title: Development of the high throughput screening assay for identification of agonists of an orphan nuclear receptor.
abstract: Retina-specific nuclear receptor (RNR), also known as PNR and NR2E3, is an orphan nuclear receptor expressed exclusively in photoreceptor cells of the retina. Here we describe homogeneous cell-based resonance energy transfer assay for identification of RNR agonists using beta-lactamase as the reporter gene. Bacterial beta-lactamase reporter construct containing GAL4 response elements was randomly integrated into the genome with subsequent selection of responsive cell pools by fluorescence-activated cell sorting. Chimeric RNR (RNR hinge and ligand-binding domains fused to GAL4 DNA-binding domain) was stably transfected into mammalian Flp-In Chinese hamster ovary cells using Flp-mediated recombination into a single pre-integrated Flp recombination target site. Since no RNR ligand could be used as a control for monitoring the development of the RNR assay, we developed a parallel cell line with the functionally related well-characterized thyroid hormone nuclear receptor. This parallel thyroid hormone nuclear receptor system was used as a "guide" in optimizing the RNR assay for ultra-high-throughput screening in 3,456-well nanoplate format. The assay was successfully used to screen a large compound collection for RNR agonists. In this study we demonstrated the feasibility of developing and optimization of the high-throughput screening-compatible assay for the orphan nuclear receptor in the absence of its cognitive ligand.
authors.affiliation: Department of Ophthalmics Research, Merck Research Laboratories, West Point, PA 19486-0004, USA.
authors.forename: Marianna
authors.initials: M
authors.lastname: Kapitskaya
authors.forename: Michael E
authors.initials: ME
authors.lastname: Cunningham
authors.forename: Raul
authors.initials: R
authors.lastname: Lacson
authors.forename: Oleg
authors.initials: O
authors.lastname: Kornienko
authors.forename: Bohumil
authors.initials: B
authors.lastname: Bednar
authors.forename: Konstantin
authors.initials: K
authors.lastname: Petrukhin
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015195:Drug Design; D005434:Flow Cytometry / Q000379:methods*; D057093:Orphan Nuclear Receptors; D004364:Pharmaceutical Preparations / Q000008:administration & dosage* / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014157:Transcription Factors / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C403073:NR2E3 protein, human; D057093:Orphan Nuclear Receptors; D004364:Pharmaceutical Preparations; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors