Selective Ah Receptor Ligands Repress Acute-Phase Response

选择性 Ah 受体配体抑制急性期反应

• 批准号: 8232259
• 负责人: Gary H. Perdew
• 金额: $ 40.62万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-10 至 2016-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8232259
• 关键词: Acute; Acute-Phase Reaction; Address; Affinity; Agonist; Algorithms; Animal Disease Models; Anti-Inflammatory Agents; Anti-inflammatory; Binding; Biochemical; Biological Assay; Blood Vessels; Cell Culture Techniques; Cell physiology; Cells; Chronic; Co-Immunoprecipitations; Complex; Computer Simulation; Crohn' s disease; DNA Binding; Development; Dioxins; Disease; Disease model; Docking; EMSA; Ear; Edema; Estrogen Receptors; Exhibits; Future; Gene Expression; Genes; Goals; Hepatocyte; Inflammation; Inflammatory; Intestines; Knowledge; Laboratories; Lead; Ligand Binding; Ligands; Liver; Malignant Neoplasms; Mediating; Mind; Modeling; Modification; Mus; Organic Synthesis; Pharmaceutical Preparations; Phase; Physiological Processes; Physiology; Property; Proteins; Receptor Signaling; Reporter; Repression; Research Personnel; Response Elements; Role; Signal Transduction; Skin; Small Interfering RNA; Structure; T-Lymphocyte; Techniques; Testing; Therapeutic; Therapeutic Uses; Toxic effect; Western Blotting; Xenobiotic Metabolism; base; chromatin immunoprecipitation; cytokine; design; effective therapy; gene induction; gene repression; hepatoma cell; in vivo; in vivo Model; meetings; mutant; neoplastic cell; programs; promoter; receptor; receptor binding; research study; response; therapeutic target

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.

描述（由申请人提供）：Ah 受体（AHR）传统上是根据其通过与二恶英响应元件（DRE）结合介导转录效应的能力来研究的。已知 AHR 参与多种生理过程，包括 T 细胞功能和肝血管发育。我们已经使用 AHR DNA 结合突变体测试了 AHR 可以在不与其同源反应元件结合的情况下调节基因表达的假设。观察到被 AHR-A78D DNA 结合突变体抑制的一类基因是急性期肝脏炎症基因（例如 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”(SAhRM)。 SGA360 是一种化合物，能够抑制细胞因子介导的急性期基因表达诱导，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，在本申请中，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。这将使用细胞培养模型（例如 Huh7 肝癌细胞）来完成。将细胞处理与 SAhRM 相结合，然后使用以下技术； siRNA、蛋白质印迹分析、染色质免疫沉淀分析、启动子分析、基于细胞的报告分析、EMSA 和免疫共沉淀分析将用于确定参与由 SGA360-AHR 复合物调节的急性期基因诱导的蛋白质。还需要开发具有更高亲和力和效力的其他 SAhRM，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配体与AHR配体结合口袋结合的计算机建模算法以及我们从之前的结构-活性研究中获得的知识来指导结构-活性研究，这将导致高亲和力选择性配体的开发具有抗炎特性。该目标将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测定、EMSA、配体竞争测定以及小鼠皮肤和肝脏炎症测定来识别增强SGA360介导的抑制的结构修饰急性期基因表达。这些研究结合在一起，将允许未来在适当的慢性炎症疾病动物模型（例如癌症、克罗恩病）中测试它们的治疗潜力。 公共健康相关性：Ah 受体 (AHR) 的激活已被证明可以抑制肝脏中细胞因子介导的急性期基因表达。已经鉴定出选择性 AHR 配体可以介导急性期基因抑制而不引起二恶英反应元件驱动的转录反应。这些研究将探索选择性AHR配体活性的机制，这可能导致AHR作为治疗慢性炎症疾病的治疗靶点的发展。还将开发更有效的选择性 AHR 配体。