Aldehyde Dehydrogenase Inhibitor Research Articles

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.

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.

Disulfiram Acts as a Potent Radio-Chemo Sensitizer in Head and Neck Squamous Cell Carcinoma Cell Lines and Transplanted Xenografts.

The poor prognosis of locally advanced and metastatic head and neck squamous cell carcinoma (HNSCC) is primarily mediated by the functional properties of cancer stem cells (CSCs) and resistance to chemoradiotherapy. We investigated whether the aldehyde dehydrogenase (ALDH) inhibitor disulfiram (DSF) can enhance the sensitivity of therapy. Cell viability was assessed by the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and apoptosis assays, and the cell cycle and reactive oxygen species (ROS) levels were evaluated by fluorescence-activated cell sorting (FACS). The radio-sensitizing effect was measured by a colony formation assay. The synergistic effects were calculated by combination index (CI) analyses. The DSF and DSF/Cu2+ inhibited the cell proliferation (inhibitory concentration 50 (IC50) of DSF and DSF/Cu2+ were 13.96 μM and 0.24 μM). DSF and cisplatin displayed a synergistic effect (CI values were <1). DSF or DSF/Cu2+ abolished the cisplatin-induced G2/M arrest (from 52.9% to 40.7% and 41.1%), and combining irradiation (IR) with DSF or DSF/Cu2+ reduced the colony formation and attenuated the G2/M arrest (from 53.6% to 40.2% and 41.9%). The combination of cisplatin, DSF or DSF/Cu2+, and IR enhanced the radio-chemo sensitivity by inducing apoptosis (42.04% and 32.21%) and ROS activity (46.3% and 37.4%). DSF and DSF/Cu2+ enhanced the sensitivity of HNSCC to cisplatin and IR. Confirming the initial data from patient-derived tumor xenograft (PDX) supported a strong rationale to repurpose DSF as a radio-chemosensitizer and to assess its therapeutic potential in a clinical setting.

Methyl-hydroxylation and subsequent oxidation to produce carboxylic acid is the major metabolic pathway of tolbutamide in chimeric TK-NOG mice transplanted with human hepatocytes

Tolbutamide is an oral anti-hyperglycaemic agent used to treat non-insulin–dependent diabetes mellitus with species-dependent metabolic profiles. In this study, we investigated tolbutamide metabolism in chimeric TK-NOG mice transplanted with human hepatocytes (humanised-liver mice). Substantial 4-hydroxytolbutamide and 4-carboxytolbutamide production was observed in hepatocytes from humanised-liver mice (Hu-Liver cells) and humans, whereas 4-carboxytolbutamide production was not detected in mouse hepatocytes. In Hu-Liver cells, 4-hydroxytolbutamide formation was inhibited by sulfaphenazole (CYP2C9 inhibitor), whereas 4-carboxytolbutamide formation was inhibited by raloxifene/ethinyloestradiol (aldehyde oxidase inhibitor) and disulfiram (aldehyde dehydrogenase inhibitor). After a single oral dose of tolbutamide (10 mg/kg), the plasma levels of 4-carboxytolbutamide and p-tolylsulfonylurea were higher in humanised-liver mice than in TK-NOG mice. Urinary excretion was the predominant route (>99% of unchanged drug and metabolites detected in excreta) of elimination in both groups. 4-Carboxytolbutamide was the most abundant metabolite in humanised-liver mouse urine, as similarly reported for humans, whereas 4-hydroxytolbutamide was predominantly excreted in TK-NOG mouse urine. These results suggest that humanised-liver mice might represent a suitable animal model for studying the successive oxidative metabolism of tolbutamide by multiple drug-metabolising enzymes. Future work is warranted to study the general nature of primary alcohol metabolism using humanised-liver mice.

Aldehyde Dehydrogenase, a Therapeutic Target in Chordoma: Analysis in 3D Cellular Models.

Chordomas are rare, slow-growing tumors of the axial skeleton. These tumors are locally aggressive and refractory to conventional therapies. Radical surgery and radiation remain the first-line treatments. Despite these aggressive treatments, chordomas often recur and second-line treatment options are limited. The mechanisms underlying chordoma radioresistance remain unknown, although several radioresistant cancer cells have been shown to respond favorably to aldehyde dehydrogenase (ALDH) inhibition. The study of chordoma has been delayed by small patient cohorts and few available models due to the scarcity of these tumors. We thus created cellular 3D models of chordoma by using low-adherence culture systems. Then, we evaluated their radiosensitivity using colony-forming and spheroid size assays. Finally, we determined whether pharmacologically inhibiting ALDH increased their radiosensitivity. We found that 3D cellular models of chordoma (derived from primary, relapse, and metastatic tumors) reproduce the histological and gene expression features of the disease. The metastatic, relapse, and primary spheroids displayed high, medium, and low radioresistance, respectively. Moreover, inhibiting ALDH decreased the radioresistance in all three models.

Activation of ALDH1A1 by omeprazole reduces cell oxidative stress damage.

Under physiological conditions, cells produce low basal levels of reactive oxygen species (ROS); however, in pathologic conditions ROS production increases dramatically, generating high concentrations of toxic unsaturated aldehydes. Aldehyde dehydrogenases (ALDHs) are responsible for detoxification of these aldehydes protecting the cell. Due to the physiological relevance of these enzymes, it is important to design strategies to modulate their activity. It was previously reported that omeprazole activation of ALDH1A1 protected Escherichia coli cells overexpressing this enzyme, from oxidative stress generated by H2 O2 . In this work, omeprazole cell protection potential was evaluated in eukaryotic cells. AS-30D cell or hepatocyte suspensions were subjected to a treatment with omeprazole and exposure to light (that is required to activate omeprazole in the active site of ALDH) and then exposed to H2 O2 . Cells showed viability similar to control cells, total activity of ALDH was preserved, while cell levels of lipid aldehydes and oxidative stress markers were maintained low. Cell protection by omeprazole was avoided by inhibition of ALDHs with disulfiram, revealing the key role of these enzymes in the protection. Additionally, omeprazole also preserved ALDH2 (mitochondrial isoform) activity, diminishing lipid aldehyde levels and oxidative stress in this organelle, protecting mitochondrial respiration and transmembrane potential formation capacity, from the stress generated by H2 O2 . These results highlight the important role of ALDHs as part of the antioxidant system of the cell, since if the activity of these enzymes decreases under stress conditions, the viability of the cell is compromised.

Combinatorial Therapeutic Effect of Inhibitors of Aldehyde Dehydrogenase and Mitochondrial Complex I, and the Chemotherapeutic Drug, Temozolomide against Glioblastoma Tumorspheres.

Resident cancer cells with stem cell-like features induce drug tolerance, facilitating survival of glioblastoma (GBM). We previously showed that strategies targeting tumor bioenergetics present a novel emerging avenue for treatment of GBM. The objective of this study was to enhance the therapeutic effects of dual inhibition of tumor bioenergetics by combination of gossypol, an aldehyde dehydrogenase inhibitor, and phenformin, a biguanide compound that depletes oxidative phosphorylation, with the chemotherapeutic drug, temozolomide (TMZ), to block proliferation, stemness, and invasiveness of GBM tumorspheres (TSs). Combination therapy with gossypol, phenformin, and TMZ induced a significant reduction in ATP levels, cell viability, stemness, and invasiveness compared to TMZ monotherapy and dual therapy with gossypol and phenformin. Analysis of differentially expressed genes revealed up-regulation of genes involved in programmed cell death, autophagy, and protein metabolism and down-regulation of those associated with cell metabolism, cycle, and adhesion. Combination of TMZ with dual inhibitors of tumor bioenergetics may, therefore, present an effective strategy against GBM by enhancing therapeutic effects through multiple mechanisms of action.

Ethanol Disrupts Hormone-Induced Calcium Signaling in Liver.

Receptor-coupled phospholipase C (PLC) is an important target for the actions of ethanol. In the ex vivo perfused rat liver, concentrations of ethanol >100 mM were required to induce a rise in cytosolic calcium (Ca2+) suggesting that these responses may only occur after binge ethanol consumption. Conversely, pharmacologically achievable concentrations of ethanol (≤30 mM) decreased the frequency and magnitude of hormone-stimulated cytosolic and nuclear Ca2+ oscillations and the parallel translocation of protein kinase C-β to the membrane. Ethanol also inhibited gap junction communication resulting in the loss of coordinated and spatially organized intercellular Ca2+ waves in hepatic lobules. Increasing the hormone concentration overcame the effects of ethanol on the frequency of Ca2+ oscillations and amplitude of the individual Ca2+ transients; however, the Ca2+ responses in the intact liver remained disorganized at the intercellular level, suggesting that gap junctions were still inhibited. Pretreating hepatocytes with an alcohol dehydrogenase inhibitor suppressed the effects of ethanol on hormone-induced Ca2+ increases, whereas inhibiting aldehyde dehydrogenase potentiated the inhibitory actions of ethanol, suggesting that acetaldehyde is the underlying mediator. Acute ethanol intoxication inhibited the rate of rise and the magnitude of hormone-stimulated production of inositol 1,4,5-trisphosphate (IP3), but had no effect on the size of Ca2+ spikes induced by photolysis of caged IP3. These findings suggest that ethanol inhibits PLC activity, but does not affect IP3 receptor function. We propose that by suppressing hormone-stimulated PLC activity, ethanol interferes with the dynamic modulation of [IP3] that is required to generate large, amplitude Ca2+ oscillations.

Aldehyde dehydrogenase inhibitors promote DNA damage in ovarian cancer and synergize with ATM/ATR inhibitors.

Rationale: Aldehyde dehydrogenase (ALDH) enzymes are often upregulated in cancer cells and associated with therapeutic resistance. ALDH enzymes protect cells by metabolizing toxic aldehydes which can induce DNA double stand breaks (DSB). We recently identified a novel ALDH1A family inhibitor (ALDHi), 673A. We hypothesized that 673A, via inhibition of ALDH1A family members, could induce intracellular accumulation of genotoxic aldehydes to cause DSB and that ALDHi could synergize with inhibitors of the ATM and ATR, proteins which direct DSB repair.Methods: We used immunofluorescence to directly assess levels of the aldehyde 4-hydroxynonenal and comet assays to evaluate DSB. Western blot was used to evaluate activation of the DNA damage response pathways. Cell counts were performed in the presence of 673A and additional aldehydes or aldehyde scavengers. ALDH inhibition results were confirmed using ALDH1A3 CRISPR knockout. Synergy between 673A and ATM or ATR inhibitors was evaluated using the Chou-Talalay method and confirmed in vivo using cell line xenograft tumor studies.Results: The ALDHi 673A cellular accumulation of toxic aldehydes which induce DNA double strand breaks. This is exacerbated by addition of exogenous aldehydes such as vitamin-A (retinaldehyde) and ameliorated by aldehyde scavengers such as metformin and hydralazine. Importantly, ALDH1A3 knockout cells demonstrated increased sensitivity to ATM/ATR inhibitors. And, ALDHi synergized with inhibitors of ATM and ATR, master regulators of the DSB DNA damage response, both in vitro and in vivo. This synergy was evident in homologous recombination (HR) proficient cell lines.Conclusions: ALDHi can be used to induce DNA DSB in cancer cells and synergize with inhibitors the ATM/ATR pathway. Our data suggest a novel therapeutic approach to target HR proficient ovarian cancer cells.

An Extract of Taro (Colocasia esculenta) Mediates Potent Inhibitory Actions on Metastatic and Cancer Stem Cells by Tumor Cell-Autonomous and Immune-Dependent Mechanisms.

