title: Development of an HTS-compatible assay for discovery of RORα 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 RORα modulators. Using the ligand binding domain (LBD) of RORα and a peptide derived from the NR1 box of the nuclear receptor coactivator PGC-1α, 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 7α-hydroxycholesterol (7α-OHC) function as modulators of the RORs. In this assay, 7α-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 RORα.
authors.affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458, USA.
authors.forename: Monica A
authors.initials: MA
authors.lastname: Istrate
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Spicer
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Jerrold A
authors.initials: JA
authors.lastname: Bernard
authors.forename: Leah M
authors.initials: LM
authors.lastname: Helvering
authors.forename: Wayne P
authors.initials: WP
authors.lastname: Bocchinfuso
authors.forename: Timothy I
authors.initials: TI
authors.lastname: Richardson
authors.forename: Richard
authors.initials: R
authors.lastname: Zink
authors.forename: Naresh
authors.initials: N
authors.lastname: Kumar
authors.forename: Chahrzad
authors.initials: C
authors.lastname: Montrose-Rafizadeh
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Dodge
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Griffin
mesh_terms: D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D057809:HEK293 Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1 / Q000378:metabolism*; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D006360:Heat-Shock Proteins; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; C486497:PPARGC1A protein, human; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; C539738:RORA protein, human; D014157:Transcription Factors