RESUMO
SRT1720, a sirtuin1-activator, and metformin (MET), an antidiabetic drug, confer health and life-span benefits when administered individually. It is unclear whether combination of the two compounds could lead to additional benefits. Groups of 56-week-old C57BL/6J male mice were fed a high-fat diet (HFD) alone or supplemented with either SRT1720 (2 g/kg food), a high dose of MET (1% wt/wt food), or a combination of both. Animals were monitored for survival, body weight, food consumption, body composition, and rotarod performance. Mice treated with MET alone did not have improved longevity, and life span was dramatically reduced by combination of MET with SRT1720. Although all groups of animals were consuming similar amounts of food, mice on MET or MET + SRT1720 showed a sharp reduction in body weight. SRT1720 + MET mice also had lower percent body fat combined with better performance on the rotarod compared to controls. These data suggest that co-treatment of SRT1720 with MET is detrimental to survival at the doses used and, therefore, risk-benefits of combining life-span-extending drugs especially in older populations needs to be systematically evaluated.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Longevidade/efeitos dos fármacos , Metformina/farmacologia , Animais , Composição Corporal , Peso Corporal , Dieta Hiperlipídica , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Masculino , Metformina/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Sirtuína 1RESUMO
The NAD+-dependent protein lysine deacylases of the Sirtuin family regulate various physiological functions, from energy metabolism to stress responses. The human Sirtuin isoforms, SIRT1-7, are considered attractive therapeutic targets for aging-related diseases, such as type 2 diabetes, inflammatory diseases and neurodegenerative disorders. We review the status of Sirtuin-targeted drug discovery and development. Potent and selective pharmacological Sirt1 activators and inhibitors are available, and initial clinical trials have been carried out. Several promising inhibitors and activators have also been described for other isoforms. Progress in understanding the mechanisms of Sirtuin modulation by such compounds provides a rational basis for further drug development.
Assuntos
Sirtuínas/fisiologia , Animais , Catálise , Desenvolvimento de Medicamentos , Humanos , Resveratrol/farmacologia , Sirtuínas/antagonistas & inibidores , Sirtuínas/químicaRESUMO
The role in longevity and healthspan of nicotinamide (NAM), the physiological precursor of NAD+, is elusive. Here, we report that chronic NAM supplementation improves healthspan measures in mice without extending lifespan. Untargeted metabolite profiling of the liver and metabolic flux analysis of liver-derived cells revealed NAM-mediated improvement in glucose homeostasis in mice on a high-fat diet (HFD) that was associated with reduced hepatic steatosis and inflammation concomitant with increased glycogen deposition and flux through the pentose phosphate and glycolytic pathways. Targeted NAD metabolome analysis in liver revealed depressed expression of NAM salvage in NAM-treated mice, an effect counteracted by higher expression of de novo NAD biosynthetic enzymes. Although neither hepatic NAD+ nor NADP+ was boosted by NAM, acetylation of some SIRT1 targets was enhanced by NAM supplementation in a diet- and NAM dose-dependent manner. Collectively, our results show health improvement in NAM-supplemented HFD-fed mice in the absence of survival effects.
Assuntos
Suplementos Nutricionais , Envelhecimento Saudável/metabolismo , Fígado , NAD/metabolismo , Niacinamida/farmacologia , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , Fígado Gorduroso/tratamento farmacológico , Inflamação/tratamento farmacológico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Longevidade , Camundongos Endogâmicos C57BL , Niacinamida/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 1/metabolismoRESUMO
The sirtuin family consists of seven NAD+-dependent enzymes affecting a broad array of regulatory protein networks by primarily catalyzing the deacetylation of key lysine residues in regulatory proteins. The enzymatic activity of SIRT1 can be enhanced by small molecule activators known as SIRT1 activator compounds (STACs). We tested the therapeutic potential of the STAC SRT3025 in two preclinical models of severe infection, the murine cecal ligation and puncture (CLP) model to induce peritonitis and intratracheal installation of Streptococcus pneumoniae to induce severe bacterial pneumonia. SRT3025 provided significant survival benefits over vehicle control in both the peritonitis and pneumococcal pneumonia models when administered with appropriate antimicrobial agents. The survival benefit of SRT3025 in the CLP model was absent in SIRT1 knockout showing the SIRT1 dependency of SRT3025's effects. SRT3025 administration promoted bacterial clearance and significantly reduced inflammatory cytokines from the lungs of animals challenged with S. pneumoniae. SRT3025 treatment was also accompanied by striking changes in the transcription profiles in multiple inflammatory and metabolic pathways in liver, spleen, small bowel, and lung tissue. Remarkably, these organ-specific changes in the transcriptome analyses were similar following CLP or pneumococcal challenge despite different sets of pathogens at disparate sites of infection. Pharmacologic activation of SIRT1 modulates the innate host response and could represent a novel treatment strategy for severe infection.
Assuntos
Anilidas/farmacologia , Sepse , Sirtuína 1/imunologia , Infecções Estreptocócicas , Streptococcus pneumoniae , Tiazóis/farmacologia , Transcrição Gênica , Animais , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Feminino , Imunidade Inata/efeitos dos fármacos , Camundongos , Sepse/tratamento farmacológico , Sepse/imunologia , Sepse/patologia , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/patologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologiaRESUMO
SIRT1, the founding member of the mammalian family of seven NAD(+)-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.
Assuntos
Lisina/metabolismo , Sirtuína 1/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Domínio Catalítico/genética , Cristalização , Cristalografia por Raios X , Medição da Troca de Deutério , Escherichia coli , Vetores Genéticos , Humanos , Espectrometria de Massas , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Sirtuína 1/genética , Sirtuína 1/metabolismo , TransfecçãoRESUMO
AIMS: The deacetylase sirtuin 1 (Sirt1) exerts beneficial effects on lipid metabolism, but its roles in plasma LDL-cholesterol regulation and atherosclerosis are controversial. Thus, we applied the pharmacological Sirt1 activator SRT3025 in a mouse model of atherosclerosis and in hepatocyte culture. METHODS AND RESULTS: Apolipoprotein E-deficient (Apoe(-/-)) mice were fed a high-cholesterol diet (1.25% w/w) supplemented with SRT3025 (3.18 g kg(-1) diet) for 12 weeks. In vitro, the drug activated wild-type Sirt1 protein, but not the activation-resistant Sirt1 mutant; in vivo, it increased deacetylation of hepatic p65 and skeletal muscle Foxo1. SRT3025 treatment decreased plasma levels of LDL-cholesterol and total cholesterol and reduced atherosclerosis. Drug treatment did not change mRNA expression of hepatic LDL receptor (Ldlr) and proprotein convertase subtilisin/kexin type 9 (Pcsk9), but increased their protein expression indicating post-translational effects. Consistent with hepatocyte Ldlr and Pcsk9 accumulation, we found reduced plasma levels of Pcsk9 after pharmacological Sirt1 activation. In vitro administration of SRT3025 to cultured AML12 hepatocytes attenuated Pcsk9 secretion and its binding to Ldlr, thereby reducing Pcsk9-mediated Ldlr degradation and increasing Ldlr expression and LDL uptake. Co-administration of exogenous Pcsk9 with SRT3025 blunted these effects. Sirt1 activation with SRT3025 in Ldlr(-/-) mice reduced neither plasma Pcsk9, nor LDL-cholesterol levels, nor atherosclerosis. CONCLUSION: We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice. Pharmacological activation of Sirt1 appears promising to be tested in patients for its effects on plasma Pcsk9, LDL-cholesterol, and atherosclerosis.
Assuntos
Arteriosclerose/prevenção & controle , Hepatócitos/metabolismo , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Sirtuína 1/metabolismo , Anilidas/farmacologia , Animais , Anticolesterolemiantes/farmacologia , Apolipoproteínas E/deficiência , Células Cultivadas , LDL-Colesterol/metabolismo , Inibidores Enzimáticos/farmacologia , Hepatócitos/efeitos dos fármacos , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9 , RNA Mensageiro/metabolismo , Receptores de LDL/efeitos dos fármacos , Tiazóis/farmacologiaRESUMO
Increased expression of SIRT1 extends the lifespan of lower organisms and delays the onset of age-related diseases in mammals. Here, we show that SRT2104, a synthetic small molecule activator of SIRT1, extends both mean and maximal lifespan of mice fed a standard diet. This is accompanied by improvements in health, including enhanced motor coordination, performance, bone mineral density, and insulin sensitivity associated with higher mitochondrial content and decreased inflammation. Short-term SRT2104 treatment preserves bone and muscle mass in an experimental model of atrophy. These results demonstrate it is possible to design a small molecule that can slow aging and delay multiple age-related diseases in mammals, supporting the therapeutic potential of SIRT1 activators in humans.
