RESUMEN
From the fruits of Cordia dichotoma, 11 new phenolic compounds, dichotomins A-K, were isolated, together with 19 known compounds. Through the analysis of detailed NMR data and HRESIMS data, the planar structures of all compounds were confirmed. Using NMR calculations, the absolute configuration of dichotomins A-K was elucidated by comparing their observed and computed electronic circular dichroism (ECD) spectra. Dichotomin H (8) and dichotomin I (9) were determined as two pairs of enantiomers. The enantiomers of compounds 8 and 9 were separated using chiral-phase high-performance liquid chromatography (HPLC), and the stereostructure of each enantiomer was determined by similarly calculating the ECD. Compounds 3, 5, 7, 17, 18, 23-25, and 27-30 increased glucose uptake by 1.04- to 2.85-folds at concentrations of 30 µg/mL. Further studies revealed that compounds 3 and 5 had a moderate effect on glucose transporter 4 (GLUT4) translocation activity in L6 cells. At 30 µg/mL, compound 3 significantly enhanced AMPK phosphorylation and GLUT4 expression. As a whole, compound 3 has the potential to be a drug candidate for the treatment of type 2 diabetes mellitus (T2DM).
Asunto(s)
Frutas , Transportador de Glucosa de Tipo 4 , Glucosa , Fenoles , Extractos Vegetales , Transportador de Glucosa de Tipo 4/metabolismo , Transportador de Glucosa de Tipo 4/genética , Frutas/química , Glucosa/metabolismo , Fenoles/química , Fenoles/farmacología , Fenoles/metabolismo , Animales , Ratas , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/metabolismo , Transporte Biológico/efectos de los fármacos , Estructura Molecular , Línea Celular , Transporte de Proteínas , Humanos , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/químicaRESUMEN
The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2ß2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.
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Proteínas Quinasas Activadas por AMP , Adenosina Difosfato , Adenosina Monofosfato , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Humanos , Regulación Alostérica , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/química , Ligandos , Fosforilación , Adenosina Trifosfato/metabolismo , Activación Enzimática , Unión ProteicaRESUMEN
Since its early days in the 19th century, medicinal chemistry has concentrated its efforts on the treatment of diseases, using tools from areas such as chemistry, pharmacology, and molecular biology. The understanding of biological mechanisms and signaling pathways is crucial information for the development of potential agents for the treatment of diseases mainly because they are such complex processes. Given the limitations that the experimental approach presents, computational chemistry is a valuable alternative for the study of these systems and their behavior. Thus, classical molecular dynamics, based on Newton's laws, is considered a technique of great accuracy, when appropriated force fields are used, and provides satisfactory contributions to the scientific community. However, as many configurations are generated in a large MD simulation, methods such as Statistical Inefficiency and Optimal Wavelet Signal Compression Algorithm are great tools that can reduce the number of subsequent QM calculations. Accordingly, this review aims to briefly discuss the importance and relevance of medicinal chemistry allied to computational chemistry as well as to present a case study where, through a molecular dynamics simulation of AMPK protein (50 ns) and explicit solvent (TIP3P model), a minimum number of snapshots necessary to describe the oscillation profile of the protein behavior was proposed. For this purpose, the RMSD calculation, together with the sophisticated OWSCA method was used to propose the minimum number of snapshots.
Asunto(s)
Simulación de Dinámica Molecular , Humanos , Química Farmacéutica , Teoría Cuántica , Algoritmos , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/químicaRESUMEN
Adenosine 5'-monophosphate activated protein kinase (AMPK) has emerged as a promising target for the discovery of drugs to treat diabetic nephropathy (DN). Herein, a series of imidazo[1,2-a]pyridines were designed and synthesized. Among them, the active compound (EC50 =11.0â nM) showed good enzyme activation and molecular docking results showed hydrogen bonding interactions with the key amino acids Asn111 and Lys29 in the active site. Meanwhile, further cellular level experiments revealed that it could reduce reactive oxygen species (ROS) levels in NRK-49F cells induced by high glucose, and Western Blot experiments also demonstrate that it can increase the levels of p-AMPK and p-ACC and decrease the levels of TGF-ß1. The results of this study extend the structural types of AMPK activators and provide novel lead compounds for the subsequent development.
