Literature Search and Review

Abstract

The use of ultra-high-throughput screens is a well-accepted mechanism to identify agonists and antagonists of target receptors. We used the Path Hunter® (DiscoveRx, Fremont, CA) to screen the entire Merck compound library for glucocorticoid receptor (GR) agonists in a 2.2-μl total reaction volume assayed in a 3,456-well plate format. This single-addition, homogeneous assay, which utilizes the principle of enzyme fragment complementation (EFC) to detect nuclear translocation of GR, an initial step of receptor activation, was used to successfully screen a large library of small molecules as indicated by an average signal-to-background ratio of approximately fourfold and an average Z-factor value of 0.45. Hits from the HTS campaign were studied in a cytokine secretion assay in primary human monocytes to gain functional information regarding these compounds in a phenotypic and physiologically relevant setting. Our data indicate that using the PathHunter assay, we successfully identified compounds that showed agonism for the GR receptor in primary human monocytes, and due to their performance in a physiologically relevant model they likely will have a better chance to evoke clinical efficacy.