RESUMEN
The study investigated the effect of exogenous melatonin and (or) curcumin treatment on the cerebral cortex of adult rats. In this context, malondialdehyde (MDA), nitric oxide (NO), glutathione (GSH), superoxide dismutase (SOD), nuclear factor E2-related factor 2 (Nrf2) and SIRT2 protein expression were examined. A total of 30 Wistar albino rats involved in the study were randomly divided into five groups. Over 30 days, the control groups received phosphate-buffered saline or dimethyl sulfoxide injections, and the treatment groups received melatonin, curcumin, or a combination of melatonin and curcumin injections. In the cerebral cortex homogenates, the MDA, GSH, and sum of NO were respectively determined by the thiobarbituric acid, modified Ellman and Griess test methods. The SOD and Nrf2 levels were examined using the ELISA method and SIRT2 protein expression using the Western blot technique. The study found that both melatonin and curcumin treatments significantly reduced lipid peroxidation and SIRT2 protein expression levels (p < 0.05) and increased the Nrf2 level in the cytoplasm (p < 0.05). The study revealed that curcumin and melatonin treatments reduced MDA and SIRT2 protein expression level and increased intracellular Nrf2, GSH, and SOD in the cortex tissue. We also found that the combined melatonin and curcumin treatment produced no synergistic effect.
Asunto(s)
Curcumina , Melatonina , Animales , Ratas , Melatonina/farmacología , Curcumina/farmacología , Sirtuina 2/metabolismo , Sirtuina 2/farmacología , Estrés Oxidativo , Factor 2 Relacionado con NF-E2/metabolismo , Ratas Wistar , Antioxidantes/farmacología , Glutatión/metabolismo , Superóxido Dismutasa/metabolismo , Corteza Cerebral/metabolismo , Malondialdehído/metabolismo , Peroxidación de Lípido , Óxido NítricoRESUMEN
Recent research on familial dysautonomia (FD) has focused on the development of therapeutics that facilitate the production of the correctly spliced, exon 20-containing, transcript in cells and individuals bearing the splice-altering, FD-causing mutation in the elongator acetyltransferase complex subunit I (ELP1) gene. We report here the ability of carnosol, a diterpene present in plant species of the Lamiaceae family, including rosemary, to enhance the cellular presence of the correctly spliced ELP1 transcript in FD patient-derived fibroblasts by upregulating transcription of the ELP1 gene and correcting the aberrant splicing of the ELP1 transcript. Carnosol treatment also elevates the level of the RNA binding motif protein 24 (RBM24) and RNA binding motif protein 38 (RBM38) proteins, two multifunctional RNA-binding proteins. Transfection-mediated expression of either of these RNA binding motif (RBMs) facilitates the inclusion of exon 20 sequence into the transcript generated from a minigene-bearing ELP1 genomic sequence containing the FD-causing mutation. Suppression of the carnosol-mediated induction of either of these RBMs, using targeting siRNAs, limited the carnosol-mediated inclusion of the ELP1 exon 20 sequence. Carnosol treatment of FD patient peripheral blood mononuclear cells facilitates the inclusion of exon 20 into the ELP1 transcript. The increased levels of the ELP1 and RBM38 transcripts and the alternative splicing of the sirtuin 2 (SIRT2) transcript, a sentinel for exon 20 inclusion in the FD-derived ELP1 transcript, are observed in RNA isolated from whole blood of healthy adults following the ingestion of carnosol-containing rosemary extract. These findings and the excellent safety profile of rosemary together justify an expedited clinical study of the impact of carnosol on the FD patient population.
