Selective Ah Receptor Ligands Repress Acute-Phase Response

描述（由申请人提供）：Ah受体（AHR）通过与二恶英反应元件（DRE）结合介导转录效应的能力一直被传统地研究。已知AHR参与广泛的生理过程，包括T细胞功能和肝脏血管发育。我们已经使用AHR DNA结合突变体验证了AHR可以在没有与其同源反应元件结合的情况下调节基因表达的假设。一类被AHR-A78D DNA结合突变体抑制的基因是急性期肝脏炎症基因（如SAA1、CRP）。接下来，鉴定了可以介导急性期基因抑制活性而不诱导dre介导的转录活性的AHR配体；这些化合物被称为“选择性Ah受体调节剂”（sahr）。其中一种化合物SGA360能够抑制细胞因子介导的急性期基因表达，而不诱导同源反应元件（DRE）效应。这种sahrm诱导的AHR依赖性抗炎活性的机制尚未确定。因此，在本应用中，第一个具体目标将确定选择性AHR配体介导的急性期反应基因表达抑制的精确机制。这将通过细胞培养模型（如Huh7肝癌细胞）来完成。将细胞治疗与SAhRMs结合，然后使用以下技术；siRNA、蛋白印迹分析、染色质免疫沉淀分析、启动子分析、基于细胞的报告基因分析、EMSA和共免疫沉淀分析将被用于确定参与由SGA360-AHR复合物调节的急性期基因诱导的蛋白。还需要开发其他具有更高亲和力和效力的SAhRMs，以增强其抗炎活性，特别是在体内模型中。因此，第二个具体目标将使用两种独立的AHR配体结合口袋的计算机建模算法和我们从先前的结构-活性研究中获得的知识来指导结构-活性研究，这将导致开发具有抗炎特性的高亲和力选择性配体。目的是利用计算机建模和配体对接程序、新化合物的有机合成、基于细胞的急性期基因抑制试验、EMSA、配体竞争试验以及小鼠皮肤和肝脏炎症试验来确定增强sga360介导的急性期基因表达抑制的结构修饰。这些研究结合在一起，将允许未来在适当的慢性炎症性疾病动物模型（如癌症、克罗恩病）中测试它们的治疗潜力。

DESCRIPTION (provided by applicant): The Ah receptor (AHR) has been traditionally studied in terms of its ability to mediate transcriptional effects through binding to dioxin-response elements (DRE). The AHR is known to be involved in a wide array of physiological processes, including T cell function and liver vascular development. We have tested the hypothesis that the AHR can regulate gene expression in the absence of binding to its cognate response element using an AHR DNA binding mutant. One class of genes that were observed to be repressed by an AHR-A78D DNA binding mutant were acute phase liver inflammatory genes (e.g. SAA1, CRP). Next, AHR ligands that can mediate acute-phase gene repression activity without inducing DRE-mediated transcriptional activity were identified; these compounds have been termed "Selective Ah receptor modulator" (SAhRM). One compound, SGA360 is capable of inhibiting cytokine-mediated induction of acute-phase gene expression without inducing a cognate response element (DRE) effect. The mechanism(s) of this SAhRM-induced AHR- dependent anti-inflammatory activity has not been established. Therefore, in this application the first specific aim will determine the precise mechanism of selective AHR ligand-mediated repression of acute-phase response gene expression. This will be accomplished using cell culture models (e.g. Huh7 hepatoma cells). A combination of cell treatment with SAhRMs followed by the use of the following techniques; siRNA, protein blot analysis, chromatin immunoprecipitation assays, promoter analysis, cell-based reporter assays, EMSA and co-immunoprecipitation analysis will be used to determine the protein(s) involved in acute-phase gene induction that is modulated by SGA360-AHR complex. There is also a need to develop additional SAhRMs with higher affinity and potency that will enhance their anti-inflammatory activity, especially in in vivo models. Thus, the second specific aim will use two independent computer modeling alogorithms of ligand binding to the AHR ligand-binding pocket and our knowledge from previous structure-activity studies to guide structure-activity studies, which will lead to the development of high affinity selective ligands that exhibit anti-inflammatory properties. The aim will utilize computer modeling and ligand docking programs, organic synthesis of new compounds, cell-based acute-phase gene repression assays, EMSA, ligand competition assays and skin and liver inflammation assays in mice to identify structural modifications that enhance SGA360-mediated repression of acute-phase gene expression. These studies taken together will allow future testing on their therapeutic potential in appropriate chronic inflammatory disease animal models (e.g. cancer, Crohn's disease). PUBLIC HEALTH RELEVANCE: Activation of the Ah receptor (AHR) has been demonstrated to repress cytokine-mediated acute phase gene expression in liver. Selective AHR ligands that can mediate acute phase gene repression without eliciting a dioxin response element-driven transcriptional response have been identified. These studies will explore the mechanism of selective AHR ligand activity, which may lead to the development of the AHR as a therapeutic target to treat chronic inflammatory diseases. Also more potent selective AHR ligands will be developed.