title: Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands.
abstract: Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10µL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results.
authors.affiliation: Department of Biology, State University of New York at New Paltz, New Paltz, New York, USA.
authors.forename: Kara
authors.initials: K
authors.lastname: DeSantis
authors.forename: Aaron
authors.initials: A
authors.lastname: Reed
authors.forename: Raneen
authors.initials: R
authors.lastname: Rahhal
authors.forename: Jeff
authors.initials: J
authors.lastname: Reinking
mesh_terms: D004958:Estradiol / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D047628:Estrogen Receptor alpha; D008024:Ligands; D004958:Estradiol