Asunto(s)
Disautonomía Familiar , Rosmarinus , Factores de Elongación Transcripcional/metabolismo , Abietanos/farmacología , Acetiltransferasas , Adulto , Proteínas Portadoras/genética , Disautonomía Familiar/tratamiento farmacológico , Disautonomía Familiar/genética , Disautonomía Familiar/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Rosmarinus/genética , Rosmarinus/metabolismo , Sirtuina 2/metabolismo , Factores de Elongación Transcripcional/genéticaRESUMEN
Oocyte in vitro maturation is crucial for in vitro embryo production technology, which provides oocytes resources for in vitro fertilization and somatic cell nuclear transfer. Previous studies proved that SIRT2, a member of the sirtuin family, plays a role in oocyte meiosis, but its role in sheep oocyte maturation and its regulating mechanism remains unknown. Firstly, we confirmed the role of Sirt2 in sheep oocytes maturation by supplementation of SIRT2 inhibitor and activator. To further explore the specific mechanism, we performed knockdown of Sirt2 in granulosa cells and then cocultured them with oocytes. Moreover, we determined the effects of Sirt2 on granulosa cell oxidative apoptosis, cell migration, and diffusion, and examined its effects on granulosa cell mitochondrial function, mitophagy, and steroid hormone levels. The results showed that supplementation of SIRT2 inhibitor decreased the oocytes maturation rate (69.28% ± 1.28 vs. 45.74% ± 4.74, p < 0.05), while resveratrol, a SIRT2 activator, increased its maturation rate (67.44% ± 1.68 vs. 78.52 ± 1.28, p < 0.05). Knockdown of Sirt2 in sheep granulosa cells also reduced the oocytes maturation rate (47.98% ± 1.43 vs. 33.60% ± 1.77, p < 0.05), and led to decreased cell migration and expansion ability, oxidative apoptosis, abnormal mitochondrial gene expression, decreased mitochondrial membrane potential and ATP level, and increased mitophagy level. Overexpression of Sirt2 improved mitochondrial membrane potential and ATP level and improved mitochondrial function. Furthermore, we found that Sirt2 knockdown in granulosa cells promotes the secretion of P4 through regulating p-ERK1/2. In conclusion the present study showed that SIRT2 is critical for sheep oocyte maturation through regulating the function of ovarian granulosa cells, especially affecting its mitochondrial function.
Asunto(s)
Técnicas de Maduración In Vitro de los Oocitos , Sirtuina 2 , Adenosina Trifosfato/metabolismo , Animales , Femenino , Células de la Granulosa/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/metabolismo , Ovinos , Sirtuina 2/genética , Sirtuina 2/metabolismoRESUMEN
Metabolic reprogramming is associated with NLRP3 inflammasome activation in activated macrophages, contributing to inflammatory responses. Tanshinone IIA (Tan-IIA) is a major constituent from Salvia miltiorrhiza Bunge, which exhibits anti-inflammatory activity. In this study, we investigated the effects of Tan-IIA on inflammation in macrophages in focus on its regulation of metabolism and redox state. In lipopolysaccharides (LPS)-stimulated mouse bone marrow-derived macrophages (BMDMs), Tan-IIA (10 µM) significantly decreased succinate-boosted IL-1ß and IL-6 production, accompanied by upregulation of IL-1RA and IL-10 release via inhibiting succinate dehydrogenase (SDH). Tan-IIA concentration dependently inhibited SDH activity with an estimated IC50 of 4.47 µM in LPS-activated BMDMs. Tan-IIA decreased succinate accumulation, suppressed mitochondrial reactive oxygen species production, thus preventing hypoxia-inducible factor-1α (HIF-1α) induction. Consequently, Tan-IIA reduced glycolysis and protected the activity of Sirtuin2 (Sirt2), an NAD+-dependent protein deacetylase, by raising the ratio of NAD+/NADH in activated macrophages. The acetylation of α-tubulin was required for the assembly of NLRP3 inflammasome; Tan-IIA increased the binding of Sirt2 to α-tubulin, and thus reduced the acetylation of α-tubulin, thus impairing this process. Sirt2 knockdown or application of Sirt2 inhibitor AGK-2 (10 µM) neutralized the effects of Tan-IIA, suggesting that Tan-IIA inactivated NLRP3 inflammasome in a manner dependent on Sirt2 regulation. The anti-inflammatory effects of Tan-IIA were observed in mice subjected to LPS challenge: pre-administration of Tan-IIA (20 mg/kg, ip) significantly attenuated LPS-induced acute inflammatory responses, characterized by elevated IL-1ß but reduced IL-10 levels in serum. The peritoneal macrophages isolated from the mice displayed similar metabolic regulation. In conclusion, Tan-IIA reduces HIF-1α induction via SDH inactivation, and preserves Sirt2 activity via downregulation of glycolysis, contributing to suppression of NLRP3 inflammasome activation. This study provides a new insight into the anti-inflammatory action of Tan-IIA from the respect of metabolic and redox regulation.
