RESUMEN
Benzoisoquinolones are aryl ring extended isoquinolinone derivatives, which are constituents of alkaloid natural products. This report describes the synthesis of novel benzoisoquinolone amino acid/peptide derivatives from the respective N-aryl amino esters/peptides through Ru-catalyzed C(sp2)-H annulation at room temperature. The N-terminal amide acts as an intrinsic directing group and coordinates with the active Ru(II) catalyst for the C-H bond activation and annulation of the aryl ring to produce benzoisoqunolone derivatives. Importantly, these benzoisoquinolinones exhibit fluorescence (QY â¼35%) in protic polar solvents, possibly due to charge transfer, and exhibit cell internalization to the cell nucleus without any significant cytotoxicity to human cell lines (HEK293T). Hence, our results are exceptional to transform standard amino acids/peptides into fluorescent peptides at room temperature in the late stage, which could be applicable for tracking specific target peptides by fluorescence microscopy.
RESUMEN
Isoindolinone is a constituent of several natural products that show a wide range of bioactivity, such as anticancer, antimicrobial, antiviral and anti-inflammatory properties. It would be interesting to explore the carbonyl group (H-bond acceptor) of isoindolinone and its structural and conformational changes. However, the synthesis of isoindolinone-comprising peptides in short steps is challenging. Herein, we have developed a synthetic methodology for introducing the isoindolinone residue to peptides via Pd-catalyzed C(sp2)-H activation/olefination, and demonstrated the conformational changes owing to the isoindolinone scaffold. Hence, isoindolinonyl peptides provide an avenue for the synthesis of novel foldamers and therapeutic agents.
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Paladio , Péptidos , Paladio/química , Catálisis , Conformación Molecular , Péptidos/químicaRESUMEN
AIM: Cabotegravir long-acting (LA) intramuscular (IM) injection is being investigated for HIV preexposure prophylaxis due to its potent antiretroviral activity and infrequent dosing requirement. A subset of healthy adult volunteers participating in a Phase I study assessing cabotegravir tissue pharmacokinetics underwent serial magnetic resonance imaging (MRI) to assess drug depot localization and kinetics following a single cabotegravir LA IM targeted injection. METHODS: Eight participants (four men, four women) were administered cabotegravir LA 600 mg under ultrasonographic-guided injection targeting the gluteal muscles. MRI was performed to determine injection-site location in gluteal muscle (IM), subcutaneous (SC) adipose tissue and combined IM/SC compartments, and to quantify drug depot characteristics, including volume and surface area, on Days 1 (≤2 hours postinjection), 3 and 8. Linear regression analysis examined correlations between MRI-derived parameters and plasma cabotegravir exposure metrics, including maximum observed concentration (Cmax ) and partial area under the concentration-time curve (AUC) through Weeks 4 and 8. RESULTS: Cabotegravir LA depot locations varied by participant and were identified in the IM compartment (n = 2), combined IM/SC compartments (n = 4), SC compartment (n = 1) and retroperitoneal cavity (n = 1). Although several MRI parameter and exposure metric correlations were determined, total depot surface area on Day 1 strongly correlated with plasma cabotegravir concentration at Days 3 and 8, Cmax and partial AUC through Weeks 4 and 8. CONCLUSION: MRI clearly delineated cabotegravir LA injection-site location and depot kinetics in healthy adults. Although injection-site variability was observed, drug depot surface area correlated with both plasma Cmax and partial AUC independently of anatomical distribution.
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Fármacos Anti-VIH , Infecciones por VIH , Imágenes de Resonancia Magnética Multiparamétrica , Adulto , Dicetopiperazinas , Femenino , Infecciones por VIH/tratamiento farmacológico , Humanos , Inyecciones Intramusculares , Cinética , Masculino , Piridonas , VoluntariosRESUMEN
Natural aromatic α-amino acid residues play critical roles in the structural and functional organization of proteins owing to π-interactions. Their aromatic residues are derived from benzenoid scaffolds. Non-benzenoid aromatic scaffolds such as tropone and tropolone are also constituents of troponoid natural products. Tropolone has also the ability to exhibit π-interactions along with additional hydrogen bonding. Thus, amino acids comprising troponyl could be potential building blocks of novel peptidomimetics. This report describes the synthesis of the L-aminotroponylalanine amino acid (ATA) and its unusual activity with the peptide coupling agent EDC. Importantly, its di-peptides form ß-sheet/-turn type secondary structures in organic solvents owing to the troponyl residue. This amino acid is an excellent scaffold for the synthesis of fluorescent amino acids such as BODIPY amino acid analogs. Nevertheless, this amino acid and its BODIPY derivatives can enter HeLa cells without exhibiting significant cytotoxicity at low concentrations (â¼50 µM). Hence, ATA and its BODIPY derivatives are promising aromatic amino acids for the construction of potential peptidomimetics and fluorescent labelling of target peptides.
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Alanina , Aminoácidos , Humanos , Células HeLa , Péptidos/farmacologíaRESUMEN
The synthetic unnatural amino acids and their peptides as peptidomimetics have shown remarkable structural and functional properties. In the repertoire of synthetic peptides, pseudopeptides have emerged as attractive small peptidomimetics that are capable of forming the characteristic secondary structures in the solid/solution phase, as in natural peptides. This report describes the synthesis and structural analyses of novel pseudopeptides as ethylenediprolyl (etpro) tetra/hexapeptides, comprising a chiral diaminedicarboxylate scaffold. Their NMR and CD spectral analyses strongly support the formation of the ß-turn-type structures in organic solvents (ACN/MeOH). Further, the single-crystal X-ray studies of tetrapseudopeptide confirm the formation of a unique self-assembly structure as ß-strand type in the solid state through hydrogen bonding. Importantly, their diamine moiety influences the formation of Cu-complexes with Cu(II) ions. A tetrapseudopeptide monocarboxylate-Cu(II) complex forms the single crystal that is studied by the single-crystal X-ray diffractometer. The crystal structure of the tetrapseudopeptide-Cu(II) complex confirms the formation of the distorted square planar geometry structure, almost like the amyloid ß(Aß)-peptide-Cu(II) complex structural geometry. Hence, these etpro-pseudopeptides are emerging peptidomimatics that form ß-turn types of structures and metal complexes mainly with Cu(II) ions. These molecules could be considered for the development of peptide-based catalysts and peptide-based therapeutic drug candidates.
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Complejos de Coordinación , Péptidos beta-Amiloides , Cobre , Cristalografía por Rayos X , Enlace de Hidrógeno , IonesRESUMEN
Isoindolinone is a constituent of various natural products and synthetic biologically active compounds. The classical multi-step synthetic methods used to prepare various indolinone derivatives are tedious and challenging. One-pot synthetic methods are attractive and economical. Transition-metal-catalyzed C-H activation is an emerging tool for synthesizing natural products and small organic molecules via reducing the number of synthetic steps necessary. This paper describes the synthesis of N-alkyl-3-methenyl chiral isoindolinone derivatives from aryl amides of L-amino acids and non-activated alkene via Pd-catalyzed C(sp2)-H olefination. Herein, the amino acid residue acts as a directing group for olefination at the aryl ring, and then cyclization occurs at the amide NH. Hence, this methodology could be helpful to transform standard amino acids into respective chiral isoindolinone derivatives.
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Alquenos/química , Amidas/química , Aminoácidos/química , Paladio/química , Ftalimidas/síntesis química , Alquilación , Catálisis , Ciclización , Ésteres , Estructura MolecularRESUMEN
Since the arrival of DNA nanotechnology nearly 40 years ago, the field has progressed from its beginnings of envisioning rather simple DNA structures having a branched, multi-strand architecture into creating beautifully complex structures comprising hundreds or even thousands of unique strands, with the possibility to exactly control the positions down to the molecular level. While the earliest construction methodologies, such as simple Holliday junctions or tiles, could reasonably be designed on pen and paper in a short amount of time, the advent of complex techniques, such as DNA origami or DNA bricks, require software to reduce the time required and propensity for human error within the design process. Where available, readily accessible design software catalyzes our ability to bring techniques to researchers in diverse fields and it has helped to speed the penetration of methods, such as DNA origami, into a wide range of applications from biomedicine to photonics. Here, we review the historical and current state of CAD software to enable a variety of methods that are fundamental to using structural DNA technology. Beginning with the first tools for predicting sequence-based secondary structure of nucleotides, we trace the development and significance of different software packages to the current state-of-the-art, with a particular focus on programs that are open source.
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ADN/química , Nanoestructuras/química , Nanotecnología/métodos , Conformación de Ácido Nucleico , Programas InformáticosRESUMEN
The pathophysiology of human immunodeficiency virus (HIV)-associated cardiomyopathy remains uncertain. We used HIV-1 transgenic (Tg26) mice to explore mechanisms by which HIV-related proteins impacted on myocyte function. Compared to adult ventricular myocytes isolated from nontransgenic (wild type [WT]) littermates, Tg26 myocytes had similar mitochondrial membrane potential (ΔΨ m ) under normoxic conditions but lower Δ Ψ m after hypoxia/reoxygenation (H/R). In addition, Δ Ψ m in Tg26 myocytes failed to recover after Ca 2+ challenge. Functionally, mitochondrial Ca 2+ uptake was severely impaired in Tg26 myocytes. Basal and maximal oxygen consumption rates (OCR) were lower in normoxic Tg26 myocytes, and further reduced after H/R. Complex I subunit and ATP levels were lower in Tg26 hearts. Post-H/R, mitochondrial superoxide (O 2â¢- ) levels were higher in Tg26 compared to WT myocytes. Overexpression of B-cell lymphoma 2-associated athanogene 3 (BAG3) reduced O 2â¢- levels in hypoxic WT and Tg26 myocytes back to normal. Under normoxic conditions, single myocyte contraction dynamics were similar between WT and Tg26 myocytes. Post-H/R and in the presence of isoproterenol, myocyte contraction amplitudes were lower in Tg26 myocytes. BAG3 overexpression restored Tg26 myocyte contraction amplitudes to those measured in WT myocytes post-H/R. Coimmunoprecipitation experiments demonstrated physical association of BAG3 and the HIV protein Tat. We conclude: (a) Under basal conditions, mitochondrial Ca 2+ uptake, OCR, and ATP levels were lower in Tg26 myocytes; (b) post-H/R, Δ Ψ m was lower, mitochondrial O 2â¢- levels were higher, and contraction amplitudes were reduced in Tg26 myocytes; and (c) BAG3 overexpression decreased O 2â¢- levels and restored contraction amplitudes to normal in Tg26 myocytes post-H/R in the presence of isoproterenol.
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Cardiomiopatías/metabolismo , Metabolismo Energético , Infecciones por VIH/complicaciones , VIH-1/genética , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Cardiomiopatías/virología , Hipoxia de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Infecciones por VIH/virología , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias Cardíacas/virología , Contracción Miocárdica , Miocitos Cardíacos/virología , Oxidación-Reducción , Estrés Oxidativo , Consumo de Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Función Ventricular IzquierdaRESUMEN
Cardiovascular disease remains a leading cause of morbidity and mortality in HIV-positive patients, even in those whose viral loads are well controlled with antiretroviral therapy. However, the underlying molecular events responsible for the development of cardiac disease in the setting of HIV remain unknown. The HIV-encoded Tat protein plays a critical role in the activation of HIV gene expression and profoundly impacts homeostasis in both HIV-infected cells and uninfected cells that have taken up released Tat via a bystander effect. Since cardiomyocyte function, including excitation-contraction coupling, greatly depends on energy provided by the mitochondria, in this study, we performed a series of experiments to assess the impact of Tat on mitochondrial function and bioenergetics pathways in a primary cell culture model derived from neonatal rat ventricular cardiomyocytes (NRVCs). Our results show that the presence of Tat in cardiomyocytes is accompanied by a decrease in oxidative phosphorylation, a decline in the levels of ATP, and an accumulation of reactive oxygen species (ROS). Tat impairs the uptake of mitochondrial Ca2+ ([Ca2+ ]m ) and the electrophysiological activity of cardiomyocytes. Tat also affects the protein clearance pathway and autophagy in cardiomyocytes under stress due to hypoxia-reoxygenation conditions. A reduction in the level of ubiquitin along with dysregulated degradation of autophagy proteins including SQSTM1/p62 and a reduction of LC3 II were detected in cardiomyocytes harboring Tat. These results suggest that, by targeting mitochondria and protein quality control, Tat significantly impacts bioenergetics and autophagy resulting in dysregulation of cardiomyocyte health and homeostasis.
