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: 8659594
• 负责人: Gary H. Perdew
• 金额: $ 1.14万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2013-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8659594
• 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; liver inflammation; 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).

说明书(由申请人提供)：传统上，人们根据其通过与二恶英反应元件(DRE)结合来调节转录效应的能力来研究AH受体(AHR)。已知AHR参与了广泛的生理过程，包括T细胞功能和肝血管发育。我们使用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炎性基因(如SAA1、CRP)。接下来，我们鉴定了AHR配体，它们可以在不诱导DRE介导的转录活性的情况下，介导急性时相基因抑制活性；这些化合物被称为“选择性AH受体调节剂”(SAHRM)。一种化合物SGA360能够抑制细胞因子介导的急性时相基因表达的诱导，而不会诱导同源反应元件(DRE)效应。这种SAHRM诱导的AHR依赖的抗炎作用的机制(S)尚未建立。因此，在这一应用中，第一个特定的目的将确定选择性AHR配体介导的急性时相反应基因表达抑制的确切机制。这将使用细胞培养模型(例如Huh7肝癌细胞)来实现。用SAhRms处理细胞，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、细胞报告分析、EMSA和免疫共沉淀分析，以确定参与SGA360-AHR复合体调控的急性时相基因诱导的蛋白质(S)。还需要开发更多具有更高亲和力和效力的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配体结合口袋的计算机模拟算法和我们以前结构-活性研究中的知识来指导结构-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。其目的将利用计算机建模和配体对接程序、新化合物的有机合成、基于细胞的急性相基因抑制分析、EMSA、配体竞争分析以及小鼠皮肤和肝脏炎症分析，以确定增强SGA360介导的急性相基因表达抑制的结构修饰。这些研究结合在一起，将使未来能够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。