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Proteínas Quinasas Activadas por AMP , Fibroblastos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas Activadas por AMP/química , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Piridinas/farmacología , Piridinas/metabolismoRESUMEN
A large part of the world's population is affected by Alzheimer's disease (AD) and diabetes mellitus type 2, which cause both social and economic impacts. These two conditions are associated with one protein, AMPK. Studies have shown that vanadium complexes, such as bis(N',N'-dimethylbiguanidato)-oxovanadium(IV), VO(metf)2·H2O, are potential agents against AD. A crucial step in drug design studies is obtaining information about the structure and interaction of these complexes with the biological targets involved in the process through molecular dynamics (MD) simulations. However, MD simulations depend on the choice of a good force field that could present reliable results. Moreover, general force fields are not efficient for describing the properties of metal complexes, and a VO(metf)2·H2O-specific force field does not yet exist; thus, the proper development of a parameter set is necessary. Furthermore, this investigation is essential and relevant given the importance for both the scientific community and the population that is affected by this neurodegenerative disease. Therefore, the present work aims to develop and validate the AMBER force field parameters for VO(metf)2·H2O since the literature lacks such information on metal complexes and investigate through classical molecular dynamics the interactions made by the complex with the protein. The proposed force field proved to be effective for describing the vanadium complex (VC), supported by different analyses and validations. Moreover, it had a great performance when compared to the general AMBER force field. Beyond that, MD findings provided an in-depth perspective of vanadium complex-protein interactions that should be taken into consideration in future studies.
Asunto(s)
Enfermedad de Alzheimer , Complejos de Coordinación , Enfermedades Neurodegenerativas , Humanos , Enfermedad de Alzheimer/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP/química , Complejos de Coordinación/uso terapéutico , Simulación de Dinámica Molecular , Vanadio/químicaRESUMEN
AIM: Currently available medicines have little to offer in terms of supporting the regeneration of injured hepatic cells. Previous experimental studies have shown that resveratrol and metformin, less specific activators of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), can effectively attenuate acute liver injury. The aim of this experimental study was to elucidate whether modulation of AMPK and SIRT1 activity can modify drug/paracetamol (APAP)-induced hepatocyte damage in vitro. METHODS: Primary rat hepatocytes were pretreated with mutual combinations of specific synthetic activators and inhibitors of SIRT1 and AMPK and followed by a toxic dose of APAP. At the end of cultivation, medium samples were collected for biochemical analysis of alanine-aminotransferase and nitrite levels. Hepatocyte viability, thiobarbituric reactive substances, SIRT1 and AMPK activity and protein expression were also assessed. RESULTS: The harmful effect of APAP was associated with decreased AMPK and SIRT1 activity and protein expression alongside enhanced oxidative stress in hepatocytes. The addition of AMPK activator (AICAR) or SIRT1 activator (CAY10591) significantly attenuated the deleterious effects of AMPK inhibitor (Compound C) on the hepatotoxicity of APAP. Furthermore, CAY10591 but not AICAR markedly decreased the deleterious effect of APAP in combination with SIRT1 inhibitor (EX-527). CONCLUSION: Our findings demonstrate that decreased AMPK activity is associated with the hepatotoxic effect of APAP which can be significantly attenuated by the administration of a SIRT1 activator. These findings suggest that differentiated modulation of AMPK and SIRT1 activity could therefore provide an interesting and novel therapeutic opportunity in the future to combat hepatocyte injury.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Enfermedad Hepática Inducida por Sustancias y Drogas , Hepatocitos , Sirtuina 1 , Animales , Ratas , Acetaminofén/toxicidad , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Ciclopentanos/farmacología , Hepatocitos/metabolismo , Sirtuina 1/metabolismo , Sirtuina 1/farmacologíaRESUMEN
