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).

描述（由申请人提供）：传统上研究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。已知AHR参与广泛的生理过程，包括T细胞功能和肝血管发育。我们已经测试的假设，即AHR可以调节基因表达的情况下，结合到其同源反应元件使用AHR DNA结合突变体。观察到被AHR-A78 D 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SAA 1、CRP）。接下来，鉴定了可以介导急性期基因阻遏活性而不诱导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）。一种化合物，SGA 360能够抑制精氨酸介导的急性期基因表达的诱导，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Huh 7肝癌细胞）完成。用SAhRM处理细胞，然后使用以下技术的组合：siRNA、蛋白质印迹分析、染色质免疫沉淀测定、启动子分析、基于细胞的报告基因测定、EMSA和共免疫沉淀分析将用于确定参与由SGA 360-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另外的SAhRM，其将增强它们的抗炎活性，特别是在体内模型中。因此，第二个具体的目标将使用两个独立的计算机建模的配体结合的AHR配体结合口袋和我们的知识，从以前的结构-活性研究，以指导结构-活性研究，这将导致发展的高亲和力选择性配体，表现出抗炎特性。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试验，EMSA，配体竞争试验以及小鼠皮肤和肝脏炎症试验，以确定增强SGA 360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性疾病动物模型（例如癌症、克罗恩病）中对其治疗潜力进行测试。