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1.
Cell ; 177(5): 1243-1251.e12, 2019 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-31080070

RESUMEN

The crystal structure of the ß2-adrenergic receptor (ß2AR) bound to the G protein adenylyl cyclase stimulatory G protein (Gs) captured the complex in a nucleotide-free state (ß2AR-Gsempty). Unfortunately, the ß2AR-Gsempty complex does not provide a clear explanation for G protein coupling specificity. Evidence from several sources suggests the existence of a transient complex between the ß2AR and GDP-bound Gs protein (ß2AR-GsGDP) that may represent an intermediate on the way to the formation of ß2AR-Gsempty and may contribute to coupling specificity. Here we present a structure of the ß2AR in complex with the carboxyl terminal 14 amino acids from Gαs along with the structure of the GDP-bound Gs heterotrimer. These structures provide evidence for an alternate interaction between the ß2AR and Gs that may represent an intermediate that contributes to Gs coupling specificity.


Asunto(s)
Adenilil Ciclasas/química , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Modelos Moleculares , Receptores Adrenérgicos beta 2/química , Humanos , Relación Estructura-Actividad
2.
Nature ; 629(8011): 474-480, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38600384

RESUMEN

The µ-opioid receptor (µOR) is an important target for pain management1 and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of Gi.


Asunto(s)
Ligandos , Conformación Proteica , Receptores Opioides mu , Humanos , beta-Arrestina 1/química , beta-Arrestina 1/metabolismo , Sitios de Unión , Transferencia Resonante de Energía de Fluorescencia , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Guanosina Difosfato/metabolismo , Guanosina Difosfato/química , Modelos Moleculares , Unión Proteica , Receptores Opioides mu/metabolismo , Receptores Opioides mu/química , Imagen Individual de Molécula
3.
Neurobiol Dis ; 184: 106228, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37454781

RESUMEN

Armcx1 is highly expressed in the brain and is located in the mitochondrial outer membrane of neurons, where it mediates mitochondrial transport. Mitochondrial transport promotes the removal of damaged mitochondria and the replenishment of healthy mitochondria, which is essential for neuronal survival after traumatic brain injury (TBI). This study investigated the role of Armcx1 and its potential regulator(s) in secondary brain injury (SBI) after TBI. An in vivo TBI model was established in male C57BL/6 mice via controlled cortical impact (CCI). Adeno-associated viruses (AAVs) with Armcx1 overexpression and knockdown were constructed and administered to mice via stereotactic cortical injection. Exogenous miR-223-3p mimic or inhibitor was transfected into cultured cortical neurons, which were then scratched to simulate TBI in vitro. It was found that Armcx1 expression decreased significantly, while miR-223-3p levels increased markedly in peri-lesion tissues after TBI. The overexpression of Armcx1 significantly reduced TBI-induced neurological dysfunction, neuronal cell death, mitochondrial dysfunction, and axonal injury, while the knockdown of Armcx1 had the opposite effect. Armcx1 was potentially a direct target of miR-223-3p. The miR-223-3p mimic obviously reduced the Armcx1 protein level, while the miR-223-3p inhibitor had the opposite effect. Finally, the miR-223-3p inhibitor dramatically improved mitochondrial membrane potential (MMP) and increased the total length of the neurites without affecting branching numbers. In summary, our results suggest that the decreased expression of Armcx1 protein in neurons after experimental TBI aggravates secondary brain injury, which may be regulated by miR-223-3p. Therefore, this study provides a potential therapeutic approach for treating TBI.


Asunto(s)
Proteínas del Dominio Armadillo , Lesiones Traumáticas del Encéfalo , MicroARNs , Proteínas Mitocondriales , Animales , Masculino , Ratones , Lesiones Traumáticas del Encéfalo/metabolismo , Muerte Celular , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Mitocondrias/metabolismo , Proteínas del Dominio Armadillo/metabolismo , Proteínas Mitocondriales/metabolismo
4.
J Biol Chem ; 296: 100538, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33722610

RESUMEN

The protein tyrosine phosphatase SHP2 mediates multiple signal transductions in various cellular pathways, controlled by a variety of upstream inputs. SHP2 dysregulation is causative of different types of cancers and developmental disorders, making it a promising drug target. However, how SHP2 is modulated by its different regulators remains largely unknown. Here, we use single-molecule fluorescence resonance energy transfer and molecular dynamics simulations to investigate this question. We identify a partially open, semiactive conformation of SHP2 that is intermediate between the known open and closed states. We further demonstrate a "multiple gear" regulatory mechanism, in which different activators (e.g., insulin receptor substrate-1 and CagA), oncogenic mutations (e.g., E76A), and allosteric inhibitors (e.g., SHP099) can shift the equilibrium of the three conformational states and regulate SHP2 activity to different levels. Our work reveals the essential role of the intermediate state in fine-tuning the activity of SHP2, which may provide new opportunities for drug development for relevant cancers.


Asunto(s)
Calgranulina A/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Piperidinas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirimidinas/metabolismo , Regulación Alostérica , Humanos , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética
5.
Biochem Biophys Res Commun ; 621: 80-87, 2022 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-35810595

RESUMEN

A fat-rich diet triggers obesity, and promotes cardiomyocyte injury. Till now, no prior investigations suggested a beneficial role of Isosteviol Sodium (STVNa) in cardiac activity in high fat diet (HFD)-exposed obese rats. However, there is evidence that STVNa accelerates healing of multiple tissue injuries. Herein, we explored the underlying mechanism behind the STVNa-based protection against HFD-induced myocardial dysfunction (MCD) in a rat model of myocardial injury. We employed dosages of 1, 10, and 20 mg/kg STVNa to treat MCD in rats fed with a HFD. Based on our results, STVNa repressed MCD (as indicated by ecocardiographic analysis), myocardium function, pathological structure, and myocardial enzymes. Mechanistically, the STVNa-mediated protection against HFD-induced MCD involved inhibition of inflammation and oxidative stress. Furthermore, using Western blot analysis, we revealed that the critical members of the Sirt1/AMPK network were markedly activated in the STVNa-treated group, relative to HFD-fed controls. Collectively, these evidences suggested that the STVNa offered strong protection against HFD-induced MCD. Moreover, this effect was mediated by the activation of the Sirt1/AMPK network, which, in turn, promoted lipid metabolism.


Asunto(s)
Cardiomiopatías , Sirtuina 1 , Proteínas Quinasas Activadas por AMP , Animales , Colesterol , Dieta Alta en Grasa/efectos adversos , Diterpenos de Tipo Kaurano , Obesidad , Ratas , Sirtuina 1/metabolismo , Sodio
6.
Nat Chem Biol ; 16(5): 507-512, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32152538

RESUMEN

The α2 adrenergic receptors (α2ARs) are G protein-coupled receptors (GPCRs) that respond to adrenaline and noradrenaline and couple to the Gi/o family of G proteins. α2ARs play important roles in regulating the sympathetic nervous system. Dexmedetomidine is a highly selective α2AR agonist used in post-operative patients as an anxiety-reducing, sedative medicine that decreases the requirement for opioids. As is typical for selective αAR agonists, dexmedetomidine consists of an imidazole ring and a substituted benzene moiety lacking polar groups, which is in contrast to ßAR-selective agonists, which share an ethanolamine group and an aromatic system with polar, hydrogen-bonding substituents. To better understand the structural basis for the selectivity and efficacy of adrenergic agonists, we determined the structure of the α2BAR in complex with dexmedetomidine and Go at a resolution of 2.9 Å by single-particle cryo-EM. The structure reveals the mechanism of α2AR-selective activation and provides insights into Gi/o coupling specificity.


Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 2/química , Dexmedetomidina/química , Receptores Adrenérgicos alfa 2/química , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Animales , Sitios de Unión , Microscopía por Crioelectrón , Dexmedetomidina/metabolismo , Dexmedetomidina/farmacología , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Insectos/citología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Complejos Multiproteicos/química , Receptores Adrenérgicos alfa 2/genética , Simpaticolíticos/química , Simpaticolíticos/farmacología
7.
Pharmacol Res ; 184: 106452, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36116706

RESUMEN

Microtubules, a highly dynamic cytoskeleton, participate in many cellular activities including mechanical support, organelles interactions, and intracellular trafficking. Microtubule organization can be regulated by modification of tubulin subunits, microtubule-associated proteins (MAPs) or agents modulating microtubule assembly. Increasing studies demonstrate that microtubule disorganization correlates with various cardiocerebrovascular diseases including heart failure and ischemic stroke. Microtubules also mediate intracellular transport as well as intercellular transfer of mitochondria, a power house in cells which produce ATP for various physiological activities such as cardiac mechanical function. It is known to all that both microtubules and mitochondria participate in the progression of cancer and Parkinson's disease. However, the interconnections between the microtubules and mitochondrial networks in cardiocerebrovascular diseases remain unclear. In this paper, we will focus on the roles of microtubules in cardiocerebrovascular diseases, and discuss the interplay of mitochondria and microtubules in disease development and treatment. Elucidation of these issues might provide significant diagnostic value as well as potential targets for cardiocerebrovascular diseases.


