Aldehyde Dehydrogenase Inhibitor Research Articles

Disulfiram in outpatient treatment of alcohol dependency

The alcohol deterrent drug disulfiram plays a minor role in the treatment of alcohol dependency in Germany. The study looks at the efficacy, tolerability and feasibility of a disulfiram outpatient treatment program in a German psychiatric hospital. Data from 190 outpatients with alcohol dependency, who had participated in a disulfiram therapy program at the psychiatric outpatient department over a period of 10 years, were analyzed with respect to efficacy and tolerability. To test for predictors, independent variables and treatment success as a dependent variable a logistic regression was carried out. After 1 year 24.2 % of the patients maintained alcohol abstinence while 55 % had had an alcohol relapse despite being in the disulfiram program. No severe complications were observed under disulfiram treatment. Therapy success was largely related to participating in treatment-specific group therapy. Disulfiram proved to be a well-tolerated medication as part of multimodal therapy of alcohol dependency. The disulfiram program was easily integrated into other health service treatment. Approximately one quarter of patients who had had an unfavorable course before, achieved abstinence, while participation in group therapy was a major predictor of treatment success. Disulfiram is a medication, which in the context of a psychosocial treatment concept, should receive a wider distribution in Germany.

Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases

Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC50 15μM) and D3 for MhpB (IC50 110μM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the β-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde.

Cyanamide Phytotoxicity in Soybean (Glycine max) Seedlings involves Aldehyde Dehydrogenase Inhibition and Oxidative Stress

The phytotoxic effect of the allelochemical cyanamide has been well-documented yet the underlying mechanism for this phenomenon has not been fully characterized. Cognizant of the putative inhibitory effect of cyanamide on aldehyde dehydrogenases (ALDHs), we hereby show that the capacity of mitochondrial preparations from cyanamide-treated soybean seedlings to oxidize acetaldehyde and succinic-semialdehyde was dose-dependently reduced to at most 55% and 70%, respectively. Cyanamide-treated plants exhibited oxidative stress (i.e. increased lipid peroxidation and H2O2 accumulation) that was exacerbated upon exposure to UV-A--symptoms reminiscent of ALDH and succinic-semialdehyde dehydrogenase (SSADH) knock-out Arabidopsis mutants. We suggest that the inhibition of mitochondrial ALDH and SSADH may be a contributory mechanism to the burst in oxidative stress mediated by cyanamide.

Increased brain nitric oxide levels following ethanol administration

Nitric oxide is a ubiquitous messenger molecule, which at elevated concentrations has been implicated in the pathogenesis of several neurological disorders. Its role in oxidative stress, attributed in particular to the formation of peroxynitrite, proceeds through its high affinity for the superoxide radical. Alcoholism has recently been associated with the induction of oxidative stress, which is generally defined as a shift in equilibrium between pro-oxidant and anti-oxidant species in the direction of the former. Furthermore, its primary metabolite acetaldehyde, has been extensively associated with oxidative damage related toxic effects following alcohol ingestion. The principal objective of this study was the application of long term in vivo electrochemistry (LIVE) to investigate the effect of ethanol (0.125, 0.5 and 2.0 g kg−1) and acetaldehyde (12.5, 50 and 200 mg kg−1) on NO levels in the nucleus accumbens of freely moving rats. Systemic administrations of ethanol and acetaldehyde resulted in a dose-dependent increases in NO levels, albeit with very differing time courses. Subsequent to this the effect on accumbal NO levels, of subjecting the animal to different drug combinations, was also elucidated. The nitric oxide synthase inhibitor L-NAME (20 mg kg−1) and acetaldehyde sequestering agent D-penicillamine (50 mg kg−1) both attenuated the increase in NO levels following ethanol (1 g kg−1) administration. Conversely, the alcohol dehydrogenase inhibitor 4-methylpyrazole (25 mg kg−1) and catalase inhibitor sodium azide (10 mg kg−1) potentiated the increase in NO levels following ethanol administration. Finally, dual inhibition of aldehyde dehydrogenase and catalase by cyanamide (25 mg kg−1) caused an attenuation of ethanol effects on NO levels. Taken together these data highlight a robust increase in brain NO levels following systemic alcohol administration which is dependent on NO synthase activity and may involve both alcohol- and acetaldehyde-dependent mechanisms.

