RESUMEN
Treatment of invasive mold infections is limited by the lack of adequate drug options that are effective against these fatal infections. High-throughput screening of molds using traditional antifungal assays of growth is problematic and has greatly limited our ability to identify new mold-active agents. Here, we present a high-throughput screening platform for use with Aspergillus fumigatus, the most common causative agent of invasive mold infections, for the discovery of novel mold-active antifungals. This assay detects cell lysis through the release of the cytosolic enzyme adenylate kinase and, thus, is not dependent on changes in biomass or metabolism to detect antifungal activity. The ability to specifically detect cell lysis is a unique aspect of this assay that allows identification of molecules that disrupt fungal cell integrity, such as cell wall-active molecules. We also found that germinating A. fumigatus conidia release low levels of adenylate kinase and that a reduction in this background allowed us to identify molecules that inhibit conidial germination, expanding the potential for discovery of novel antifungal compounds. Here, we describe the validation of this assay and proof-of-concept pilot screens that identified a novel antifungal compound, PIK-75, that disrupts cell wall integrity. This screening assay provides a novel platform for high-throughput screens with A. fumigatus for the identification of anti-mold drugs. IMPORTANCE Fungal infections caused by molds have the highest mortality rates of human fungal infections. These devastating infections are hard to treat and available antifungal drugs are often not effective. Therefore, the identification of new antifungal drugs with mold activity is critical. Drug screening with molds is challenging and there are limited assays available to identify new antifungal compounds directly with these organisms. Here, we present an assay suitable for use for high-throughput screening with a common mold pathogen. This assay has exciting future potential for the identification of new drugs to treat these fatal infections.
Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Adenilato Quinasa/antagonistas & inhibidores , Aspergilosis/tratamiento farmacológico , Aspergillus fumigatus/enzimología , Aspergillus fumigatus/crecimiento & desarrollo , Pared Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Prueba de Estudio Conceptual , Bibliotecas de Moléculas Pequeñas/farmacología , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/enzimologíaRESUMEN
We have identified a range of food phytochemicals that inhibit Janus Kinase 2 (JAK2) and Adenosine Monophosphate Kinase (AMPK). A mutated and dysregulated form of JAK2, a tyrosine kinase, is associated with several diseases including Crohn's disease. Using an in vitro, time-resolved fluorescence (TR-FRET) assay, we tested 49 different types of food extracts, plus 10 concentrated fractions of increasing hydrophobicity from each extract, to find foods containing JAK2 inhibitors. The food extracts tested included grains, meat, fish, shellfish, dairy products, herbs, mushrooms, hops, fruits and vegetables. Several fruits were potent inhibitors of JAK2: blackberry, boysenberry, feijoa, pomegranate, rosehip and strawberry, which all contain ellagitannins, known inhibitors of kinases. These fruits are in the Rosales and Myrtales plant orders. No other foods gave >1% of the maximal JAK2 inhibitory activities of these fruits. AMPK, a sensor and regulator of energy metabolism in cells, is a serine-threonine kinase which is reported to be activated by various flavonoid phytochemicals. Using a TR-FRET assay, we tested various fruit extracts for AMPK activation and inhibition. Ellagitannin containing foods scored highly as AMPK inhibitors. Despite several reports of AMPK activation in whole cells by phytochemicals, no extracts or pure compounds activated AMPK in our assay.
Asunto(s)
Adenilato Quinasa/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/aislamiento & purificación , Inhibidores Enzimáticos/aislamiento & purificación , Frutas/química , Alimentos Funcionales , Janus Quinasa 2/antagonistas & inhibidores , Fitoquímicos/aislamiento & purificación , Actinidia/química , Adenilato Quinasa/metabolismo , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Enfermedad de Crohn/dietoterapia , Enfermedad de Crohn/enzimología , Enfermedad de Crohn/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Análisis de los Alimentos , Humanos , Taninos Hidrolizables/aislamiento & purificación , Taninos Hidrolizables/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Lythraceae/química , Mutación , Nueva Zelanda , Fitoquímicos/química , Fitoquímicos/metabolismo , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/aislamiento & purificación , Inhibidores de Proteínas Quinasas/metabolismo , Rosales/químicaRESUMEN
Here, we have investigated the effect of metformin pretreatment in the rat models of global cerebral ischemia. Cerebral ischemia which leads to brain dysfunction is one of the main causes of neurodegeneration and death worldwide. Metformin is used in clinical drug therapy protocols of diabetes. It is suggested that metformin protects cells under hypoxia and ischemia in non-neuronal contexts. Protective effects of metformin may be modulated via activating the AMP activated protein kinase (AMPK). Our results showed that induction of 30 min global cerebral I/R injury using 4-vesseles occlusion model led to significant cell death in the rat brain. Metformin pretreatment (200 mg kg/once/day, p.o., 2 weeks) attenuated apoptotic cell death and induced mitochondrial biogenesis proteins in the ischemic rats, analyzed using histological and Western blot assays. Besides, inhibition of AMPK by compound c showed that metformin resulted in apoptosis attenuation via AMPK activation. Interestingly, AMPK activation was also involved in the induction of mitochondrial biogenesis proteins using metformin, inhibition of AMPK by compound c reversed such effect, further supporting the role of AMPK upstream of mitochondrial biogenesis proteins. In summary, Metformin pretreatment is able to modulate mitochondrial biogenesis and apoptotic cell death pathways through AMPK activation in the context of global cerebral ischemia, conducting the outcome towards neuroprotection.
