Modulation of glucocorticoid receptor-mediated gene induction by chemicals

我们实验室的各种研究表明，GR介导的基因诱导参数(Amax、EC50和PAA)可以通过改变相关辅因子的浓度来调节。最近对三个可形成三元复合体(GR、共激活因子和共调节因子)的辅因子的研究表明，调节蛋白的不同区域并不等同地影响所有参数，只能选择性地改变一到两个参数(Awpari和Simons，2012，Mol Cell Endocrinol，355,121-134)。我们之前对人外周血单核细胞(PBMC)的研究证实，辅因子浓度的变化会影响内源性和外源性GR调节基因的诱导参数(Luo和Simons Jr.，2009，人类免疫学，70,785-789)。这些结果有力地支持了我们的假设，即一个或多个GR诱导参数的调节是人类生理的相关特征。 已报道的参与类固醇调节基因表达的辅因子数量超过350个，而且仍在增长。关于内源性基因的各种报道表明，提高内分泌治疗的特异性的一个可行的方法是选择性地改变一个或多个特定辅因子的细胞内丰度。不幸的是，尽管基因工程可能会带来回报，但事实证明，操纵蛋白质水平是困难的。另一种可能更简单的方法是寻找能够改变调节辅因子的活性和/或丰度的化学物质。为此，我们正在与Chris Austin和Kyle Brimacombe(NCGC，NIH)合作，使用高通量筛选来识别改变GR调节基因诱导的Amax、EC50和/或PAA的化学物质。据我们所知，到目前为止还没有进行过这样的筛查，部分原因是除了奥斯汀博士之外，没有人被设置为遵循一个以上的参数。 我们构建了用于高通量筛选的双报告质粒。本报告含有GR可诱导的荧光素酶和非GR可诱导的绿色荧光蛋白(GFP)作为内对照。该报告的荧光素酶活性在瞬时转入细胞后，受到外源辅因子的调节，与我们早期研究中看到的简单的GR诱导的荧光素酶报告相同。在高通量分析中完成了信号输出的优化。已经完成了对包含1280个化合物的LOPAC文库的筛选和数据分析。最有希望的10种化合物正在我们的实验室使用我们新开发的竞争分析方法进行更详细的检测。这个测试将允许根据明确的动力学定义的机制对调节剂的类型和化学物质的作用位置进行分类。到目前为止，高通量筛选和更精确的竞争分析之间有相当好的一致性。这一信息将与我们已经检查过的各种辅因子的作用部位相匹配，以努力确定每种化学物质的目标辅因子。将对该化学品和假定的目标进行直接竞争分析，以确认初步结论。 上述研究提供了以前无法获得的关于GR反式激活活性的那些辅助因子的潜在化学调节器的分子信息，也可能提供了GR本身的分子信息。它们也构成了确定类固醇激素作用中特定步骤的化学抑制物或激活剂的合理方法。这些调节化学物质将成为药物干预的诱人线索，旨在更精确地控制内分泌治疗的发展、分化、动态平衡和内分泌治疗。这些综合发现有助于我们的长期目标，即以有利于人类健康的方式在分子水平上定义类固醇激素的作用。

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.