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。 使用可形成三元复合物的三种辅因子（GR、共激活剂和共调节剂）的更近期的研究揭示，调节蛋白的不同区域不同等地影响所有参数，并且可选择性地仅改变一个或两个参数（Awasthi和Simons，2012，Mol Cell Endocrinol，355，121 - 134）。 我们先前在人外周单核细胞（PBMC）中的研究证实，辅因子浓度的变化影响内源性以及外源性GR调节基因的诱导参数（Luo和Simons Jr.，2009，Human Immunology，70，785 - 789）。 这些结果为我们的假设提供了强有力的支持，即一个或多个GR诱导参数的调节是人类生理学的相关特征。 糖皮质激素类固醇影响几乎每一种组织类型，因此被广泛用于治疗各种人类病理。 然而，许多副作用的严重性限制了糖皮质激素治疗的频率和持续时间。 在控制对糖皮质激素的脱靶反应的众多方法中，小分子和药物提供了几个优点。 在这里，我们描述了一个新的，扩展的高通量筛选在完整的细胞，以确定地塞米松诱导的糖皮质激素受体（GR）转录活性的小分子调节剂。 该测定的新颖性在于其监测GR最大活性（Amax）和EC 50的变化，或地塞米松剂量-反应曲线的位置。 在筛选1280种化学品后，选择Amax或EC 50绝对值变化最大的10种进行进一步检查。 在一个完全不同的系统中观察到60 - 90%的化学品的质量相同的行为，这表明其他系统也会受到这些化学品的类似影响。 在最近描述的竞争试验中对十种化学物质进行的额外分析确定了它们的动力学定义机制和作用位点。 尽管对GR诱导产物水平的影响不同，但一些化学物质具有相似的作用机制。 这些联合检测提供了一种简单的方法，可以识别许多新的药物，可以改变GR反式激活的方式，可能是临床上有用的。 上述研究提供了以前无法获得的有关GR反式激活活性的那些辅因子的潜在化学调节剂的分子信息，并且可能是GR本身的化学调节剂。 它们也构成了一个合理的方法，以确定化学抑制剂或激活剂的具体步骤，类固醇激素的行动。 这种调节化学品将是有吸引力的药物干预，旨在更精确地控制内分泌疗法在发展，分化，稳态，和内分泌治疗。 最后，这种方法确定了GR活性的下游效应物，其应该具有减少副作用的临床期望的性质。 这些综合研究结果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. 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.