RESUMEN
The NAD+-dependent deacetylase SIRT6 is an emerging cancer drug target, whose inhibition sensitizes cancer cells to chemo-radiotherapy and has pro-differentiating effects. Here we report on the identification of novel SIRT6 inhibitors with a salicylate-based structure. The new SIRT6 inhibitors show improved potency and specificity compared to the hit inhibitor identified in an in silico compound screen. As predicted based on SIRT6 biological roles, the new leads increase histone 3 lysine 9 acetylation and glucose uptake in cultured cells, while blocking TNF-α production and T lymphocyte proliferation. Notably, the new SIRT6 inhibitors effectively sensitize pancreatic cancer cells to gemcitabine. Finally, studies of compound fingerprinting and pharmacokinetics defined the drug-like properties of one of the new SIRT6 inhibitors, potentially allowing for subsequent in vivo proof-of-concept studies. In conclusion, new SIRT6 inhibitors with a salicylate-like structure were identified, which are active in cells and could potentially find applications in disease conditions, including cancer and immune-mediated disorders.
Asunto(s)
Sistemas de Liberación de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Salicilatos/química , Sirtuinas/antagonistas & inhibidores , Acetilación/efectos de los fármacos , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Concentración 50 Inhibidora , Ratones , Estructura Molecular , Salicilatos/farmacología , Sirtuinas/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the "membrane-access" and the "acyl chain-binding" pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH's mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.
Asunto(s)
Amidohidrolasas/química , Amidohidrolasas/metabolismo , Ácidos Araquidónicos/química , Ácidos Araquidónicos/metabolismo , Endocannabinoides/química , Endocannabinoides/metabolismo , Alcamidas Poliinsaturadas/química , Alcamidas Poliinsaturadas/metabolismo , Amidohidrolasas/genética , Sitios de Unión , Catálisis , Biología Computacional , Humanos , Simulación de Dinámica Molecular , Mutación , Conformación ProteicaRESUMEN
The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high inâ vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents.
Asunto(s)
Amidas/química , Benzoxazoles/química , Ceramidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacologíaRESUMEN
Systemic and intraplaque biomarkers have been widely investigated in clinical cohorts as promising surrogate parameters of cardiovascular vulnerability. In this pilot study, we investigated if systemic and intraplaque levels of calcification biomarkers were affected by treatment with a statin in a cohort of patients with severe carotid stenosis and being asymptomatic for ischemic stroke. Patients on statin therapy had reduced serum osteopontin (OPN), RANKL/osteoprotegerin (OPG) ratio, and MMP-9/pro-MMP-9 activity as compared to untreated patients. Statin-treated patients exhibited increased levels of collagen and reduced neutrophil infiltration in downstream portions of carotid plaques as compared to untreated controls. In upstream plaque portions, OPG content was increased in statin-treated patients as compared to controls. Other histological parameters (such as lipid, macrophage, smooth muscle cell, and MMP-9 content) as well as RANKL, RANK, and OPG mRNA levels did not differ between the two patient groups. Serum RANKL/OPG ratio positively correlated with serum levels of neutrophilic products, intraplaque neutrophil, and MMP-9 content within downstream portions of carotid plaques. In conclusion, statin treatment was associated with improvement in serum RANKL levels and reduced neutrophil activity both systemically and in atherosclerotic plaques.
Asunto(s)
Estenosis Carotídea/patología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , FN-kappa B/metabolismo , Neutrófilos/citología , Ligando RANK/sangre , Anciano , Aterosclerosis/tratamiento farmacológico , Atorvastatina , Estudios de Cohortes , Femenino , Fluorobencenos/uso terapéutico , Ácidos Heptanoicos/uso terapéutico , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Activación Neutrófila , Neutrófilos/efectos de los fármacos , Osteopontina/metabolismo , Osteoprotegerina/metabolismo , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , ARN Mensajero/metabolismo , Factores de Riesgo , Rosuvastatina Cálcica , Simvastatina/uso terapéutico , Sulfonamidas/uso terapéuticoRESUMEN
Intracellular NAD(+) levels ([NAD(+)](i)) are important in regulating human T lymphocyte survival, cytokine secretion, and the capacity to respond to antigenic stimuli. NAD(+)-derived Ca(2+)-mobilizing second messengers, produced by CD38, play a pivotal role in T cell activation. Here we demonstrate that [NAD(+)](i) modifications in T lymphocytes affect intracellular Ca(2+) homeostasis both in terms of mitogen-induced [Ca(2+)](i) increase and of endoplasmic reticulum Ca(2+) store replenishment. Lowering [NAD(+)](i) by FK866-mediated nicotinamide phosphoribosyltransferase inhibition decreased the mitogen-induced [Ca(2+)](i) rise in Jurkat cells and in activated T lymphocytes. Accordingly, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores was greatly reduced in these cells in the presence of FK866. When NAD(+) levels were increased by supplementing peripheral blood lymphocytes with the NAD(+) precursors nicotinamide, nicotinic acid, or nicotinamide mononucleotide, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores as well as cell responsiveness to mitogens in terms of [Ca(2+)](i) elevation were up-regulated. The use of specific siRNA showed that the changes of Ca(2+) homeostasis induced by NAD(+) precursors are mediated by CD38 and the consequent ADPR-mediated TRPM2 gating. Finally, the presence of NAD(+) precursors up-regulated important T cell functions, such as proliferation and IL-2 release in response to mitogens.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , ADP-Ribosa Cíclica/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Mitógenos/farmacología , NAD/metabolismo , Linfocitos T/metabolismo , Canales Catiónicos TRPM/metabolismo , Acrilamidas/farmacología , Señalización del Calcio/fisiología , Proliferación Celular/efectos de los fármacos , ADP-Ribosa Cíclica/genética , Citocinas/antagonistas & inhibidores , Citocinas/genética , Citocinas/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Activación del Canal Iónico/fisiología , Células Jurkat , NAD/genética , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Piperidinas/farmacología , Linfocitos T/citología , Canales Catiónicos TRPM/genética , Tapsigargina/farmacologíaRESUMEN
Cytokine secretion by cancer cells contributes to cancer-induced symptoms and angiogenesis. Studies show that the sirtuin SIRT6 promotes inflammation by enhancing TNF expression. Here, we aimed to determine whether SIRT6 is involved in conferring an inflammatory phenotype to cancer cells and to define the mechanisms linking SIRT6 to inflammation. We show that SIRT6 enhances the expression of pro-inflammatory cyto-/chemokines, such as IL8 and TNF, and promotes cell migration in pancreatic cancer cells by enhancing Ca(2+) responses. Via its enzymatic activity, SIRT6 increases the intracellular levels of ADP-ribose, an activator of the Ca(2+) channel TRPM2. In turn, TRPM2 and Ca(2+) are shown to be involved in SIRT6-induced TNF and IL8 expression. SIRT6 increases the nuclear levels of the Ca(2+)-dependent transcription factor, nuclear factor of activated T cells (NFAT), and cyclosporin A, a calcineurin inhibitor that reduces NFAT activity, reduces TNF and IL8 expression in SIRT6-overexpressing cells. These results implicate a role for SIRT6 in the synthesis of Ca(2+)-mobilizing second messengers, in the regulation of Ca(2+)-dependent transcription factors, and in the expression of pro-inflammatory, pro-angiogenic, and chemotactic cytokines. SIRT6 inhibition may help combat cancer-induced inflammation, angiogenesis, and metastasis.
