RESUMEN
Screening of the Merck compound collection identified 6 as an unusually simple, low molecular weight hit with moderate affinity for GABAA receptors. The structural novelty of 6, compared to our advanced series of GABAA alpha5 inverse agonists, made it an attractive molecule for further exploration. This paper will describe the evolution of 6 into a new series of ligands with nanomolar affinity and functional selectivity for GABAA alpha5 receptor subtypes.
Asunto(s)
Piridazinas/síntesis química , Receptores de GABA-A/efectos de los fármacos , Animales , Línea Celular , Humanos , Técnicas In Vitro , Ligandos , Masculino , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Técnicas de Placa-Clamp , Subunidades de Proteína/fisiología , Piridazinas/farmacocinética , Piridazinas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/fisiología , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
This report presents a highly automated procedure for the determination of drug concentrations in plasma samples. The method is generic, in that it has been applied without adaptation to many different drug candidate molecules, but is also flexible, in that variations in the nature and number of samples to be analyzed can be readily accommodated. The method includes preparation of dilutions of analyte stock solutions, spiking these into control plasma to generate analytical standards, and preparation of samples suitable for analysis by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) by precipitation of plasma proteins with acetonitrile, centrifugation, and dilution of the supernatants with HPLC buffer. All of these steps, apart from centrifugation, are performed without manual intervention on an automated liquid-handling workstation using 96-well plates. Analysis is by HPLC/MS/MS, using a generic HPLC gradient. Commercially available software was used for optimization of parameters for analysis by HPLC/MS/MS, integration of chromatographic peaks, and quantification of drug concentrations. The use of this methodology in our laboratory has greatly facilitated the analysis of small sample sets for a large number of analytes, a situation regularly encountered in an early drug discovery environment.