Asunto(s)
Microtúbulos , Tubulina (Proteína) , Adenosina Trifosfato/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitocondrias/metabolismo , Tubulina (Proteína)/metabolismo
8.
J Cell Mol Med ; 25(2): 1166-1177, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33336505

RESUMEN

Pathological hypertrophy contributes to heart failure and there is not quite effective treatment to invert this process. Isosteviol has been shown to protect the heart against ischaemia-reperfusion injury and isoproterenol-induced cardiac hypertrophy, but its effect on pressure overload-induced cardiac hypertrophy is still unknown. Pressure overload induced by transverse aortic constriction (TAC) causes cardiac hypertrophy in rats to mimic the pathological condition in human. This study examined the effects of isosteviol sodium (STVNa) on cardiac hypertrophy by the TAC model and cellular assays in vitro. Cardiac function test, electrocardiogram analysis and histological analysis were conducted. The effects of STVNa on calcium transient of the adult rat ventricular cells and the proliferation of neonatal rat cardiac fibroblasts were also studied in vitro. Cardiac hypertrophy was observed after 3-week TAC while the extensive cardiac dysfunction and electronic remodelling were observed after 9-week TAC. Both STVNa and sildenafil (positive drug) treatment reversed the two process, but STVNa appeared to be more superior in some aspects and did not change calcium transient considerably. STVNa also reversed TAC-induced cardiac fibrosis in vivo and TGF-ß1-induced fibroblast proliferation in vitro. Moreover, STVNa, but not sildenafil, reversed impairment of the autonomic nervous system induced by 9-week TAC.


Asunto(s)
Aorta/fisiopatología , Cardiotónicos/farmacología , Diterpenos de Tipo Kaurano/farmacología , Remodelación Ventricular/efectos de los fármacos , Actinas/metabolismo , Animales , Aorta/diagnóstico por imagen , Aorta/efectos de los fármacos , Sistema Nervioso Autónomo/efectos de los fármacos , Sistema Nervioso Autónomo/fisiopatología , Peso Corporal/efectos de los fármacos , Cardiomegalia/fisiopatología , Constricción , Electrocardiografía , Fenómenos Electrofisiológicos/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Fibrosis , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Tamaño de los Órganos/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Presión , Ratas Wistar
9.
J Am Chem Soc ; 143(42): 17566-17576, 2021 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-34663067

RESUMEN

The ß2-adrenergic receptor (ß2AR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. ß2AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length ß2AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length ß2AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length ß2ARs with distinct modification patterns, including a full-length ß2AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length ß2AR receptors, we found that different phosphorylation patterns mediate different interactions with ß-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between ß2AR and ß-arrestin1. Access to full-length ß2AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of ß2AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.


Asunto(s)
Procesamiento Proteico-Postraduccional , Receptores Adrenérgicos beta 2/química , Regulación Alostérica , Aminoaciltransferasas/química , Proteínas Bacterianas/química , Cisteína Endopeptidasas/química , Humanos , Fosforilación , Receptores Adrenérgicos beta 2/metabolismo , Staphylococcus aureus/enzimología , Ubiquitinación , beta-Arrestina 1/metabolismo
10.
J Cell Mol Med ; 24(18): 10866-10875, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32757458

RESUMEN

Cardiomyocyte dysfunction is attributed to excess oxidative damage, but the molecular pathways involved in this process have not been completely elucidated. Evidence indicates that isosteviol sodium (STVNa) has cardioprotective effects. We therefore aimed to identify the effect of STVNa on cardiomyocytes, as well as the potential mechanisms involved in this process. We established two myocardial hypertrophy models by treating H9c2 cells with high glucose (HG) and isoprenaline (ISO). Our results showed that STVNa reduced H9c2 mitochondrial damage by attenuating oxidative damage and altering the morphology of mitochondria. The results also indicated that STVNa had a positive effect on HG- and ISO-induced damages via mitochondrial biogenesis. The protective effects of STVNa on cardiomyocytes were associated with the regulation of the SIRT1/PGC-1α signalling pathway. Importantly, the effects of STVNa involved different methods of regulation in the two models, which was confirmed by experiments using an inhibitor and activator of SIRT1. Together, the results provide the basis for using STVNa as a therapy for the prevention of cardiomyocyte dysfunctions.


