Figure 1

A high-through chemical screen to identify GR modulators. (A) Establishment of a stable reporter cell line A549/NF-κB-luc to assay tethered trans-repression of NF-κB activation by GR. A plasmid in which five tandem NF-κB responsive elements (5 × NF-κB-RE) was put upstream of mini promoter (MP) to control Luc2P expression was stably transfected into A549 cells. The individual colonies were selected, expanded and characterized. (B) A549/NF-κB-luc reporter cells were sensitive to IL-1β in NFκB activation and sensitive to Dex in NF-κB repression by GR. Cells were treated with 5 ng/mL IL-1β ± Dex as indicated for 18 h and Luciferase assay was performed. (C) Schematic representation of the chemical screen design. A549/NF-κB-luc reporter cells were seeded in 384 well plates, and library compounds were added to the cell plates. One hour later, 5 ng/mL IL-1β was added to assay I plates and 5 ng/mL IL-1β + 2.5 nM Dex was added to assay II plates. Luciferase assay was performed after 18 h treatment and MAD based Z scores for each well and ratio II/I of each tested compound were calculated. The inhibitor and enhancer hits were selected based on predefined criteria (see Results). (D) Summary of the GR inhibitors and enhancers identified in the screen.