The taro plant, Colocasia esculenta, contains bioactive proteins with potential as cancer therapeutics. Several groups have reported anti-cancer activity in vitro and in vivo of taro-derived extracts (TEs). We reported that TE inhibits metastasis in a syngeneic murine model of Triple-Negative Breast Cancer (TNBC).Purpose:We sought to confirm our earlier studies in additional models and to identify novel mechanisms by which efficacy is achieved.Methods:We employed a panel of murine and human breast and ovarian cancer cell lines to determine the effect of TE on tumor cell viability, migration, and the ability to support cancer stem cells. Two syngeneic models of TNBC were employed to confirm our earlier report that TE potently inhibits metastasis. Cancer stem cell assays were employed to determine the ability of TE to inhibit tumorsphere-forming ability and to inhibit aldehyde dehydrogenase activity. To determine if host immunity contributes to the mechanism of metastasis inhibition, efficacy was assessed in immune-compromised mice.Results:We demonstrate that viability of some, but not all cell lines is inhibited by TE. Likewise, tumor cell migration is inhibited by TE. Using 2 immune competent, syngeneic models of TNBC, we confirm our earlier findings that tumor metastasis is potently inhibited by TE. We also demonstrate, for the first time, that TE directly inhibits breast cancer stem cells. Administration of TE to mice elicits expansion of several spleen cell populations but it was not known if host immune cells contribute to the mechanism by which TE inhibits tumor cell dissemination. In novel findings, we now show that the ability of TE to inhibit metastasis relies on immune T-cell-dependent, but not B cell or Natural Killer (NK)-cell-dependent mechanisms. Thus, both tumor cell-autonomous and host immune factors contribute to the mechanisms underlying TE efficacy. Our long-term goal is to evaluate TE efficacy in clinical trials. Most of our past studies as well as many of the results reported in this report were carried out using an isolation protocol described earlier (TE). In preparation for a near future clinical trial, we have now developed a strategy to isolate an enriched taro fraction, TE-method 2, (TE-M2) as well as a more purified subfraction (TE-M2F1) which can be scaled up under Good Manufacturing Practice (GMP) conditions for evaluation in human subjects. We demonstrate that TE-M2 and TE-M2F1 retain the anti-metastatic properties of TE.Conclusions:These studies provide further support for the continued examination of biologically active components of Colocasia esculenta as potential new therapeutic entities and identify a method to isolate sufficient quantities under GMP conditions to conduct early phase clinical studies.

Visualization of Necroptotic Cell Death through Transmission Electron Microscopy.

Transmission electron microscopy (TEM) is an all-in-one tool to visualize the complex systems of any specimen that is 1nm in size or smaller. The current chapter provides detailed guidelines for imaging morphological changes during programmed cell necrosis using TEM as a single-step methodology. In this protocol, a novel aldehyde dehydrogenase inhibitor is used to induce cell programmed necrosis in ovarian cancer cell lines (A2780 and SKOV3). This process is followed by gradient dehydration with ethanol, chemical fixation, sampled grid preparation, and staining with 0.75% uranyl formate. Following fixation and grid preparation, cells are imaged using TEM. The resulting images reveal morphological changes consistent with necrotic morphology, including swelling of cells and organelles, appearance of vacuoles, and plasma membrane rupture followed by leakage of cellular contents. The current approach allows a single-step methodology for characterization of cell-programmed necrosis in cells based on morphology.

Aldehyde Dehydrogenases and Prostate Cancer: Shedding Light on Isoform Distribution to Reveal Druggable Target.

Prostate cancer represents the most common malignancy diagnosed in men, and is the second-leading cause of cancer death in this population. In spite of dedicated efforts, the current therapies are rarely curative, requiring the development of novel approaches based on innovative molecular targets. In this work, we validated aldehyde dehydrogenase 1A1 and 1A3 isoform expressions in different prostatic tissue-derived cell lines (normal, benign and malignant) and patient-derived primary prostate tumor epithelial cells, demonstrating their potential for therapeutic intervention using a small library of aldehyde dehydrogenase inhibitors. Compound 3b, 6-(4-fluorophenyl)-2-phenylimidazo [1,2-a]pyridine exhibited not only antiproliferative activity in the nanomolar range against the P4E6 cell line, derived from localized prostate cancer, and PC3 cell lines, derived from prostate cancer bone metastasis, but also inhibitory efficacy against PC3 colony-forming efficiency. Considering its concomitant reduced activity against normal prostate cells, 3b has the potential as a lead compound to treat prostate cancer by means of a still untapped molecular target.

EXTH-61. COMBINATION OF DISULFIRAM AND COPPER INDUCES NPL4 AGGREGATION, TARGETS CD133-NESTIN CELLS AND EXTENDS SURVIVAL IN MEDULLOBLASTOMA GROUP 3 MODELS

Abstract BACKGROUND Medulloblastoma (MB), the most common brain malignancy among children, is classified into four molecular subtypes, WNT-driven, Shh-driven, Group 3 and 4. The aim of this study was to assess the effects of the combination of Disulfiram, an inhibitor of Aldehyde Dehydrogenase, and Copper in Shh-driven and Group 3 MB, in vitro and in vivo. METHODS The mechanisms of action and anti-cancer stem-cell effects of disulfiram were evaluated with clonogenic assays, flow-cytometry (FC), western blotting (WB), and immunofluorescence (IF) using established MB cell-lines - ONS76, UW228, D425med, D341 and D283- representing the main molecular subtypes. Survival, tumor volume, nuclear protein localization protein-4-expression (NPL4) and markers of proliferation/apoptosis were assessed in multiple models of Group 3 MB in vivo and ex vivo with WB and immunohistochemistry (IHC). RESULTS Significant in vitro cytotoxicity was demonstrated at nanomolar concentrations of DSF in all lines. DSF/Cu++ induced cell death (increased AnnV/PI, cleaved-Poly(ADP-ribose)polymerase fraction, and Apoptosis Inducing Factor on WB/FC/IF, in vitro and ex vivo) through NPL4 accumulation in cell nucleus and intracellular buildup of poly-ubiquitylated proteins. DNA damage was also detected with WB and H2AX foci on immunofluorescence. Flow-cytometry analysis demonstrated a significant reduction in ALDH, Nestin- and CD133-positive cells in Group 3 lines, confirmed with WB in vitro and ex vivo. DSF/Cu++-toxicity was tested in vivo in multiple dose-escalating trials, and the combination significantly prolonged survival and reduced tumor volume in D425 and D341 xenografts. IHC showed lower Ki67 and increased Cleaved-Caspase-3 expression, higher NPL4-positive cells and no difference in NeuN and GFAP-positive cells after treatment. CONCLUSIONS DSF/Cu++ demonstrated a potent therapeutic effect on Shh-driven and Group 3 MB cell lines by determining apoptosis, targeting cancer stem cells and inducing DNA damage. Our data suggest that this combination may serve as a novel treatment, alone or with existing therapies, for pediatric MB.

Long Term Pharmacotherapy for Alcohol Use Disorder

The psychopharmacology of alcohol dependence is today poised at interesting crossroads. Three major drugs Naltrexone, Disulfiram and Acamprosate have been tried and tested in various trials and have many meta-analyses each to support them. While Naltrexone may reduce craving, Acamprosate scores on cost effectiveness worldwide with Disulfiram being an alcohol deterrent drug. Studies support, refute and criticize the use of each of these drugs. Combining one or more of them is also a trend seen. The most important factor in efficacy has been the combination of psychosocial treatment with medication. In this article, we are going to discuss about long term pharmacological management of chronic alcoholism.

Mechanism of cystathionine-β-synthase inhibition by disulfiram: The role of bis(N,N-diethyldithiocarbamate)-copper(II)

