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. The number of reported cofactors involved in steroid-regulated gene expression that can modify Amax, EC50, and PAA is greater than 350 and still growing. A variety of reports with endogenous genes indicate that a viable method for increasing the specificity of endocrine therapies is to selectively alter the intracellular abundance of one or more specific cofactors. Unfortunately, despite the possible rewards of genetic engineering, manipulating the levels of proteins is proving difficult. An alternative, and potentially simpler approach is to seek chemicals that can alter the activity and/or abundance of the modulatory cofactors. To this end, we are collaborating with Chris Austin and Kyle Brimacombe (NCGC, NIH) to use high-throughput screening to identify chemicals that alter the Amax, EC50, and/or PAA of GR-regulated gene induction. Such a screen has, as far as we are aware, not been performed up to now, partially because nobody other than Dr. Austin is set up to follow more than one parameter. We have constructed a dual reporter plasmid for the high-throughput screening. This reporter contains a GR-inducible Luciferase and a non-GR-inducible green fluorescence protein (GFP) as an internal control. The Luciferase activity of this reporter, after transient transfection into cells, is modulated by exogenous cofactors in the same manner as seen with the simple GR-inducible Luciferase reporter of our earlier studies. Optimization of signal output has been accomplished in the high-throughput assay. Screening and data analysis of the LOPAC library of 1280 compounds has been completed. The most promising 10 compounds are being examined in greater detail in our laboratory using our newly developed competition assay. This assay will permit the classification of both the type of modulator, in terms of unambiguous kinetically-defined mechanisms, and the position of action of the chemical. So far, there is reasonably good agreement between the high throughput screening and the more precise competition assay. This information will be matched with the sites of action of a variety of cofactors that we have already examined in an effort to determine the target cofactor of each chemical. Direct competition assays of the chemical and the putative target will be run to confirm initial conclusions. 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. 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.
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