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 ? ligand binding domain (ER? LBD). Compounds that interact specifically with ER? 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 ER?, with no false positive results.

SUBMITTER: DeSantis K 

PROVIDER: S-EPMC3309076 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature