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1.
Sci Rep ; 12(1): 15440, 2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104373

RESUMO

Nicotinamide N-methyltransferase (NNMT) is a metabolic regulator that catalyzes the methylation of nicotinamide (Nam) using the co-factor S-adenosyl-L-methionine to form 1-methyl-nicotinamide (MNA). Overexpression of NNMT and the presence of the active metabolite MNA is associated with a number of diseases including metabolic disorders. We conducted a high-throughput screening campaign that led to the identification of a tricyclic core as a potential NNMT small molecule inhibitor series. Elaborate medicinal chemistry efforts were undertaken and hundreds of analogs were synthesized to understand the structure activity relationship and structure property relationship of this tricyclic series. A lead molecule, JBSNF-000028, was identified that inhibits human and mouse NNMT activity, reduces MNA levels in mouse plasma, liver and adipose tissue, and drives insulin sensitization, glucose modulation and body weight reduction in a diet-induced obese mouse model of diabetes. The co-crystal structure showed that JBSNF-000028 binds below a hairpin structural motif at the nicotinamide pocket and stacks between Tyr-204 (from Hairpin) and Leu-164 (from central domain). JBSNF-000028 was inactive against a broad panel of targets related to metabolism and safety. Interestingly, the improvement in glucose tolerance upon treatment with JBSNF-000028 was also observed in NNMT knockout mice with diet-induced obesity, pointing towards the glucose-normalizing effect that may go beyond NNMT inhibition. JBSNF-000028 can be a potential therapeutic option for metabolic disorders and developmental studies are warranted.


Assuntos
Doenças Metabólicas , Nicotinamida N-Metiltransferase , Animais , Humanos , Camundongos , Glucose , Doenças Metabólicas/tratamento farmacológico , Niacinamida/metabolismo , Niacinamida/farmacologia , Nicotinamida N-Metiltransferase/metabolismo , Obesidade/tratamento farmacológico
2.
Artigo em Inglês | MEDLINE | ID: mdl-34909677

RESUMO

Non-alcoholic fatty liver disease (NAFLD) and Non-alcoholic steatohepatitis (NASH) are chronic liver disorders, the prevalence of which is increasing worldwide. Long term High Fat Diet (HFD) induced NASH animal models closely mimic the characteristics of human NASH and hence used by investigators as a model system for studying the mechanism of action of new drugs. Bempedoic acid (ETC-1002), a ATP citrate lyase (ACLY) inhibitor that lowers the LDL cholesterol was recently approved by US FDA for the treatment of heterozygous familial hypercholesterolemia (HeFH) and established atherosclerotic cardiovascular disease (ASCVD). ACLY is one of the genes modulated in NASH patients and hence we studied the effect of ACLY inhibitor Bempedoic acid in long term HFD induced NASH animal model to understand the pharmacological benefits and the associated mechanism of action of this newly approved drug in NASH. Mice fed with 60% Kcal High Fat Diet for 32 weeks were used for the study and the animals were given Bempedoic acid for 5 weeks at doses of 10 â€‹mg â€‹kg-1, po, qd, and 30 â€‹mg â€‹kg-1, po, qd. Bempedoic acid treatment resulted in inhibition of body weight gain and improved the glycemic control. Bempedoic acid treated group showed statistically significant reduction in plasma ALT, AST, hepatic triglycerides (TG) and total cholesterol (TC), along with statistically significant reduction in steatosis score by histological analysis. Hepatic gene expression analysis showed significant reduction in inflammatory and fibrotic genes such as Mcp-1/Ccl2, Timp-1 & Col1α1. Histological analysis showed significant improvement in NAS score. Overall, Bempedoic acid alleviated HFD induced Non-Alcoholic Steatohepatitis through inhibition of body weight gain, improvement in glycemic control, reduction of hepatic triglycerides & total cholesterol, modulation of inflammatory & fibrotic genes, and improvement in NAS score. Hence, Bempedoic acid can be a potential therapeutic option for metabolic syndrome and NASH.

