NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

NCGC Assay Overview:
The glucocorticoid receptor (GR) is a cytoplasmic receptor that belongs to the nuclear receptor family of ligand-dependent transcription factors. Upon glucocorticoid binding to its receptor, the glucocorticoid-GR complex translocates into the nucleus, where it binds as a dimer to specific DNA sequences (glucocorticoid response elements), enhancing or suppressing transcription of a wide variety of glucocorticoid-responsive genes. In addition to their important roles in normal physiology and metabolism, glucocorticoids are administered exogenously as treatments for a wide variety of allergic, autoimmune, and neoplastic conditions. Identification of compounds that modulate aspects of GR signalling would be useful in dissecting glucocorticoid response pathways as well as developing GR modulators with more targeted clinical effects.

Traditionally, GR modulators have been identified using receptor binding or direct transactivation assays. However, assays for glucocorticoid-dependent nuclear translocation can also be used to screen for compounds affecting GR signaling.
Immunocytochemical staining has been used to assay for GR translocation, but is not suitable for high throughout screening due to the multiple reagent addition and wash steps required. Recently, a novel cell-based GR assay in Enzyme Fragment Complementation (EFC) format was developed by DiscoveRx (Fremont, CA). The assay uses EFC of b-galactosidase (b-gal) as an indicator of GR-translocation in engineered CHO-K1 biosensor cells. The enzyme acceptor (EA) fragment of b-gal resides in the nucleus , as designed through the use of a proprietary set of sequence additions and modifications. The small peptide enzyme donor (ED) fragment of b-gal is fused directly to the C-terminus of GR, and is localized in the cytoplasm is the absence of receptor signaling. Upon binding to a GR ligand, the complex translocates to the nucleus, where intact enzyme activity is restored by complementation and b-gal activity is detected. We optimized this GR-EFC assay in homogenous 1536-well plate assay format for high throughput screening.

NCGC Assay Protocol Summary:

CHO-K1 cells stably expressing NLS-enzyme acceptor fragment (EA) of b-gal and GR-enzyme donor (ED) fragment of b-gal were maintained in F12 medium (Invitrogen, Carlsbad, CA) at 37 Celsius degrees under a humidified atmosphere containing 5% CO2 and 95% air. The medium contained 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin 50 ug/ml streptomycin, and 250 ug/ml hygromycin and 500 ug/ml G418 (Invitrogen).

GR-translocation was measured beta-gal enzyme activity using the PathHunter Detection Kit containing cell membrane permeablizing reagent and beta-gal substrate (DiscoveRx, Fremont, CA). All compounds were screened as titrations from 0.6 nM to 46 uM final concentration. The assay was performed in 1536-well format as follows:
5 uL cell suspension (1,000 cells)/well
23 nL compound
Incubation at 37 Celsius degrees for two hours
1.25 uL 1X detection buffer from the kit supplied by DiscoveRx
Incubation at room temperature for one hour
Read plate in luminescence mode in ViewLux plate reader (PerkinElmer)

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, GR-EFC, GREFC, qHTS, NCGC

References:

Fung, et al. "Homogeneous Cell-Based Assay to Measure Nuclear Translocation Using b-Galactosidase Enzyme Fragment Complementation" ASSAY Drug Devel.Technol. 2006, 4(3), 263-272

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive

2. Within each curve class, compounds are ranked by potency