z cells, which was much higher than those of NP69-SV40T cells. DSF-Cu caused the obvious changes of cell morphology and membrane ultrastructure in CNE-2Z cells. The roughness decreased and stiffness increased significantly in CNE-2Z cells, which correlated with the rearrangement of intracellular F-actin, FLNa and α-tubulin structures in CNE-2Z cells. And the adhesion force of CNE-2Z cells was also increased accompanied with the increased E-cadherin expression. However, these results could not be observed in the NP69-SV40T cells even the concentration of DSF reached up to 400nM. Finally, the detection of cell wound scratch assay confirmed DSF-Cu could inhibit the migration of CNE-2Z cells, but no effect on NP69-SV40T cells. These findings demonstrated the selective cytotoxicity of DSF-Cu in CNE-2Z cells may attribute to the different mechanical properties and cytoskeleton rearrangement from the normal nasopharyngeal epithelial cells.

Effective elimination of adult B-lineage acute lymphoblastic leukemia by disulfiram/copper complex in vitro and in vivo in patient-derived xenograft models.

Disulfiram (DS), a clinically used drug to control alcoholism, has displayed promising anti-cancer activity against a wide range of tumors. Here, we demonstrated that DS/copper (Cu) complex effectively eliminated adult B-ALL cells in vitro and in vivo in patient-derived xenograft (PDX) humanized mouse models, reflected by inhibition of cell proliferation, induction of apoptosis, suppression of colony formation, and reduction of PDX tumor growth, while sparing normal peripheral blood mononuclear cells. Mechanistically, these events were associated with disruption of mitochondrial membrane potential and down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Further analysis on B-ALL patients' clinical characteristics revealed that the ex vivo efficacy of DS/Cu in primary samples was significantly correlated to p16 gene deletion and peripheral blood WBC counts at diagnosis, while age, LDH level, extramedullary infiltration, status post intensive induction therapy, immune phenotype, risk category, and Ph chromosome had no effect. Together, these findings indicate that disulfiram, particularly when administrated in combination with copper, might represent a potential repurposing agent for treatment of adult B-ALL patients, including those clinically characterized by one or more adverse prognostic factors.

Disulfiram and its novel derivative sensitize prostate cancer cells to the growth regulatory mechanisms of the cell by re-expressing the epigenetically repressed tumor suppressor-estrogen receptor ß.

Estrogen Receptor-ß (ER-ß), a tumor-suppressor in prostate cancer, is epigenetically repressed by hypermethylation of its promoter. DNA-methyltransferases (DNMTs), which catalyze the transfer of methyl-groups to CpG islands of gene promoters, are overactive in cancers and can be inhibited by DNMT-inhibitors to re-express the tumor suppressors. The FDA-approved nucleoside DNMT-inhibitors like 5-Azacytidine and 5-Aza-deoxycytidine carry notable concerns due to their off-target toxicity, therefore non-nucleoside DNMT inhibitors are desirable for prolonged epigenetic therapy. Disulfiram (DSF), an antabuse drug, inhibits DNMT and prevents proliferation of cells in prostate and other cancers, plausibly through the re-expression of tumor suppressors like ER-ß. To increase the DNMT-inhibitory activity of DSF, its chemical scaffold was optimized and compound-339 was discovered as a doubly potent DSF-derivative with similar off-target toxicity. It potently and selectively inhibited cell proliferation of prostate cancer (PC3/DU145) cells in comparison to normal (non-cancer) cells by promoting cell-cycle arrest and apoptosis, accompanied with inhibition of total DNMT activity, and re-expression of ER-ß (mRNA/protein). Bisulfite-sequencing of ER-ß promoter revealed that compound-339 demethylated CpG sites more efficaciously than DSF, restoring near-normal methylation status of ER-ß promoter. Compound-339 docked on to the MTase domain of DNMT1 with half the energy of DSF. In xenograft mice-model, the tumor volume regressed by 24% and 50% after treatment with DSF and compound-339, respectively, with increase in ER-ß expression. Apparently both compounds inhibit prostate cancer cell proliferation by re-expressing the epigenetically repressed tumor-suppressor ER-ß through inhibition of DNMT activity. Compound-339 presents a new lead for further study as an anti-prostate cancer agent. © 2015 Wiley Periodicals, Inc.

Altering APP proteolysis: increasing sAPPalpha production by targeting dimerization of the APP ectodomain.