3.
Molecules ; 26(4)2021 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-33668468

RESUMO

Nicotinamide-N-methyltransferase (NNMT) is a cytosolic enzyme catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to nicotinamide (Nam). It is expressed in many tissues including the liver, adipose tissue, and skeletal muscle. Its expression in several cancer cell lines has been widely discussed in the literature, and recent work established a link between NNMT expression and metabolic diseases. Here we describe our approach to identify potent small molecule inhibitors of NNMT featuring different binding modes as elucidated by X-ray crystallographic studies.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/enzimologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Animais , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Niacinamida/metabolismo , Nicotinamida N-Metiltransferase/metabolismo , Ratos , Especificidade por Substrato/efeitos dos fármacos
4.
J Biol Methods ; 7(3): e135, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32934967

RESUMO

Hepatic steatosis is a metabolic disease, characterized by selective and progressive accumulation of lipids in liver, leading to progressive non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and cirrhosis. The existing in vitro models of hepatic steatosis to elucidate the molecular mechanisms behind the onset of hepatic steatosis and to profile small molecule modulators uses lipid loaded primary hepatocytes, and cell lines like HepG2. The limitation of these models includes high variability between the different donor samples, reproducibility, and translatability to physiological context. An in vitro human hepatocyte derived model that mimics the pathophysiological changes seen in hepatic steatosis may provide an alternative tool for pre-clinical drug discovery research. We report the development of an in vitro experimental model of hepatic steatosis using human induced pluripotent stem cell (iPSC) derived hepatocytes like cells (HLC), loaded with lipids. Our data suggests that HLC carry some of the functional characteristics of primary hepatocytes and are amenable for development of an in vitro steatosis model using lipid loading method. The in vitro experimental model of hepatic steatosis was further characterized using biomarker analysis and validated using telmisartan. With some refinement and additional validation, our in vitro steatosis model system may be useful for profiling small molecule inhibitors and studying the mechanism of action of new drugs.

5.
Biomed Chromatogr ; 34(5): e4790, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31883352

RESUMO

Ceramides are known to be involved in various biological processes with their physiological levels elevated in various disease conditions such as diabetes, Alzheimer's, atherosclerosis. To facilitate the rapid screening of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 inhibition in HepG2 cells, a RapidFire coupled to tandem mass spectrometry (RF-MS/MS) method has been developed. The RF platform provides an automated solid-phase extraction system that gave a throughput of 12.6 s per sample to an MS/MS system using electrospray ionization under the positive ion mode. Chromatographic separation of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 was achieved using a ternary gradient on C8 type E cartridge. The MS/MS ion transitions monitored were 538.2 → 264.2, 650.7 → 264.2, 648.6 → 264.2, 566.4 → 264.2, 510.4 → 264.2, 594.4 → 264.2, 622.5 → 264.2, and 552.3 → 250.2 for Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, d18:1/22:0, and the internal standard (Cer d17:1/18:0), respectively. The RF-MS/MS methodology showed an excellent performance with an average Z' value of 0.5-0.7. This is the first report of an RF-MS/MS assay for screening of ceramides which is amenable for high-throughput screening.


Assuntos
Ceramidas/química , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas em Tandem/métodos , Ceramidas/isolamento & purificação , Células Hep G2 , Humanos , Extração em Fase Sólida
6.
J Med Chem ; 62(21): 9837-9873, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31589440

RESUMO

Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that methylates nicotinamide (NAM) using cofactor S-adenosylmethionine (SAM). NNMT overexpression has been linked to diabetes, obesity, and various cancers. In this work, structure-based rational design led to the development of potent and selective alkynyl bisubstrate inhibitors of NNMT. The reported nicotinamide-SAM conjugate (named NS1) features an alkyne as a key design element that closely mimics the linear, 180° transition state geometry found in the NNMT-catalyzed SAM → NAM methyl transfer reaction. NS1 was synthesized in 14 steps and found to be a high-affinity, subnanomolar NNMT inhibitor. An X-ray cocrystal structure and SAR study revealed the ability of an alkynyl linker to span the methyl transfer tunnel of NNMT with ideal shape complementarity. The compounds reported in this work represent the most potent and selective NNMT inhibitors reported to date. The rational design principle described herein could potentially be extended to other methyltransferase enzymes.


