A high-throughput chemical screen identifies novel inhibitors and enhancers of anti-inflammatory functions of the glucocorticoid receptor

Abstract

Glucocorticoids (GCs)—ligands of the glucocorticoid receptor (GR)—are widely used to treat inflammatory diseases, but suffer from significant side effects and poor responsiveness in certain patient populations. Identification of chemical GR modulators may provide insights into the regulatory mechanisms of anti-inflammatory functions of GR and help improve GC-based therapy. Here we report the development and application of a high-throughput screening to identify compounds that either enhance or suppress the anti-inflammatory effect of GR function. Using a cell-based GR activity assay that measures Dexamethasone (Dex)-mediated NF-κB repression, we have screened ~8,000 compounds and identified several compounds that suppressed GR activity, including multiple GSK3β inhibitors and anti-cancer agent camptothecin. Notably, we also identified two kinase IKK2 inhibitors, including TPCA-1, as GR enhancers that improve the anti-inflammatory effect of GR. In particular, TPCA-1 augmented the activity of Dex in NF-κB repression by attenuating GR down-regulation. Consistent with the observation, siRNA-mediated IKK2 knockdown decreased GR down-regulation and increased GR expression. Together, our results identified chemical compounds as novel modulators of GR and revealed an unexpected role for IKK2 in GR down-regulation. Furthermore, we have established a high-throughput screening platform for discovering GR-modulating compounds that may be repurposed to improve current GC-based therapies.

Expression of SMARCD1 interacts with age in association with asthma control on inhaled corticosteroid therapy

“…To investigate whether SMARCD1 plays a role in the inflammatory response as well as in the anti-inflammatory action of corticosteroids in vitro, we knocked down SMARCD1 in a human lung epithelial cell line (similar to the replication samples which were enriched for airway epithelium) stably expressing nuclear factor-kappa B (NF-κB) luciferase reporter [24]. We transfected this reporter cell line with two different SMARCD1 siRNAs.…”

“…In vitro verification A549/NF-κB-luc reporter cells were transfected with either scramble control or one of two SMARCD1 small interfering RNAs (siRNA). These cells are responsive to IL-1β stimulation, which is reduced by dexamethasone, a glucocorticoid receptor agonist, indicating that these cells exhibit glucocorticoid-mediated tethered transrepression of NF-κB [24]. 48 h post-transfection, the cells were stimulated with 5 ng/mL IL-1β ± 5 nM Dexamethasone.…”

Drug Repurposing to Treat Glucocorticoid Resistance in Asthma

“…The top 10 available drug compounds with the highest weighted mean negative connectivity scores were screened, and five were chosen for in vitro validation based on safety data in humans, mechanistic plausibility, and drug availability. Functionally validation was performed using a previously described glucocorticoid-mediated tethered transrepression of NF-κB activity assay [ 33 ]. A549/NF-κB-luc reporter cells were treated with the selected compounds at indicated concentrations for 1 h. Standard drug concentrations were used (1 µM or 1µg/mL) and increased up to the limit where no cell death occurred.…”

“…A549/NF-κB-luc reporter cells were treated with the selected compounds at indicated concentrations for 1 h. Standard drug concentrations were used (1 µM or 1µg/mL) and increased up to the limit where no cell death occurred. These A549/NF-κB-luc reporter cells are responsive to interleukin (IL)-1β stimulation, which is reduced by dexamethasone, a glucocorticoid receptor agonist, indicating that these cells exhibit glucocorticoid-mediated tethered transrepression of NF-κB [ 33 ]. One hour post-treatment, the cells were stimulated with 5 ng/mL IL-1β ± 2 nM dexamethasone.…”

“…Together, these data suggest that γ-linolenic acid, brinzolamide, and INCA-6 can each act as independent anti-inflammatory agents and synergistically augment the anti-inflammatory effects of corticosteroids. inflammatory effect of glucocorticoids using an assay based on the tethered transrepression of NF-κB activity by dexamethasone [33]. γ-linolenic acid, brinzolamide, and INCA-6 significantly reduced IL-1β induced luciferase activity in A549/NF-κB-luc reporter cells (Figure 1).…”