One of the events associated with Alzheimer's disease is the dysregulation of α- versus ß-cleavage of the amyloid precursor protein (APP). The product of α-cleavage (sAPPα) has neuroprotective properties, while Aß1-42 peptide, a product of ß-cleavage, is neurotoxic. Dimerization of APP has been shown to influence the relative rate of α- and ß- cleavage of APP. Thus finding compounds that interfere with dimerization of the APP ectodomain and increase the α-cleavage of APP could lead to the development of new therapies for Alzheimer's disease. Examining the intrinsic fluorescence of a fragment of the ectodomain of APP, which dimerizes through the E2 and Aß-cognate domains, revealed significant changes in the fluorescence of the fragment upon binding of Aß oligomers--which bind to dimers of the ectodomain--and Aß fragments--which destabilize dimers of the ectodomain. This technique was extended to show that RERMS-containing peptides (APP(695) 328-332), disulfiram, and sulfiram also inhibit dimerization of the ectodomain fragment. This activity was confirmed with small angle x-ray scattering. Analysis of the activity of disulfiram and sulfiram in an AlphaLISA assay indicated that both compounds significantly enhance the production of sAPPα by 7W-CHO and B103 neuroblastoma cells. These observations demonstrate that there is a class of compounds that modulates the conformation of the APP ectodomain and influences the ratio of α- to ß-cleavage of APP. These compounds provide a rationale for the development of a new class of therapeutics for Alzheimer's disease.

Comparison of ivermectin and benzyl benzoate lotion for scabies in Nigerian patients.

Few studies have compared ivermectin directly with topical agents in developing countries. We compared the effectiveness of oral ivermectin (200 microg/kg) with topical 25% benzyl benzoate and monosulfiram soap in 210 subjects of age 5 to 65 years with scabies. Subjects with persistent lesions after 2 weeks received a second course of treatment. All lesions had resolved after 2 weeks in 77 of 98 (79%) subjects treated with ivermectin and in 60 of 102 (59%) subjects treated topically (P = 0.003). The improvement in severity score was greater in the ivermectin group than in the topical treatment group (P < 0.001). The overall cure rate after 4 weeks was 95% in the ivermectin group and 86% in the topical treatment group (P = 0.04). Compared with topical benzyl benzoate and monosulfiram in the treatment of scabies, ivermectin was at least as effective and led to more rapid improvement.

[Profuse scabies: kinetic curves of parasitologic cure with an association of benzyl benzoate and sulfiram]. / Gale profuse: cinétique de guérison parasitologique avec l'association benzoate de benzyl et sulfiram.

BACKGROUND: In vitro exposure to benzyl benzoate (25 p. 100) kills Sarcoptes scabiei within three hours. The aim of our study was to determine in vivo elimination of Sarcopte scabiei with a benzyl benzoate-sulfiram association. METHODS: Medical charts of patients hospitalized for disseminated scabies from 1993 to 1999 were reviewed retrospectively. The diagnosis of scabies was confirmed by microscopic determination. Parasitological examinations were conducted every day or every two days until negative results. Patients were treated by successive applications of benzyl benzoate until parasitological cure. RESULTS: Twenty patients were included in the study. The median delay of parasitological cure was seven days. After 15 days, 95 p. 100 of patients were cured. Two cutaneous side-effects were reported. DISCUSSION: Despite immediate in vitro efficacy, benzyl benzoate action is delayed in vivo. The time of parasitological negativation after one application of benzyl benzoate is unknown. Therefore, it is not currently possible to determine whether our therapeutic regimen was excessive or not.

Inhibitory effects of seven organosulphur compounds on clinical isolates of Candida species in vitro.

Thirty clinical isolates of Candida albicans and 10 other Candida species were tested for susceptibility to 6 substituted dithiocarbamates and one dimercaptosuccinate. Dimethyldithiocarbamate, sodium pyrrolidine dithiocarbamate, and sodium diethyldithiocarbamate showed dose-dependent antifungal activity which was partially reversed by the addition of zinc, copper, or iron sulfate with greatest reversal at 2:1 metal to dithiocarbamate molar ratio. Anaerobiosis also interfered with dithiocarbamate antifungal activity.

[Integrated treatment of alcoholism. Evaluation of its efficacy]. / Trattamento integrato dell'alcolismo. Valutazione dell'efficacia.

Patients with alcohol addiction are analyzed after one year from the beginning of therapy. Treatment is carried out by an Anti-addiction Service working in territory, an Hospital medical ward, anti-alcoholic clubs and Hospital voluntary groups. The method is described at first, then the answers of investigated patients, the abstinence period, the attendance to help groups, the use of drugs (disulfiram) and the emerged problems are considered and analyzed. More than one year later, patients with alcohol addiction who have followed this method are still in abstinence and have changed their habit from alcoholic to unalcoholic one, as just they have done for the behaviour and the relationship.