Assuntos
Alcinos/química , Alcinos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/metabolismo , Alcanos/química , Alcinos/metabolismo , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Humanos , Células K562 , Simulação de Acoplamento Molecular , Nicotinamida N-Metiltransferase/química , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Temperatura
7.
Acta Trop ; 185: 212-218, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29802846

RESUMO

Parasitic worms are receiving much attention as a potential new therapeutic approach to treating autoimmune and allergic conditions but concerns remain regarding their safety. As an alternative strategy, we have focused on the use of defined parasitic worm products and recently taken this one step further by designing drug-like small molecule analogues of one such product, ES-62, which is anti-inflammatory by virtue of covalently attached phosphorylcholine moieties. Previously, we have shown that ES-62 mimics are efficacious in protecting against disease in mouse models of rheumatoid arthritis, systemic lupus erythematosus and skin and lung allergy. Given the potential role of chronic inflammation in fibrosis, in the present study we have focused our attention on lung fibrosis, a debilitating condition for which there is no cure and which in spite of treatment slowly gets worse over time. Two mouse models of fibrosis - bleomycin-induced and LPS-induced - in which roles for inflammation have been implicated were adopted. Four ES-62 analogues were tested - 11a and 12b, previously shown to be active in mouse models of allergic and autoimmune disease and 16b and AIK-29/62 both of which are structurally related to 11a. All four compounds were found to significantly reduce disease development in both fibrosis models, as shown by histopathological analysis of lung tissue, indicating their potential as treatments for this condition.


Assuntos
Anti-Inflamatórios/uso terapêutico , Proteínas de Helminto/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL
8.
Sci Rep ; 8(1): 3660, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29483571

RESUMO

Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/enzimologia , Nicotinamida N-Metiltransferase/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicotinamida N-Metiltransferase/antagonistas & inibidores
9.
Bioorg Med Chem Lett ; 28(5): 922-925, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29433927

RESUMO

Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and reduced the formation of 1-methyl-nicotinamide (MNA), the primary metabolite of NA by ∼80% at 2 h when dosed in mice orally at 50 mg/kg.


Assuntos
Inibidores Enzimáticos/farmacologia , Niacinamida/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Niacinamida/síntese química , Niacinamida/química , Nicotinamida N-Metiltransferase/metabolismo , Relação Estrutura-Atividade
10.
Biochem Biophys Res Commun ; 491(2): 416-422, 2017 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-28720493

RESUMO

Nicotinamide N-methyltransferase (NNMT) is a S-adenosyl-l-methionine (SAM)-dependent enzyme that catalyzes N-methylation of nicotinamide (NA) and other pyridines to form N-methyl pyridinium ions. Here we report the first ternary complex X-ray crystal structures of monkey NNMT and mouse NNMT in bound form with the primary endogenous product, 1-methyl nicotinamide (MNA) and demethylated cofactor, S-adenosyl-homocysteine (SAH) determined at 2.30 Å and 1.88 Å respectively. The structural fold of these enzymes is identical to human NNMT. It is known that the primary endogenous product catalyzed by NNMT, MNA is a specific inhibitor of NNMT. Our data clearly indicates that the MNA binds to the active site and it would be trapped in the active site due to the formation of the bridge between the pole (long helix, α3) and long C-terminal loop. This might explain the mechanism of MNA acting as a feedback inhibitor of NNMT.


Assuntos
Retroalimentação Fisiológica , Niacinamida/análogos & derivados , Nicotinamida N-Metiltransferase/química , S-Adenosilmetionina/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Macaca mulatta , Camundongos , Modelos Moleculares , Niacinamida/química , Niacinamida/metabolismo , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/genética , Nicotinamida N-Metiltransferase/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
11.
Sci Rep ; 5: 16025, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26531810

RESUMO

Mathematical models of metabolism from bacterial systems biology have proven their utility across multiple fields, for example metabolic engineering, growth phenotype simulation, and biological discovery. The usefulness of the models stems from their ability to compute a link between genotype and phenotype, but their ability to accurately simulate gene-gene interactions has not been investigated extensively. Here we assess how accurately a metabolic model for Escherichia coli computes one particular type of gene-gene interaction, synthetic lethality, and find that the accuracy rate is between 25% and 43%. The most common failure modes were incorrect computation of single gene essentiality and biological information that was missing from the model. Moreover, we performed virtual and biological screening against several synthetic lethal pairs to explore whether two-compound formulations could be found that inhibit the growth of Gram-negative bacteria. One set of molecules was identified that, depending on the concentrations, inhibits E. coli and S. enterica serovar Typhimurium in an additive or antagonistic manner. These findings pinpoint specific ways in which to improve the predictive ability of metabolic models, and highlight one potential application of systems biology to drug discovery and translational medicine.


