A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors

The identification of agonist and antagonist leads for G-protein-coupled receptors (GPCRs) is of critical importance to the pharmaceutical and biotechnology industries. We report on the utilization of a novel, high-density, well-less screening platform known as microarrayed compound screening (muARCS) that tests 8640 compounds in the footprint of a standard microliter plate for the identification of novel agonists for a specific G-protein-coupled receptor. Although receptors coupled to the Galpha(q) protein can readily be assessed by fluorescence-based Ca2+ release measurements, many GPCRs that are coupled to Galpha(s) or Galpha(i/o) proteins are not amenable to functional evaluation in such a high-throughput manner. In this study, the human dopamine D-4.4 receptor, which normally couples through the Galpha(i/o) protein to inhibit adenylate cyclase and to reduce levels of intracellular cAMP, was coupled to intracellular Ca2+ release by stably coexpressing this receptor with a chimeric G(alphaqo5) protein in HEK-293 cells. In muARCS format, the cells expressing D-4.4 receptor and Galphaq(o5) protein were preloaded with fluo-4, cast into a 1% agarose gel, placed above the compound sheets, and imaged successively using a ViewLux charge-coupled device imaging system. Dopamine and other agonists evoked an increase in fluorescence response that appeared as bright spots in a time- and concentration-dependent manner. Utilizing this technology, a library of 260,000 compounds was rapidly screened and led to the identification of several novel agonists. These agonists were further characterized using a fluorometric imaging plate reader assay. Excellent confirmation rates coupled with enhanced efficiency and throughput enable muARCS to serve as an alternative platform for the screening and identification of novel GPCR agonists. (C) 2003 Elsevier Inc. All rights reserved.