Asunto(s)
Cardiotónicos/farmacología , Diterpenos de Tipo Kaurano/farmacología , Miocitos Cardíacos/efectos de los fármacos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/fisiología , Transducción de Señal/efectos de los fármacos , Sirtuina 1/fisiología , Animales , Carbazoles/farmacología , Línea Celular , ADN Mitocondrial/metabolismo , ADN Mitocondrial/fisiología , ADN Mitocondrial/ultraestructura , Glucosa/toxicidad , Hipertrofia , Isoproterenol/toxicidad , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Biogénesis de Organelos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Resveratrol/farmacología , Sirtuina 1/efectos de los fármacos
11.
Biol Pharm Bull ; 43(8): 1172-1178, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32741937

RESUMEN

The sodium salt of isosteviol (STVNa) is a beyerane diterpene synthesized through acid hydrolysis of stevioside. STVNa improves multiple types of tissue injuries. However, it is not known how isosteviol sodium affects high-fat and high cholesterol diet (HFD)-induced kidney. Therefore, in this study we examined the potential molecular mechanism underlying STVNa mediated protective effect against high fat/high cholesterol-induced kidney dysfunction in HFD-induced kidney injury. Sprague-Dawley (SD) rats were allocated into six groups: the normal group, HFD group and HFD treated with three doses of STVNa, fenofibrate treatment group. The results indicated that HFD induced kidney injury evident by a 60% increase in serum creatinine (CRE) leves. In addition, there was a significant accumulation of triglycerides (approx. 60%), fatty acids (approx. 50%) and total cholesterol (approx. 2.5 fold) in the kidneys. STVNa inhibited HFD-induced kidney injury evident by reducing the increased levels of serum CRE. Specifically, STVNa attenuated HFD-induced kidney injury by inhibiting inflammation, oxidative stress, and apoptosis. These findings indicate that STVNa has a therapeutic potential for HFD-induced kidney dysfunction. The mechanisms of this pharmacological effect are through the inhibition of inflammation, oxidative stress and apoptosis.


Asunto(s)
Apoptosis/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Diterpenos de Tipo Kaurano/farmacología , Inflamación/prevención & control , Enfermedades Renales/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Animales , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/etiología , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley
12.
Int J Mol Sci ; 21(2)2020 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-31968660

RESUMEN

Recent data show that cardiac hypertrophy contributes substantially to the overall heart failure burden. Mitochondrial dysfunction is a common feature of cardiac hypertrophy. Recent studies have reported that isosteviol inhibits myocardial ischemia-reperfusion injury in guinea pigs and H9c2 cells. This work investigated the protective mechanisms of isosteviol sodium (STVNa) against isoproterenol (Iso)-induced cardiac hypertrophy. We found that STVNa significantly inhibited H9c2 cell and rat primary cardiomyocyte cell surface, restored mitochondrial membrane potential (MMP) and morphological integrity, and decreased the expression of mitochondrial function-related proteins Fis1 and Drp1. Furthermore, STVNa decreased reactive oxygen species (ROS) levels and upregulated the expression of antioxidant factors, Thioredoxin 1 (Trx1) and Peroxiredoxin 2 (Prdx2). Moreover, STVNa restored the activity of histone deacetylase 4 (HDAC4) in the nucleus. Together, our data show that STVNa confers protection against Iso-induced myocardial hypertrophy primarily through the Prdx2/ROS/Trx1 signaling pathway. Thus, STVNA is a potentially effective treatment for cardiac hypertrophy in humans.


