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

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 (R) technology to develop a high-throughput screening (HTS)-compatible assay to facilitate the discovery of ROR alpha modulators. Using the ligand binding domain (LBD) of RORa and a peptide derived from the NR1 box of the nuclear receptor coactivator PGC-1 alpha, 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 alpha-hydroxycholesterol (7 alpha-OHC) function as modulators of the RORs. In this assay, 7 alpha-OHC produced a concentration-response curve with an EC50 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 (TM) 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 alpha. (Journal of Biomolecular Screening 2011;16:183-191)