Assuntos
Osso e Ossos/efeitos dos fármacos , Compostos Heterocíclicos com 2 Anéis/farmacologia , Envelhecimento , Animais , Composição Corporal , Índice de Massa Corporal , Osso e Ossos/metabolismo , Dieta , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de SobrevidaRESUMO
The prevention or delay of the onset of age-related diseases prolongs survival and improves quality of life while reducing the burden on the health care system. Activation of sirtuin 1 (SIRT1), an NAD(+)-dependent deacetylase, improves metabolism and confers protection against physiological and cognitive disturbances in old age. SRT1720 is a specific SIRT1 activator that has health and lifespan benefits in adult mice fed a high-fat diet. We found extension in lifespan, delayed onset of age-related metabolic diseases, and improved general health in mice fed a standard diet after SRT1720 supplementation. Inhibition of proinflammatory gene expression in both liver and muscle of SRT1720-treated animals was noted. SRT1720 lowered the phosphorylation of NF-κB pathway regulators in vitro only when SIRT1 was functionally present. Combined with our previous work, the current study further supports the beneficial effects of SRT1720 on health across the lifespan in mice.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Sirtuína 1/metabolismo , Animais , Dieta , Longevidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sirtuína 1/genética , Análise de Sobrevida , TranscriptomaRESUMO
Sirtuin 1 is a nicotinamide adenine dinucleotide-dependent protein deacetylase which regulates longevity and improves metabolism. Activation of Sirtuin 1 confers beneficial effects in models of neurodegenerative diseases. We and others have provided convincing evidence that overexpression of Sirtuin 1 plays a neuroprotective role in mouse models of Huntington's disease. In this study, we report that SRT2104, a small molecule Sirtuin 1 activator, penetrated the blood-brain barrier, attenuated brain atrophy, improved motor function, and extended survival in a mouse model of Huntington's disease. These findings imply a novel therapeutic strategy for Huntington's disease by targeting Sirtuin 1.
RESUMO
SIRT1 is an NAD (+) -dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carbazóis/farmacologia , Regulação da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Processamento de Proteína Pós-Traducional , Sirtuína 1/metabolismo , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítios de Ligação , Linhagem Celular Tumoral , Genes Reporter , Humanos , Luciferases/genética , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Especificidade por SubstratoRESUMO
Type 1 diabetes is caused by autoimmune-mediated ß cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to ß cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes.
Assuntos
Autoimunidade/genética , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença/genética , Sirtuína 1/genética , Análise de Variância , Sequência de Bases , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Imunoprecipitação , Masculino , Dados de Sequência Molecular , Mutagênese , Mutação de Sentido Incorreto/genética , Óxido Nítrico/metabolismo , Linhagem , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , SuíçaRESUMO
A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.
Assuntos
Sirtuína 1/química , Sirtuína 1/metabolismo , Estilbenos/farmacologia , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células Cultivadas , Ativação Enzimática , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Dados de Sequência Molecular , Mioblastos/efeitos dos fármacos , Mioblastos/enzimologia , Estrutura Terciária de Proteína , Resveratrol , Sirtuína 1/genética , Estilbenos/química , Especificidade por SubstratoRESUMO
Circadian rhythms govern a wide variety of physiological and metabolic functions in many organisms, from prokaryotes to humans. We previously reported that silent information regulator 1 (SIRT1), a NAD(+)-dependent deacetylase, contributes to circadian control. In addition, SIRT1 activity is regulated in a cyclic manner in virtue of the circadian oscillation of the coenzyme NAD(+). Here we used specific SIRT1 activator compounds both in vitro and in vivo. We tested a variety of compounds to show that the activation of SIRT1 alters CLOCK:BMAL1-driven transcription in different systems. Activation of SIRT1 induces repression of circadian gene expression and decreases H3 K9/K14 acetylation at corresponding promoters in a time-specific manner. Specific activation of SIRT1 was demonstrated in vivo using liver-specific SIRT1-deficient mice, where the effect of SIRT1 activator compounds was shown to be dependent on SIRT1. Our findings demonstrate that SIRT1 can fine-tune circadian rhythm and pave the way to the development of pharmacological strategies to address a broad range of therapeutic indications.
Assuntos
Ritmo Circadiano/genética , Ativadores de Enzimas/farmacologia , Sirtuína 1/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Linhagem Celular , Cromatina/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Knockout , NAD/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Chronic inflammation is a major contributing factor in the pathogenesis of many age-associated diseases. One central protein that regulates inflammation is NF-κB, the activity of which is modulated by post-translational modifications as well as by association with co-activator and co-repressor proteins. SIRT1, an NAD(+)-dependent protein deacetylase, has been shown to suppress NF-κB signaling through deacetylation of the p65 subunit of NF-κB resulting in the reduction of the inflammatory responses mediated by this transcription factor. The role of SIRT1 in the regulation of NF-κB provides the necessary validation for the development of pharmacological strategies for activating SIRT1 as an approach for the development of a new class of anti-inflammatory therapeutics. We report herein the development of a quantitative assay to assess compound effects on acetylated p65 protein in the cell. We demonstrate that small molecule activators of SIRT1 (STACs) enhance deacetylation of cellular p65 protein, which results in the suppression of TNFα-induced NF-κB transcriptional activation and reduction of LPS-stimulated TNFα secretion in a SIRT1-dependent manner. In an acute mouse model of LPS-induced inflammation, the STAC SRTCX1003 decreased the production of the proinflammatory cytokines TNFα and IL-12. Our studies indicate that increasing SIRT1-mediated NF-κB deacetylation using small molecule activating compounds is a novel approach to the development of a new class of therapeutic anti-inflammatory agents.