Background: Abnormal proliferation of vascular smooth muscle cells (VSMCs) in the intimal region is a key event in the development of neointimal hyperplasia. 10-G, a bioactive compound found in ginger, exerted inhibitory effects on the proliferation of several cancer cells. However, the effect and mechanism of 10-G on neointimal hyperplasia are not clear. Purpose: To explore the suppressive effects of 10-G on the proliferation and migration of VSMCs, and investigate the underlying mechanisms. Methods: In vivo, a left common carotid artery ligation mouse model was used to observe the effects of neointimal formation through immunohistochemistry and hematoxylin-eosin staining. In vitro, the cell proliferation and migration of HASMCs and A7r5 cells were detected by MTS assay, EdU staining, wound healing assay, Transwell assay, and western blotting as well. Molecular docking, molecular dynamics simulations and surface plasmon resonance imaging were collectively used to evaluate the interaction of 10-G with AMP-activated protein kinase (AMPK). Compound C and si-AMPK were used to inhibit the expression of AMPK. Results: Treatment with 10-G significantly reduced neointimal hyperplasia in the left common carotid artery ligation mouse model. MST and EdU staining showed that 10-G inhibited the proliferation of VSMC cells A7r5 and HASMC. We also found that 10-G altered the expression of proliferation-related proteins, including CyclinD1, CyclinD2, CyclinD3, and CDK4. Molecular docking revealed that the binding energy between AMPK and 10-G is -7.4 kcal mol-1. Molecular simulations suggested that the binding between 10-G and AMPK is stable. Surface plasmon resonance imaging analysis also showed that 10-G has a strong binding affinity to AMPK (KD = 6.81 × 10-8 M). 10-G promoted AMPKα phosphorylation both in vivo and in vitro. Blocking AMPK by an siRNA or AMPK inhibitor pathway partly abolished the anti-proliferation effects of 10-G on VSMCs. Conclusion: These data showed that 10-G might inhibit neointimal hyperplasia and suppress VSMC proliferation by the activation of AMPK as a natural AMPK agonist.
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Proteínas Quinasas Activadas por AMP/metabolismo , Catecoles/farmacología , Alcoholes Grasos/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Neointima/patología , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/química , Animales , Catecoles/química , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Activación Enzimática , Alcoholes Grasos/química , Humanos , Hiperplasia , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Simulación del Acoplamiento Molecular , Músculo Liso Vascular/efectos de los fármacos , Fosforilación , Conformación Proteica , Ratas , Transducción de Señal , Resonancia por Plasmón de Superficie , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The adenosine monophosphate activated protein kinase (AMPK) is critical in the regulation of important cellular functions such as lipid, glucose, and protein metabolism; mitochondrial biogenesis and autophagy; and cellular growth. In many diseases-such as metabolic syndrome, obesity, diabetes, and also cancer-activation of AMPK is beneficial. Therefore, there is growing interest in AMPK activators that act either by direct action on the enzyme itself or by indirect activation of upstream regulators. Many natural compounds have been described that activate AMPK indirectly. These compounds are usually contained in mixtures with a variety of structurally different other compounds, which in turn can also alter the activity of AMPK via one or more pathways. For these compounds, experiments are complicated, since the required pure substances are often not yet isolated and/or therefore not sufficiently available. Therefore, our goal was to develop a screening tool that could handle the profound heterogeneity in activation pathways of the AMPK. Since machine learning algorithms can model complex (unknown) relationships and patterns, some of these methods (random forest, support vector machines, stochastic gradient boosting, logistic regression, and deep neural network) were applied and validated using a database, comprising of 904 activating and 799 neutral or inhibiting compounds identified by extensive PubMed literature search and PubChem Bioassay database. All models showed unexpectedly high classification accuracy in training, but more importantly in predicting the unseen test data. These models are therefore suitable tools for rapid in silico screening of established substances or multicomponent mixtures and can be used to identify compounds of interest for further testing.
