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1.
Toxicol Sci ; 136(1): 216-41, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23956101

RESUMEN

The bile salt export pump (BSEP) is expressed at the canalicular domain of hepatocytes, where it serves as the primary route of elimination for monovalent bile acids (BAs) into the bile canaliculi. The most compelling evidence linking dysfunction in BA transport with liver injury in humans is found with carriers of mutations that render BSEP nonfunctional. Based on mounting evidence, there appears to be a strong association between drug-induced BSEP interference and liver injury in humans; however, causality has not been established. For this reason, drug-induced BSEP interference is best considered a susceptibility factor for liver injury as other host- or drug-related properties may contribute to the development of hepatotoxicity. To better understand the association between BSEP interference and liver injury in humans, over 600 marketed or withdrawn drugs were evaluated in BSEP expressing membrane vesicles. The example of a compound that failed during phase 1 human trials is also described, AMG 009. AMG 009 showed evidence of liver injury in humans that was not predicted by preclinical safety studies, and BSEP inhibition was implicated. For 109 of the drugs with some effect on in vitro BSEP function, clinical use, associations with hepatotoxicity, pharmacokinetic data, and other information were annotated. A steady state concentration (C(ss)) for each of these annotated drugs was estimated, and a ratio between this value and measured IC50 potency values were calculated in an attempt to relate exposure to in vitro potencies. When factoring for exposure, 95% of the annotated compounds with a C(ss)/BSEP IC50 ratio ≥ 0.1 were associated with some form of liver injury. We then investigated the relationship between clinical evidence of liver injury and effects to multidrug resistance-associated proteins (MRPs) believed to play a role in BA homeostasis. The effect of 600+ drugs on MRP2, MRP3, and MRP4 function was also evaluated in membrane vesicle assays. Drugs with a C(ss)/BSEP IC50 ratio ≥ 0.1 and a C(ss)/MRP IC50 ratio ≥ 0.1 had almost a 100% correlation with some evidence of liver injury in humans. These data suggest that integration of exposure data, and knowledge of an effect to not only BSEP but also one or more of the MRPs, is a useful tool for informing the potential for liver injury due to altered BA transport.


Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Hígado/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Análisis por Conglomerados , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Farmacocinética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Medición de Riesgo , Factores de Riesgo , Pruebas de Toxicidad/métodos
2.
Curr Protoc Toxicol ; Chapter 23: Unit 23.5, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23169270

RESUMEN

The use of plasma membrane vesicles that overexpress the bile salt export pump (BSEP) or multidrug resistance-associated protein 2, 3, or 4 (MRP2-4) with an in vitro vacuum filtration system offers a rapid and reliable means for screening drug candidates for their effects on transporter function in hepatocytes and thus their potential for causing drug-induced liver injury (DILI). Comparison of transporter activity in the presence and absence of ATP allows for determination of a specific assay window for each transporter. This window is used to determine the degree to which each test compound inhibits transporter activity. This assay battery is helpful for prioritizing and rank-ordering compounds within a chemical series with respect to each other and in the context of known inhibitors of transporter activity and/or liver injury. This model can be used to influence the drug development process at an early stage and provide rapid feedback regarding the selection of compounds for advancement to in vivo safety evaluations. A detailed protocol for the high-throughput assessment of ABC transporter function is provided, including specific recommendations for curve-fitting to help ensure consistent results.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Bioensayo/métodos , Vesículas Transportadoras/fisiología , Membrana Celular , Evaluación Preclínica de Medicamentos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Modelos Lineales , Conteo por Cintilación
3.
Toxicol Sci ; 118(2): 485-500, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20829430

