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1.
Nat Chem Biol ; 11(5): 347-354, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25848931

RESUMEN

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.


Asunto(s)
Aminoaciltransferasas/efectos de los fármacos , Aminoaciltransferasas/genética , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , ARN Interferente Pequeño , Aminoaciltransferasas/antagonistas & inhibidores , Animales , Células Cultivadas , Biología Computacional , Drosophila , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteína Huntingtina , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Pez Cebra , Cadena B de alfa-Cristalina/metabolismo
2.
EMBO Mol Med ; 7(2): 127-39, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25535254

RESUMEN

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection.


Asunto(s)
Anticonvulsivantes/administración & dosificación , Antituberculosos/administración & dosificación , Autofagia/efectos de los fármacos , Carbamazepina/administración & dosificación , Inositol/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Tuberculosis/fisiopatología , Animales , Línea Celular , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Tuberculosis/inmunología , Tuberculosis/metabolismo , Pez Cebra
3.
Neuropharmacology ; 59(3): 149-59, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20450924

RESUMEN

Treatment of the autoimmune demyelinating disease multiple sclerosis (MS) requires therapies that both limit and repair damage. While several immunomodulatory treatments exist to limit damage there are currently no treatments that promote the regenerative process of remyelination. A rapid way of screening potential pro-remyelination compounds is therefore required. The use of larval zebrafish in a drug reprofiling screen allows rapid in vivo screening and has been used successfully in the past as an efficient way of identifying new indications for existing drugs. A novel screening platform for potential pro-myelination compounds was developed using zebrafish larvae. Two percent of compounds screened from reprofiling libraries altered oligodendrocyte lineage cell recruitment and/or proliferation, as measured by the numbers of dorsally migrated spinal cord olig2(+) cells. Selective screening identified three compounds that altered levels of myelination, as measured by whole larvae myelin basic protein (mbp) transcript levels; the src family kinase inhibitor PP2, a biogenic amine and a thioxanthene. As well as many previously unrecognised compounds, identified compounds included those with previously known effects on myelin and/or the oligodendrocyte lineage, such as a PPAR agonist, steroid hormones and src family kinase inhibitors. As well as providing methods for further assessment of potentially beneficial compounds, this screen has highlighted 25 targets that are able to alter oligodendrocyte lineage cell recruitment or proliferation and/or mbp transcript levels in vivo and are worthy of further investigation for their potential effects on remyelination.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Modelos Animales , Proteína Básica de Mielina/metabolismo , Oligodendroglía/metabolismo , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Antiinflamatorios no Esteroideos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Recuento de Células/métodos , Diferenciación Celular/fisiología , Evaluación Preclínica de Medicamentos , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/genética , Proteína Básica de Mielina/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Oligodendroglía/efectos de los fármacos , Piroxicam/análogos & derivados , Piroxicam/farmacología , Médula Espinal/citología , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
4.
J Pharmacol Toxicol Methods ; 58(1): 59-68, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18585469

RESUMEN

INTRODUCTION: Safety pharmacology is integral to the non-clinical safety assessment of new chemical entities prior to first administration to humans. The zebrafish is a well established model organism that has been shown to be relevant to the study of human diseases. The potential role of zebrafish in safety pharmacology was evaluated using reference compounds in three models assessing cardiac, visual and intestinal function. METHODS: Compound toxicity was first established in zebrafish to determine the non toxic concentration of a blinded set of 16 compounds. In the cardiac assay, zebrafish larvae at 3 days post fertilisation (d.p.f.) were exposed to compounds for 3 h before measurement of the atrial and ventricular rates. To investigate visual function, the optomotor response was assessed in 8 d.p.f. larvae following a 5 day compound exposure. In the intestinal function assay, the number of gut contractions was measured in 7 d.p.f. larvae after a 1 h compound exposure. Finally, compound uptake was determined for 9 of the 16 compounds to measure the concentration of compound absorbed by the zebrafish larvae. RESULTS: Seven compounds out of nine produced an expected effect that was statistically significant in the cardiac and visual functions assays. In the gut contraction assay, six out of ten compounds showed a statistically significant effect that was also the expected result whilst two displayed anticipated but non-significant effects. The compound uptake method was used to determine larval tissue concentrations and allowed the identification of false negatives when compound was poorly absorbed into the zebrafish. DISCUSSION: Overall, results generated in three zebrafish larvae assays demonstrated a good correlation between the effects of compounds in zebrafish and the data available from other in vivo models or known clinical adverse effects. These results suggest that for the cardiac, intestinal and visual function, zebrafish assays have the potential to predict adverse drug effects and supports their possible role in early safety assessment of novel compounds.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Pruebas de Toxicidad/métodos , Animales , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/fisiología , Corazón/efectos de los fármacos , Corazón/fisiología , Humanos , Larva/efectos de los fármacos , Larva/metabolismo , Modelos Animales , Preparaciones Farmacéuticas/metabolismo , Especificidad de la Especie , Factores de Tiempo , Visión Ocular/efectos de los fármacos , Visión Ocular/fisiología , Pez Cebra/fisiología
5.
Neuropsychopharmacology ; 33(5): 1206-15, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17581529

RESUMEN

Habituation, where a response is reduced when exposed to a continuous stimulus is one of the simplest forms of non-associative learning and has been shown in a number of organisms from sea slugs to rodents. However, very little has been reported in the zebrafish, a model that is gaining popularity for high-throughput compound screens. Furthermore, since most of the studies involving learning and memory in zebrafish have been conducted in adults, we sought to determine if zebrafish larvae could display non-associative learning and whether it could be modulated by compounds identified in previous rodent studies. We demonstrated that zebrafish larvae (7 days post fertilization) exhibit iterative reduction in a startle response to a series of acoustic stimuli. Furthermore, this reduction satisfied criteria for habituation: spontaneous recovery, more rapid reductions in startle to shorter intertrial intervals and dishabituation. We then investigated the pathways mediating this behavior using established compounds in learning and memory. Administration of rolipram (PDE4 inhibitor), donepezil (acetylcholinesterase inhibitor), and memantine (N-methyl-D-aspartic acid (NMDA) receptor antagonist) all increased the acoustic startle response and decreased habituation in the larvae, similar to previous rodent studies. Further studies demonstrated that NMDA blocked the memantine response and the effect of donepezil was blocked by mecamylamine but not atropine suggesting that the donepezil response was mediated by nicotinic rather than muscarinic receptors. Zebrafish larvae possess numerous advantages for medium to high-throughput screening; the model described herein therefore offers the potential to screen for additional compounds for further study on cognition function.


Asunto(s)
Habituación Psicofisiológica , Larva/fisiología , Reflejo de Sobresalto/fisiología , Estimulación Acústica/métodos , Factores de Edad , Análisis de Varianza , Animales , Área Bajo la Curva , Conducta Animal/fisiología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Inhibidores Enzimáticos/administración & dosificación , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Inhibición Psicológica , Memantina/farmacología , N-Metilaspartato/farmacología , Reflejo de Sobresalto/efectos de los fármacos , Pez Cebra
6.
J Biomol Screen ; 10(8): 823-31, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16234346

RESUMEN

Osteoporosis and diseases of bone loss are a major public health problem for the present and the future since longevity and prevalence of the disease are increasing in all parts of the world. The bisphosphonates, widely used in the treatment of osteoporosis, act by inhibiting bone resorption. However, there are few agents that promote or increase bone formation in patients who have suffered substantial bone loss. To facilitate the identification of novel anabolic therapies, the authors have developed a rapid, high-throughput in vivo screen using larval zebrafish (Danio rerio) in which they are able to identify agents with anabolic effects in the skeleton within a 6-day time period. Vitamin D3 analogs and intermittent parathyroid hormone (PTH) result in dose-dependent increases in the formation of mineralized bone, whereas continuous exposure to PTH results in net bone loss. Because this model is fast, economical, and genetically tractable, it provides a powerful adjunct to mammalian models for the identification of new anabolic bone agents and offers the potential for genetic elucidation of pathways important in osteoblastic activity.


Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Calcificación Fisiológica/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Pez Cebra , Animales , Conservadores de la Densidad Ósea/uso terapéutico , Remodelación Ósea/efectos de los fármacos , Remodelación Ósea/fisiología , Calcificación Fisiológica/fisiología , Colecalciferol/análogos & derivados , Colecalciferol/farmacología , Colecalciferol/uso terapéutico , Ácido Etidrónico/farmacología , Ácido Etidrónico/uso terapéutico , Estudios de Factibilidad , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Modelos Animales , Osteoblastos/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Hormona Paratiroidea/farmacología , Hormona Paratiroidea/uso terapéutico
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