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Metabolismo Energético , VIH-1/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Autofagia , Calcio/metabolismo , Canales de Calcio/metabolismo , Hipoxia de la Célula , Células Cultivadas , Interacciones Huésped-Patógeno , Potenciales de la Membrana , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias Cardíacas/virología , Mitofagia , Miocitos Cardíacos/virología , Fosforilación Oxidativa , Cultivo Primario de Células , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Proteína Sequestosoma-1/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid protein that is found predominantly in the heart, skeletal muscle, and many cancers. Deletions and truncations in BAG3 that result in haplo-insufficiency have been associated with the development of dilated cardiomyopathy. To study the cellular and molecular events attributable to BAG3 haplo-insufficiency we generated a mouse in which one allele of BAG3 was flanked by loxP recombination sites (BAG3fl/+ ). Mice were crossed with α-MHC-Cre mice in order to generate mice with cardiac-specific haplo-insufficiency (cBAG3+/-) and underwent bi-weekly echocardiography to assess their cardiac phenotype. By 10 weeks of age, cBAG3+/- mice demonstrated increased heart size and diminished left ventricular ejection fraction when compared with non-transgenic littermates (Cre-/- BAG3fl/+ ). Contractility in adult myocytes isolated from cBAG3+/- mice were similar to those isolated from control mice at baseline, but showed a significantly decreased response to adrenergic stimulation. Intracellular calcium ([Ca2+ ]i ) transient amplitudes in myocytes isolated from cBAG3+/- mice were also similar to myocytes isolated from control mice at baseline but were significantly lower than myocytes from control mice in their response to isoproterenol. BAG3 haplo-insufficiency was also associated with decreased autophagy flux and increased apoptosis. Taken together, these results suggest that mice in which BAG3 has been deleted from a single allele provide a model that mirrors the biology seen in patients with heart failure and BAG3 haplo-insufficiency.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores Adrenérgicos beta/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Adrenérgicos/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Calcio/metabolismo , Cardiomiopatía Dilatada/metabolismo , Insuficiencia Cardíaca/metabolismo , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , FenotipoRESUMEN
RATIONALE: SUMOylation plays an important role in cardiac function and can be protective against cardiac stress. Recent studies show that SUMOylation is an integral part of the ubiquitin proteasome system, and expression of the small ubiquitin-like modifier (SUMO) E2 enzyme UBC9 improves cardiac protein quality control. However, the precise role of SUMOylation on other protein degradation pathways, particularly autophagy, remains undefined in the heart. OBJECTIVE: To determine whether SUMOylation affects cardiac autophagy and whether this effect is protective in a mouse model of proteotoxic cardiac stress. METHODS AND RESULTS: We modulated expression of UBC9, a SUMO E2 ligase, using gain- and loss-of-function in neonatal rat ventricular cardiomyocytes. UBC9 expression seemed to directly alter autophagic flux. To confirm this effect in vivo, we generated transgenic mice overexpressing UBC9 in cardiomyocytes. These mice have an increased level of SUMOylation at baseline and, in confirmation of the data obtained from neonatal rat ventricular cardiomyocytes, demonstrated increased autophagy, suggesting that increased UBC9-mediated SUMOylation is sufficient to upregulate cardiac autophagy. Finally, we tested the protective role of SUMOylation-mediated autophagy by expressing UBC9 in a model of cardiac proteotoxicity, induced by cardiomyocyte-specific expression of a mutant α-B-crystallin, mutant CryAB (CryAB(R120G)), which shows impaired autophagy. UBC9 overexpression reduced aggregate formation, decreased fibrosis, reduced hypertrophy, and improved cardiac function and survival. CONCLUSIONS: The data showed that increased UBC9-mediated SUMOylation is sufficient to induce relatively high levels of autophagy and may represent a novel strategy for increasing autophagic flux and ameliorating morbidity in proteotoxic cardiac disease.