Inhibition of human alcohol and aldehyde dehydrogenases by aspirin and salicylate: Assessment of the effects on first-pass metabolism of ethanol

Previous studies have reported that aspirin significantly reduced the first-pass metabolism (FPM) of ethanol in humans thereby increasing adverse effects of alcohol. The underlying causes, however, remain poorly understood. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition profiles by aspirin and its major metabolite salicylate of ethanol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and acetaldehyde oxidation by ALDH1A1 and ALDH2, at pH 7.5 and 0.5mM NAD+. Competitive inhibition pattern was found to be a predominant type among the ADHs and ALDHs studied, although noncompetitive and uncompetitive inhibitions were also detected in a few cases. The inhibition constants of salicylate for the ADHs and ALDHs were considerably lower than that of aspirin with the exception of ADH1A that can be ascribed to a substitution of Ala-93 at the bottom of substrate pocket as revealed by molecular docking experiments. Kinetic inhibition equation-based simulations show at higher therapeutic levels of blood plasma salicylate (1.5mM) that the decrease of activities at 2–10mM ethanol for ADH1A/ADH2 and ADH1B2/ADH1B3 are predicted to be 75–86% and 31–52%, respectively, and that the activity decline for ALDH1A1 and ALDH2 at 10–50μM acetaldehyde to be 62–73%. Our findings suggest that salicylate may substantially inhibit hepatic FPM of alcohol at both the ADH and ALDH steps when concurrent intaking aspirin.

Rotenone decreases intracellular aldehyde dehydrogenase activity: implications for the pathogenesis of Parkinson's disease.

Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson's disease (PD). Mechanisms of relatively selective rotenone-induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the 'catecholaldehyde hypothesis,' buildup of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F-labeled catechols were measured in PC12 cells incubated with rotenone (0-1000 nM, 180 min), without or with F-dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose dependently increased DOPAL, F-DOPAL, and 3,4-dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD(+) was supplied, whereas the direct-acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone-induced toxicity in dopaminergic neurons. We report that rotenone, a mitochondrial complex I inhibitor that produces an animal model of Parkinson's disease, increases intracellular levels of the toxic dopamine metabolite 3,4-dihydroxyphenyl-acetaldehyde (DOPAL), via decreased DOPAL metabolism by aldehyde dehydrogenase (ALDH) and decreased vesicular sequestration of cytoplasmic dopamine by the vesicular monoamine transporter (VMAT). The results provide a novel mechanism for rotenone-induced toxicity in dopaminergic neurons.

N-Acetyl-S-(N,N-diethylcarbamoyl) cysteine in rat nucleus accumbens, medial prefrontal cortex, and in rat and human plasma after disulfiram administration

Disulfiram (DSF), a treatment for alcohol use disorders, has shown some clinical effectiveness in treating addiction to cocaine, nicotine, and pathological gambling. The mechanism of action of DSF for treating these addictions is unclear but it is unlikely to involve the inhibition of liver aldehyde dehydrogenase (ALDH2). DSF is a pro-drug and forms a number of metabolites, one of which is N-acetyl-S-(N,N-diethylcarbamoyl) cysteine (DETC-NAC). Here we describe a LCMS/MS method on a QQQ type instrument to quantify DETC-NAC in plasma and intracellular fluid from mammalian brain. An internal standard, the N,N-di-isopropylcarbamoyl homolog (MIM: 291>128) is easily separable from DETC-NAC (MIM: 263>100) on C18 RP media with a methanol gradient. The method's linear range is 0.5–500nM from plasma and dialysate salt solution with all precisions better than 10% RSD. DETC-NAC and internal standards were recovered at better than 95% from all matrices, perchloric acid precipitation (plasma) or formic acid addition (salt) and is stable in plasma or salt at low pH for up to 24h. Stability is observed through three freeze-thaw cycles per day for 7 days. No HPLC peak area matrix effect was greater than 10%. A human plasma sample from a prior analysis for S-(N,N-diethylcarbamoyl) glutathione (CARB) was found to have DETC NAC as well. In other human plasma samples from 62.5mg/d and 250mg/d dosing, CARB concentration peaks at 0.3 and 4nM at 3h followed by DETC-NAC peaks of 11 and 70nM 2h later. Employing microdialysis sampling, DETC-NAC levels in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and plasma of rats treated with DSF reached 1.1, 2.5 and 80nM at 6h. The correlation between the appearance and long duration of DETC-NAC concentration in rat brain and the persistence of DSF-induced changes in neurotransmitters observed by Faiman et al. (Neuropharmacology, 2013, 75C, 95–105) is discussed.

