RESUMO
BACKGROUND: Organ fibrosis due to excessive production of extracellular matrix by resident fibroblasts is estimated to contribute to >45% of deaths in the Western world, including those due to cardiovascular diseases such as heart failure. Here, we screened for small molecule inhibitors with a common ability to suppress activation of fibroblasts across organ systems. METHODS: High-content imaging of cultured cardiac, pulmonary, and renal fibroblasts was used to identify nontoxic compounds that blocked induction of markers of activation in response to the profibrotic stimulus, transforming growth factor-ß1. SW033291, which inhibits the eicosanoid-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase, was chosen for follow-up studies with cultured adult rat ventricular fibroblasts and human cardiac fibroblasts (CF), and for evaluation in mouse models of cardiac fibrosis and diastolic dysfunction. Additional mechanistic studies were performed with CFs treated with exogenous eicosanoids. RESULTS: Nine compounds, including SW033291, shared a common ability to suppress transforming growth factor-ß1-mediated activation of cardiac, pulmonary, and renal fibroblasts. SW033291 dose-dependently inhibited transforming growth factor-ß1-induced expression of activation markers (eg, α-smooth muscle actin and periostin) in adult rat ventricular fibroblasts and normal human CFs, and reduced contractile capacity of the cells. Remarkably, the 15-hydroxyprostaglandin dehydrogenase inhibitor also reversed constitutive activation of fibroblasts obtained from explanted hearts from patients with heart failure. SW033291 blocked cardiac fibrosis induced by angiotensin II infusion and ameliorated diastolic dysfunction in an alternative model of systemic hypertension driven by combined uninephrectomy and deoxycorticosterone acetate administration. Mechanistically, SW033291-mediated stimulation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase signaling was required for the compound to block CF activation. Of the 12 exogenous eicosanoids that were tested, only 12(S)-hydroxyeicosatetraenoic acid, which signals through the G protein-coupled receptor, GPR31, recapitulated the suppressive effects of SW033291 on CF activation. CONCLUSIONS: Inhibition of degradation of eicosanoids, arachidonic acid-derived fatty acids that signal through G protein-coupled receptors, is a potential therapeutic strategy for suppression of pathological organ fibrosis. In the heart, we propose that 15-hydroxyprostaglandin dehydrogenase inhibition triggers CF-derived autocrine/paracrine signaling by eicosanoids, including 12(S)-hydroxyeicosatetraenoic acid, to stimulate extracellular signal-regulated kinase 1/2 and block conversion of fibroblasts into activated cells that secrete excessive amounts of extracellular matrix and contribute to heart failure pathogenesis.
Assuntos
Insuficiência Cardíaca , Camundongos , Ratos , Humanos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/metabolismo , Insuficiência Cardíaca/metabolismo , Fibroblastos/metabolismo , Fibrose , Células CultivadasRESUMO
Heart failure is the one of the leading causes of death in the United States. Heart failure is a complex syndrome caused by numerous diseases, including severe myocardial infarction (MI). MI occurs after an occlusion of a cardiac artery causing downstream ischemia. MI is followed by cardiac remodeling involving extensive remodeling and fibrosis, which, if the original insult is severe or prolonged, can ultimately progress into heart failure. There is no "cure" for heart failure because therapies to regenerate dead tissue are not yet available. Previous studies have shown that in both post-MI and post-ischemia-reperfusion (I/R) models of heart failure, administration of cortical bone stem cell (CBSC) treatment leads to a reduction in scar size