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Cardiomiopatías/metabolismo , Miocitos Cardíacos/metabolismo , Sumoilación , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Autofagia , Cardiomiopatías/genética , Células Cultivadas , Ratones , Ratas , Ratas Sprague-Dawley , Enzimas Ubiquitina-Conjugadoras/genética , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Mitochondrial abnormalities impact the development of myofibrillar myopathies. Therefore, understanding the mechanisms underlying the removal of dysfunctional mitochondria from cells is of great importance toward understanding the molecular events involved in the genesis of cardiomyopathy. Earlier studies have ascribed a role for BAG3 in the development of cardiomyopathy in experimental animals leading to the identification of BAG3 mutations in patients with heart failure which may play a part in the onset of disease development and progression. BAG3 is co-chaperone of heat shock protein 70 (HSP70), which has been shown to modulate apoptosis and autophagy, in several cell models. In this study, we explore the potential role of BAG3 in mitochondrial quality control. We demonstrate that siRNA mediated suppression of BAG3 production in neonatal rat ventricular cardiomyocytes (NRVCs) significantly elevates the level of Parkin, a key component of mitophagy. We found that both BAG3 and Parkin are recruited to depolarized mitochondria and promote mitophagy. Suppression of BAG3 in NRVCs significantly reduces autophagy flux and eliminates clearance of Tom20, an essential import receptor for mitochondria proteins, after induction of mitophagy. These observations suggest that BAG3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in BAG3 expression impact cardiomyocyte function. J. Cell. Physiol. 232: 797-805, 2017. © 2016 Wiley Periodicals, Inc.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Mitocondrias/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Línea Celular , Metabolismo Energético , Técnicas de Silenciamiento del Gen , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitofagia/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Transporte de Proteínas/efectos de los fármacos , Ratas Sprague-Dawley , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
In an attempt to develop potent anti-tubulin agents against most dreadful disease cancer, a library of 28 novel triazole tethered isatin-coumarin hybrids were synthesized by click chemistry approach. Synthesized hybrids were characterized and evaluated against a panel of human cancer cell lines viz. THP-1, COLO-205, HCT-116 and PC-3. Biological assay unveiled that, compounds A-1 to A-6, B-1 to B-4 and C-1 to C-3 displayed significant inhibitory potential against THP-1, COLO-205 and HCT-116 cell lines which were more sensitive towards the designed hybrids. PC-3 among these cell lines was found to be almost resistant. Established SAR revealed marked dependence of the cytotoxic activity on the type of substituent on isatin and the length of carbon-bridge connecting isatin moiety with triazole ring. Unsubstituted isatin and two carbon-bridge were found to be crucial for cytotoxicity. Three most potent hybrids (A-1, A-2 and B-1) were further tested for tubulin polymerization inhibition. Among these three compounds, A-1 found to be endowed with most prominent tubulin polymerization inhibition potential with IC50 value of 1.06µM which was further confirmed by using confocal microscopy. Possible binding interactions between the most potent hybrid molecule A-1 and tubulin were also divulged by molecular modeling studies.
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Antineoplásicos/farmacología , Cumarinas/farmacología , Diseño de Fármacos , Isatina/farmacología , Triazoles/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cumarinas/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Isatina/química , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad , Triazoles/químicaRESUMEN
A library of forty 7,8-benzoflavone derivatives was synthesized and evaluated for their inhibitory potential against cholesterol esterase (CEase). Among all the synthesized compounds seven benzoflavone derivatives (A-7, A-8, A-10, A-11, A-12, A-13, A-15) exhibited significant inhibition against CEase in in vitro enzymatic assay. Compound A-12 showed the most promising activity with IC50 value of 0.78nM against cholesterol esterase. Enzyme kinetic studies carried out for A-12, revealed its mixed-type inhibition approach. Molecular protein-ligand docking studies were also performed to figure out the key binding interactions of A-12 with the amino acid residues of the enzyme's active site. The A-12 fits well at the catalytic site and is stabilized by hydrophobic interactions. It completely blocks the catalytic assembly of CEase and prevents it to participate in ester hydrolysis mechanism. The favorable binding conformation of A-12 suggests its prevailing role as CEase inhibitor.
