Novel assays monitoring direct glucocorticoid receptor protein activity exhibit high predictive power for ligand activity on endogenous gene targets

Abstract

Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and auto-immune diseases. Unfortunately, their use is hampered by many side effects and therapy resistance. Efforts to find more selective glucocorticoid receptor (GR) agonists and modulators (called SEGRAMs) that are able to separate anti-inflammatory effects via gene repression from metabolic effects via gene activation, have been unsuccessful so far. In this study, we characterized a set of functionally diverse GR ligands in A549 cells, first using a panel of luciferase-based reporter gene assays evaluating GR-driven gene activation and gene repression. We expanded this minimal assay set with novel luciferase-based read-outs monitoring GR protein levels, GR dimerization and GR Serine 211 (Ser211) phosphorylation status and compared their outcomes with compound effects on the mRNA levels of known GR target genes in A549 cells and primary hepatocytes. We found that luciferase reporters evaluating GR-driven gene activation and gene repression were not always reliable predictors for effects on endogenous target genes. Remarkably, our novel assay monitoring GR Ser211 phosphorylation levels proved to be the most reliable predictor for compound effects on almost all tested endogenous GR targets, both driven by gene activation and repression. The integration of this novel assay in existing screening platforms running both in academia and industry may therefore boost chances to find novel GR ligands with an actual improved therapeutic benefit.

Glucocorticoids rescue cell surface trafficking of R451C Neuroligin3 and enhance synapse formation

“…The mechanism of action of GCs usually relies on pleiotropic effects of the GC receptor (GR) on several transcriptional pathways. GR activity is modulated by posttranslational modifications, such as phosphorylation at Ser211, which is increased after GCs administration 30 . To investigate the involvement of the GR in mediating the effects of GCs on R451C NLGN3 protein levels, we analyzed the phosphorylation of the receptor at Ser211 in lysates of cells expressing either WT or R451C NLGN3, untreated or treated with AD, D, PSP, or DEX.…”