RESUMEN
Several potent inhibitors of hepatitis C virus (HCV) NS3/4A protease have been identified that show great clinical potential against genotype 1. Due to the tremendous genetic diversity that exists among HCV isolates, development of broad spectrum inhibitors is challenging. With a limited number of lab strains available for preclinical testing, new tools are required for assessing protease inhibitor activity. We developed a chimeric replicon system for evaluating NS3 protease inhibitor activity against naturally occurring isolates. NS3/4A genes were cloned from the plasma of HCV-infected individuals and inserted into lab strain replicons, replacing the native sequences. The chimeric reporter replicons were transfected into Huh 7.5 cells, their replication monitored by luciferase assays, and their susceptibilities to inhibitors determined. Viable chimeras expressing heterologous genotypes 1, 2, 3, and 4 protease domains were identified that exhibited varying susceptibilities to inhibitors. Protease inhibitor spectrums observed against the chimeric replicon panel strongly correlated with published enzymatic and clinical results. This cell-based chimeric replicon system can be used to characterize the activities of protease inhibitors against diverse natural isolates and may improve the ability to predict dose and clinical efficacy.
Asunto(s)
Antivirales/farmacología , Hepacivirus/genética , Inhibidores de Proteasas/química , Replicón , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/química , Secuencia de Bases , Sitios de Unión , Carbamatos/química , Carbamatos/farmacología , Línea Celular , Clonación Molecular , Transferencia Resonante de Energía de Fluorescencia/métodos , Variación Genética , Vectores Genéticos/genética , Genotipo , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , Humanos , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Pruebas de Sensibilidad Microbiana , Oligopéptidos/química , Oligopéptidos/farmacología , Filogenia , Inhibidores de Proteasas/farmacología , Quinolinas/química , Quinolinas/farmacología , Análisis de Secuencia de Proteína/métodos , Tiazoles/química , Tiazoles/farmacología , Proteínas no Estructurales Virales/química , Replicación ViralRESUMEN
With the sequence of the human genome at hand, target discovery strategies are needed that can rapidly identify novel gene products involved in human disease pathways. In this article, the authors describe a cell-based, high-throughput assay that can identify gene products capable of modulating the vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha) signaling pathways in human endothelial cells. The assay uses real-time PCR technology to measure downstream reporter mRNA transcripts induced upon cytokine stimulation in a 96-well plate format and has been adapted for use with recombinant adenoviruses. The authors specifically demonstrate modulation of cytokine-driven reporter transcripts using drug inhibitors and through adenoviral-mediated expression of known signaling intermediates of the respective pathways. In addition, they have used an arrayed library of 350 recombinant adenoviruses to screen for novel modulators of the VEGF and TNFalpha pathways. The high-throughput screening capacity and sensitivity of this system make it a useful tool for new drug target identification.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Endotelio Vascular/efectos de los fármacos , Genómica/métodos , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Adenoviridae/genética , Bioensayo , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Biblioteca de Genes , Técnicas de Transferencia de Gen , Genes Reporteros , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Tromboplastina/genética , Tromboplastina/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Analysis of the Ah Receptor Signal Transduction Pathway (Michael S. Denison, Jane M. Rohers, S. Renee Rushing. Carol L. Jones, Selwyna C. Tetangico, and Sharon Heath-Pagliuso, University of California, Davis, California).The protocols in this unit will allow researchers to detect the Ah receptor and characterize its functional activities (i.e., ligand binding, transformation and DNA binding, and gene expression) in their biological test system and to use these methods to detect chemical and biochemical events that affect this signaling system.