BackgroundHydrogen sulfide (H2S) is an endogenous mammalian gasotransmitter. Cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) are the principal enzymes responsible for its biogenesis. A recent yeast screen suggested that disulfiram (a well-known inhibitor of aldehyde dehydrogenase and a clinically used drug in the treatment of alcoholism) may inhibit CBS in a cell-based environment. However, prior studies have not observed any direct inhibition of CBS by disulfiram. We investigated the potential role of bioconversion of disulfiram to bis(N,N-diethyldithiocarbamate)-copper(II) complex (CuDDC) in the inhibitory effect of disulfiram on H2S production and assessed its effect in two human cell types with high CBS expression: HCT116 colon cancer cells and Down syndrome (DS) fibroblasts. MethodsH2S production from recombinant human CBS, CSE and 3-MST was measured using the fluorescent H2S probe AzMC. Mouse liver homogenate (a rich source of CBS) was also employed to measure H2S biosynthesis. The interaction of copper with accessible protein cysteine residues was evaluated using the DTNB method. Cell proliferation and viability were measured using the BrdU and MTT methods. Cellular bioenergetics was evaluated by Extracellular Flux Analysis. ResultsWhile disulfiram did not exert any significant direct inhibitory effect on any of the H2S-producing enzymes, its metabolite, CuDDC was a potent inhibitor of CBS and CSE. The mode of its action is likely related to the complexed copper molecule. In cell-based systems, the effects of disulfiram were variable. In colon cancer cells, no significant effect of disulfiram was observed on H2S production or proliferation or viability. In contrast, in DS fibroblasts, disulfiram inhibited H2S production and improved proliferation and viability. Copper, on its own, failed to have any effects on either cell type, likely due to its low cell penetration. CuDDC inhibited H2S production in both cell types studied and exerted the functional effects that would be expected from a CBS inhibitor: inhibition of cell proliferation of cancer cells and a bell-shaped effect (stimulation of proliferation at low concentration and inhibition of these responses at higher concentration) in DS cells. Control experiments using a chemical H2S donor showed that, in addition to inhibiting CBS and CSE, part of the biological effects of CuDDC relates to a direct reaction with H2S, which occurs through its complexed copper. ConclusionsDisulfiram, via its metabolite CuDDC acts as an inhibitor of CBS and a scavenger of H2S, which, in turn, potently suppresses H2S levels in various cell types. Inhibition of H2S biosynthesis may explain some of the previously reported actions of disulfiram and CuDDC in vitro and in vivo. Disulfiram or CuDDC may be considered as potential agents for the experimental therapy of various pathophysiological conditions associated with H2S overproduction.

Aldh inhibitor restores auditory function in a mouse model of human deafness.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

Investigating the Molecular Basis for the Selective Inhibition of Aldehyde Dehydrogenase 2 by the Isoflavonoid Daidzin.

ALDH-2 has been considered an important molecular target for the treatment of drug addiction due to its involvement in the metabolism of the neurotransmitter dopamine: however, the molecular basis for the selective inhibition of ALDH-2 versus ALDH-1 should be better investigated to enable a more pragmatic approach to the design of novel ALDH-2 selective inhibitors. In the present study, we investigated the molecular basis for the selective inhibition of ALDH-2 by the antioxidant isoflavonoid daidzin (IC50 = 0.15 μM) compared to isoform 1 of ALDH through molecular dynamics studies and semiempirical calculations of the enthalpy of interaction. The applied methodology consisted of performing the molecular docking of daidzin in the structures of ALDH-1 and ALDH-2 and submitting the lower energy complexes obtained to semiempirical calculations and dynamic molecular simulations. Daidzin in complex with ALDH-2 presented directed and more specific interactions, resulting in stronger bonds in energetic terms and, therefore, in enthalpic gain. Moreover, the hydrophobic subunits of daidzin, in a conformationally more restricted environment (such as the catalytic site of ALDH-2), promote the better organization of the water molecules when immersed in the solvent, also resulting in an entropic gain. The molecular basis of selective inhibition of ALDH-2 by isoflavonoids and related compounds could be related to a more favorable equilibrium relationship between enthalpic and entropic features. The results described herein expand the available knowledge regarding the physiopathological and therapeutic mechanisms associated with drug addiction.

