Identification of Nuclear Receptor Ligands using Differential Scanning Fluorimetry

Abstract

Identification of ligands that interact with nuclear receptors (NRs) 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 NR ligand candidate chemical library to identify interactors to several well characterized human NRs. Compounds that interact specifically with NR Ligand Binding Domains stabilize the protein and result in an elevation of the thermal denaturation point as monitored by the environmentally sensitive dye SYPRO orange. We present results validating DSF as a new tool for identifying NR ligands and screening candidate compounds and also present results of orphan NRs currently under investigation.