Asunto(s)
Abietanos/uso terapéutico , Antiinflamatorios/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Inflamación/prevención & control , Macrófagos/efectos de los fármacos , Succinato Deshidrogenasa/antagonistas & inhibidores , Acetilación/efectos de los fármacos , Animales , Glucólisis/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamasomas/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Lipopolisacáridos , Masculino , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 2/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
The localization of NQO1 near acetylated microtubules has led to the hypothesis that NQO1 may work in concert with the NAD+-dependent deacetylase SIRT2 to regulate acetyl α-tubulin (K40) levels on microtubules. NQO1 catalyzes the oxidation of NADH to NAD+ and may supplement levels of NAD+ near microtubules to aid SIRT2 deacetylase activity. While HDAC6 has been shown to regulate the majority of microtubule acetylation at K40, SIRT2 is also known to modulate microtubule acetylation (K40) in the perinuclear region. In this study we examined the potential roles NQO1 may play in modulating acetyl α-tubulin levels. Knock-out or knock-down of NQO1 or SIRT2 did not change the levels of acetyl α-tubulin in 16HBE human bronchial epithelial cells and 3T3-L1 fibroblasts; however, treatment with a mechanism-based inhibitor of NQO1 (MI2321) led to a short-lived temporal increase in acetyl α-tubulin levels in both cell lines without impacting the intracellular pools of NADH or NAD+. Inactivation of NQO1 by MI2321 resulted in lower levels of NQO1 immunostaining on microtubules, consistent with redox-dependent changes in NQO1 conformation as evidenced by the use of redox-specific, anti-NQO1 antibodies in immunoprecipitation studies. Given the highly dynamic nature of acetylation-deacetylation reactions at α-tubulin K40 and the crowded protein environment surrounding this site, disruption in the binding of NQO1 to microtubules may temporally disturb the physical interactions of enzymes responsible for maintaining the microtubule acetylome.
Asunto(s)
Microtúbulos , Tubulina (Proteína) , Células 3T3-L1 , Acetilación , Animales , Humanos , Ratones , Microtúbulos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Oxidación-Reducción , Sirtuina 2/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Sirtuins (SIRTs) are a class of nicotinamide adenine dinucleotide (NAD+)-dependent protein histone deacetylases (HDACs) that are evolutionarily conserved from bacteria to mammals. This group of enzymes catalyses the reversible deacetylation of lysine residues in the histones or non-histone substrates using NAD+ as a cosubstrate. Numerous studies have demonstrated that the aberrant enzymatic activity of SIRTs has been linked to various diseases like diabetes, cancer, and neurodegenerative disorders. Previously, we performed a pharmacophore-based virtual screening campaign and an aryloxybenzamide derivative (1) displaying SIRT1/2 inhibitory effect was identified as a hit compound. In the current study, the hit-to-lead optimization on the hit compound was explored in order to improve the SIRT binding and inhibition. Fourteen compounds, ten of which were new, have been synthesized and subjected to in vitro biological evaluation for their inhibitory activity against SIRT1-3. By the structural modifications performed, a significant improvement was observed in selective SIRT1 inhibition for ST01, ST02, and ST11 compared to that of the hit compound. The highest SIRT2 inhibitory activity was observed for ST14, which was designed according to compatibility with pharmacophore model developed for SIRT2 inhibitors and thus, providing the interactions required with key residues in SIRT2 active site. Furthermore, ST01, ST02, ST11, and ST14 were subjected to in vitro cytotoxicity assay against MCF-7 human breast cancer cell line to determine the influence of the improvement in SIRT1/2 inhibition along with the structural modifications on the cytotoxic properties of the compounds. The cytotoxicity of the compounds was found to be correlated with their SIRT inhibitory profiles indicating the effects of SIRT1/2 inhibition on cancer cell viability. Overall, this study provides structural insights for further inhibitor improvement.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Sirtuina 1/antagonistas & inhibidores , Sirtuina 2/antagonistas & inhibidores , Sirtuina 3/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Benzamidas/síntesis química , Benzamidas/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Humanos , Células MCF-7 , Simulación del Acoplamiento Molecular , Estructura Molecular , Sirtuina 1/metabolismo , Sirtuina 2/metabolismo , Sirtuina 3/metabolismo , Relación Estructura-ActividadRESUMEN
It has been widely reported that advanced maternal age impairs oocyte quality. To date, various molecules have been discovered to be involved in this process. However, prevention of fertility issues associated with maternal age is still a challenge. In the present study, we find that both in vitro supplement and in vivo administration of melatonin are capable of alleviating the meiotic phenotypes of aged oocytes, specifically the spindle/chromosome disorganization and aneuploidy generation. Furthermore, we identify SIRT2 as a critical effector mediating the effects of melatonin on meiotic structure in old oocytes. Candidate screening shows that SIRT2-controlled deacetylation of histone H4K16 is essential for maintaining the meiotic apparatus in oocytes. Importantly, non-acetylatable-mimetic mutant H4K16R partially rescues the meiotic deficits in oocytes from reproductive aged mice. In contrast, overexpression of acetylation-mimetic mutant H4K16Q abolishes the beneficial effects of melatonin on the meiotic phenotypes in aged oocytes. To sum up, our data uncover that melatonin alleviates advanced maternal aged-associated meiotic defects in oocytes through the SIRT2-depenendet H4K16 deacetylation pathway.
Asunto(s)
Envejecimiento/metabolismo , Histonas/metabolismo , Meiosis/efectos de los fármacos , Melatonina/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Sirtuina 2/metabolismo , Acetilación , Factores de Edad , Envejecimiento/genética , Animales , Suplementos Dietéticos , Expresión Génica , Edad Materna , Ratones , Modelos BiológicosRESUMEN
Silybin is one of the effective, traditional Chinese medicines used as a hepatoprotective agent in nonalcoholic fatty liver disease (NAFLD) therapy worldwide, and the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome has been recognized as an important factor involved in NAFLD development. However, little is known about the mechanisms of silybin in the regulation of high-fat diet (HFD)-induced liver inflammation. In our study, we found that silybin inhibited endoplasmic reticulum stress and NLRP3 inflammasome activation in the livers of HFD-fed mice and in cultured hepatocytes. Phosphorylation of inositol-requiring enzyme (IRE)1α and eIF2α, expression of thioredoxin-interacting protein and cleaved caspase-1, and release of IL-1ß were reduced by silybin. In addition, silybin inhibited the approach of calreticulin and translocase of outer membrane 20 (Tom20), prevented assembly of the NLRP3 inflammasome complex, and suppressed the accumulation of acetylated α-tubulin in the perinuclear region. Both MEC-17 and sirtuin 2 (SIRT2) were influenced by palmitate and silybin, whereas histone deacetylase 6 was not affected. In addition, supplementing NAD+ directly or increasing NAD+ concentration with silybin could maintain the activity of SIRT2. The anti-inflammatory effect of silybin was blocked by SIRT2 silencing or by the SIRT2 inhibitor AGK2, as evidenced by NLRP3/ASC colocalization, AC-α-tubulin expression, and IL-1ß release. These findings indicate that the NAD+/SIRT2 pathway is an important mediator through which silybin prevents NLRP3 inflammasome activation during NAFLD.-Zhang, B., Xu, D., She, L., Wang, Z., Yang, N., Sun, R., Zhang, Y., Yan, C., Wei, Q., Aa, J., Liu, B., Wang, G., Xie, Y. Silybin inhibits NLRP3 inflammasome assembly through the NAD+/SIRT2 pathway in mice with nonalcoholic fatty liver disease.
Asunto(s)
Inflamasomas/metabolismo , NAD/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Transducción de Señal , Silimarina/farmacología , Sirtuina 2/metabolismo , Animales , Caspasa 1/metabolismo , Endorribonucleasas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Furanos/farmacología , Silenciador del Gen , Interleucina-1beta/metabolismo , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Quinolinas/farmacología , Receptores de Superficie Celular/metabolismo , Silibina , Sirtuina 2/antagonistas & inhibidoresRESUMEN
Nicotinamide (NAM), a form of vitamin B3, is a byproduct and noncompetitive inhibitor of the deacetylation reaction catalyzed by Sirtuins. These represent a family of evolutionarily conserved NAD+-dependent deacetylases that are well-known critical regulators of metabolism and aging and whose founding member is Sir2 of Saccharomyces cerevisiae. Here, we investigated the effects of NAM supplementation in the context of yeast chronological aging, the established model for studying aging of postmitotic quiescent mammalian cells. Our data show that NAM supplementation at the diauxic shift results in a phenocopy of chronologically aging sir2Δ cells. In fact, NAM-supplemented cells display the same chronological lifespan extension both in expired medium and extreme Calorie Restriction. Furthermore, NAM allows the cells to push their metabolism toward the same outcomes of sir2Δ cells by elevating the level of the acetylated Pck1. Both these cells have the same metabolic changes that concern not only anabolic pathways such as an increased gluconeogenesis but also respiratory activity in terms both of respiratory rate and state of respiration. In particular, they have a higher respiratory reserve capacity and a lower non-phosphorylating respiration that in concert with a low burden of superoxide anions can affect positively chronological aging.
Asunto(s)
Carbono/metabolismo , Niacinamida/metabolismo , Consumo de Oxígeno/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo , Eliminación de Gen , Niacinamida/farmacología , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/genéticaRESUMEN
BACKGROUND: Javamide-I and-II are phenolic amide compounds found in Coffea sp. Previous study suggested that javamide-II may be a potent compound with Sirt1/2 inhibition activity. PURPOSE: However, the effects of javamide-I and the its O-methyl ester on Sirt inhibition, p53 acetylation and cell death have not been investigated. METHODS: The isolation and synthesis of javamide-I and its O-methyl ester analogue were confirmed by NMR. Their potential effects on Sirt1/2/3, p53 acetylation and cell death were examined using sirt assay, silico analysis, Western blot, caspase 3/7 and apoptotic assay methods. RESULTS: Javamide-I and its O-methyl ester demonstrated a similar inhibition pattern; Sirt1 (IC50 of 19µM) better than Sirt2 (IC50 of 104µM) and Sirt3 (IC50 of 160µM). However, javamide-I and its O-methyl ester were found to inhibit Sirt1 better than Sirt2, which is different from javamide-II able to inhibit Sirt2 stronger than Sirt1. In silico analysis, javamide-I and its O-methyl ester showed a competitive binding pattern against NAD+, which was also supported by the kinetic analysis with Ki=20.1µM for javamide-I and 19.5µM for the O-methyl ester. However, the O-methyl ester increased p53 acetylation better than javamide-I in monocytic THP-1 cells. Since caspase 3/7 activation is often followed by the p53 activation, we investigated their effects on caspase 3/7, and found that O-methyl ester again activated caspase 3/7 greater than javamide-I in the cells, eventually leading to apoptotic cell death. CONCLUSION: These data suggest that the O-methyl modification in javamide-I may play a critical role in increasing p53 acetylation, activating caspase, and eventually inducing the THP-1 cell death.
Asunto(s)
Coffea/química , Indoles/farmacología , Fenoles/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Activación Enzimática/efectos de los fármacos , Humanos , Indoles/química , Concentración 50 Inhibidora , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Fenoles/química , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/metabolismo , Sirtuina 2/antagonistas & inhibidores , Sirtuina 2/metabolismoRESUMEN
Recent studies suggest that Sirt inhibition may have beneficial effects on several human diseases such as neurodegenerative diseases and cancer. Coffee is one of most popular beverages with several positive health effects. Therefore, in this paper, potential Sirt inhibitors were screened using coffee extract. First, HPLC was utilized to fractionate coffee extract, then screened using a Sirt1/2 inhibition assay. The screening led to the isolation of a potent Sirt1/2 inhibitor, whose structure was determined as javamide-II (N-caffeoyltryptophan) by NMR. For confirmation, the amide was chemically synthesized and its capacity of inhibiting Sirt1/2 was also compared with the isolated amide. Javamide-II inhibited Sirt2 (IC50; 8.7 µM) better than Sirt1(IC50; 34µM). Since javamide-II is a stronger inhibitor for Sirt2 than Sirt1. The kinetic study was performed against Sirt2. The amide exhibited noncompetitive Sirt2 inhibition against the NAD+ (Ki = 9.8 µM) and showed competitive inhibition against the peptide substrate (Ki = 5.3 µM). Also, a docking simulation showed stronger binding pose of javamide-II to Sirt2 than AGK2. In cellular levels, javamide-II was able to increase the acetylation of total lysine, cortactin and histone H3 in neuronal NG108-15 cells. In the same cells, the amide also increased the acetylation of lysine (K382) in p53, but not (K305). This study suggests that Javamide-II found in coffee may be a potent Sirt1/2 inhibitor, probably with potential use in some conditions of human diseases.
Asunto(s)
Ácidos Cafeicos/química , Ácidos Cafeicos/farmacología , Café/química , Sirtuina 1/antagonistas & inhibidores , Sirtuina 2/antagonistas & inhibidores , Triptófano/análogos & derivados , Acetilación/efectos de los fármacos , Ácidos Cafeicos/síntesis química , Ácidos Cafeicos/aislamiento & purificación , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Diseño de Fármacos , Histonas/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Sirtuina 1/metabolismo , Sirtuina 2/metabolismo , Triptófano/síntesis química , Triptófano/química , Triptófano/aislamiento & purificación , Triptófano/farmacología , Tubulina (Proteína)/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Sirtuins are important regulators of lysine acylation, which is implicated in cellular metabolism and transcriptional control. This makes the sirtuin class of enzymes interesting targets for development of small molecule probes with pharmaceutical potential. To achieve detailed profiling and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2 as a long chain deacylase enzyme. Novel enzymatic activities of SIRT2 were thus established in vitro, which warrant further investigation, and two known inhibitors, suramin and SirReal2, were profiled against substrates containing ε-N-acyllysine modifications of varying length.