Asunto(s)
Calcio/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/metabolismo , NAD/metabolismo , Neoplasias Pancreáticas/metabolismo , Sirtuinas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Citocinas/metabolismo , Humanos , Inflamación , Interleucina-8/metabolismo , Ratones , FN-kappa B/metabolismo , ARN Interferente Pequeño/metabolismo , Retroviridae/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
G proteins are an important class of regulatory switches in all living systems. They are activated by guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP. This activity makes GEFs attractive targets for modulating disease-relevant G-protein-controlled signalling networks. GEF inhibitors are therefore of interest as tools for elucidating the function of these proteins and for therapeutic intervention; however, only one small molecule GEF inhibitor, brefeldin A (BFA), is currently available. Here we used an aptamer displacement screen to identify SecinH3, a small molecule antagonist of cytohesins. The cytohesins are a class of BFA-resistant small GEFs for ADP-ribosylation factors (ARFs), which regulate cytoskeletal organization, integrin activation or integrin signalling. The application of SecinH3 in human liver cells showed that insulin-receptor-complex-associated cytohesins are required for insulin signalling. SecinH3-treated mice show increased expression of gluconeogenic genes, reduced expression of glycolytic, fatty acid and ketone body metabolism genes in the liver, reduced liver glycogen stores, and a compensatory increase in plasma insulin. Thus, cytohesin inhibition results in hepatic insulin resistance. Because insulin resistance is among the earliest pathological changes in type 2 diabetes, our results show the potential of chemical biology for dissecting the molecular pathogenesis of this disease.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Resistencia a la Insulina , Hígado/efectos de los fármacos , Hígado/metabolismo , Triazoles/farmacología , Factores de Ribosilacion-ADP/metabolismo , Animales , Aptámeros de Nucleótidos , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Humanos , Insulina/metabolismo , Ratones , Transducción de Señal/efectos de los fármacos , Triazoles/aislamiento & purificaciónRESUMEN
Acid ceramidase (AC) hydrolyzes ceramides, which are central lipid messengers for metabolism and signaling of sphingolipids. A growing body of evidence links deregulation of sphingolipids to several diseases, including cancer. Indeed, AC expression is abnormally high in melanoma cells. AC inhibition may thus be key to treating malignant melanoma. Here, we have used a systematic scaffold exploration to design a general pharmacophore for AC inhibition. This pharmacophore comprises a 6 + 5 fused ring heterocycle linked to an aliphatic substituent via a urea moiety. We have thus identified the novel benzimidazole derivatives 10, 21, 27, and 30, which are highly potent AC inhibitors. Their chemical and metabolic stabilities are comparable or superior to those of previously reported AC inhibitors. Moreover, they are potent against endogenous AC in intact melanoma cells. These novel inhibitors merit further characterization and can serve as a promising starting point for the discovery of new antimelanoma therapeutics.
Asunto(s)
Ceramidasa Ácida/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ceramidasa Ácida/metabolismo , Animales , Antineoplásicos/sangre , Bencimidazoles/sangre , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Estabilidad de Medicamentos , Inhibidores Enzimáticos/sangre , Células HEK293 , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , RatonesRESUMEN
The Yes-associated protein, YAP, is a transcriptional co-activator, mediating the Epithelial to Mesenchymal Transition program in pancreatic ductal adenocarcinoma (PDAC). With the aim to identify compounds that can specifically modulate YAP functionality in PDAC cell lines, we performed a small scale, drug-based screening experiment using YAP cell localization as the read-out. We identified erlotinib as an inducer of YAP cytoplasmic localization, an inhibitor of the TEA luciferase reporter system and the expression of the bona fide YAP target gene, Connective Tissue Growth Factor CTGF. On the other hand, BIS I, an inhibitor of PKCδ and GSK3ß, caused YAP accumulation into the nucleus. Activation of ß-catenin reporter and interfering experiments show that inhibition of the PKCδ/GSK3ß pathway triggers YAP nuclear accumulation inducing YAP/TEAD transcriptional response. Inhibition of GSK3ß by BIS I reduced the expression levels of SMADs protein and reduced YAP contribution to EMT. Notably, BIS I reduced proliferation, migration and clonogenicity of PDAC cells in vitro, phenocopying YAP genetic down-regulation. As shown by chromatin immunoprecipitation experiments and YAP over-expressing rescue experiments, BIS I reverted YAP-dependent EMT program by modulating the expression of the YAP target genes E-cadherin, vimentin, CTGF and of the newly identified target, CD133. In conclusion, we identified two different molecules, erlotinib and BIS I, modulating YAP functionality although via different mechanisms of action, with the second one specifically inhibiting the YAP-dependent EMT program in PDAC cell lines.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Indoles/farmacología , Maleimidas/farmacología , Neoplasias Pancreáticas/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Clorhidrato de Erlotinib/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Humanos , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Smad/biosíntesis , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Dendritic cells (DCs) are antigen-presenting cells that critically influence decisions about immune activation or tolerance. Impaired DC function is at the core of common chronic disorders and contributes to reduce immunocompetence during aging. Knowledge on the mechanisms regulating DC generation and function is necessary to understand the immune system and to prevent disease and immunosenescence. Here we show that the sirtuin Sirt6, which was previously linked to healthspan promotion, stimulates the development of myeloid, conventional DCs (cDCs). Sirt6-knockout (Sirt6KO) mice exhibit low frequencies of bone marrow cDC precursors and low yields of bone marrow-derived cDCs compared to wild-type (WT) animals. Sirt6KO cDCs express lower levels of class II MHC, of costimulatory molecules, and of the chemokine receptor CCR7, and are less immunostimulatory compared to WT cDCs. Similar effects in terms of differentiation and immunostimulatory capacity were observed in human monocyte-derived DCs in response to SIRT6 inhibition. Finally, while Sirt6KO cDCs show an overall reduction in their ability to produce IL-12, TNF-α and IL-6 secretion varies dependent on the stimulus, being reduced in response to CpG, but increased in response to other Toll-like receptor ligands. In conclusion, Sirt6 plays a crucial role in cDC differentiation and function and reduced Sirt6 activity may contribute to immunosenescence.
Asunto(s)
Diferenciación Celular , Células Dendríticas/enzimología , Inmunosenescencia , Sirtuinas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Genotipo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Inmunosenescencia/efectos de los fármacos , Interleucina-12/inmunología , Interleucina-12/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Noqueados , Fenotipo , Receptores CCR7/inmunología , Receptores CCR7/metabolismo , Sirtuinas/antagonistas & inhibidores , Sirtuinas/deficiencia , Sirtuinas/genética , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The fatty acid ethanolamides (FAEs) are lipid mediators present in all organisms and involved in highly conserved biological functions, such as innate immunity, energy balance, and stress control. They are produced from membrane N-acylphosphatidylethanolamines (NAPEs) and include agonists for G protein-coupled receptors (e.g., cannabinoid receptors) and nuclear receptors (e.g., PPAR-α). Here, we report the crystal structure of human NAPE-hydrolyzing phospholipase D (NAPE-PLD) at 2.65 Å resolution, a membrane enzyme that catalyzes FAE formation in mammals. NAPE-PLD forms homodimers partly separated by an internal â¼ 9-Å-wide channel and uniquely adapted to associate with phospholipids. A hydrophobic cavity provides an entryway for NAPE into the active site, where a binuclear Zn(2+) center orchestrates its hydrolysis. Bile acids bind with high affinity to selective pockets in this cavity, enhancing dimer assembly and enabling catalysis. These elements offer multiple targets for the design of small-molecule NAPE-PLD modulators with potential applications in inflammation and metabolic disorders.