Abstract 575: Disulfiram plus copper synergizes with etoposide for the treatment of Merkel cell carcinoma

Abstract Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with limited treatment options. Currently two immunotherapies are approved for the treatment of MCC, the PD-L1 inhibitor avelumab and the PD-1 inhibitor pembrolizumab. Unfortunately, these treatments only have durable benefit for less than half of patients that can be treated with immunotherapy. Therefore, new and effective treatments are needed for MCC. To address this, we conducted a high-throughput drug screening of ~4,000 small molecules. Our drug screen identified disulfiram (DSF), an aldehyde dehydrogenase inhibitor used in the treatment of alcoholism, as an agent that selectively reduced MCC cell viability. Recent studies suggest that DSF complexed with copper can have anti-cancer effects. Consistent with this, the addition copper at physiological levels increased the potency of DSF in MCC cells lines. Next, we demonstrated that disulfiram combined with copper synergized with the topoisomerase II inhibitor etoposide, lowering the effective dose of etoposide required to reduce MCC cell viability. Mechanistically, combining DSF, copper and etoposide was cytotoxic but did not induce apoptosis in MCC cell lines. This combination increased the formation of double stranded DNA breaks while disulfiram plus copper induced autophagy, which could mechanistically be an indication of immunogenic cell death signaling. Taken together, our data suggest that disulfiram combined with copper and etoposide could be repurposed for the effective treatment of advanced MCC. One strong advantage of this combination is that all agents can be given orally and at dosages with minimal adverse effects. Citation Format: Natasha Tremayne Hill, Tara Gelb, Dan Urban, Tyler Kellenberger, Matthew Hall, Isaac Brownell. Disulfiram plus copper synergizes with etoposide for the treatment of Merkel cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 575.

In vitro metabolic profile and drug-drug interaction assessment of secnidazole, a high-dose 5-nitroimidazole antibiotic for the treatment of bacterial vaginosis.

A single‐dose oral granule formulation of secnidazole 2 g (SOLOSEC™) has been approved in the US as a treatment for bacterial vaginosis. Available data on the likelihood of in vitro drug‐drug and alcohol‐drug interactions are limited. Secnidazole was incubated with cultured human hepatocytes over a range of concentrations (0‐10 000 μmol/L) to assess metabolic profiling. Cytochrome P450 (CYP) and aldehyde dehydrogenase inhibition over a similar concentration range were evaluated in human liver microsomes (HLMs) or recombinant enzymes using competition or time‐dependent inactivation assays. Secnidazole exhibited very low metabolism in HLMs at concentrations up to 6400 µmol/L. Secnidazole was found to be metabolized to a limited extent predominantly by CYP3A4 and CYP3A5 among a panel of cDNA‐expressed enzymes. Secnidazole inhibited CYP2C19 and CYP3A4, with IC50 values of 3873 and 3722 µmol/L, respectively. Secnidazole did not exhibit time‐dependent inhibition. There was no inhibition (IC50 value >5000 µmol/L) observed for any other CYP enzyme or with human recombinant aldehyde dehydrogenase 2 (ALDH2). These results are the first reported observation of the metabolism and drug‐drug interaction profile for secnidazole and demonstrate that the agent has minimal to no potential drug interactions of concern.

Alkyl and Aryl Derivatives Based on p-Coumaric Acid Modification and Inhibitory Action against Leishmania braziliensis and Plasmodium falciparum

In low-income populations, neglected diseases are the principal cause of mortality. Of these, leishmaniasis and malaria, being parasitic, protozoan infections, affect millions of people worldwide and are creating a public health problem. The present work evaluates the leishmanicidal and antiplasmodial action of a series of twelve p-coumaric acid derivatives. Of the tested derivatives, eight presented antiparasitic activities 1–3, 8–12. The hexyl p-coumarate derivative (9) (4.14 ± 0.55 μg/mL; selectivity index (SI) = 2.72) showed the highest leishmanicidal potency against the Leishmania braziliensis amastigote form. The results of the molecular docking study suggest that this compound inhibits aldehyde dehydrogenase (ALDH), mitogen-activated kinase protein (MPK4), and DNA topoisomerase 2 (TOP2), all of which are key enzymes in the development of Leishmania braziliensis. The data indicate that these enzymes interact via Van der Waals bonds, hydrophobic interactions, and hydrogen bonds with phenolic and aliphatic parts of this same compound. Of the other compounds analyzed, methyl p-coumarate (64.59 ± 2.89 μg/mL; IS = 0.1) demonstrated bioactivity against Plasmodium falciparum. The study reveals that esters presenting a p-coumarate substructure are promising for use in synthesis of derivatives with good antiparasitic profiles.