Asunto(s)
Acetamidas/farmacología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/química , Lisina/metabolismo , Sirtuina 2/antagonistas & inhibidores , Sirtuina 2/metabolismo , Suramina/farmacología , Tiazoles/farmacología , Acetamidas/síntesis química , Acetamidas/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Lisina/análogos & derivados , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos , Suramina/síntesis química , Suramina/química , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
BACKGROUND: Seven isoforms of histone deacetylase Class III have been reported - Sirtuin (SIRT) 1-7. We recently demonstrated that EX-527, an inhibitor of SIRT1, reduces mortality in a mouse model of lethal-cecal-ligationand- puncture (CLP)-induced septic shock. Our present study was aimed at determining whether selective inhibition of SIRT2, with AGK2, would decrease animal death and attenuate the inflammatory response in a septic model. METHODS: Experiment I: C57BL/6J mice were intraperitoneally given either AGK2 (82 mg/kg) in dimethyl sulfoxide (DMSO) or DMSO alone, and 2 h later subjected to CLP. Survival was monitored for 240 hours. Experiment II: mice treated the same way as Experiment I, were grouped into (i) DMSO vehicle, and (ii) AGK2, with sham mice (operating but without any treatment) serving as controls. Peritoneal fluid and peripheral blood were examined at 24 and 48 hours for cytokine production. Samples of blood at 48 h were also allocated to assess coagulability using Thrombelastography (TEG). Morphological changes of bone marrow were evaluated from long bones (femurs and tibias) with hematoxylin and eosin (H&E) staining. Bone marrow atrophy was quantified by a blinded pathologist. Experiment III: cytokines in supernatant of the cultured normal primary splenocytes were measured after the cells were stimulated by lipopolysaccharide and treated with or without AGK2 (10 µM) for 6 hours. RESULTS: AGK2 significantly reduced mortality and decreased levels of cytokines in blood (TNF-α: 298.3±24.6 vs 26.8±2.8 pg/ml, p=0.0034; IL-6: 633.4±82.8 vs 232.6±133.0 pg/ml, p=0.0344) and peritoneal fluid (IL-6: 704.8±67.7 vs 391.4±98.5 pg/ml, p=0.033) compared to vehicle control. Also, AGK2 suppressed the TNF-α and IL-6 production in the cultured splenocytes (TNF-α: 68.1±6.4 vs 23.9±2.8 pg/ml, p=0.0009; IL-6: 73.1±4.2 vs 49.6±3.0 pg/ml; p=0.0051). The TEG data showed that the mice subjected to CLP displayed prolonged fibrin formation and fibrin cross-linkage time, slower clot formation, decreased platelet function, and clot rigidity. AGK2 treatment was associated with dramatic improvements in fibrin cross-linkage and clot formation times, without a significant impact on the clot initiation parameters or platelet function. Additionally, AGK2 significantly attenuated the bone marrow atrophy (58.3±6.5 vs 30.0±8.2%, p=0.0262). CONCLUSION: Selective inhibition of SIRT2 significantly improves survival, and attenuates sepsis-associated "cytokine storm", coagulopathy, and bone marrow atrophy in a mouse model of lethal septic shock.
Asunto(s)
Furanos/administración & dosificación , Inhibidores de Histona Desacetilasas/administración & dosificación , Quinolinas/administración & dosificación , Choque Séptico/tratamiento farmacológico , Sirtuina 2/antagonistas & inhibidores , Animales , Atrofia/prevención & control , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Células Cultivadas , Citocinas/sangre , Evaluación Preclínica de Medicamentos , Inyecciones Intraperitoneales , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , Choque Séptico/sangre , Choque Séptico/enzimología , Choque Séptico/inmunología , Sirtuina 2/metabolismoRESUMEN
A series of bicyclic pyrazole carboxamides was synthesized and tested for inhibitory activity against the class III deacetylase sirtuin enzymes. Moderate to low micromolar inhibitory activities were obtained against SIRT1 and SIRT2. These bicyclic pyrazole compounds represent a new class of sirtuin inhibitors with a preference for SIRT1 over SIRT2.