Asunto(s)
Fosfolipasa D/química , Ácidos y Sales Biliares/fisiología , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Hidrólisis , Modelos Moleculares , Fosfatidiletanolaminas/biosíntesis , Fosfolipasa D/fisiología , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de ProteínaRESUMEN
The NAD(+)-dependent sirtuin SIRT6 is highly expressed in human breast, prostate, and skin cancer where it mediates resistance to cytotoxic agents and prevents differentiation. Thus, SIRT6 is an attractive target for the development of new anticancer agents to be used alone or in combination with chemo- or radiotherapy. Here we report on the identification of novel quinazolinedione compounds with inhibitory activity on SIRT6. As predicted based on SIRT6's biological functions, the identified new SIRT6 inhibitors increase histone H3 lysine 9 acetylation, reduce TNF-α production and increase glucose uptake in cultured cells. In addition, these compounds exacerbate DNA damage and cell death in response to the PARP inhibitor olaparib in BRCA2-deficient Capan-1 cells and cooperate with gemcitabine to the killing of pancreatic cancer cells. In conclusion, new SIRT6 inhibitors with a quinazolinedione-based structure have been identified which are active in cells and could potentially find applications in cancer treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores Enzimáticos/farmacología , Ftalazinas/farmacología , Piperazinas/farmacología , Quinazolinonas/farmacología , Sirtuinas/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/síntesis química , Protocolos de Quimioterapia Combinada Antineoplásica/química , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Ftalazinas/química , Piperazinas/química , Quinazolinonas/síntesis química , Quinazolinonas/química , Sirtuinas/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
SIRT6 is an NAD(+)-dependent deacetylase with a role in the transcriptional control of metabolism and aging but also in genome stability and inflammation. Broad therapeutic applications are foreseen for SIRT6 inhibitors, including uses in diabetes, immune-mediated disorders, and cancer. Here we report on the identification of the first selective SIRT6 inhibitors by in silico screening. The most promising leads show micromolar IC50s, have significant selectivity for SIRT6 versus SIRT1 and SIRT2, and are active in cells, as shown by increased acetylation at SIRT6 target lysines on histone 3, reduced TNF-α secretion, GLUT-1 upregulation, and increased glucose uptake. Taken together, these results show the value of these compounds as starting leads for the development of new SIRT6-targeting therapeutic agents.
Asunto(s)
Inhibidores de Histona Desacetilasas/química , Sirtuinas/antagonistas & inhibidores , Acetilación , Animales , Línea Celular , Simulación por Computador , Furanos/química , Furanos/farmacología , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Piridinas/química , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Ratas , Salicilatos/química , Salicilatos/farmacología , Sirtuinas/química , Relación Estructura-Actividad , Tiazoles/química , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Pharmacological treatments targeting CXC chemokines and the associated neutrophil activation and recruitment into atherosclerotic plaques hold promise for treating cardiovascular disorders. Therefore, we investigated whether FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with anti-inflammatory properties that we recently found to reduce neutrophil recruitment into the ischaemic myocardium, would exert beneficial effects in a mouse atherosclerosis model. Atherosclerotic plaque formation was induced by carotid cast implantation in ApoE-/- mice that were fed with a Western-type diet. FK866 or vehicle were administrated intraperitoneally from week 8 until week 11 of the diet. Treatment with FK866 reduced neutrophil infiltration and MMP-9 content and increased collagen levels in atherosclerotic plaques compared to vehicle. No effect on other histological parameters, including intraplaque lipids or macrophages, was observed. These findings were associated with a reduction in both systemic and intraplaque CXCL1 levels in FK866-treated mice. In vitro, FK866 did not affect MMP-9 release by neutrophils, but it strongly reduced CXCL1 production by endothelial cells which, in the in vivo model, were identified as a main CXCL1 source at the plaque level. CXCL1 synthesis inhibition by FK866 appears to reflect interference with nuclear factor-κB signalling as shown by reduced p65 nuclear levels in endothelial cells pre-treated with FK866. In conclusion, pharmacological inhibition of NAMPT activity mitigates inflammation in atherosclerotic plaques by reducing CXCL1-mediated activities on neutrophils. These results support further assessments of NAMPT inhibitors for the potential prevention of plaque vulnerability.