Assuntos
Antibacterianos/farmacologia , Escherichia coli O157/genética , Genes Letais/genética , Klebsiella pneumoniae/genética , Salmonella typhimurium/genética , Biologia de Sistemas/métodos , Yersinia pestis/genética , Antibacterianos/síntese química , Combinação de Medicamentos , Descoberta de Drogas , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/metabolismo , Doenças Transmitidas por Alimentos/microbiologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/metabolismo , Testes de Sensibilidade Microbiana , Modelos Biológicos , Modelos Teóricos , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Yersinia pestis/crescimento & desenvolvimento , Yersinia pestis/metabolismo
12.
J Microbiol Methods ; 118: 173-5, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26432950

RESUMO

A 384-well-based antibacterial assay amenable for high-throughput screening and combination testing is described. The assay uses 100-500nL of test compounds and tolerates up to 2.5% dimethyl sulfoxide concentrations. It can be used for screening compound libraries and testing combinatory/synergistic/antagonistic effects of antibiotics, small molecules, and natural product extracts.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana/métodos
13.
Front Microbiol ; 6: 958, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26441892

RESUMO

Mathematical models of biochemical networks form a cornerstone of bacterial systems biology. Inconsistencies between simulation output and experimental data point to gaps in knowledge about the fundamental biology of the organism. One such inconsistency centers on the gene aldA in Escherichia coli: it is essential in a computational model of E. coli metabolism, but experimentally it is not. Here, we reconcile this disparity by providing evidence that aldA and prpC form a synthetic lethal pair, as the double knockout could only be created through complementation with a plasmid-borne copy of aldA. Moreover, virtual and biological screening against the two proteins led to a set of compounds that inhibited the growth of E. coli and Salmonella enterica serovar Typhimurium synergistically at 100-200 µM individual concentrations. These results highlight the power of metabolic models to drive basic biological discovery and their potential use to discover new combination antibiotics.

14.
Biomol Detect Quantif ; 4: 1-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27077032

RESUMO

The successful discovery and subsequent development of small molecule inhibitors of drug targets relies on the establishment of robust, cost-effective, quantitative, and physiologically relevant in vitro assays that can support prolonged screening and optimization campaigns. The current study illustrates the process of developing and validating an enzymatic assay for the discovery of small molecule inhibitors using alkaline phosphatase from bovine intestine as model target. The assay development workflow includes an initial phase of optimization of assay materials, reagents, and conditions, continues with a process of miniaturization and automation, and concludes with validation by quantitative measurement of assay performance and signal variability. The assay is further evaluated for dose-response and mechanism-of-action studies required to support structure-activity-relationship studies. Emphasis is placed on the most critical aspects of assay optimization and other relevant considerations, including the technology, assay materials, buffer constituents, reaction conditions, liquid handling equipment, analytical instrumentation, and quantitative assessments. Examples of bottlenecks encountered during assay development and strategies to address them are provided.

15.
Biochem Biophys Res Commun ; 386(4): 750-6, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19563780

RESUMO

A chemical inhibitor library of 84 compounds was screened to investigate the signaling pathway(s) leading to activation of Nrf2 in response to nitric oxide (NO). We identified the protein kinase C delta (PKCdelta) inhibitor rottlerin as the only compound that reduced NO-induced ARE-luciferase reporter activity and diminished NO-induced up-regulation of two Nrf2/ARE-regulated proteins - NAD(P)H:quinone oxidoreductase-1 (NQO1) and hemeoxygenase-1 (HO-1) in SH-Sy5y cells. Rottlerin also sensitized neuroblastoma cells and mouse primary cortical neurons to NO-induced apoptosis. Stable over-expression of PKCdelta augmented NO-induced, ARE-dependent gene expression of HO-1 in SH-Sy5y cells, which were more protected from NO killing. Conversely, NO-induced ARE-dependent gene expression was reduced in PKCdelta-knockdown SH-EP cells, which displayed greater sensitivity to apoptosis. PKCdelta(-/-) cortical neurons exhibited increased NO-induced apoptosis and less HO-1 mRNA and protein induction compared with wild type neurons. Hence, PKCdelta is an important positive modulator of NO-induced Nrf2/ARE-dependent signaling that counteracts NO-mediated apoptosis in neuronal cells.