Asunto(s)
Cardiomegalia/tratamiento farmacológico , Diterpenos de Tipo Kaurano/farmacología , Histona Desacetilasas/metabolismo , Peroxirredoxinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/metabolismo , Animales , Cardiomegalia/inducido químicamente , Insuficiencia Cardíaca/metabolismo , Histona Desacetilasas/genética , Humanos , Isoproterenol/efectos adversos , Mitocondrias/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos
13.
J Proteome Res ; 18(8): 3099-3118, 2019 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-31265301

RESUMEN

Stroke is a leading cause of mortality and disability, and ischemic stroke accounts for more than 80% of the disease occurrence. Timely reperfusion is essential in the treatment of ischemic stroke, but it is known to cause ischemia-reperfusion (I/R) injury and the relevant studies have mostly focused on the acute phase. Here we reported on a global proteomic analysis to investigate the development of cerebral I/R injury in the subacute and long-term phases. A rat model was used, with 2 h-middle cerebral artery occlusion (MCAO) followed with 1, 7, and 14 days of reperfusion. The proteins of cerebral cortex were analyzed by SDS-PAGE, whole-gel slicing, and quantitative LC-MS/MS. Totally 5621 proteins were identified, among which 568, 755, and 492 proteins were detected to have significant dys-regulation in the model groups with 1, 7, and 14 days of reperfusion, respectively, when compared with the corresponding sham groups (n = 4, fold change ≥1.5 or ≤0.67 and p ≤ 0.05). Bioinformatic analysis on the functions and reperfusion time-dependent dys-regulation profiles of the proteins exhibited changes of structures and biological processes in cytoskeleton, synaptic plasticity, energy metabolism, inflammation, and lysosome from subacute to long-term phases of cerebral I/R injury. Disruption of cytoskeleton and synaptic structures, impairment of energy metabolism processes, and acute inflammation responses were the most significant features in the subacute phase. With the elongation of reperfusion time to the long-term phase, a tendency of recovery was detected on cytoskeleton, while inflammation pathways different from the subacute phase were activated. Also, lysosomal structures and functions might be restored. This is the first work reporting the proteome changes that occurred at different time points from the subacute to long-term phases of cerebral I/R injury and we expect it would provide useful information to improve the understanding of the mechanisms involved in the development of cerebral I/R injury and suggest candidates for treatment.


Asunto(s)
Isquemia Encefálica/genética , Proteoma/genética , Proteómica , Daño por Reperfusión/genética , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Corteza Cerebral , Cromatografía Liquida , Modelos Animales de Enfermedad , Metabolismo Energético/genética , Humanos , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/patología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Proteoma/metabolismo , Ratas , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Espectrometría de Masas en Tándem
14.
Org Biomol Chem ; 17(6): 1519-1530, 2019 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-30681116

RESUMEN

We report the identification of 14 novel anticancer agents through established computational anticancer cell-based models. Among these novel hits, the compound G03 exhibits stronger inhibitory effects on the proliferation of MCF-7, HepG2, MDA-MB-231, HCTT116, and HeLa as compared with the FDA-approved sorafenib, with IC50 values of 4.61, 3.20, 2.82, 2.98, and 2.90 µM, respectively. The tubulin protein was validated to be a target of G03 using SPR, tubulin polymerization, immunofluorescence, and western blot assays. G03 is a novel structurally simple anticancer agent with unusual microtubule-stabilizing effects. Our study demonstrated the identification of bioactive small molecules by computational phenotypic modeling, which represents a feasible route toward innovative leads for chemical biology and medicinal chemistry.


Asunto(s)
Antineoplásicos/farmacología , Bioensayo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Simulación del Acoplamiento Molecular , Antineoplásicos/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Conformación Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Interfaz Usuario-Computador
15.
J Stroke Cerebrovasc Dis ; 28(1): 175-184, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30539754

RESUMEN

BACKGROUND: Stevioside, isolated from the herb Stevia rebaudiana, has been widely used as a food sweetener all over the world. Isosteviol Sodium (STV-Na), an injectable formulation of isosteviol sodium salt, has been proved to possess much greater solubility and bioavailability and exhibit protective effects against cerebral ischemia injury in vivo by inhibiting neuron apoptosis. However, the underlying mechanisms of the neuroprotective effects STV-Na are still not completely known. In the present study, we investigated the effects of STV-Na on neuronal cell death caused by hypoxia in vitro and its underlying mechanisms. METHODS: We used cobalt chloride (CoCl2) to expose mouse neuroblastoma N2a cells to hypoxic conditions in vitro. RESULTS: Our results showed that pretreatment with STV-Na (20 µM) significantly attenuated the decrease of cell viability, lactate dehydrogenase release and cell apoptosis under conditions of CoCl2-induced hypoxia. Meanwhile, STV-Na pretreatment significantly attenuated the upregulation of intracellular Ca2+ concentration and reactive oxygen species production, and inhibited mitochondrial depolarization in N2a cells under conditions of CoCl2-induced hypoxia. Furthermore, STV-Na pretreatment significantly downregulated expressions of nitric oxide synthase, interleukin-1ß, tumor necrosis factor-α, interleukin-6, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) signalings in N2a cells under conditions of CoCl2-induced hypoxia. CONCLUSIONS: Taken together, STV-Na protects neural cells against hypoxia-induced apoptosis through inhibiting MAPK and NF-κB pathways.