Methylmercury impairs canonical dopamine metabolism in rat undifferentiated pheochromocytoma (PC12) cells by indirect inhibition of aldehyde dehydrogenase.

The environmental neurotoxicant methylmercury (MeHg) disrupts dopamine (DA) neurochemical homeostasis by stimulating DA synthesis and release. Evidence also suggests that DA metabolism is independently impaired. The present investigation was designed to characterize the DA metabolomic profile induced by MeHg, and examine potential mechanisms by which MeHg inhibits the DA metabolic enzyme aldehyde dehydrogenase (ALDH) in rat undifferentiated PC12 cells. MeHg decreases the intracellular concentration of 3,4-dihydroxyphenylacetic acid (DOPAC). This is associated with a concomitant increase in intracellular concentrations of the intermediate metabolite 3,4-dihydroxyphenylaldehyde (DOPAL) and the reduced metabolic product 3,4-dihydroxyethanol. This metabolomic profile is consistent with inhibition of ALDH, which catalyzes oxidation of DOPAL to DOPAC. MeHg does not directly impair ALDH enzymatic activity, however MeHg depletes cytosolic levels of the ALDH cofactor NAD(+), which could contribute to impaired ALDH activity following exposure to MeHg. The observation that MeHg shunts DA metabolism along an alternative metabolic pathway and leads to the accumulation of DOPAL, a reactive species associated with protein and DNA damage, as well as cell death, is of significant consequence. As a specific metabolite of DA, the observed accumulation of DOPAL provides evidence for a specific mechanism by which DA neurons may be selectively vulnerable to MeHg.

Diethylaminobenzaldehyde is a covalent, irreversible inactivator of ALDH7A1.

There is growing interest in aldehyde dehydrogenases (ALDHs) because of their overexpression in cancer stem cells and the ability to mediate resistance to cancer drugs. Here, we report the first crystal structure of an aldehyde dehydrogenase complexed with the inhibitor 4-diethylaminobenzaldehyde (DEAB). Contrary to the widely held belief that DEAB is a reversible inhibitor of ALDHs, we show that DEAB irreversibly inactivates ALDH7A1 via formation of a stable, covalent acyl-enzyme species.

Disulfiram’s Antineoplastic Effects on Ovarian Cancer

Objective: Aldehyde dehydrogenase (ALDH) enzymatic activity identifies ovarian cancer stem-like cells. We investigated the antineoplastic activity of the ALDH inhibitor Disulfiram on bulk ovarian cancer cells and CD133 + /ALDH + cancer stem-like cells. Study Design: Ovarian cancer cell lines, human ovarian surface epithelial cells, and mesenchymal stem cells were treated with increasing concentrations of Disulfiram and/or Cisplatin in vitro. Treated cells were assessed for viability or FACS-analyzed for either percentage of ovarian cancer stem-like cells or induction of apoptosis. Disulfiram’s impact on cancer stem-like cells was tested in vitro using tumor sphere formation assays and in vivo using tumor initiation assays with in vitro-treated A2780 cells in NSG mice. Finally, Disulfiram’s in vivo activity was assessed versus CD133 + /ALDH + cell-initiated tumor xenografts. Results: Disulfiram demonstrated antineoplastic activity against multiple ovarian cancer cell lines. While Disulfiram had limited in vitro toxicity against human ovarian surface epithelial cells or mesenchymal stem cells (IC50 of ~15 µM and >30 µM, respectively), its antineoplastic activity against cell lines was comparable to Cisplatin (IC50 ~1.5 μM). Disulfiram-mediated cell death was due, at least in part, to induction of apoptosis. Disulfiram activity was additive with chemotherapy. Disulfiram demonstrated selective depletion of CD44 + cells but not the CD133 + cancer stem-like cells. Disulfiram had no therapeutic impact on tumor initiation studies or in vivo therapy of whole cell line or stem cell-initiated tumor xenografts. Conclusions: In biologically relevant concentrations, Disulfiram has clear antineoplastic activity against ovarian cancer cells in vitro. Disulfiram selectively depleted CD44+ but not CD133+ ovarian cancer stem-like cells in vitro. However, Disulfiram had no significant activity in vivo. Thus, improved and more selective ALDH inhibitors may be required to target ovarian cancer stem cells.

