Abstract
σ receptors represent a potential drug target for numerous therapeutic indications including cancer, depression, psychostimulant abuse, and stroke. Most published radioligand binding studies for σ receptors utilize a low throughput method employing a "cell harvester." Higher throughput methods are required to facilitate efficient screening of large numbers of novel compounds. In this study, a series of reference compounds was analyzed with a new medium-throughput 96-well filtration method and the results were compared to those obtained using the conventional cell harvester-based method. The 96-well assay utilized rat liver membranes for the determination of both known σ receptor subtypes (σ(1) and σ(2)) because this tissue contains high densities of both subtypes and fulfills criteria required for reliable use with the 96-well format. The new method gave comparable K(i) values for reference ligands analyzed in parallel with samples prepared in rat brain membranes and processed on the traditional cell harvester. For σ(1) receptors, equivalent affinity values were observed for both methods/tissues. For σ(2) receptors, approximately 2-fold higher affinities were observed for most compounds in liver, as compared to brain membranes, but excellent correlation with brain-derived values was maintained. To further demonstrate the utility of the new method it was used to screen a novel series of 2(3H)-benzothiazolone compounds, resulting in the identification of several analogues with nanomolar affinity and greater than 50-fold specificity for σ(1) versus σ(2) receptors.