Development of an HTS-compatible assay for discovery of ROR? modulators using AlphaScreen® technology.

ABSTRACT: The retinoid acid receptor-related orphan receptors (RORs) represent important targets for the treatment of metabolic and immune disorders. Here the authors describe the application of AlphaScreen(®) technology to develop a high-throughput screening (HTS)-compatible assay to facilitate the discovery of ROR? modulators. Using the ligand binding domain (LBD) of ROR? and a peptide derived from the NR1 box of the nuclear receptor coactivator PGC-1?, a 384-well format assay was developed exhibiting high sensitivity, requiring only low nanomolar concentration of reagents. Recently, it was shown that oxysterols such as 7?-hydroxycholesterol (7?-OHC) function as modulators of the RORs. In this assay, 7?-OHC produced a concentration-response curve with an EC(50) of 162 nM, a Z' factor of 0.6, and a signal-to-background (S/B) ratio of 4.2, demonstrating that the assay is HTS compatible. Validation of the assay was afforded by screening against the Sigma LOPAC1280™ library in a 384-well format. In summary, the results presented here demonstrate that this assay can be used to screen large chemical libraries to discover novel modulators of ROR?.

SUBMITTER: Istrate MA 

PROVIDER: S-EPMC4434603 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature