A variety of studies from our laboratory have demonstrated that the parameters of GR-mediated gene induction (Amax, EC50, and PAA) can be modulated by changing the concentrations of involved cofactors. More recent studies with three cofactors that can form a ternary complex (GR, a coactivator, and a comodulator) revealed that different regions of the modulatory proteins do not affect all parameters equally and can selectively alter just one or two parameters (Awasthi and Simons, 2012, Mol Cell Endocrinol, 355, 121-134). Our previous studies in human peripheral mononuclear cells (PBMCs) confirmed that changes in cofactor concentration affect the induction parameters of endogenous, as well as exogenous, GR-regulated genes (Luo and Simons Jr., 2009, Human Immunology, 70, 785-789). These results provide strong support for our hypothesis that the modulation of one or more GR induction parameters is a relevant feature of human physiology. Glucocorticoid steroids affect almost every tissue-type and thus are widely used to treat a variety of human pathologies. 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 (Amax) and EC50, or the position of the dexamethasone dose-response curve. Upon screening 1280 chemicals, ten with the greatest change in the absolute value of Amax or EC50 were selected for further examination. Qualitatively identical behaviors for 60-90% 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 ten 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 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. The above studies are providing previously unobtainable molecular information about potential chemical modulators of those cofactors of GR transactivation activity, and possibly of the GR itself. They also constitute a rational approach to identifying chemical inhibitors or activators of specific steps in steroid hormone action. Such modulatory chemicals would be attractive leads for pharmaceutical interventions aimed at more precise control of endocrine therapies during development, differentiation, homeostasis, and endocrine therapies. Finally, this approach identifies downstream effectors of GR activity, which should have the clinically desirable properties of reduced side effects. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level in a manner that benefits human health.
| Blackford Jr, John A; Brimacombe, Kyle R; Dougherty, Edward J et al. (2014) Research resource: modulators of glucocorticoid receptor activity identified by a new high-throughput screening assay. Mol Endocrinol 28:1194-206 |
| Simons Jr, S Stoney; Edwards, Dean P; Kumar, Raj (2014) Minireview: dynamic structures of nuclear hormone receptors: new promises and challenges. Mol Endocrinol 28:173-82 |
| Simons Jr, S Stoney; Kumar, Raj (2013) Variable steroid receptor responses: Intrinsically disordered AF1 is the key. Mol Cell Endocrinol 376:81-4 |
| Blackford Jr, John A; Guo, Chunhua; Zhu, Rong et al. (2012) Identification of location and kinetically defined mechanism of cofactors and reporter genes in the cascade of steroid-regulated transactivation. J Biol Chem 287:40982-95 |
