Miniaturization of Cell-Based β -Lactamase-Dependent FRET Assays to Ultra-High Throughput Formats to Identify Agonists of Human Liver X Receptors
AbstractActivation of liver X receptors (LXRs) induces reverse cholesterol transport and increases high-density lipoprotein cholesterol in vivo . Here, we describe novel, functional, homogeneous cell-based fluorescence resonance energy transfer assays for identifying agonists of LXRs using β -lactamase as the reporter gene. Stable Chinese hamster ovary cell lines expressing LXR α -GAL4 or LXR β -GAL4 fusion proteins that regulate β -lactamase transcription from upstream 7 × UAS GAL4 DNA binding sequences were generated and characterized. Synthetic and natural ligands of LXR dose-dependently activated the expression of β -lactamase in a subtype-specific manner. These assays were used to demonstrate that a 1-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXR α receptors. The β -lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation. Clonal LXR β -GAL4- β -lactamase cells were miniaturized into an ultra high throughput (3,456-well nanoplates) screening format.