Miniaturization of Cell-Based β -Lactamase-Dependent FRET Assays to Ultra-High Throughput Formats to Identify Agonists of Human Liver X Receptors

Activation 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 LXRα-GAL4 or LXRβ-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 LXRα receptors. The β-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation. Clonal LXRβ-GAL4-β-lactamase cells were miniaturized into an ultra high throughput (3,456-well nanoplates) screening format.