Fate and distribution of [14C]S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine, an antimutagenic mixed disulfide from disulfiram, in rats.

S-(N,N-Diethyldithiocarbamoyl)-N-acetyl-L-cysteine (AC-DDTC) is a mixed disulfide from disulfiram and N-acetyl-L-cysteine, which possesses putative anticarcinogenic and antimutagenic properties. The present study describes the absorption, distribution, metabolism and excretion of 14C-labeled AC-DDTC in rats. AC-DDTC was well absorbed after oral administration. Based on the excretion of radioactivity in urine, the minimum absorption was about 73%. The rate of absorption was very rapid, with the peak level of radioactivity in plasma after 15 min of administration. Mean Cmax value for N,N-diethyldithiocarbamate (DDTC) after oral dose of AC-DDTC (20 mg/kg) was 3.8 +/- 0.2 nmol/ml at 15 min and the mean residence time was 47.1 +/- 2.8 min. After oral administration of [14C]AC-DDTC, radioactivity was distributed relatively rapidly. Maximum concentrations were observed in the liver (0.443% dose/g), kidneys (0.496% dose/g), oesophagus (0.313% dose/g) and in the adrenals (0.364% dose/g) at 30 min to 1 h after dosing. Liver was the only organ which contains a considerable amount of radioactivity (0.091% dose/g) 24 h after dosing. Two metabolites of AC-DDTC following oral administration were identified in the plasma and liver by GC and HPLC using extractive alkylation technique, namely DDTC and its methyl ester. Urinary excretion was a major route of elimination of radioactivity derived from [14C]AC-DDTC, in that about 73% of the dose was recovered in urine whereas only 14% was found in feces over 7 days.

The effects of two inositol (1,4,5)-trisphosphate 5-phosphatase inhibitors on thyrotropin releasing hormone stimulated inositol (1,4,5)-trisphosphate production in GH3 cells.

The effects of FLA99 and EWP840, two disulfiram analogs which potently inhibit Ins(1,4,5)P3 5-phosphatase, upon basal and thyrotropin-releasing hormone (TRH)-stimulated inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) levels were investigated. Neither test compound affected the characteristics of the [3H]Ins(1,4,5)P3 binding site used in the competitive protein binding assay of Ins(1,4,5)P3 levels. In rat GH3 pituitary cell suspensions, TRH (100 nM) produced a large and time-dependent increase in Ins(1,4,5)P3 concentration, the maximum response being obtained within 5 seconds of stimulation in these cells. Neither FLA99 (100, 300 and 1000 microM) nor EWP840 (100 microM) produced obvious effects on the Ins(1,4,5)P3 response to TRH stimulation. Higher concentrations of EWP840 (300 and 1000 microM) abolished the Ins(1,4,5)P3 response to TRH stimulation. The lack of effect of the 5-phosphatase inhibition in the cells may indicate that 5-phosphatase is not the major metabolic pathway of this second messenger in this cell line under the assay conditions used.

Photolysis of sulfiram: a mechanism for its disulfiram-like reaction.

Sulfiram, a drug applied topically to treat scabies, produces effects similar to those of disulfiram after subsequent ingestion of ethanol. Disulfiram, used in aversion therapy in the treatment of alcoholism, inhibits hepatic aldehyde dehydrogenase (ALDH) causing an accumulation of acetaldehyde after ethanol ingestion. The increased tissue levels of acetaldehyde cause a spectrum of undesirable side-effects including flushing, nausea, vomiting, and tachycardia, which are referred to as the disulfiram reaction. Previous studies have shown that in vitro sulfiram is a very weak inhibitor of ALDH, but solutions of sulfiram markedly increase in potency with time. In the present study, fresh solutions of sulfiram were exposed to fluorescent room light under ambient conditions and analyzed at timed intervals by HPLC. At least eight products, including disulfiram, were formed in the light-exposed sulfiram solutions, but not in solutions kept in the dark. Structural characterization of two of the photolysis products was obtained by on-line microbore HPLC-mass spectrometry (mu LC-MS) and on-line microbore HPLC-tandem mass spectrometry (mu LC-MS/MS) using continuous flow-liquid secondary ion mass spectrometry (CF-LSIMS) as the primary ionization method. Sulfiram was converted to disulfiram at an initial rate of 0.7%/hr, and the formation of disulfiram correlated with the increase in ALDH inhibition in vitro. The results of this investigation show that while sulfiram is a weak inhibitor of ALDH in vitro, it is readily photoconverted to disulfiram, a very potent inhibitor of ALDH, which may explain the adverse reaction to ethanol after sulfiram therapy.