Asunto(s)
Adenilato Quinasa/fisiología , Isquemia Encefálica/prevención & control , Encéfalo/efectos de los fármacos , Metformina/farmacología , Fármacos Neuroprotectores/farmacología , Factores de Transcripción/fisiología , Adenilato Quinasa/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/patología , Isquemia Encefálica/patología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Hipocampo/patología , Masculino , Metformina/administración & dosificación , Metformina/uso terapéutico , Recambio Mitocondrial/efectos de los fármacos , Factor 1 Relacionado con NF-E2/biosíntesis , Factor 1 Relacionado con NF-E2/genética , Fármacos Neuroprotectores/uso terapéutico , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Premedicación , Pirazoles/farmacología , Pirimidinas/farmacología , Ratas , Daño por Reperfusión/prevención & control , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The adenine monophosphate (AMP) activated protein kinase (AMPK), is a heterotrimeric complex that is activated by an increase in the AMP/ATP ratio, and is considered to be a cellular energy sensor that contributes to regulate energy balance and caloric intake. AMPK is activated by LKB1 hinase and it can phophorylate several enzymes involved in anabolism to prevent further ATP consumption, and induces some catabolic enzymes to increase ATP generation. Furthermore, AMPK regulates the expression of genes involved in lipogenesis and mitochondrial biogenesis, among others. AMPK is distributed in most organs including, liver, skeletal muscle, heart and hypothalamus; and even in adipose cells. In addition, AMPK is activated in the hypothalamus stimulating appetite due to energy depletion. AMPK also participates in glycolysis regulation, glucose uptake, lipid oxidation, fatty acid synthesis, cholesterol synthesis and gluconeogenesis, and it has been considered as a possible target enzyme in the treatment of some diseases such as obesity, type 2 diabetes and hepatic steatosis. This review provides a general overview of AMPK structure, its activators and its function in the organism.
Asunto(s)
Adenilato Quinasa/fisiología , Metabolismo Energético/fisiología , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adenilato Quinasa/antagonistas & inhibidores , Adenilato Quinasa/química , Adipoquinas/fisiología , Tejido Adiposo/metabolismo , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/fisiopatología , Activación Enzimática/efectos de los fármacos , Hígado Graso/tratamiento farmacológico , Hígado Graso/enzimología , Hígado Graso/fisiopatología , Glucosa/metabolismo , Humanos , Hipoglucemiantes/farmacología , Hipotálamo/metabolismo , Lipogénesis/fisiología , Hígado/metabolismo , Miocardio/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/enzimología , Obesidad/fisiopatología , Especificidad de Órganos , Fosforilación , Esfuerzo Físico/fisiología , Procesamiento Proteico-PostraduccionalRESUMEN
Obesity is an epidemic in Western society, and causes rapidly accelerating rates of type 2 diabetes and cardiovascular disease. The evolutionarily conserved serine/threonine kinase, AMP-activated protein kinase (AMPK), functions as a 'fuel gauge' to monitor cellular energy status. We investigated the potential role of AMPK in the hypothalamus in the regulation of food intake. Here we report that AMPK activity is inhibited in arcuate and paraventricular hypothalamus (PVH) by the anorexigenic hormone leptin, and in multiple hypothalamic regions by insulin, high glucose and refeeding. A melanocortin receptor agonist, a potent anorexigen, decreases AMPK activity in PVH, whereas agouti-related protein, an orexigen, increases AMPK activity. Melanocortin receptor signalling is required for leptin and refeeding effects on AMPK in PVH. Dominant negative AMPK expression in the hypothalamus is sufficient to reduce food intake and body weight, whereas constitutively active AMPK increases both. Alterations of hypothalamic AMPK activity augment changes in arcuate neuropeptide expression induced by fasting and feeding. Furthermore, inhibition of hypothalamic AMPK is necessary for leptin's effects on food intake and body weight, as constitutively active AMPK blocks these effects. Thus, hypothalamic AMPK plays a critical role in hormonal and nutrient-derived anorexigenic and orexigenic signals and in energy balance.