and improved cardiac function. Our first study investigated the ability of mouse CBSC-derived exosomes (mCBSC-dEXO) to recapitulate mouse CBSCs (mCBSC) therapeutic effects in a 24-h post-I/R model. This study showed that injection of mCBSCs and mCBSC-dEXOs into the ischemic region of an infarct had a protective effect against I/R injury. mCBSC-dEXOs recapitulated the effects of CBSC treatment post-I/R, indicating exosomes are partly responsible for CBSC's beneficial effects. To examine if exosomes decrease fibrotic activation, adult rat ventricular fibroblasts (ARVFs) and adult human cardiac fibroblasts (NHCFs) were treated with transforming growth factor ß (TGFß) to activate fibrotic signaling before treatment with mCBSC- and human CBSC (hCBSC)-dEXOs. hCBSC-dEXOs caused a 100-fold decrease in human fibroblast activation. To further understand the signaling mechanisms regulating the protective decrease in fibrosis, we performed RNA sequencing on the NHCFs after hCBSC-dEXO treatment. The group treated with both TGFß and exosomes showed a decrease in small nucleolar RNA (snoRNA), known to be involved with ribosome stability.NEW & NOTEWORTHY Our work is noteworthy due to the identification of factors within stem cell-derived exosomes (dEXOs) that alter fibroblast activation through the hereto-unknown mechanism of decreasing small nucleolar RNA (snoRNA) signaling within cardiac fibroblasts. The study also shows that the injection of stem cells or a stem-cell-derived exosome therapy at the onset of reperfusion elicits cardioprotection, emphasizing the importance of early treatment in the post-ischemia-reperfusion (I/R) wounded heart.
Assuntos
Osso Cortical/citologia , Exossomos/transplante , Fibroblastos/patologia , Infarto do Miocárdio/cirurgia , Traumatismo por Reperfusão Miocárdica/cirurgia , Miocárdio/patologia , Transplante de Células-Tronco , Remodelação Ventricular , Animais , Células Cultivadas , Modelos Animais de Doenças , Exossomos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Ratos , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury that is part of a complex and highly organized wound-healing process. Cortical bone stem cell (CBSC) therapy after MI has been shown to reduce adverse structural and functional remodeling of the heart after MI in both mouse and swine models. The basis for these CBSC treatment effects on wound healing are unknown. The present experiments show that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably macrophage colony-stimulating factor (M-CSF) and transforming growth factor-ß, but not IL-4. CBSC therapy increased the number of galectin-3+ macrophages, CD4+ T cells, and fibroblasts in the heart while decreasing apoptosis in an in vivo swine model of MI. Macrophages treated with CBSC medium in vitro polarized to a proreparative phenotype are characterized by increased CD206 expression, increased efferocytic ability, increased IL-10, TGF-ß, and IL-1RA secretion, and increased mitochondrial respiration. Next generation sequencing revealed a transcriptome significantly different from M2a or M2c macrophage phenotypes. Paracrine factors from CBSC-treated macrophages increased proliferation, decreased α-smooth muscle actin expression, and decreased contraction by fibroblasts in vitro. These data support the idea that CBSCs are modulating the immune response to MI to favor cardiac repair through a unique macrophage polarization that ultimately reduces cell death and alters fibroblast populations that may result in smaller scar size and preserved cardiac geometry and function.NEW & NOTEWORTHY Cortical bone stem cell (CBSC) therapy after myocardial infarction alters the inflammatory response to cardiac injury. We found that cortical bone stem cell therapy induces a unique macrophage phenotype in vitro and can modulate macrophage/fibroblast cross talk.
Assuntos
Mediadores da Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Infarto do Miocárdio/cirurgia , Miocárdio/metabolismo , Comunicação Parácrina , Transplante de Células-Tronco , Células-Tronco/metabolismo , Cicatrização , Animais , Apoptose , Células Cultivadas , Osso Cortical/citologia , Modelos Animais de Doenças , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibrose , Humanos , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/metabolismo , Miocárdio/imunologia , Fenótipo , Transdução de Sinais , Suínos , Porco Miniatura , Linfócitos T/imunologia , Linfócitos T/metabolismo , TranscriptomaRESUMO
OBJECTIVE: Our recent work demonstrates that PTEN (phosphatase and tensin homolog) is an important regulator of smooth muscle cell (SMC) phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Ang II (angiotensin II)-induced pathological vascular remodeling, suggesting that pharmacological PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Approach and Results: To identify novel PTEN activators, we conducted a high-throughput screen using a fluorescence based PTEN promoter-reporter assay. After screening ≈3400 compounds, 11 hit compounds were chosen based on level of activity and mechanism of action. Following in vitro confirmation, we focused on 5-azacytidine, a DNMT1 (DNA methyltransferase-1) inhibitor, for further analysis. In addition to PTEN upregulation, 5-azacytidine treatment increased expression of genes associated with a differentiated SMC phenotype. 5-Azacytidine treatment also maintained contractile gene expression and reduced inflammatory cytokine expression after PDGF (platelet-derived growth factor) stimulation, suggesting 5-azacytidine blocks PDGF-induced SMC de-differentiation. However, these protective effects were lost in PTEN-deficient SMCs. These findings were confirmed in vivo using carotid ligation in SMC-specific PTEN knockout mice treated with 5-azacytidine. In wild type controls, 5-azacytidine reduced neointimal formation and inflammation while maintaining contractile protein expression. In contrast, 5-azacytidine was ineffective in PTEN knockout mice, indicating that the protective effects of 5-azacytidine are mediated through SMC PTEN upregulation. CONCLUSIONS: Our data indicates 5-azacytidine upregulates PTEN expression in SMCs, promoting maintenance of SMC differentiation and reducing pathological vascular remodeling in a PTEN-dependent manner.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , PTEN Fosfo-Hidrolase/fisiologia , Remodelação Vascular/efeitos dos fármacos , Animais , Azacitidina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , PTEN Fosfo-Hidrolase/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Regiões Promotoras GenéticasRESUMO
RATIONALE: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown. OBJECTIVE: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. METHODS AND RESULTS: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-ß (transforming growth factor-ß) signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin (Postn)-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the Sertad4 (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for Sertad4 in TGF-ß-mediated cardiac fibroblast activation. CONCLUSIONS: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for Sertad4. The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.
Assuntos
Cromatina/metabolismo , Insuficiência Cardíaca/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miofibroblastos/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Azepinas/farmacologia , Azepinas/uso terapêutico , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Elementos Facilitadores Genéticos , Epigênese Genética , Matriz Extracelular/metabolismo , Feminino , Fibrose , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/genética , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Ligação Proteica , RNA Polimerase II/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcriptoma , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Triazóis/farmacologia , Triazóis/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.
Assuntos
Nefropatias Diabéticas/enzimologia , Fibroblastos/enzimologia , Glomérulos Renais/enzimologia , MAP Quinase Quinase Quinase 5/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Feminino , Fibroblastos/patologia , Fibrose , Humanos , Glomérulos Renais/patologia , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase Quinase 5/genética , Masculino , Camundongos , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Distribuição Aleatória , Ratos Sprague-DawleyAssuntos
Epigenômica , Cardiopatias/patologia , Fibrose , Cardiopatias/tratamento farmacológico , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , RNA Polimerase II/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
Transforming growth factor ß (TGF-ß) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2ß]pyridazin 6-amine) with a low IC(50) value of 0.7 µM. Compound 8 also inhibited the TGF-ß-induced nuclear translocation of SMAD with an EC(50) value of 0.8 µM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.
Assuntos
Ensaios de Triagem em Larga Escala , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Difosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Simulação por Computador , Transferência Ressonante de Energia de Fluorescência , Fluorimunoensaio , Humanos , Cinética , Conformação Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Smad/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The synthesis and preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D (PKD) are reported. Notably, optimized compounds in this class have been found to be active in cellular assays of phosphorylation-dependant HDAC5 nuclear export, orally bioavailable, and highly selective versus a panel of additional putative histone deacetylase (HDAC) kinases. Therefore these compounds could provide attractive tools for the further study of PKD/HDAC5 signaling.
Assuntos
Azóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Azóis/síntese química , Azóis/química , Azóis/farmacocinética , Disponibilidade Biológica , Histona Desacetilases/metabolismo , Concentração Inibidora 50 , Estrutura Molecular , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
A novel 2,6-naphthyridine was identified by high throughput screen (HTS) as a dual protein kinase C/D (PKC/PKD) inhibitor. PKD inhibition in the heart was proposed as a potential antihypertrophic mechanism with application as a heart failure therapy. As PKC was previously identified as the immediate upstream activator of PKD, PKD vs PKC selectivity was essential to understand the effect of PKD inhibition in models of cardiac hypertrophy and heart failure. The present study describes the modification of the HTS hit to a series of prototype pan-PKD inhibitors with routine 1000-fold PKD vs PKC selectivity. Example compounds inhibited PKD activity in vitro, in cells, and in vivo following oral administration. Their effects on heart morphology and function are discussed herein.
Assuntos
Aminopiridinas/síntese química , Naftiridinas/síntese química , Proteína Quinase C/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Administração Oral , Aminopiridinas/farmacocinética , Aminopiridinas/farmacologia , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Cardiomegalia/tratamento farmacológico , Cardiomegalia/patologia , Núcleo Celular/metabolismo , Histona Desacetilases/metabolismo , Isoenzimas/antagonistas & inibidores , Masculino , Modelos Moleculares , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Células Musculares/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Naftiridinas/farmacocinética , Naftiridinas/farmacologia , Fosforilação , Ligação Proteica , Ratos , Ratos Endogâmicos Dahl , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The synthesis and biological evaluation of potent and selective PKD inhibitors are described herein. The compounds described in the present study selectively inhibit PKD among other putative HDAC kinases. The PKD inhibitors of the present study blunt phosphorylation and subsequent nuclear export of HDAC4/5 in response to diverse agonists. These compounds further establish the central role of PKD as an HDAC4/5 kinase and enhance the current understanding of cardiac myocyte signal transduction. The in vivo efficacy of a representative example compound on heart morphology is reported herein.
Assuntos
2,2'-Dipiridil/análogos & derivados , Aminopiridinas/síntese química , Naftiridinas/síntese química , Piperazinas/síntese química , Proteína Quinase C/antagonistas & inibidores , 2,2'-Dipiridil/síntese química , 2,2'-Dipiridil/farmacocinética , 2,2'-Dipiridil/farmacologia , Transporte Ativo do Núcleo Celular , Administração Oral , Aminopiridinas/farmacocinética , Aminopiridinas/farmacologia , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/farmacocinética , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Cardiomegalia/tratamento farmacológico , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Núcleo Celular/metabolismo , Histona Desacetilases/metabolismo , Isoenzimas/antagonistas & inibidores , Masculino , Modelos Moleculares , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Células Musculares/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Naftiridinas/farmacocinética , Naftiridinas/farmacologia , Fosforilação , Piperazinas/farmacocinética , Piperazinas/farmacologia , Ligação Proteica , Ratos , Ratos Endogâmicos Dahl , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Class IIa histone deacetylases (HDACs) repress genes involved in pathological cardiac hypertrophy. The anti-hypertrophic action of class IIa HDACs is overcome by signals that promote their phosphorylation-dependent nuclear export. Several kinases have been shown to phosphorylate class IIa HDACs, including calcium/calmodulin-dependent protein kinase (CaMK), protein kinase D (PKD) and G protein-coupled receptor kinase (GRK). However, the identity of the kinase(s) responsible for phosphorylating class IIa HDACs during cardiac hypertrophy has remained controversial. We describe a novel and selective small molecule inhibitor of PKD, bipyridyl PKD inhibitor (BPKDi). BPKDi blocks signal-dependent phosphorylation and nuclear export of class IIa HDACs in cardiomyocytes and concomitantly suppresses hypertrophy of these cells. These studies define PKD as a principal cardiac class IIa HDAC kinase.
