FIG. 5.

Effect of Compound 11a on interaction of nuclear receptors with transcriptional corepressors. (A) Addition of Compound 11a at indicated concentrations did not affect the NR2E3–RetCOR interaction in the TR-FRET assay. Biotin at 10 μM was used as a surrogate positive control to mimic the effect of an agonist as described in the Methods section. (B) Increasing concentrations of Compound 11a (structure shown in the inset) were added to CHO cells cotransfected with the GAL4-responsive pGL4.35 luciferase reporter along with GAL-NCOR and VP16-NR2E3_LBD plasmids. In the absence of ligand, NR2E3–NCOR interaction brings the VP16 activation domain to the vicinity of the luciferase promoter-stimulating luciferase gene expression. Compound 11a dose-dependently decreased luciferase expression. (C, D) Evaluation of Compound 11a added at 20 μM on TRβ–NCOR (C), and RARα–NCOR (D) interactions in the mammalian 2-hybrid assay. pGL4.35 reporter and a GAL-NCOR plasmid along with VP16-TRβ_LBD (C) or VP16-RARα_LBD (D) plasmids were used in transfections. Control nuclear receptor agonists, 3,3′,5-triiodo-l-thyronine (T3) and 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl benzoic acid (TTNPB), specifically induced the NCOR release from TRβ (C) or RARα (D), respectively. Compound 11a nonspecifically inhibited luciferase expression in TRβ–NCOR (C) and RARα–NCOR (D) transfections. Control experiments shown in (C, D) include transfections conducted with the pGL4.35 reporter alone, as well as with pGL4.35 plus GAL-NCOR and pGL4.35 plus VP16-TRβ_LBD (C) or VP16- RARα_LBD (D) constructs. The numbers over the bars indicate luciferase activity in CHO cell extracts expressed in arbitrary light units.