RESUMEN
A high-throughput screen at 100 microM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Imidazolidinas/síntesis química , Imidazolidinas/farmacología , Piperidinas/síntesis química , Piperidinas/farmacología , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Enlace de Hidrógeno , Imidazolidinas/química , Modelos Moleculares , Estructura Molecular , Piperidinas/química , Relación Estructura-ActividadRESUMEN
Safe and efficient methods for in vivo delivery of transgenes of interest must be developed so that the promise of these therapies can be practically used in the clinic. In this work, we describe the use of electrostimulation to enhance the in vivo efficiency of plasmid DNA delivery. The method was optimized to work over a range of moderate frequencies, utilizing low field strengths and simple symmetrical waveforms. After studying several parameters of delivery in mice, we demonstrate how this methodology can be employed to significantly improve both gene expression (over 16-fold) and the immunogenicity of HIV-1 vaccines (over 28-fold) compared to naked DNA in non-human primates. Compared to an efficient viral Ad5 vector system, the gene expression levels of DNA+electrostimulation were surprisingly within a factor of four of the viral delivery system.
Asunto(s)
Vacunas contra el SIDA/inmunología , Electroporación/métodos , Vectores Genéticos , Plásmidos/genética , Transgenes , Vacunas de ADN/inmunología , Vacunas contra el SIDA/genética , Fosfatasa Alcalina/genética , Animales , Línea Celular , Expresión Génica , Técnicas de Transferencia de Gen , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Humanos , Macaca , Ratones , Ratones Endogámicos BALB C , Vacunas de ADN/genéticaRESUMEN
This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.