Asunto(s)
Compuestos Bicíclicos con Puentes/química , Pirazoles/química , Sirtuina 1/antagonistas & inhibidores , Sirtuina 2/antagonistas & inhibidores , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Simulación de Dinámica Molecular , Unión Proteica , Pirazoles/síntesis química , Pirazoles/metabolismo , Sirtuina 1/metabolismo , Sirtuina 2/metabolismo , Relación Estructura-ActividadRESUMEN
Sirtuins are NAD(+)-dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ϵ-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated microtiter plates and make it possible to screen for inhibitors of either smSirt2 or its human isoform (hSirt2) for selectivity studies. We also present hits from a pilot screen with inhibitors showing an IC50 lower than 50 µM. Binding of the hits to their targets is rationalized by docking studies using a homology model of smSirt2.
Asunto(s)
Fluorometría/métodos , Schistosoma mansoni/enzimología , Sirtuina 2/metabolismo , Animales , Sitios de Unión , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Epigénesis Genética/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Conformación Molecular , Niacinamida/química , Niacinamida/farmacología , Unión Proteica , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/genética , Sirtuina 2/antagonistas & inhibidores , Sirtuina 2/genética , Relación Estructura-Actividad , Tirfostinos/química , Tirfostinos/farmacologíaRESUMEN
Sirtuins, NAD(+) -dependent histone deacetylases (HDACs), have recently emerged as potential therapeutic targets for the treatment of a variety of diseases. The discovery of potent and isoform-selective inhibitors of this enzyme family should provide chemical tools to help determine the roles of these targets and validate their therapeutic value. Herein, we report the discovery of a novel class of highly selective SIRT2 inhibitors, identified by pharmacophore screening. We report the identification and validation of 3-((2-methoxynaphthalen-1-yl)methyl)-7-((pyridin-3-ylmethyl)amino)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (ICL-SIRT078), a substrate-competitive SIRT2 inhibitor with a Ki value of 0.62 ± 0.15 µM and more than 50-fold selectivity against SIRT1, 3 and 5. Treatment of MCF-7 breast cancer cells with ICL-SIRT078 results in hyperacetylation of α-tubulin, an established SIRT2 biomarker, at doses comparable with the biochemical IC50 data, while suppressing MCF-7 proliferation at higher concentrations. In concordance with the recent reports that suggest SIRT2 inhibition is a potential strategy for the treatment of Parkinson's disease, we find that compound ICL-SIRT078 has a significant neuroprotective effect in a lactacystin-induced model of Parkinsonian neuronal cell death in the N27 cell line. These results encourage further investigation into the effects of ICL-SIRT078, or an optimised derivative thereof, as a candidate neuroprotective agent in in vivo models of Parkinson's disease.
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Inhibidores de Histona Desacetilasas/química , Fármacos Neuroprotectores/química , Pirimidinonas/química , Sirtuina 2/antagonistas & inhibidores , Tiofenos/química , Animales , Sitios de Unión , Línea Celular , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Evaluación Preclínica de Medicamentos , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Células MCF-7 , Simulación del Acoplamiento Molecular , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Unión Proteica , Estructura Terciaria de Proteína , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , Ratas , Sirtuina 2/metabolismo , Relación Estructura-Actividad , Tiofenos/farmacología , Tiofenos/uso terapéuticoRESUMEN
Acetylation of α-tubulin was studied in cultures of human hepatocytes under the influence of selective inhibitors of histone deacetylases HDAC6 and SIRT-2 - tubastatin A and 2-(3-phenethoxyphenylamino)benzamide, respectively. It was found that in hepatocyte cell line HepG2 acetylated α-tubulin is accumulated preferentially on inhibition of HDAC6 but not of SIRT-2. Under the same conditions, no acetylation of α-tubulin was observed in hepatocyte cell line Huh7. However, the inhibition of HDAC6 with tubastatin A led to hyperacetylation of α-tubulin and simultaneously to decrease in viral RNA concentration in hepatocyte cell line Huh7-luc/neo, which supports propagation of the full genome replicon of hepatitis C virus. The correlation between these two processes points to HDAC6 as a promising cellular target for therapy of hepatitis C.