Assuntos
Apoptose , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/enzimologia , Óxido Nítrico/metabolismo , Proteína Quinase C-delta/metabolismo , Animais , Células Cultivadas , Técnicas de Química Combinatória , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter/efeitos dos fármacos , Heme Oxigenase-1/genética , Luciferases/genética , Camundongos , NAD(P)H Desidrogenase (Quinona) , NADPH Desidrogenase/genética , Neurônios/citologia , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Inibidores de Proteínas Quinases/farmacologia
16.
Nucleic Acids Res ; 35(16): 5439-51, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17702766

RESUMO

Toxic nitric oxide (NO) levels can regulate gene expression. Using a novel protein/DNA array, we show that toxic NO levels regulate the binding of trans-factors to various cis-elements in neuroblastoma cells, including CRE and those recognized by the transcription factors AP1, AP2, Brn-3a, EGR, E2F1 and SP1. Functionality of some of the cis-elements was confirmed by electro mobility shift and reporter assays. Interestingly, CREB, AP-1, Brn-3a, EGR and E2F1 can control mammalian cell viability. NO induced the anti-apoptotic Bcl-2 protein and its mRNA prior to the onset of death of 30-60% of the cells. Promoter analysis of the bcl-2 gene confirmed the involvement of a CRE in NO-dependent bcl-2 transcription. Neuroblastoma cells over-expressing bcl-2 became much more resistant to NO-induced apoptosis; conversely, Bcl-2 knockdown cells were rendered markedly more sensitive to NO. Together these results suggest that Bcl-2 counteracts NO-induced apoptosis in a fraction of the cell population. Thus, NO stimulates the binding of many trans-factors to their cognate cis-elements, some of which can regulate cell viability through transcriptional activation of target genes. Our results emphasize that a DNA/protein array approach can reveal novel, global transcription factor activities stimulated by cell death-regulating molecules.


Assuntos
Apoptose/genética , Óxido Nítrico/metabolismo , Elementos de Resposta , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular , AMP Cíclico/metabolismo , Genes bcl-2 , Humanos , Neuroblastoma , Análise de Sequência com Séries de Oligonucleotídeos , Análise Serial de Proteínas
17.
FASEB J ; 20(14): 2624-6, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17142801

RESUMO

Tissue reoxygenation following hypoxia is associated with ischemia-reperfusion injury (IRI) and may signal the development of ischemic preconditioning, an adaptive state that is protective against subsequent IRI. Here we used microarray RNA analysis of in vivo and in vitro models of IRI to delineate the underlying molecular mechanisms. Microarray analysis of renal tissue after ischemia-reperfusion revealed a number of highly up-regulated antioxidant genes including aldehyde dehydrogenases (ALDH1A1 and ALDH1A7), glutathione S-transferases (GSTM5, GSTA2 and GSTP1), and NAD(P)H quinone oxidoreductase (NQO1). The transcription factor NF-E2-related factor-2 (Nrf2), a master regulator of this antioxidant response, is also elevated in IRI. Furthermore, microarray analysis of renal epithelial cells exposed to hypoxia/reoxygenation identified Nrf2 to be up-regulated on reoxygenation. We also reveal a reoxygenation-specific nuclear accumulation of Nrf2 protein and subsequent activation of a NQO1 promoter reporter construct. Attenuating reactive oxygen species (ROS) in reoxygenation using the antioxidant N-acetyl cysteine results in inhibition of Nrf-2 activation. mRNA levels for Nrf2-dependent genes were detected in human liver biopsy 1 h after transplantation. These results indicate that reoxygenation-dependent Nrf-2 activity facilitates ischemic preconditioning through the induction of antioxidant gene expression and that ROS may be critical in signaling this event.


Assuntos
Nefropatias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Fígado/metabolismo , Transplante de Fígado , Camundongos , NAD(P)H Desidrogenase (Quinona) , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Fator 2 Relacionado a NF-E2/genética , Regiões Promotoras Genéticas , Transdução de Sinais , Regulação para Cima
18.
EMBO J ; 24(15): 2815-26, 2005 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-16001080

RESUMO

Apoptosis-inducing factor (AIF) exhibits reactive oxygen species (ROS)-generating NADH oxidase activity of unknown significance, which is dispensable for apoptosis. We knocked out the aif gene in two human colon carcinoma cell lines that displayed lower mitochondrial complex I oxidoreductase activity and produced less ROS, but showed increased sensitivity to peroxide- or drug-induced apoptosis. AIF knockout cells failed to form tumors in athymic mice or grow in soft agar. Only AIF with intact NADH oxidase activity restored complex I activity and anchorage-independent growth of aif knockout cells, and induced aif-transfected mouse NIH3T3 cells to form foci. AIF knockdown in different carcinoma cell types resulted in lower superoxide levels, enhanced apoptosis sensitivity and loss of tumorigenicity. Antioxidants sensitized AIF-expressing cells to apoptosis, but had no effect on tumorigenicity. In summary, AIF-mediated resistance to chemical stress involves ROS and probably also mitochondrial complex I. AIF maintains the transformed state of colon cancer cells through its NADH oxidase activity, by mechanisms that involve complex I function. On both counts, AIF represents a novel type of cancer drug target.