Be careful what you eat and drink; Coprinic and Kounis syndrome in a patient with the Samter`s triad

Allergic reactions to chemicals, food products or even insect bites are encountered all over the world with a variety of manifestations. Symptoms range from the development of a minor rash to life threatening anaphylactic reactions. Sometimes, acute coronary syndrome (ACS) can be registered in such patients, which may have a serious impact on the course and management of the allergic reaction. Mushroom poisoning constitutes the main portion of plant toxicities in Spain. Depending on the type of mushroom, the adverse effects range from mild gastrointestinal (GI) symptoms to major cytotoxic effects resulting in organ failure and death. Coprinopsis atramentaria poisoning is rare but with serious consequences. We report a case of a 27 year old patient with a previous history of mild persistent asthma, nasal polyps and aspirin allergy that arrived to the Emergency Department with dizziness and temor after consuming beer at a party. On arrival to the hospital, the patient suddenly collapsed, with an electrocardiogram (ECG) showing ST elevation in the inferior leads and 3rd degree atrioventricular (AV) block accompanied by urticarial-like lesions. After administration of treatment, the patient and ECG abnormalities improved. A detailed medical history showed that the patient had eaten scrambled mushrooms and beer. Expert analysis demonstrated that the combined consumption of Coprinopsis atramentaria and alcoholic beverages produced a disulfiram-like reaction (Coprinic syndrome) and type I Kounis syndrome. No case like this has been previously reported.

Disulfiram modulates stemness and metabolism of brain tumor initiating cells in atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumors (AT/RT) are among the most malignant pediatric brain tumors. Cells from brain tumors with high aldehyde dehydrogenase (ALDH) activity have a number of characteristics that are similar to brain tumor initiating cells (BTICs). This study aimed to evaluate the therapeutic potential of ALDH inhibition using disulfiram (DSF) against BTICs from AT/RT. Primary cultured BTICs from AT/RT were stained with Aldefluor and isolated by fluorescence activated cell sorting. The therapeutic effect of DSF against BTICs from AT/RT was confirmed in vitro and in vivo. AT/RT cells displayed a high expression of ALDH. DSF demonstrated a more potent cytotoxic effect on ALDH(+) AT/RT cells compared with standard anticancer agents. Notably, treatment with DSF did not have a considerable effect on normal neural stem cells or fibroblasts. DSF significantly inhibited the ALDH enzyme activity of AT/RT cells. DSF decreased self-renewal ability, cell viability, and proliferation potential and induced apoptosis and cell cycle arrest in ALDH(+) AT/RT cells. Importantly, DSF reduced the metabolism of ALDH(+) AT/RT cells by increasing the nicotinamide adenine dinucleotide ratio of NAD(+)/NADH and regulating Silent mating type Information Regulator 2 homolog 1 (SIRT1), nuclear factor-kappaB, Lin28A/B, and miRNA let-7g. Animals in the DSF-treated group demonstrated a reduction of tumor volume (P < .05) and a significant survival benefit (P = .02). Our study demonstrated the therapeutic potential of DSF against BTICs from AT/RT and suggested the possibility of ALDH inhibition for clinical application.

Abstract 3749: Novel aldehyde dehydrogenase inhibitors as potential anti-chemoresistance drugs for head and neck cancers