Assuntos
Anti-Inflamatórios/farmacologia , Benzimidazóis/farmacologia , Inflamação/tratamento farmacológico , Processamento de Proteína Pós-Traducional , Sirtuína 1/metabolismo , Tiazóis/farmacologia , Fator de Transcrição RelA/metabolismo , Acetilação , Animais , Anti-Inflamatórios/química , Benzimidazóis/química , Linhagem Celular , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-12/biossíntese , Interleucina-12/imunologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/genética , Bibliotecas de Moléculas Pequenas , Tiazóis/química , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Sirt1 is an NAD(+)-dependent deacetylase that extends lifespan in lower organisms and improves metabolism and delays the onset of age-related diseases in mammals. Here we show that SRT1720, a synthetic compound that was identified for its ability to activate Sirt1 in vitro, extends both mean and maximum lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, enhanced locomotor activity and normalization of gene expression profiles and markers of inflammation and apoptosis, all in the absence of any observable toxicity. Using a conditional SIRT1 knockout mouse and specific gene knockdowns we show SRT1720 affects mitochondrial respiration in a Sirt1- and PGC-1α-dependent manner. These findings indicate that SRT1720 has long-term benefits and demonstrate for the first time the feasibility of designing novel molecules that are safe and effective in promoting longevity and preventing multiple age-related diseases in mammals.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Longevidade/efeitos dos fármacos , Obesidade/fisiopatologia , Animais , Apoptose/efeitos dos fármacos , Composição Corporal/efeitos dos fármacos , Gorduras na Dieta/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/efeitos dos fármacosAssuntos
Envelhecimento , Sirtuína 1/fisiologia , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Fármacos Antiobesidade/farmacologia , Fármacos Antiobesidade/uso terapêutico , Catálise , Diabetes Mellitus Tipo 2/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/uso terapêutico , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Cinética , Sirtuína 1/antagonistas & inibidores , Especificidade por SubstratoRESUMO
Inhibitors of Rho kinase (ROCK) are a relatively new class of drugs with potential benefits in oncology, neurology, and fibrotic and cardiovascular diseases. ROCK inhibitors modulate many cellular functions, some of which are similar to the pleiotropic effects of statins, suggesting additive or synergistic properties. Studies to date have used compounds that inhibit both isoforms of ROCK, ROCK1 and ROCK2. This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2 inhibitor SLx-2119 in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. Cells were treated with each compound for 24 h, after which total RNA was isolated and genome-wide gene-expression profiles were obtained with Illumina arrays. Because of the known effect of statins on the actin cytoskeleton and on connective tissue growth factor, a prominent growth factor involved in tissue fibrosis, the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and connective tissue growth factor mRNA were also examined in cultures of smooth muscle cells with a fibrotic phenotype, isolated from biopsies of human intestine with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected expression of genes belonging to the same biological processes, individual genes were mostly different, consistent with synergistic or additive properties. Both SLx-2119 and atorvastatin reduced connective tissue growth factor mRNA and remodeled the actin cytoskeleton in fibrosis-derived smooth muscle cells, suggesting that both compounds have antifibrotic properties. These results form the basis for further studies to assess the possible therapeutic benefit of combined treatments.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Ácidos Heptanoicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Sequência de Aminoácidos , Atorvastatina , Linhagem Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Músculo Liso/fisiologia , Reação em Cadeia da Polimerase/métodosRESUMO
Indomethacin, a nonselective cyclooxygenase (COX) inhibitor, was modified in three distinct regions in an attempt both to increase cyclooxygenase-2 (COX-2) selectivity and to enhance drug safety by covalent attachment of an organic nitrate moiety as a nitric oxide donor. A human whole-blood COX assay shows the modifications on the 3-acetic acid part of the indomethacin yielding an amide-nitrate derivative 32 and a sulfonamide-nitrate derivative 61 conferred COX-2 selectivity. Along with their respective des-nitrate analogs, for example, 31 and 62, the nitrates 32 and 61 were effective antiinflammatory agents in the rat air-pouch model. After oral dosing, though, only 32 increased nitrate and nitrite levels in rat plasma, indicating that its nitrate tether served as a nitric oxide donor in vivo. In a rat gastric injury model, examples 31 and 32 both show a 98% reduction in gastric lesion score compared to that of indomethacin. In addition, the nitrated derivative 32 inducing 85% fewer gastric lesions when coadministered with aspirin as compared to the combination of aspirin and valdecoxib.