Assuntos
Histona Desacetilases/metabolismo , Miocárdio/enzimologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Immunoblotting , Imunoprecipitação , Fosforilação , Transporte Proteico , Ratos , Ratos Sprague-DawleyRESUMO
Histone deacetylase 5 (HDAC5) represses expression of nuclear genes that promote cardiac hypertrophy. Agonism of a variety of G protein coupled receptors (GPCRs) triggers phosphorylation-dependent nuclear export of HDAC5 via the CRM1 nuclear export receptor, resulting in derepression of pro-hypertrophic genes. A cell-based high-throughput screen of a commercial compound collection was employed to identify compounds with the ability to preserve the nuclear fraction of GFP-HDAC5 in primary cardiomyocytes exposed to GPCR agonists. A hit compound potently inhibited agonist-induced GFP-HDAC5 nuclear export in cultured neonatal rat ventricular myocytes (NRVMs). A small set of related compounds was designed and synthesized to evaluate structure-activity relationship (SAR). The results demonstrated that inhibition of HDAC5 nuclear export was a result of compounds irreversibly reacting with a key cysteine residue in CRM1 that is required for its function. CRM1 inhibition by the compounds also resulted in potent suppression of cardiomyocyte hypertrophy. These studies define a novel class of anti-hypertrophic compounds that function through irreversible inhibition of CRM1-dependent nuclear export.
Assuntos
Cardiomegalia/tratamento farmacológico , Histona Desacetilases/metabolismo , Carioferinas/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Amidas/farmacologia , Compostos de Anilina/farmacologia , Animais , Cardiomegalia/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Inibidores de Histona Desacetilases , Histona Desacetilases/química , Humanos , Carioferinas/metabolismo , Microscopia de Fluorescência , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Proteína Exportina 1RESUMO
An early step in sphingolipid biosynthesis, the reduction of 3-ketosphinganine, is catalyzed in the yeast Saccharomyces cerevisiae by Tsc10p (TSC10 (YBR265W)). We have identified orthologs of TSC10 in two clinically important fungal pathogens, Candida albicans and Aspergillus fumigatus. The translated sequences of the putative C. albicans ortholog, KSR1 (orf6.5112), and the putative A. fumigatus ortholog, ksrA, show significant homology to the yeast protein. All three proteins contain the signature motifs of NAD(P)H-dependent oxidoreductases in the short-chain dehydrogenase/reductase family and a conserved putative substrate-binding domain. Despite being essential in S. cerevisiae, we demonstrate that the C. albicans ortholog, KSR1, is not required for cell viability. However, ksr1 null mutants produce lower levels of inositolphosphorylceramides, are significantly more sensitive than the wildtype to an inhibitor of a subsequent step in sphingolipid biosynthesis, and are defective for the transition from yeast to filamentous growth, a key virulence determinant. Recombinant, purified Ksr1p and KsrA can carry out the reduction of 3-ketosphinganine in an NADPH-dependent manner. Molecular modeling of Ksr1p with bound substrates suggests that a significant portion of the aliphatic chain of 3-ketosphinganine protrudes from the enzyme. Guided by this molecular model, we developed shorter, water-soluble derivatives of 3-ketosphinganine that are substrates for 3-ketosphinganine reductase.
Assuntos
Oxirredutases do Álcool/metabolismo , Aspergillus fumigatus/enzimologia , Candida albicans/enzimologia , Esfingolipídeos/biossíntese , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Sequência de Bases , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Primers do DNA , Genoma Fúngico , Genótipo , Cinética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The role of the Lys68*:Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase. Two double-mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence (C) and impact (I) formalism, previously defined for functional chimeric analysis. Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context. Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters. The differential sensitivity of k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) to salt bridge mutagenic replacements is shown by a linear-free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former. Thus, k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) are 133 and 442 mM(-1)s(-1) for the wild-type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM(-1)s(-1) for K68E). A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes. The more deeply rooted tree indicates that the Csb variant was the ancestral specie.