Abstract The 19 human aldehyde dehydrogenase (ALDH) gene products are enzymes that metabolize a wide range of reactive aldehydes, including those generated by the metabolism of drugs, alcohol, cigarette smoke and organic compounds. These toxic aldehydes are metabolized into respective non-reactive, non-toxic acids. We reasoned that expression of high levels of ALDH support cancer stem cells growth and chemoresistance by detoxifying metabolic aldehydes and aldehydic intermediates of common chemotherapy drugs. Thus, inhibitors of ALDHs (Aldis) may potentiate current treatments. Many malignant cancer cells and cancer stem cells (CSCs) are resistant to chemotherapy, which can lead to secondary relapses. Further, because head and neck cancer (HNC) stem cells express high levels of ALDHs, Aldis may be useful in increasing sensitivity of these tumors to chemotherapy. Testing several different HNC cell lines demonstrated that SCC4 cells are more resistant to cisplatin treatment and PCI13 cells are less resistant (e.g., at 100μM cisplatin, 30±2% of SCC4 cells survived vs. 15±6% of PCI13 cells; n=16). After cisplatin treatment (15μM for 8 days), ALDH activity increased in SCC4 more than in PCI13 (35±5% vs. 2±0.5% increase) as compared with non-treated cells measured. Co-treatment of cisplatin with Aldi-6 reduced ALDH activity in SCC4 by 67% as measured by Aldefluor assay. When cells were treated with Aldi-6 (an ALDH1, 2 and 3 inhibitor) together with cisplatin, Aldi-6 increased cell death by 30% in SCC4 and by 10% in PCI13 compared with cells treated with cisplatin alone (n=8 each). ALDH3A1 protein levels (but not ALDH1 or 2) increased with cisplatin treatment by ∼100 fold and 25 fold, respectively, in SCC4 and PCI13 as compared with non-treated cells measured by Western blot. Based on this data, we tested the efficacy of Aldi-6 in vivo. Tumor (SCC4)-bearing mice were divided into 4 groups and were treated for two weeks with vehicle, Aldi-6 (8mg/kg/day, delivered via an osmotic pump), cisplatin (2mg/kg/BW, ip injection, 2/week) or cisplatin+Aldi-6. Aldi-6 alone resulted in smaller tumors (average tumor volume (mm3): vehicle, 2,945±112; Aldi6, 773±481). Aldi-6 also enhanced cisplatin reduction of tumor size by more than 60% as compared with cisplatin treatment alone (average tumor volume (mm3): cisplatin, 1,560; Aldi6+cisplatin, 597±112; n=3-6). In conclusion, we found that increased ALDH3A1 levels in HNCs following cisplatin treatment in culture and in vivo may contribute to tumor growth and therefore, ALDH3A1 inhibitors may be suitable as chemotherapeutic agents alone; ALDH3A1 inhibitors may also reduce chemoresistance to cisplatin. Understanding the role of ALDH in HNC not only provides insight into the chemoresistance of this disease, but it may also provide novel strategies for therapy. Citation Format: Jeewon Kim, JuneHo Shin, Che-Hong Chen, Leslie Cruz, Lovisa Farnebo, Jieying Yang, John B. Sunwoo, Daria Mochly-Rosen. Novel aldehyde dehydrogenase inhibitors as potential anti-chemoresistance drugs for head and neck cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3749. doi:10.1158/1538-7445.AM2014-3749

Abstract 5513: Targeting brain tumor initiating cells in atypical teratoid/rhabdoid tumors: Aldehyde dehydrogenase inhibition with disulfiram

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are one of the most malignant pediatric brain tumors with a dismal prognosis. Cells with high aldehyde dehydrogenase (ALDH) activity from brain tumors have a number of characteristics that are expected of brain tumor initiating cells (BTICs). This study aimed to evaluate the therapeutic potential of ALDH inhibition using disulfiram (DSF) against BTICs from AT/RT. Primary cultured BTICs from AT/RT were stained with Aldefluor and isolated by fluorescence activated cell sorting. Therapeutic effect of DSF against BTICs from AT/RT was confirmed using in vitro and in vivo studies. AT/RT showed high expression of ALDH. DSF significantly inhibited ALDH enzyme activity of AT/RT cells. DSF decreased self-renewal ability, cell viability, proliferation potential and induced apoptosis and cell cycle arrest on ALDH+ AT/RT cells. DSF showed more potent cytotoxic effect to ALDH+ AT/RT cells compared to standard anti-cancer agents including ifosfamide, carboplatin and etoposide. Importantly, DSF reduced metabolite (metabolism?) of ALDH+ AT/RT cells by increasing NAD+ ratio and regulating SIRT1, NF-κB, Lin28A/B and miRNA let-7g. Notably, treatment with DSF did not have considerable effect on the normal neural stem cells and fibroblasts. Animals in the DSF-treated group demonstrated a significant survival benefit (105 days of median survival in the DSF-treated group versus 91 days in the control group, p = 0.0219). Our study demonstrated the therapeutic potential of DSF against BTICs from AT/RT and suggested the possibility of ALDH inhibition for clinical application. Note: This abstract was not presented at the meeting. Citation Format: Seung Ah Choi, Jung Won Choi, Kyu-Chang Wang, Ji Hoon Phi, Ji Yeoun Lee, Yong Hwy Kim, Young-Hoon Kim, Sung-Hye Park, Seung-Ki Kim. Targeting brain tumor initiating cells in atypical teratoid/rhabdoid tumors: Aldehyde dehydrogenase inhibition with disulfiram. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5513. doi:10.1158/1538-7445.AM2014-5513

Abstract 3982: Repurposing disulfiram for treatment of relapsed/refractory acute myeloid leukemia

Abstract Background Despite toxic treatments, acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children while significantly less in older adults. For patients with relapsed AML refractory to chemotherapy, there is little chance for survival. Therefore, the need for novel treatment modalities continues to be critically important. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor that has been used for decades for the treatment of alcoholism without major side effects. While DS has been reported to have anticancer cytotoxicity that is in part copper (Cu2+)-dependent, the mechanisms are still not fully elucidated. The goal of the present study was to investigate whether DS has significant cytotoxicity against AML. Methods and Results In the present study the IC50 were determined for DS alone or in combination with 1 µM Cu2+ in a panel of 16 AML cell lines using Cell Titer Glo assay. DS/Cu2+ resulted in significant killing of all cell lines (IC50 between 20-75 nM) while the response to DS alone showed significant variation (IC50 from 20 nM - 7 µM). Cu2+ alone was not toxic. Cytarabine (Ara-C), a key drug in AML treatment, was compared with DS/Cu2+. The AML cell lines THP1 (acute monocytic leukemia), UT7-epo and CMY (acute megakaryoblastic leukemia) were profoundly resistant to Ara-C (IC50 between 1.2-2.4 µM) but showed extreme sensitivity to DS/Cu2+ (IC50 between 31-46 nM). Although DS is an inhibitor of ALDH, we demonstrated that the cytotoxicity of DS or DS/Cu2+ was not directly associated with the level of ALDH or its inhibition. AML cell lines were treated with different doses of DS or DS/Cu2+ and ALDH activity and cell death were determined by flow cytometry. The percentage of cell death was dose-dependent and occurred irrespective of ALDH activity in cell lines with high endogenous ALDH activity (MV-4-11, CMY, CMK and K562) compared to those with low or undetectable ALDH activity (Kasumi-1, Molm-13, NB4 and KG-1a). As DS-mediated cytotoxicity has been associated in previous reports with proteasome inhibition, the effects of DS and DS/Cu2+ were compared to Bortezumib. Western blot analysis showed that DS induced PARP cleavage similar to Bortezumib, suggesting that proteasome inhibition may be contributing to DS or DS/Cu2+ cytotoxicity. Conclusion These data demonstrate that multiple AML cell lines are sensitive to DS and to DS/Cu2+, including some that show extreme resistance to Ara-C, such as CMY from a patient with Down Syndrome. This DS sensitivity may provide a less toxic and efficacious treatment approach to be tested in this group of patients with poor outcome following relapse. The cytotoxic activity of DS does not appear to be solely dependent on ALDH inhibition. Ongoing studies are focusing on determining the mechanisms of cell death, and cytotoxic efficacy in in vitro and xenograft models using patient AML samples, with the goal of developing a clinical trial in patients with relapsed/refractory AML. Citation Format: Eiman A. Aleem, David O. Azorsa, Ranjan Bista, Oliver B. Pepper, David W. Lee, Daniel H. Wai, Robert J. Arceci. Repurposing disulfiram for treatment of relapsed/refractory acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3982. doi:10.1158/1538-7445.AM2014-3982

Abstract 222: Disulfiram targets glioblsatoma stem like cells by modulating aldehyde dehydrogenase and hypoxia-NF-κB pathway