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Proteínas Quinasas Activadas por AMP/química , Modelos Teóricos , Relación Estructura-Actividad Cuantitativa , Proteínas Quinasas Activadas por AMP/metabolismo , Algoritmos , Aprendizaje Profundo , Activación Enzimática , Humanos , Aprendizaje Automático , Curva ROC , Reproducibilidad de los Resultados , Relación Estructura-Actividad , Máquina de Vectores de SoporteRESUMEN
As the central node between nutrition signaling input and the metabolic pathway, AMP-activated protein kinase (AMPK) is tightly regulated to maintain energy homeostasis. Subcellular compartmentalization of AMPK is one of the critical regulations that enables AMPK to access proper targets and generate appropriate responses to specific perturbations and different levels of stress. One of the characterized localization mechanisms is RanGTPase-driven CRM1 that recognizes the nuclear export sequence (NES) on the α subunit to translocate AMPK into the cytoplasm. Nuclear localization putatively employs RanGTPase-driven importin that might recognize the nuclear localization signal (NLS) present on the AMPKα2 kinase domain. Nucleo-cytoplasmic shuttling of AMPK is influenced by multiple factors, such as starvation, exercise, heat shock, oxidant, cell density, and circadian rhythm. Tissue-specific localization, which distributes AMPK trimers with different combinations, has also been shown to be vital in maintaining tissue-specific metabolism. Tissue-specific and subcellular distribution of AMPK might be attributed to differences in the expression of the subunit, the stabilization by protein regulators, tissue activity, and the localization of AMPK activators. Considering the importance of AMPK localization in coordinating signaling and metabolism, further research is due to fully elucidate the largely unknown complex mechanism underlying this regulation.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Metabolismo Energético , Proteínas Quinasas Activadas por AMP/química , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Ritmo Circadiano , Citoplasma/metabolismo , Respuesta al Choque Térmico , Humanos , Carioferinas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína Exportina 1RESUMEN
Myocardial fibrosis is a characteristic of various cardiomyopathies, and myocardial fibroblasts play a central role in this process. Gelsolin (GSN) is an actin severing and capping protein that regulates actin assembly and may be involved in fibroblast activation. While the role of GSN in mechanical stress-mediated cardiac fibrosis has been explored, its role in myocardial fibrosis in the absence of mechanical stress is not defined. In this study, we investigated the role of GSN in myocardial fibrosis induced by Angiotensin II (Ang II), a profibrotic hormone that is elevated in cardiovascular disease. We utilized mice lacking GSN (Gsn-/- ) and cultured primary adult cardiac fibroblasts (cFB). In vivo, Ang II infusion in mice resulted in significantly less severe myocardial fibrosis in Gsn-/- compared with Gsn+/+ mice, along with diminished activation of the TGFß1-Smad2/3 pathway, and reduced expression of cardiac extracellular matrix proteins (collagen, fibronectin, periostin). Moreover, Gsn-deficient hearts exhibited suppressed activity of the AMPK pathway and its downstream effectors, mTOR and P70S6Kinase, which could contribute to the suppressed TGFß1 activity. In vitro, the Ang II-induced activation of cFBs was reduced in Gsn-deficient fibroblasts evident from decreased expression of αSMA and periostin, diminished actin filament turnover; which also exhibited reduced activity of the AMPK-mTOR pathway, and P70S6K phosphorylation. AMPK inhibition compensated for the loss of GSN, restored the levels of G-actin in Gsn-/- cFBs and promoted activation to myofibroblasts by increasing αSMA and periostin levels. This study reveals a novel role for GSN in mediating myocardial fibrosis by regulating the AMPK-mTOR-P70S6K pathway in cFB activation independent from mechanical stress-induced factors.
Asunto(s)
Angiotensina II/farmacología , Fibroblastos/efectos de los fármacos , Fibrosis/patología , Gelsolina/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/metabolismo , Actinas/metabolismo , Animales , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/metabolismo , Gelsolina/deficiencia , Gelsolina/genética , Homeostasis , Masculino , Ratones , Miocardio/metabolismo , Miocardio/patología , Miofibroblastos/efectos de los fármacos , Miofibroblastos/patología , Fosforilación , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
5'AMP-activated protein kinase (AMPK) is known as metabolic sensor in mammalian cells that becomes activated by an increasing adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio. The heterotrimeric AMPK protein comprises three subunits, each of which has multiple phosphorylation sites, playing an important role in the regulation of essential molecular pathways. By phosphorylation of downstream proteins and modulation of gene transcription AMPK functions as a master switch of energy homeostasis in tissues with high metabolic turnover, such as the liver, skeletal muscle, and adipose tissue. Regulation of AMPK under conditions of chronic caloric oversupply emerged as substantial research target to get deeper insight into the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Evidence supporting the role of AMPK in NAFLD is mainly derived from preclinical cell culture and animal studies. Dysbalanced de novo lipogenesis has been identified as one of the key processes in NAFLD pathogenesis. Thus, the scope of this review is to provide an integrative overview of evidence, in particular from clinical studies and human samples, on the role of AMPK in the regulation of primarily de novo lipogenesis in human NAFLD.
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Proteínas Quinasas Activadas por AMP/metabolismo , Lipogénesis , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteínas Quinasas Activadas por AMP/química , Humanos , Insulina/metabolismo , Modelos Biológicos , Enfermedad del Hígado Graso no Alcohólico/epidemiología , Transducción de SeñalRESUMEN
Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/inmunología , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Microscopía por Crioelectrón , Humanos , Fragmentos Fab de Inmunoglobulinas , Modelos Moleculares , Fosforilación , Conformación Proteica , Dominios Proteicos , Ingeniería de ProteínasRESUMEN
Our recent in vivo human studies showed that colonic administration of sodium acetate (SA) resulted in increased circulating acetate levels, which was accompanied by increments in whole-body fat oxidation in overweight-obese men. Since skeletal muscle has a major role in whole-body fat oxidation, we aimed to investigate effects of SA on fat oxidation and underlying mechanisms in human primary skeletal muscle cells (HSkMC). We investigated the dose (0-5 mmol/L) and time (1, 4, 20, and 24 h) effect of SA on complete and incomplete endogenous and exogenous oxidation of 14C-labeled palmitate in HSkMC derived from a lean insulin sensitive male donor. Both physiological (0.1 and 0.25 mmol/L) and supraphysiological (0.5, 1 and 5 mmol/L) concentrations of SA neither increased endogenous nor exogenous fat oxidation over time in HSkMC. In addition, no effect of SA was observed on Thr172-AMPKα phosphorylation. In conclusion, our previously observed in vivo effects of SA on whole-body fat oxidation in men may not be explained via direct effects on HSkMC fat oxidation. Nevertheless, SA-mediated effects on whole-body fat oxidation may be triggered by other mechanisms including gut-derived hormones or may occur in other metabolically active tissues.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Palmitatos/metabolismo , Acetato de Sodio/farmacología , Proteínas Quinasas Activadas por AMP/química , Secuencias de Aminoácidos , Células Cultivadas , Humanos , Insulina/metabolismo , Masculino , Persona de Mediana Edad , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacosRESUMEN
Two series of tetrahydrocarbazole derivatives have been designed and synthesized based on ZG02, a promising candidate developed in our previous studies. The newly prepared compounds were screened for glucose consumption activity in HepG2 cell lines. Aza-tetrahydrocarbazole compound 12b showed the most potent hypoglycemic activity with a 45% increase in glucose consumption when compared to the solvent control, which had approximately 1.2-fold higher activity than the positive control compounds (metformin and ZG02). An investigation of the potential mechanism indicated that 12b may exhibit hypoglycemic activity via activation of the AMPK pathway. Metabolic stability assays revealed that 12b showed good stability profiles in both artificial gastrointestinal fluids and blood plasma from SD rats. An oral glucose tolerance test (OGTT) was performed and the results further confirmed that 12b was a potent hypoglycemic agent.
Asunto(s)
Carbazoles/química , Diseño de Fármacos , Hipoglucemiantes/síntesis química , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Sitios de Unión , Carbazoles/farmacología , Carbazoles/uso terapéutico , Estabilidad de Medicamentos , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Semivida , Células Hep G2 , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Trypanosoma cruzi, the etiological agent of Chagas disease, has a digenetic life cycle. In its passage from the insect vector to the mammalian host, and vice versa, it must be prepared to cope with abrupt changes in environmental conditions, such as carbon source, pH, temperature and osmolarity, in order to survive. Sensing and signaling pathways that allow the parasite to adapt, have unique characteristics with respect to their hosts and other free-living organisms. Many of the canonical proteins involved in these transduction pathways have not yet been found in the genomes of these parasites because they present divergences either at the functional, structural and/or protein sequence level. All of this makes these pathways promising targets for therapeutic drugs. The AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by environmental stresses such as osmotic stress, hypoxia, ischaemia and exercise that results in reduction of ATP and increase of AMP levels. Thus, AMPK is regarded as a fuel gauge, functioning both as a nutrient and an energy sensor, to maintain energy homeostasis and, eventually, to protect cells from death by nutrient starvation. In the present study we report the characterization of AMPK complexes for the first time in T. cruzi and propose the function of TcAMPK as a novel regulator of nutritional stress in epimastigote forms. We show that there is phosphotransferase activity specific for SAMS peptide in epimastigotes extracts, which is inhibited by Compound C and is modulated by carbon source availability. In addition, TcAMPKα2 subunit has an unprecedented functional substitution (Ser x Thr) at the activation loop and its overexpression in epimastigotes led to higher autophagic activity during prolonged nutritional stress. Moreover, the over-expression of the catalytic subunits resulted in antagonistic phenotypes associated with proliferation. Together, these results point to a role of TcAMPK in autophagy and nutrient sensing, key processes for the survival of trypanosomatids and for its life cycle progression.
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Proteínas Quinasas Activadas por AMP/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/metabolismo , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Autofagia , Metabolismo Energético , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Estrés Fisiológico , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
α-Amyrin, a natural pentacyclic triterpene, has an antihyperglycemic effect in mice and dual PPARδ/γ action in 3T3-L1 adipocytes, and potential in the control of type 2 diabetes (T2D). About 80% of glucose uptake occurs in skeletal muscle cells, playing a significant role in insulin resistance (IR) and T2D. Peroxisome-proliferator activated receptors (PPARs), in particular PPARδ and PPARγ, are involved in the regulation of lipids and carbohydrates and, along with adenosine-monophosphate (AMP) - activated protein kinase (AMPK) and protein kinase B (Akt), are implicated in translocation of glucose transporter 4 (GLUT4); however, it is still unknown whether α-amyrin can affect these pathways in skeletal muscle cells. Our objective was to determine the action of α-amyrin in PPARδ, PPARγ, AMPK, and Akt in C2C12 myoblasts. The expression of PPARδ, PPARγ, fatty acid transporter protein (FATP), and GLUT4 was quantified using reverse transcription quantitative PCR and Western blot. α-Amyrin increased these markers along with phospho-AMPK (p-AMPK) but not p-Akt. Molecular docking showed that α-amyrin acts as an AMPK-allosteric activator, and may be related to GLUT4 translocation, as evidenced by confocal microscopy. These data support that α-amyrin could have an insulin-mimetic action in C2C12 myoblasts and should be considered as a bioactive molecule for new multitarget drugs with utility in T2D and other metabolic diseases.
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Proteínas Quinasas Activadas por AMP/fisiología , Transportador de Glucosa de Tipo 4/metabolismo , Mioblastos/efectos de los fármacos , PPAR delta/fisiología , PPAR gamma/fisiología , Triterpenos Pentacíclicos/farmacología , Proteínas Quinasas Activadas por AMP/química , Animales , Células Cultivadas , Proteínas de Transporte de Ácidos Grasos/fisiología , Ratones , Simulación del Acoplamiento Molecular , Mioblastos/metabolismo , Triterpenos Pentacíclicos/química , Transporte de Proteínas/efectos de los fármacosRESUMEN
Physical exercise elicits physiological metabolic perturbations such as energetic and oxidative stress; however, a diverse range of cellular processes are stimulated in response to combat these challenges and maintain cellular energy homeostasis. AMP-activated protein kinase (AMPK) is a highly conserved enzyme that acts as a metabolic fuel sensor and is central to this adaptive response to exercise. The complexity of AMPK's role in modulating a range of cellular signalling cascades is well documented, yet aside from its well-characterised regulation by activation loop phosphorylation, AMPK is further subject to a multitude of additional regulatory stimuli. Therefore, in this review we comprehensively outline current knowledge around the post-translational modifications of AMPK, including novel phosphorylation sites, as well as underappreciated roles for ubiquitination, sumoylation, acetylation, methylation and oxidation. We provide insight into the physiological ramifications of these AMPK modifications, which not only affect its activity, but also subcellular localisation, nutrient interactions and protein stability. Lastly, we highlight the current knowledge gaps in this area of AMPK research and provide perspectives on how the field can apply greater rigour to the characterisation of novel AMPK regulatory modifications.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/metabolismo , Metabolismo Energético , Procesamiento Proteico-Postraduccional , Proteínas Quinasas Activadas por AMP/genética , Acetilación , Animales , Homeostasis , Humanos , Metilación , Oxidación-Reducción , Estrés Oxidativo , Fosforilación , Dominios Proteicos , Transducción de Señal/genética , UbiquitinaciónRESUMEN
Luteinizing hormone (LH) via protein kinase A (PKA) triggers ovulation and formation of the corpus luteum, which arises from the differentiation of follicular granulosa and theca cells into large and small luteal cells, respectively. The small and large luteal cells produce progesterone, a steroid hormone required for establishment and maintenance of pregnancy. We recently reported on the importance of hormone-sensitive lipase (HSL, also known as LIPE) and lipid droplets for appropriate secretory function of the corpus luteum. These lipid-rich intracellular organelles store cholesteryl esters, which can be hydrolyzed by HSL to provide cholesterol, the main substrate necessary for progesterone synthesis. In the present study, we analyzed dynamic posttranslational modifications of HSL mediated by PKA and AMP-activated protein kinase (AMPK) as well as their effects on steroidogenesis in luteal cells. Our results revealed that AMPK acutely inhibits the stimulatory effects of LH/PKA on progesterone production without reducing levels of STAR, CYP11A1, and HSD3B proteins. Exogenous cholesterol reversed the negative effects of AMPK on LH-stimulated steroidogenesis, suggesting that AMPK regulates cholesterol availability in luteal cells. AMPK evoked inhibitory phosphorylation of HSL (Ser565). In contrast, LH/PKA decreased phosphorylation of AMPK at Thr172, a residue required for its activation. Additionally, LH/PKA increased phosphorylation of HSL at Ser563, which is crucial for enzyme activation, and decreased inhibitory phosphorylation of HSL at Ser565. The findings indicate that LH and AMPK exert opposite posttranslational modifications of HSL, presumptively regulating cholesterol availability for steroidogenesis.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Lúteas/citología , Células Lúteas/enzimología , Progesterona/metabolismo , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Secuencias de Aminoácidos , Animales , Bovinos , Colesterol/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Activación Enzimática , Femenino , Células Lúteas/metabolismo , Hormona Luteinizante/metabolismo , Fosforilación , Transducción de SeñalRESUMEN
The adenosine monophosphateactivated protein kinase (AMPK) is a promising target in drug development for various metabolic diseases. In the present study, the aim was to discover natural direct AMPK activators from natural sources, thus a virtual screening for direct AMPK activators was conducted by combining ligandbased and structurebased screening. A commonfeature pharmacophore model (HipHop1) was generated with two hydrogen bond acceptor lipid features and one hydrophobic region feature. A total of 1,235 natural products were screened using the HipHop1 hypothesis and CDOCKER protocol successively. According to the docking score, seven hit compounds were selected for AMPK activation assays. Ultimately, ()catechin (compound 522) and licochalcone A (compound 1148) exhibited the highest AMPK activation activity. These findings may contribute to the development of AMPK activators from medicinal plants.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Activadores de Enzimas/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Diseño de Fármacos , Humanos , Ligandos , Relación Estructura-Actividad CuantitativaRESUMEN
AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance. In response to metabolic stress, it acts to redress energy imbalance through promotion of ATP-generating catabolic processes and inhibition of ATP-consuming processes, including cell growth and proliferation. While findings that AMPK was a downstream effector of the tumour suppressor LKB1 indicated that it might act to repress tumourigenesis, more recent evidence suggests that AMPK can either suppress or promote cancer, depending on the context. Prior to tumourigenesis AMPK may indeed restrain aberrant growth, but once a cancer has arisen, AMPK may instead support survival of the cancer cells by adjusting their rate of growth to match their energy supply, as well as promoting genome stability. The two isoforms of the AMPK catalytic subunit may have distinct functions in human cancers, with the AMPK-α1 gene often being amplified, while the AMPK-α2 gene is more often mutated. The prevalence of metabolic disorders, such as obesity and Type 2 diabetes, has led to the development of a wide range of AMPK-activating drugs. While these might be useful as preventative therapeutics in individuals predisposed to cancer, it seems more likely that AMPK inhibitors, whose development has lagged behind that of activators, would be efficacious for the treatment of pre-existing cancers.