Assuntos
Inibidores de Ciclo-Oxigenase 2/síntese química , Indometacina/análogos & derivados , Indometacina/síntese química , Doadores de Óxido Nítrico/síntese química , Animais , Aspirina/efeitos adversos , Celecoxib , Inibidores de Ciclo-Oxigenase 2/efeitos adversos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Desenho de Fármacos , Sinergismo Farmacológico , Feminino , Mucosa Gástrica/patologia , Humanos , Ácidos Hidroxâmicos/efeitos adversos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Indometacina/efeitos adversos , Indometacina/farmacologia , Masculino , Doadores de Óxido Nítrico/efeitos adversos , Doadores de Óxido Nítrico/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/patologia , Relação Estrutura-Atividade , Sulfonamidas/farmacologiaRESUMO
This investigation examined a possible correlation between lipopolysaccharide (LPS)-induced pulmonary neutrophilia and cough. Conscious male guinea pigs were acutely exposed to aerosolized LPS and thereafter at various times challenged with citric acid aerosol (CA; 250mM) to induce cough followed by bronchoalveolar lavage (BAL) to quantitate inflammatory cell accumulation. LPS caused a hyporesponsive cough at 24h post-LPS with neutrophilia apparent from 2h post-LPS. By 96h post-LPS both cough and neutrophilia had returned towards normal. Dexamethasone (DEX, 2mgkg(-1)/day for 3 days prior) did not affect the cough hyporesponsiveness at 24h; however it attenuated LPS-induced BAL fluid neutrophilia. Since LPS can stimulate inducible nitric oxide synthase (iNOS) we hypothesized that the cough hyporesponsiveness may involve nitric oxide. To investigate this we treated animals with an aerosolized iNOS inhibitor 1400W (1mM) immediately prior to LPS. 1400W had no significant effect on either cough hyporesponsiveness or BAL fluid neutrophilia at 24h post-LPS. Despite differing effects on neutrophilia, these findings clearly indicate that neither DEX nor iNOS inhibition had any direct effect on LPS-induced cough hyporesponsiveness. The mechanism underlying the LPS-induced cough hyporesponsiveness does not appear to be directly linked to LPS-induced neutrophilic inflammation.
Assuntos
Amidinas/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Benzilaminas/uso terapêutico , Tosse/prevenção & controle , Dexametasona/uso terapêutico , Pneumopatias/prevenção & controle , Neutrófilos/efeitos dos fármacos , Administração por Inalação , Aerossóis , Amidinas/administração & dosagem , Animais , Anti-Inflamatórios/administração & dosagem , Benzilaminas/administração & dosagem , Líquido da Lavagem Broncoalveolar/citologia , Ácido Cítrico/administração & dosagem , Ácido Cítrico/toxicidade , Tosse/induzido quimicamente , Dexametasona/administração & dosagem , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/uso terapêutico , Cobaias , Injeções Intraperitoneais , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/toxicidade , Pneumopatias/induzido quimicamente , Pneumopatias/imunologia , Masculino , Nebulizadores e Vaporizadores , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/patologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
A series of glycolamide naproxen prodrugs containing a nitrate group as a nitric oxide (NO) donor moiety has been synthesized. These compounds were evaluated for their anti-inflammatory activity, naproxen release, and gastric tolerance. Compounds 4a, 4b, 5a, 5b, 7b, and 7c exhibited anti-inflammatory activity equivalent to that of the parent NSAID, naproxen-Na, in the rat carrageenan paw edema model. At equimolar doses relative to naproxen-Na, the NO-donor glycolamide derivatives 4a, 4b, 5a, 5b, 7b, and 7c were gastro-sparing in the rat. Naproxen formation from these NO-donor glycolamides varied among the structures examined, with the N-substituent on the amide group having a particular influence, and demonstrated their prodrug nature. Compound 7b was selected for exemplary demonstration that the glycolamide nitrates can be bioactivated to release NO. These data open the possibility that naproxen glycolamide nitrates may represent a safer alternative to naproxen as anti-inflammatory medicines.