Janneke Simon¹, Jonathan Thommis², Karolien de Bosscher², Onno Meijer¹, Jan Kroon¹ 
 
(1) LUMC, Department of Endocrinology , Leiden, The Netherlands. (2) VIB Center for Medical Biotechnology, Ghent, Belgium.  

Synthetic glucocorticoids (GCs) like dexamethasone (DEX) are widely used for their strong anti-inflammatory actions. Although highly effective, prolonged exposure to synthetic GCs is associated with metabolic, musculoskeletal and neurocognitive side effects. Underlying this dual effect are different mechanisms through which the liganded glucocorticoid receptor (GR) can exert its transcriptional effect. Transrepression, where the GR-GC complex tethers to other transcription factors like NFkB, is believed to drive its anti-inflammatory effects. Transactivation, which entails direct binding of GR dimers to glucocorticoid response elements (GRE) on the chromatin, is believed largely responsible for GC-induced side effects. Dissociating these mechanisms with single ligands to favor transrepression over transactivation has been pursued for decades, with only limited results. 

We aim to identify molecular pathways that affect GR signaling in a dissociative manner by decreasing DEX-induced GR transactivation while maintaining or enhancing GR transrepression. To this end, we optimized a high-throughput in vitrodrug screening assay using three complementary A549 cell lines expressing firefly-luciferase reporter constructs. We used A549-NFkB_Luc cells to monitor GR transrepression as a measure for anti-inflammatory efficacy, whereas A549-GRE_Luc cells monitor GR transactivation as a proxy for GC-induced side effects. Constitutive luciferase activity of the A549-NEO-Luc cells allows for cytotoxicity monitoring. We demonstrate assay reproducibility and GR-mediated responsiveness, as validation runs showed an additive induction of DEX-induced GRE-Luc activity by other synthetic GCs. Additionally, the GR antagonist RU486 diminished both transrepressive and transactivating effects of DEX. 

To date, we screened 1002 drugs, of which 35 decreased DEX-induced transactivation while maintaining or enhancing anti-inflammatory efficacy without inducing toxicity. In conclusion, we developed a promising pipeline for identifying drugs exerting dissociative effects on GR signaling in vitro.