RESUMEN

The bile salt export pump (BSEP) is an efflux transporter, driving the elimination of endobiotic and xenobiotic substrates from hepatocytes into the bile. More specifically, it is responsible for the elimination of monovalent, conjugated bile salts, with little or no assistance from other apical transporters. Disruption of BSEP activity through genetic disorders is known to manifest in clinical liver injury such as progressive familial intrahepatic cholestasis type 2. Drug-induced disruption of BSEP is hypothesized to play a role in the development of liver injury for several marketed or withdrawn therapeutics. Unfortunately, preclinical animal models have been poor predictors of the liver injury associated with BSEP interference observed for humans, possibly because of interspecies differences in bile acid composition, differences in hepatobiliary transporter modulation or constitutive expression, as well as other mechanisms. Thus, a BSEP-mediated liver liability may go undetected until the later stages of drug development, such as during clinical trials or even postlicensing. In the absence of a relevant preclinical test system for BSEP-mediated liver injury, the toxicological relevance of available in vitro models to human health rely on the use of benchmark compounds with known clinical outcomes, such as marketed or withdrawn drugs. In this study, membrane vesicles harvested from BSEP-transfected insect cells were used to assess the activity of more than 200 benchmark compounds to thoroughly investigate the relationship between interference with BSEP function and liver injury. The data suggest a relatively strong association between the pharmacological interference with BSEP function and human hepatotoxicity. Although the most accurate translation of risk would incorporate pharmacological potency, pharmacokinetics, clearance mechanisms, tissue distribution, physicochemical properties, indication, and other drug attributes, the additional understanding of a compound's potency for BSEP interference should help to limit or avoid BSEP-related liver liabilities in humans that are not often detected by standard preclinical animal models.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Evaluación Preclínica de Medicamentos/métodos , Xenobióticos/toxicidad , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Animales , Bioensayo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratas , Reproducibilidad de los Resultados , Spodoptera/citología , Transfección
4.
Assay Drug Dev Technol ; 7(3): 281-93, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19604106

RESUMEN

The ability to combine primary hit identification assays with target profiling would significantly streamline the current drug discovery process. Working towards this end, we report here the development of a microarray-based ligand binding assay that supports multiplexed analysis of G protein-coupled receptor systems in a 96-well microplate format that is compatible with the equipment and infrastructure typical of high-throughput screening laboratories. A prototype microarray was generated by pin-printing seven different receptors within the wells of a specially coated glass-bottom microplate and assaying with a cocktail of fluorescent ligands. Development of the multiplexed system included optimization of methods for depositing receptor membrane proteins and establishing a generic set of assay conditions that simultaneously satisfied the pharmacology requirements of all of the receptor systems included on the array. The multiplexed system is shown to produce valid pharmacological results as evidenced by its ability to report K(i) values for receptor-specific fluorescent ligands and rank ordered potencies for diagnostic displacing compounds comparable to values generated by conventional simplexed assays. Moreover, the results of a 40-compound mini-screen confirmed that the assay accurately identifies valid hits. The results suggest the assay may be immediately suitable for routine profiling tasks and demonstrate the potential of the format for high-throughput multiplexed drug discovery.


Asunto(s)
Evaluación Preclínica de Medicamentos , Análisis por Micromatrices/métodos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Tampones (Química) , Interpretación Estadística de Datos , Dimetilsulfóxido/química , Diseño de Fármacos , Perfilación de la Expresión Génica , Humanos , Indicadores y Reactivos , Ligandos , Unión Proteica , Proteínas/química , Receptores de Droga/química , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solventes
5.
Assay Drug Dev Technol ; 7(2): 133-42, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19505230

RESUMEN

The G protein-coupled receptor (GPCR) G2A (for G2 accumulation) was identified as a stress-inducible antiproliferative cell cycle regulator. Targeted G2A gene deletion in mice resulted in systemic lupus erythematosus-like and atherosclerotic lesion phenotypes. These findings suggested that G2A may be a therapeutic target for cancers and autoimmune and cardiovascular diseases. The G2A receptor is cytotoxic upon ectopic expression, and its cognate ligand has not been identified, making it difficult to generate a cell line for screening using a conventional approach. The function of human G2A remains obscure. Here we show that by using an inducible T-REx (Invitrogen, Carlsbad, CA) expression system an inducible G2A functional cell-based beta-lactamase reporter assay could be developed using the constitutive activity of the receptor. Furthermore, G2A expression levels can be controlled under this inducible system to avoid the expression artifacts of conventional approaches using constitutive expression vectors. This stable cell line expressing the human G2A receptor was screened against a chemical library containing 740,000 compounds, and small molecules showing selective agonistic activity on G2A were identified. We believe the strategy employed here for G2A should be applicable to other "intractable" GPCRs where target gene expression results in cytotoxic and/or high constitutive activities.