Abstract Introduction: Glioblastoma Multiforme (GBM) is the most common lethal brain tumor associated with dismal survival rate. It is widely accepted that GBM contains a small population of cancer cells (∼1%) expressing CSC markers that promotes therapeutic resistance. High levels of aldehyde dehydrogenase (ALDH) activity, a characteristic feature of CSCs, play a significant role in chemoresistance. Many recent studies have elucidated that intra-tumoral hypoxia induces CSC phenotypes in tumour via epithelial-to-mesenchymal transition (EMT). Hypoxia inducible factors (HIFs) are master transcriptional regulators under hypoxic conditions. However NF-κB, another key transcription factor, is also highly up-regulated in hypoxia induced CSCs. There are no clear insights on how HIFs and NF-κB together orchestrates the anti-apoptotic signalling, chemo-radiation resistance and maintenance of stemness in CSC phenotypes. Disulfiram (DS), an anti-alcoholism drug, in combination with copper (Cu) effectively reverses chemoresistance in many cancers. DS/Cu is a strong inhibitor of NF-κB and ALDH and induces apoptosis. In this study we used an in vitro GBM CSC model and NF-κB-p65 and HIFs transfected GBM cell lines to investigate the relationship between hypoxia induced HIFs, NF-κB activation and ALDH activity and their role in chemoresistance. We also examined the cytotoxicity of DS/Cu on GBM CSCs and its effect on the above pathways. Results In comparison with adhered cells, the GBM cellsgrown as spheres in specialized neurosphere medium (NS) or normal DMEM (SUS) showed high proportion of hypoxic cells and elevated levels of CSC and EMT markers (ALDH, CD133, CD44, OCT4, SOX2, NANOG, E-cadherin to N-cadherin switch) suggesting hypoxia induced EMT phenotypes inCSCs. Increased levels of HIFs, ALDH (1A3 and 3A1) and NF-κB activity were detected in CSCs. CSCs are highly resistant to conventional anticancer drugs like temozolomide, paclitaxel, vincristine and doxorubicin. GBM cells transfected with NF-κB-p65 displayed CSC markers, chemoresistance and nuclear translocation of HIFs. Although transfection of HIFs induced expression of CSC markers, the cells did not show NF-κB activation and are not resistant to anticancer drugs. These results indicate the pivotal role of NF-κB in maintaining CSC characteristics and chemoresistance. DS is cytotoxic to both GBM cell lines and the CSCs derived from them in a Cu-dependent manner.DS/Cu inhibits NF-κB and ALDH activity and triggers intrinsic apoptotic pathway. After short exposure (2 hours) to low concentration of DS/Cu, the CSCs are completely eradicated in the cell culture. Conclusions: NF-κB plays pivotal role in chemoresistance. DS/Cu inhibits NF-κB, eradicates CSCs and reverses resistance in GBM.DS is an FDA approved drug with low/no toxicity to normal tissues and can freely pass through the BBB. Further study may lead to quick translation of DS into clinical trials. Citation Format: Vinodh Kannappan, Peng Liu, Sarah Brown, Xiuwu Bian, Tawari Patricia Erebi, Angel L. Armesilla, John L. Darling, Weiguang Wang. Disulfiram targets glioblsatoma stem like cells by modulating aldehyde dehydrogenase and hypoxia-NF-κB pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 222. doi:10.1158/1538-7445.AM2014-222

SY04-3 * PHARMACOTHERAPY FOR ALCOHOLISM IN KOREA

Alcohol drinking is popular in the Republic of Korea (Korea). Psychiatric epidemiologic surveys conducted in Korea revealed that while prevalence rates of most psychiatric disorders were significantly lower compared to those found in the US, the alcohol dependence rate was substantially higher in Korea than in both Western and other Asian countries. Moreover, the rates of alcohol drinking in men and women have been increasing in Korea during the last several decades, especially among women. Excessive alcohol consumption causes more than 4,500 deaths annually in Korea, and the alcohol-induced death rate was 9.1 per 100,000 individuals in 2010. Currently, there are three medications approved by The Korea Ministry of Food and Drug Safety (MFDS) to treat alcohol dependence in Korea: Disulfiram (an acetaldehyde dehydrogenase inhibitor), oral naltrexone (an opioid antagonist) and acamprosate (an Nmethyl-D-aspartate receptor (NMDA) modulator). However, Disulfiram has not been produced nor merchandised in Korea. Chlordiazepoxide has been approved by MFDS to treat alcohol withdrawal symptoms. The MFDS does not yet approve the use of long-acting injectable naltrexone and nalmefene.

Enrichment of chemical libraries docked to protein conformational ensembles and application to aldehyde dehydrogenase 2.