Assuntos
Apoptose/fisiologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Complexo I de Transporte de Elétrons/fisiologia , Flavoproteínas/fisiologia , Proteínas de Membrana/fisiologia , Animais , Fator de Indução de Apoptose , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Dano ao DNA , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Proteínas de Membrana/deficiência , Camundongos , Camundongos Nus , Mitocôndrias/enzimologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Células NIH 3T3 , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo
19.
J Biol Chem ; 280(17): 16891-900, 2005 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-15734732

RESUMO

The antioxidant response element (ARE) and Nrf2 are known to regulate the expression and coordinated induction of genes encoding detoxifying enzymes including NAD(P)H:quinone oxidoreductase1 (NQO1) in response to antioxidants. In this report, we demonstrate that overexpression of the transcription factor Bach1 in Hep-G2 cells negatively regulated NQO1 gene expression and induction in response to antioxidant t-BHQ. Bandshift and supershift assays revealed that Bach1 binds to the ARE as a heterodimer with small Maf proteins but not as a homodimer or heterodimer with Nrf2. The transfection and ChIP assays revealed that Bach1 and Nrf2 competed with each other to regulate ARE-mediated gene expression. Heme, a negative regulator of Bach1 relieved the Bach1 repression of NQO1 gene expression in transfected cells. The transcription of Bach1 and Nrf2 did not change in response to t-BHQ. Immunofluorescence assays and Western blot analysis revealed that both Bach1 and Nrf2 localized in the cytoplasm and nucleus of the untreated cells. The treatment of cells with t-BHQ resulted in the nuclear accumulation of both Bach1 and Nrf2. Interestingly, the t-BHQ-induced nuclear accumulation of Bach1 was significantly delayed over that of Nrf2. These results led to the conclusion that a balance of Nrf2 versus Bach1 inside the nucleus influences up- or down-regulation of ARE-mediated gene expression. The results further suggest that antioxidant-induced delayed accumulation of Bach1 contributes to the down-regulation of ARE-regulated genes, presumably to reduce the antioxidant enzymes to normal levels.


Assuntos
Antioxidantes/metabolismo , Proteínas de Ligação a DNA/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Ligação Competitiva , Northern Blotting , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Citoplasma/metabolismo , Dimerização , Regulação para Baixo , Proteínas de Grupos de Complementação da Anemia de Fanconi , Regulação da Expressão Gênica , Genes Reporter , Heme/química , Humanos , Cinética , Luciferases/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Fator 2 Relacionado a NF-E2 , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-maf , RNA Mensageiro/metabolismo , Elementos de Resposta , Frações Subcelulares , Fatores de Tempo , Transcrição Gênica , Transfecção , Regulação para Cima
20.
Bioessays ; 26(6): 656-64, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15170863

RESUMO

A distinct group of receptors including DCC, UNC5, RET and Ptc1 is known to function in ligand-dependent neuronal growth and differentiation or axon guidance. Acting as "dependence receptors", they may also regulate neuronal cell survival by inducing apoptosis in the absence of cognate ligand. Receptor-initiated apoptosis requires proteolytic (caspase) cleavage and exposure of a pro-apoptotic region in the cytoplasmic domains of the receptors. In contrast, classical apoptosis induced by growth factor or cytokine deprivation involves loss of survival signaling without receptor cleavage. DCC, UNC5, RET and Ptc1 are downregulated or mutated in diverse cancers, and show properties characteristic of tumor suppressors, consistent with their ability to promote neuronal cell death. Dysfunctional dependence receptors have been linked to the loss of specific neurons in certain inherited and neurodegenerative diseases. Dependence receptor-initiated apoptosis represents a novel paradigm for the controlled removal of specific cells during neural development and elimination of malignant cells that have strayed beyond regions of ligand availability.


Assuntos
Apoptose , Morte Celular , Animais , Caspases/metabolismo , Citoplasma/metabolismo , Humanos , Ligantes , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo
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