Research resource: modulators of glucocorticoid receptor activity identified by a new high-throughput screening assay

Abstract

Glucocorticoid steroids affect almost every type of tissue and thus are widely used to treat a variety of human pathological conditions. However, the severity of numerous side effects limits the frequency and duration of glucocorticoid treatments. Of the numerous approaches to control off-target responses to glucocorticoids, small molecules and pharmaceuticals offer several advantages. Here we describe a new, extended high-throughput screen in intact cells to identify small molecule modulators of dexamethasone-induced glucocorticoid receptor (GR) transcriptional activity. The novelty of this assay is that it monitors changes in both GR maximal activity (A(max)) and EC(50) (the position of the dexamethasone dose-response curve). Upon screening 1280 chemicals, 10 with the greatest changes in the absolute value of A(max) or EC(50) were selected for further examination. Qualitatively identical behaviors for 60% to 90% of the chemicals were observed in a completely different system, suggesting that other systems will be similarly affected by these chemicals. Additional analysis of the 10 chemicals in a recently described competition assay determined their kinetically defined mechanism and site of action. Some chemicals had similar mechanisms of action despite divergent effects on the level of the GR-induced product. These combined assays offer a straightforward method of identifying numerous new pharmaceuticals that can alter GR transactivation in ways that could be clinically useful.

Figure 1.

Optimization of GR transactivation in high-throughput assay. A, Dose-response curve for GR-mediated induction of luciferase by Dex. The assay was conducted as described in Materials and Methods. Data are plotted as a percentage of the maximal response. The curve represents the best fit to a Michaelis-Menten plot. The dashed lines indicate Dex concentrations required for 10% and 90% of full induction. B, Relative dose-response curves for 4 known GR ligands: Dex, dexamethasone-21-mesylate (Dex-21-Mes), cortisol, and aldosterone. Assays were conducted as in panel A. Data are plotted as relative luciferase activity (±SD) and best fit to a Michaelis-Menten plot.

Figure 2.

Layout of GR modulation qHTS assay. The dosing array for the GR modulation screen is shown, with final Dex concentrations along the left and compound library concentrations along the top.

Figure 3.

GREtkLUC acts as an accelerator at the CLS. Values of A_max and EC₅₀ from competition assays of the indicated amounts of GREtkLUC plus AC93253 (A), sanguinarine (Sang; B), and stattic (C) were determined and plotted as described in Materials and Methods. Similar results were obtained in 4, 8, and 3 additional independent experiments, respectively.

Figure 4.

Chemicals acting as a competitive decelerator at or before the CLS. Values of A_max and EC₅₀ from competition assays of the indicated concentrations of GREtkLUC plus AC93253 (A), sanguinarine (B), and stattic (C) were determined and plotted as described in Materials and Methods. Similar results were obtained in 4, 8, and 3 additional independent experiments, respectively.

Figure 5.

GREtkLUC acts as an accelerator at the CLS in assay with nocodazole (Nocod) acting as an accelerator after the CLS. Values of A_max and EC₅₀ from competition assays of the indicated amounts of GREtkLUC and nocodazole were determined and plotted as 1/EC₅₀ vs GREtkLUC (A), A_max/EC₅₀ vs GREtkLUC (B), and A_max/EC₅₀ vs nocodazole (C) as described in Materials and Methods. Similar results were obtained in 2 additional independent experiments.