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Antivirales/farmacología , Hepacivirus/fisiología , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Acetilación , Evaluación Preclínica de Medicamentos , Células Hep G2 , Hepacivirus/efectos de los fármacos , Hepatocitos/virología , Histona Desacetilasa 6 , Histona Desacetilasas/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Replicón/genética , Sirtuina 2/metabolismo , Tubulina (Proteína)/metabolismo , Replicación ViralRESUMEN
OBJECTIVE: Increasing evidence suggests that cinnamon has many health benefits when used in herbal medicine and as a dietary ingredient. The aim of this study was to investigate the effects of an aqueous extract of cinnamon, high in type A polyphenols, on molecular targets in rat C6 glioma cells that underlie their protective effects. METHODS: C6 rat glioma cells were seeded in 35-mm culture dishes or six-well plates, then were incubated with cinnamon polyphenols at doses of 10 and 20 µg/mL for 24 h. The targeting protein expression, secretion, and phosphorylation were evaluated by immunoprecitation/immunoblotting and immunofluorescence imaging. RESULTS: Cinnamon polyphenols significantly enhanced secretion of S100ß, a Ca(2+)-binding protein, and increased intracellular S100ß expression after 24 h of incubation, in rat C6 glioma cells. Cinnamon polyphenols also enhanced protein levels of sirtuin 1, 2, and 3, deacetylases important in cell survival, and the tumor suppressor protein, p53, and inhibited the inflammatory factors, tumor necrosis factor alpha, and phospho-p65, a subunit of nuclear factor-κß. Cinnamon polyphenols also up-regulated levels of phospho-p38, extracellular signal-regulated protein and mitogen-activated protein and kinase-activated protein kinases that may be important for prosurvival functions. CONCLUSION: Our results indicate that the effects of cinnamon polyphenols on upregulating prosurvival proteins, activating mitogen-activated protein kinase pathways, and decreasing proinflammatory cytokines may contribute to their neuroprotective effects.
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Cinnamomum zeylanicum/química , Polifenoles/farmacología , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Sirtuina 1/metabolismo , Sirtuina 2/metabolismo , Animales , Línea Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Glioma/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Ratas , Subunidad beta de la Proteína de Unión al Calcio S100/genética , Sirtuina 1/genética , Sirtuina 2/genética , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies. In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells (MSC) with Nf2 inactivation as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.
Asunto(s)
Neurofibromatosis 2/tratamiento farmacológico , Neurofibromatosis 2/patología , Neurofibromina 2/genética , Células de Schwann/metabolismo , Células de Schwann/patología , Sirtuina 2/antagonistas & inhibidores , Acetilación , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Genes Supresores de Tumor , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Necrosis , Neurofibromatosis 2/genética , Neurofibromatosis 2/metabolismo , Neurofibromina 2/metabolismo , Células de Schwann/efectos de los fármacos , Transducción de Señal , Sirtuina 2/metabolismoRESUMEN
Black chokeberry (Aronia melanocarpa) is a rich source of polyphenols. The hypolipidemic effects of polyphenol-rich black chokeberry extract (CBE) have been reported, but underlying mechanisms have not been well characterized. We investigated the effect of CBE on the expression of genes involved in intestinal lipid metabolism. Caco-2 cells were incubated with 50 or 100 µg/ml of CBE for 24 h for quantitative realtime polymerase chain reaction analysis. Expression of genes for cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase and sterol regulatory element binding protein 2), apical cholesterol uptake (Niemann-Pick C1 Like 1 and scavenger receptor class B Type 1) and basolateral cholesterol efflux [ATP-binding cassette transporter A1 (ABCA1)] was significantly decreased by CBE compared with control. Western blot analysis confirmed that CBE inhibited expression of these proteins. In contrast, CBE markedly induced mRNA and/or protein levels of ABCG5 and ABCG8 that mediate apical cholesterol efflux to the intestinal lumen. Furthermore, CBE significantly increased mRNA and protein levels of low-density lipoprotein (LDL) receptor, and cellular LDL uptake. Expression of genes involved in lipid metabolism and lipoprotein assembly, including sterol regulatory element-binding protein 1c, fatty acid synthase and acyl-CoA oxidase 1, was significantly decreased by CBE in a dose-dependent manner. Concomitantly, CBE significantly increased sirtuin 1, 3 and 5 mRNA levels, while it decreased SIRT-2. Our data suggest that hypolipidemic effects of CBE may be attributed, at least in part, to increased apical efflux of LDL-derived cholesterol and to decreased chylomicron formation in the intestine; and specific isoforms of SIRT may play an important role in this process.