Development of an HTS-Compatible Assay for Discovery of ROR alpha Modulators Using AlphaScreen (R) Technology

作者：Istrate Monica A; Spicer Timothy P; Wang Yan; Bernard Jerrold A; Helvering Leah M; Bocchinfuso Wayne P; Richardson Timothy I; Zink Richard; Kumar Naresh; Montrose Rafizadeh Chahrzad; Dodge Jeffrey; Hodder Peter; Griffin Patrick R^*

来源：Journal of Biomolecular Screening, 2011, 16(2): 183-191.

DOI：10.1177/1087057110389040

• nuclear receptor (NR)
• retinoid acid receptor-related orphan receptor (ROR)
• peroxisome proliferator-activated receptor gamma coactivator-1 alpha
• 7 alpha-hydroxycholesterol (7 alpha-OHC)
• AlphaScreen (R) technology
• assay performance
• high-throughput screening (HTS)

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 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 (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)

• 出版日期2011-2