Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands.

Kara DeSantis, Aaron Reed, Raneen Rahhal, Jeff Reinking

Nuclear receptor signaling 2012

filter terms:

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.

Citation

Kara DeSantis, Aaron Reed, Raneen Rahhal, Jeff Reinking. Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands. Nuclear receptor signaling. 2012;10:e002