Asunto(s)
Proteínas de Ciclo Celular/agonistas , Proteínas de Ciclo Celular/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , beta-Lactamasas/metabolismo , Apoptosis , Proteínas de Ciclo Celular/fisiología , Línea Celular , Citometría de Flujo , Humanos , Interferencia de ARN , Receptores Acoplados a Proteínas G/fisiología , beta-Lactamasas/genética
6.
J Biomol Screen ; 13(8): 748-54, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18460694

RESUMEN

The measurement of ligand receptor binding parameters for G-protein-coupled receptors is indispensable in the drug discovery process. Traditional ligand receptor binding assays require scale-up of cells and membrane preparations, which is an expensive and time-consuming process. In this report, the authors describe the development of a homogeneous live-cell binding assay for GPCRs using a fluorophore-labeled nonpeptide ligand. The model assay used Cy3B-labeled telenzepine and Chinese hamster ovary cells expressing M1 muscarinic acetylcholine receptors. This homogeneous live-cell fluorescence binding assay format is superior to the traditional binding methods because it measures binding of a ligand to intact receptors on living cells. The assay requires no washing or separation steps, thereby allowing a real-time kinetic readout for the determination of ligand association and dissociation from the intact receptors. The results also suggest that miniaturization is feasible without compromising the data quality.


Asunto(s)
Bioensayo/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Atropina/metabolismo , Bencenosulfonatos/metabolismo , Células CHO , Carbocianinas/metabolismo , Cricetinae , Cricetulus , Colorantes Fluorescentes/metabolismo , Ligandos , Miniaturización , Antagonistas Muscarínicos/química , Antagonistas Muscarínicos/metabolismo , Parasimpatolíticos/metabolismo , Pirenzepina/análogos & derivados , Pirenzepina/metabolismo , Quinuclidinil Bencilato/química , Quinuclidinil Bencilato/metabolismo , Receptor Muscarínico M1/metabolismo , Receptores Acoplados a Proteínas G/genética
7.
Assay Drug Dev Technol ; 6(1): 83-94, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18336088

RESUMEN

The Epic cell assay technology (Corning Inc., Corning, NY) uses a resonant waveguide grating optical biosensor to measure cellular response to ligands manifested through dynamic mass redistribution (DMR) of cellular contents. The DMR measurement is a noninvasive, label-free assay that can be used to assess the pharmacological properties of compounds. In this study, a panel of 12 compounds was evaluated against two G protein-coupled receptor (GPCR) targets in recombinant expressed cell lines using the Corning Epic system in 384-well microplates. The evaluation was performed in a double-blinded fashion such that the identity and properties of both the GPCR targets and compounds were unknown to the researchers at the time of the study. Analysis of the DMR response from cell stimulation was used to identify compounds that functioned as agonists or antagonists and to evaluate the associated efficacy and potency. DMR results were shown to have good agreement with data obtained from cyclic AMP and calcium flux assays for compounds evaluated. A further analysis was performed and successfully identified the signaling pathways that the two GPCRs activated. In addition, the DMR measurement was able to detect responses from an endogenous receptor in these cells. The Epic DMR technology provides a generic platform amenable to pharmacological evaluation of cellular responses to GPCR activation in a label-free live cell assay format.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/estadística & datos numéricos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Animales , Técnicas Biosensibles , Células CHO , Calcio/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Cinética , Modelos Estadísticos , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes , Transducción de Señal/efectos de los fármacos
8.
Bioorg Med Chem Lett ; 16(19): 5066-72, 2006 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-16887348

RESUMEN

Melanin-concentrating hormone (MCH) is a cyclic 19 amino acid orexigenic neuropeptide. The action of MCH on feeding is thought to involve the activation of its respective G protein-coupled receptor MCH-R1. Consequently, antagonists that block MCH regulated MCH-R1 activity may provide a viable approach to the treatment of diet-induced obesity. This communication reports the discovery of a novel MCH-R1 receptor antagonist, the biarylether 7, identified through high throughput screening. The solid-phase synthesis and structure-activity relationship of related analogs is described.


Asunto(s)
Éteres/síntesis química , Receptores de la Hormona Hipofisaria/antagonistas & inhibidores , Receptores de Somatostatina/antagonistas & inhibidores , Técnicas Químicas Combinatorias , Evaluación Preclínica de Medicamentos , Éteres/farmacología , Humanos , Concentración 50 Inhibidora , Obesidad/tratamiento farmacológico , Relación Estructura-Actividad
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