title: Miniaturization of cell-based beta-lactamase-dependent FRET assays to ultra-high throughput formats to identify agonists of human liver X receptors.
abstract: 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 beta-lactamase as the reporter gene. Stable Chinese hamster ovary cell lines expressing LXRalpha-GAL4 or LXRbeta-GAL4 fusion proteins that regulate beta-lactamase transcription from upstream 7 x UAS GAL4 DNA binding sequences were generated and characterized. Synthetic and natural ligands of LXR dose-dependently activated the expression of beta-lactamase in a subtype-specific manner. These assays were used to demonstrate that a 1-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXRalpha receptors. The beta-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation. Clonal LXRbeta-GAL4-beta-lactamase cells were miniaturized into an ultra high throughput (3456-well nanoplates) screening format.
authors.affiliation: Department of Atherosclerosis and Endocrinology, Merck Research Laboratories, Rahway, NJ 07065, USA.
authors.forename: Jayne
authors.initials: J
authors.lastname: Chin
authors.forename: Alan D
authors.initials: AD
authors.lastname: Adams
authors.forename: Aileen
authors.initials: A
authors.lastname: Bouffard
authors.forename: Ahren
authors.initials: A
authors.lastname: Green
authors.forename: Raul G
authors.initials: RG
authors.lastname: Lacson
authors.forename: Todd
authors.initials: T
authors.lastname: Smith
authors.forename: Paul A
authors.initials: PA
authors.lastname: Fischer
authors.forename: John G
authors.initials: JG
authors.lastname: Menke
authors.forename: Carl P
authors.initials: CP
authors.lastname: Sparrow
authors.forename: Lyndon J
authors.initials: LJ
authors.lastname: Mitnaul
mesh_terms: D000818:Animals; D001565:Benzoates / Q000737:chemistry / Q000494:pharmacology; D001596:Benzylamines / Q000737:chemistry / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D000071518:Liver X Receptors; D008904:Miniaturization / Q000379:methods*; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists* / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D001565:Benzoates; D001596:Benzylamines; D004268:DNA-Binding Proteins; C473027:GW 3965; D000071518:Liver X Receptors; C000605213:NR1H3 protein, human; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D001618:beta-Lactamases