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Benzoflavonas/química , Inhibidores Enzimáticos/química , Esterol Esterasa/antagonistas & inhibidores , Benzoflavonas/síntesis química , Benzoflavonas/metabolismo , Sitios de Unión , Dominio Catalítico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Cinética , Simulación del Acoplamiento Molecular , Unión Proteica , Esterol Esterasa/metabolismo , Relación Estructura-ActividadRESUMEN
Bag5 is a member of the BAG family of molecular chaperone regulators and is unusual in that it consists of five BAG domains, which function as modulators of chaperone activity. Bag family proteins play a key role in cellular as well as in cardiac function and their differential expression is reported in heart failure. In this study, we examined the importance of a Bag family member protein, Bag5, in cardiomyocytes during endoplasmic reticulum (ER) stress. We found that expression of Bag5 in cardiomyocytes is significantly increased with the induction of ER stress in a time dependent manner. We have taken gain-in and loss-of functional approaches to characterize Bag5 protein function in cardiomyocytes. Adenoviral mediated expression of Bag5 significantly decreased cell death as well as improved cellular viability in ER stress. Along with this, ER stress-induced CHOP protein expression is significantly decreased in cells that overexpress Bag5. Conversely, we found that siRNA-mediated knockdown of Bag5 caused cell death, increased cytotoxicity, and decreased cellular viability in cardiomyocytes. Mechanistically, we found that Bag5 protein expression is significantly increased in the ER during ER stress and that this in turn modulates GRP78 protein stability and reduces ER stress. This study suggests that Bag5 is an important regulator of ER function and so could be exploited as a tool to improve cardiomyocyte function under stress conditions. J. Cell. Biochem. 117: 1813-1821, 2016. © 2016 Wiley Periodicals, Inc.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Estrés del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Choque Térmico/genética , Estabilidad Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
RATIONALE: Impairment of proteasomal function is pathogenic in several cardiac proteinopathies and can eventually lead to heart failure. Loss of proteasomal activity often results in the accumulation of large protein aggregates. The ubiquitin proteasome system (UPS) is primarily responsible for cellular protein degradation, and although the role of ubiquitination in this process is well studied, the function of an ancillary post-translational modification, SUMOylation, in protein quality control is not fully understood. OBJECTIVE: To determine the role of ubiquitin-conjugating enzyme 9 (UBC9), a small ubiquitin-like modifier-conjugating enzyme, in cardiomyocyte protein quality control. METHODS AND RESULTS: Gain- and loss-of-function approaches were used to determine the importance of UBC9. Overexpression of UBC9 enhanced UPS function in cardiomyocytes, whereas knockdown of UBC9 by small interfering RNA caused significant accumulations of aggregated protein. UPS function and relative activity was analyzed using a UPS reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein (GFPu). Subsequently, the effects of UBC9 on UPS function were tested in a proteotoxic model of desmin-related cardiomyopathy, caused by cardiomyocyte-specific expression of a mutated αB crystallin, CryAB(R120G). CryAB(R120G) expression leads to aggregate formation and decreased proteasomal function. Coinfection of UBC9-adenovirus with CryAB(R120G) virus reduced the proteotoxic sequelae, decreasing overall aggregate concentrations. Conversely, knockdown of UBC9 significantly decreased UPS function in the model and resulted in increased aggregate levels. CONCLUSIONS: UBC9 plays a significant role in cardiomyocyte protein quality control, and its activity can be exploited to reduce toxic levels of misfolded or aggregated proteins in cardiomyopathy.