Asunto(s)
Apoptosis/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Diterpenos de Tipo Kaurano/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Apoptosis/fisiología , Calcio/metabolismo , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cobalto/toxicidad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Neuronas/metabolismo , Neuronas/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos
16.
Biol Pharm Bull ; 41(4): 575-584, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29607930

RESUMEN

Previous reports have indicated that isosteviol sodium (STVNa) has neuroprotective effects against acute focal cerebral ischemia in rats; however, the exact underlying mechanisms and ideal treatment paradigm are not known. To find a reasonable method for STVNa administration and to determine its possible therapeutic mechanisms, we characterized the protective effects of single-dose and multiple-dose STVNa in cerebral ischemic/reperfusion (I/R) injury in rats. Single and multiple treatments with 10 mg/kg STVNa were administered intraperitoneally after injury to investigate its neuroprotective effects. Neurobehavioral deficits and infarct volume were assessed 7 d after ischemia. Both STVNa treatments reduced infarct volumes, improved neurological behaviors, preserved cellular morphology, enhanced neuronal survival, and suppressed cell apoptosis. Multiple treatments performed better than single treatment. Reactive astrogliosis was apparent at 7 d after injury and was significantly inhibited by multiple STVNa treatments but not single treatment. These results indicate that STVNa exerts neuroprotection by different mechanisms in the acute and delayed phases of I/R. Specifically, STVNa neuroprotection in the delayed phase of injury was found to be accompanied with the inhibition of astrogliosis.


Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Diterpenos de Tipo Kaurano/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Daño por Reperfusión/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Isquemia Encefálica/patología , Masculino , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas Sprague-Dawley , Daño por Reperfusión/patología
17.
Bioorg Med Chem ; 25(1): 360-371, 2017 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-27856236

RESUMEN

A series of novel chalcone-rivastigmine hybrids were designed, synthesized, and tested in vitro for their ability to inhibit human acetylcholinesterase and butyrylcholinesterase. Most of the target compounds showed hBChE selective activity in the micro- and submicromolar ranges. The most potent compound 3 exhibited comparable IC50 to the commercially available drug (rivastigmine). To better understand their structure activity relationships (SAR) and mechanisms of enzyme-inhibitor interactions, kinetic and molecular modeling studies including molecular docking and molecular dynamics (MD) simulations were carried out. Furthermore, compound 3 blocks the formation of reactive oxygen species (ROS) in SH-SY5Y cells and shows the required druggability and low cytotoxicity, suggesting this hybrid is a promising multifunctional drug candidate for Alzheimer's disease (AD) treatment.


Asunto(s)
Chalconas/farmacología , Inhibidores de la Colinesterasa/farmacología , Rivastigmina/análogos & derivados , Rivastigmina/farmacología , Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Butirilcolinesterasa/metabolismo , Dominio Catalítico , Línea Celular Tumoral , Chalconas/síntesis química , Chalconas/toxicidad , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/toxicidad , Humanos , Enlace de Hidrógeno , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Rivastigmina/síntesis química , Rivastigmina/toxicidad , Relación Estructura-Actividad
18.
Acta Biochim Biophys Sin (Shanghai) ; 49(11): 1022-1028, 2017 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-29036266