Molecular recognition is a complex process that involves a large ensemble of structures of the receptor and ligand. Yet, most structure-based virtual screening is carried out on a single structure typically from X-ray crystallography. Explicit-solvent molecular dynamics (MD) simulations offer an opportunity to sample multiple conformational states of a protein. Here we evaluate our recently developed scoring method SVMSP in its ability to enrich chemical libraries docked to MD structures of seven proteins from the Directory of Useful Decoys (DUD). SVMSP is a target-specific rescoring method that combines machine learning with statistical potentials. We find that enrichment power as measured by the area under the ROC curve (ROC-AUC) is not affected by increasing the number of MD structures. Among individual MD snapshots, many exhibited enrichment that was significantly better than the crystal structure, but no correlation between enrichment and structural deviation from crystal structure was found. We followed an innovative approach by training SVMSP scoring models using MD structures (SVMSPMD). The resulting models were applied to two difficult cases (p38 and CDK2) for which enrichment was not better than random. We found remarkable increase in enrichment power, particularly for p38, where the ROC-AUC increased by 0.30 to 0.85. Finally, we explored approaches for a priori identification of MD snapshots with high enrichment power from an MD simulation in the absence of active compounds. We found that the use of randomly selected compounds docked to the target of interest using SVMSP led to notable enrichment for EGFR and Src MD snapshots. SVMSP rescoring of protein–compound MD structures was applied for the search of small-molecule inhibitors of the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2). Rank-ordering of a commercial library of 50 000 compounds docked to MD structures of ALDH2 led to five small-molecule inhibitors. Four compounds had IC50s below 5 μM. These compounds serve as leads for the design and synthesis of more potent and selective ALDH2 inhibitors.

The cancer stem cell marker aldehyde dehydrogenase is required to maintain a drug-tolerant tumor cell subpopulation.

Selective kinase inhibitors have emerged as an important class of cancer therapeutics, and several such drugs are now routinely used to treat advanced-stage disease. However, their clinical benefit is typically short-lived because of the relatively rapid acquisition of drug resistance following treatment response. Accumulating preclinical and clinical data point to a role for a heterogeneous response to treatment within a subpopulation of tumor cells that are intrinsically drug-resistant, such as cancer stem cells. We have previously described an epigenetically determined reversibly drug-tolerant subpopulation of cancer cells that share some properties with cancer stem cells. Here, we define a requirement for the previously established cancer stem cell marker ALDH (aldehyde dehydrogenase) in the maintenance of this drug-tolerant subpopulation. We find that ALDH protects the drug-tolerant subpopulation from the potentially toxic effects of elevated levels of reactive oxygen species (ROS) in these cells, and pharmacologic disruption of ALDH activity leads to accumulation of ROS to toxic levels, consequent DNA damage, and apoptosis specifically within the drug-tolerant subpopulation. Combining ALDH inhibition with other kinase-directed treatments delayed treatment relapse in vitro and in vivo, revealing a novel combination treatment strategy for cancers that might otherwise rapidly relapse following single-agent therapy.

Nitric oxide as a target for the hypotensive and vasorelaxing effects induced by (Z)-ethyl 12-nitrooxy-octadec-9-enoate in rats

The cardiovascular effects induced by a new organic nitrate were investigated in rats. The (Z)-ethyl 12-nitrooxy-octadec-9-enoate (NCOE) was synthesized from ricinoleic acid, the major compound of the castor oil. NCOE induced significant and dose-dependent hypotension and bradycardia in normotensive rats. In rats pretreated with NCOE (60mg/kg, i.v., once a day) for 4 consecutive days, hypotension induced by the nitrate was similar to that observed in rats that were not pretreated with the compound. The vasorelaxation induced by the compound was concentration-dependent (10−10–10−3M) in rat mesenteric artery rings, pre-contracted with phenylephrine (1μM), with or without endothelium. Pre-incubation with PTIO (300μM), a free radical form of NO (NO) scavenger, attenuated the NCOE vasorelaxation potency. However, in the presence of l-cysteine (3mM), a reduced form of NO (NO−) scavenger, NCOE response was potentiated. NCOE effect was not changed in the presence of an inhibitor of cytochrome P450, proadifen (10μM). On the other hand, the vasodilation was reduced in the presence of mitochondrial aldehyde dehydrogenase inhibitor (mtALDH), cyanamide (1mM); soluble guanylyl cyclase inhibitor (sGC), ODQ (10μM); and non-selective K+ channels blocker, TEA (3mM). In addition the NCOE-induced vasorelaxation was reduced by BKCa (iberiotoxin, 100nM) and KATP selective (glibenclamide, 10μM) blockers, however the effect was not modified by a KV blocker (4-aminopyridine, 1mM). Furthermore, NCOE increased NO levels in rat aortic smooth muscle cultured cells, detected by NO-sensitive probe DAF-2DA, by flow cytometry. These results together suggest that NCOE induces short-lasting hypotension and bradycardia, and promotes vasorelaxation due to NO release through the compound metabolism via mtALDH and consequent sGC, KATP and BKCa activation. Furthermore, the compound was not able to induce tolerance.