Asunto(s)
Acrilamidas/farmacología , Antiinflamatorios/farmacología , Aterosclerosis/tratamiento farmacológico , Arterias Carótidas/efectos de los fármacos , Enfermedades de las Arterias Carótidas/tratamiento farmacológico , Quimiocina CXCL1/metabolismo , Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Infiltración Neutrófila/efectos de los fármacos , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piperidinas/farmacología , Placa Aterosclerótica , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Arterias Carótidas/enzimología , Arterias Carótidas/inmunología , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/enzimología , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/inmunología , Enfermedades de las Arterias Carótidas/patología , Células Cultivadas , Colágeno/metabolismo , Citocinas/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nicotinamida Fosforribosiltransferasa/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factor de Transcripción ReIA/metabolismoRESUMEN
Sirtuins are a family of NAD+-dependent enzymes that was proposed to control organismal life span about a decade ago. While such role of sirtuins is now debated, mounting evidence involves these enzymes in numerous physiological processes and disease conditions, including metabolism, nutritional behavior, circadian rhythm, but also inflammation and cancer. SIRT1, SIRT2, SIRT3, SIRT6, and SIRT7 have all been linked to carcinogenesis either as tumor suppressor or as cancer promoting proteins. Here, we review the biological rationale for the search of sirtuin inhibitors and activators for treating cancer and the experimental approaches to their identification.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Sirtuinas/efectos de los fármacos , Animales , Diseño de Fármacos , Descubrimiento de Drogas/métodos , Humanos , Terapia Molecular Dirigida , Neoplasias/patología , Sirtuinas/metabolismoRESUMEN
Nicotinamide adenine dinucleotide (NAD+) biosynthesis from nicotinamide is used by mammalian cells to replenish their NAD+ stores and to avoid unwanted nicotinamide accumulation. Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in this biosynthetic pathway, almost invariably leads to intracellular NAD+ depletion and, when protracted, to ATP shortage and cell demise. Cancer cells and activated immune cells express high levels of NAMPT and are highly susceptible to NAMPT inhibitors, as shown by the activity of these agents in models of malignant and inflammatory disorders. As the spectrum of conditions which could benefit from pharmacological NAMPT inhibition becomes broader, the mechanisms accounting for their activity are also eventually becoming apparent, including the induction of autophagy and the impairment of Ca2+--and NF-κB-dependent signaling. Here, we discuss the rationales for exploiting NAMPT inhibitors in cancer and inflammatory diseases and provide an overview of the preclinical and clinical studies in which these agents have been evaluated.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/metabolismo , Animales , Humanos , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Inflamación/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Niacinamida/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
AIMS: Nicotinamide phosphoribosyltransferase (Nampt) is a key enzyme for nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, and recent evidence indicates its role in inflammatory processes. Here, we investigated the potential effects of pharmacological Nampt inhibition with FK866 in a mouse myocardial ischemia/reperfusion model. In vivo and ex vivo mouse myocardial ischemia/reperfusion procedures were performed. RESULTS: Treatment with FK866 reduced myocardial infarct size, neutrophil infiltration, and reactive oxygen species (ROS) generation within infarcted hearts in vivo in a mouse model of ischemia and reperfusion. The benefit of FK866 was not shown in the Langendorff model (ex vivo model of working heart without circulating leukocytes), suggesting a direct involvement of these cells in cardiac injury. Sera from FK866-treated mice showed reduced circulating levels of the neutrophil chemoattractant CXCL2 and impaired capacity to prime migration of these cells in vitro. The release of CXCL8 (human homolog of murine chemokine CXCL2) by human peripheral blood mononuclear cells (PBMCs) and Jurkat cells was also reduced by FK866, as well as by sirtuin (SIRT) inhibitors and SIRT6 silencing, implying a pivotal role for this NAD(+)-dependent deacetylase in the production of this chemokine. INNOVATION: The pharmacological inhibition of Nampt might represent an effective approach to reduce neutrophilic inflammation- and oxidative stress-mediated tissue damage in early phases of reperfusion after a myocardial infarction. CONCLUSIONS: Nampt inhibition appears as a new strategy to dampen CXCL2-induced neutrophil recruitment and thereby reduce neutrophil-mediated tissue injury in mice.