RESUMEN

The development of cardiac hypertrophy is a complicated process, which undergoes a transition from compensatory hypertrophy to heart failure, and the identification of new biomarkers and targets for this disease is greatly needed. Here we investigated the development of isoproterenol (ISO)-induced cardiac hypertrophy in an in vitro experimental model. After the induction of hypertrophy with ISO treatment in H9c2 cells, cell surface area, cell viability, cellular reactive oxygen species (ROS), and nitric oxide (NO) levels were tested. Our data showed that the cell viability, mitochondrial membrane potential, and NO/ROS balance varied during the development of cardiac hypertrophy in H9c2 cells. It was also found that the expression of thioredoxin1 (Trx1) and peroxiredoxin2 (Prdx2) was decreased during the cardiac hypertrophy of H9c2 cells. These results suggest a critical role for Trx1 and Prdx2 in the cardiac hypertrophy of H9c2 cells and in the transition from compensated hypertrophy to de-compensated hypertrophy in H9c2 cells, and our findings may have important implications for the management of this disease.


Asunto(s)
Cardiomegalia/etiología , Isoproterenol/farmacología , Óxido Nítrico/análisis , Peroxirredoxinas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/fisiología , Animales , Cardiomegalia/metabolismo , Cardiomegalia/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Óxido Nítrico/fisiología , Peroxirredoxinas/análisis , Ratas , Tiorredoxinas/análisis
19.
J Stroke Cerebrovasc Dis ; 26(11): 2603-2614, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28784277

RESUMEN

BACKGROUND: Isosteviol sodium (STVNa) has been reported to have neuroprotective effects against ischemia/reperfusion (I/R) injury in rats. Furthermore, recanalization treatments, including thrombolytic therapy, have several limitations. Excessive inflammation and apoptosis contribute to the pathogenesis of ischemic brain damage. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is critical to these processes and is associated with cerebral ischemia. Therefore, we studied the potential therapeutic effects and mechanisms of STVNa on permanent cerebral ischemia in mice. METHODS: Permanent middle cerebral artery occlusion (pMCAO) was established via the suture method, followed by intravenous STVNa (7.5, 15, 30, 45, and 60 mg/kg). Neurobehavioral deficits, infarct volume, and histology were examined 24 hours after cerebral ischemia. In addition, the messenger RNA (mRNA) expression of NF-κB-related genes was detected using real-time quantitative polymerase chain reaction (qPCR). RESULTS: STVNa (30 mg/kg) had significant neuroprotective effects 24 hours after pMCAO, including the reduction of the infarct volume and the improvement of the neurological severity score. Immunohistochemistry demonstrated that STVNa significantly increased the number of restored neurons and decreased the number of astrocytes. qPCR also demonstrated that the mRNA expression of inhibitor of nuclear factor kappa-B kinase-α, inhibitor of nuclear factor kappa-B kinase-ß, NF-κB, inhibitor of NF-κB-α, tumor necrosis factor-α, interleukin-1 beta, Bcl2-associated X protein, and caspase-3 were significantly downregulated, whereas B-cell CLL/lymphoma 2 mRNA was upregulated with STVNa treatment compared with vehicle. CONCLUSIONS: These findings demonstrate a neuroprotective role of STVNa during cerebral ischemia, which may result from interactions with the NF-κB signaling pathway and the associated inflammatory and apoptotic responses.


Asunto(s)
Apoptosis/efectos de los fármacos , Lesiones Encefálicas/prevención & control , Diterpenos de Tipo Kaurano/uso terapéutico , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , FN-kappa B/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Animales , Lesiones Encefálicas/etiología , Isquemia Encefálica/complicaciones , Caspasa 3/metabolismo , Circulación Cerebrovascular/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , Examen Neurológico
20.
Int J Mol Sci ; 18(6)2017 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-28632150

RESUMEN

Centrosome aberrations have been implicated in the development and progression of breast cancer. Our previous worked show that centrosomal protein 70 (Cep70) regulates breast cancer growth and metastasis. However, it remains elusive whether Cep70 is implicated in the sensitivity of the anti-microtubule drug paclitaxel in breast cancer. Here we provide evidence that Cep70 is a mediator of paclitaxel sensitivity in breast cancer. Cell proliferation assays show that Cep70 expression correlates with paclitaxel sensitivity in breast cancer cell lines. In addition, paclitaxel sensitivity varies when altering Cep70 expression level. Mechanistic studies reveal that Cep70 interacts with tubulin, and promotes the ability of paclitaxel to stimulate microtubule assembly. These data demonstrate that Cep70 mediates paclitaxel sensitivity in breast cancer.


Asunto(s)
Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Paclitaxel/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Células MCF-7 , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo
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