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Miocitos Cardíacos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Mutación , Miocitos Cardíacos/citología , Cultivo Primario de Células , Procesamiento Proteico-Postraduccional , Proteolisis , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Sumoilación , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitinación , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Keeping in view the limitations associated with currently available anticancer drugs, molecular hybrids of mono carbonyl curcumin and isatin tethered by triazole ring have been synthesized and evaluated for in vitro cytotoxicity against THP-1, COLO-205, HCT-116, A549, HeLa, CAKI-I, PC-3, MiaPaca-2 human cancer cell lines. The results revealed that the compounds SA-1 to SA-9, SB-2, SB-3, SB-4, SB-7 and SC-2 showed a good range of IC50 values against THP-1, COLO-205, HCT-116 and PC-3 cell lines, while the other four cell lines among these were found to be almost resistant. Structure activity relationship revealed that the nature of Ring X and substitution at position R influences the activity. Methoxy substituted phenyl ring as Ring X and H as R were found to be the ideal structural features. The most potent compounds (SA-2, SA-3, SA-4, SA-7) were further tested for tubulin inhibition. Compound SA-2 was found to significantly inhibit the tubulin polymerization (IC50=1.2 µM against HCT-116). Compound SA-2, moreover, lead to the disruption of microtubules as confirmed by immunofluorescence technique. The significant cytotoxicity and tubulin inhibition by SA-2 was streamlined by molecular modeling studies where it was docked at the curcumin binding site of tubulin.
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Curcumina/química , Diseño de Fármacos , Isatina/química , Triazoles/química , Moduladores de Tubulina/síntesis química , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Microscopía Fluorescente , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
Tuberculosis (TB) is still a major health concern worldwide. The increasing incidences of multi-drug-resistant tuberculosis (MDR-TB) necessitate the development of new anti-TB drugs acting via novel mode of action. The search of newer drugs for TB led to the identification of several quinoline-based antimycobacterial agents against both the drug-sensitive and MDR-TB. These agents have been designed by substituting quinoline scaffold with diverse chemical functionalities as well as by modifying quinoline/quinolone-based antibacterial drugs. Several of quinoline/quinolone derivatives displayed excellent antimycobacterial activity and were found free of cytotoxicity. This review highlights the critical aspects of design and structure-activity relationship of quinoline- and quinolone-based antimycobacterial agents.
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Antibacterianos/química , Antibacterianos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Quinolinas/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Quinolinas/farmacología , Relación Estructura-ActividadRESUMEN
Cardiac myosin binding protein C (cMyBP-C) is an integral sarcomeric protein that associates with the thick, thin, and titin filament systems in the contractile apparatus. Three different isoforms of MyBP-C exist in mammalian muscle: slow skeletal (MyBPC1), fast skeletal (MyBP-C2, with several variants), and cardiac (cMyBP-C). Genetic screening studies show that mutations in MYBPC3 occur frequently and are responsible for as many as 30-35 % of identified cases of familial hypertrophic cardiomyopathy. The function of cMyBP-C is stringently regulated by its post-translational modification. In particular, the addition of phosphate groups occurs with high frequency on certain serine residues that are located in the cardiac-specific regulatory M domain. Phosphorylation of this domain has been extensively studied in vitro and in vivo. Phosphorylation of the M domain can regulate the manner in which actin and myosin interact, affecting the cross bridge cycle and ultimately, cardiac hemodynamics.
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Proteínas Portadoras/metabolismo , Contracción Miocárdica/fisiología , Actinas/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hemodinámica , Humanos , Miocardio/metabolismo , Miofibrillas/metabolismo , Miosinas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Sarcómeros/metabolismoRESUMEN
In view of reported xanthine oxidase inhibitory potential of naphthopyrans and flavones, naphthoflavones as hybrids of the two were designed, synthesized and evaluated for in vitro xanthine oxidase inhibitory activity in the present study. The results of the assay revealed that the naphthoflavones possess promising inhibitory potential against the enzyme with IC50 values ranging from 0.62 to 41.2 µM. Structure activity relationship indicated that the nature and placement of substituents on the phenyl ring at 2nd position remarkably influences the inhibitory activity. Substitution of halo and nitro groups at ortho and para position of the phenyl ring (2nd position) remarkably favored the activity. NF-4 with p-fluoro phenyl ring was the most potent inhibitor with IC50 value of 0.62 µM. Enzyme kinetics study was also performed to investigate the inhibition mechanism and it was found that the naphthoflavones displayed mixed type inhibition. The basis of significant inhibition of xanthine oxidase by NF-4 was rationalized by molecular modeling studies.