Screening a plant extract library for inhibitors of cholecystokinin receptor CCK1 pathways.

This study describes the screening of a plant extract library for inhibitors of signal transduction pathways mediated by the cholecystokinin receptor, CCK1. CCK1 receptors are coupled to Galpha(q/11)-proteins, localized mainly in the gastrointestinal tract, and implicated in the regulation of various digestive functions. A primary screen was performed using a cell-based assay that used the beta-lactamase gene reporter controlled by the transcriptional activator NFAT. The assay was validated with the CCK1 receptor antagonist, lorglumide, and automated by the use of a liquid-handling robot MultiProbe II. Off-target hits were triaged by counterscreening against gene reporter cells activated by a combination of thapsigargin and phorbol ester. Purification of active compounds was guided by the beta-lactamase gene reporter and Ca2+ mobilization assays. Pure compounds were characterized by Ca2+ mobilization, radioligand binding, inositol-1 phosphate formation, and Eu-GTP binding assays. The selectivity of inhibition was tested against a panel of Galpha(q/11), Galpha(s), and Galpha(i/0)-coupled receptors. These studies led to the identification of a novel Galpha(q/11)-selective inhibitor.