Screening of glucocorticoid receptor small-molecule regulators using cognate site

描述（由申请人提供）：糖皮质激素受体（GR）是核受体（NR）超家族的成员，有助于调节炎症和免疫抑制相关基因，是哮喘、关节炎、狼疮和其他慢性疾病的主要治疗靶点。GR药物设计的主要挑战是必须增强GR介导的抗炎和免疫抑制作用，同时下调有害的皮质类固醇副作用，包括糖尿病、体重增加、高血压和骨质疏松症。GR介导的基因转录中最关键的相互作用发生在其糖皮质激素反应元件（GRE）DNA位点。GRE的变异决定了差异基因转录，但尚未对GR相互作用进行全面分析，探索GRE的每一种排列。我们已经开发了同源位点标识符（CSI）的DNA微阵列包含双链体DNA内的12个碱基对的DNA序列的每一个排列。我们的建议将集中在了解如何与配体和异二聚体蛋白质的相互作用影响GR识别的DNA，扩大CSI技术发现新的药物设计，小分子筛选和人工转录因子的发展目标。第一阶段提案的具体目标是：1. GR响应于受体与配体和异二聚体伴侣AP-1的相互作用的DNA结合偏好将使用CSI分析来表征。这些研究将确定潜在的新药靶点和DNA结合特异性的关键变化。2. DNA结合小分子聚酰胺，由N-甲基吡咯和N-甲基咪唑对组成，可以精确靶向DNA位点，将被设计用于破坏GR-GRE和GR-AP1-DNA相互作用。这些分子将可用作治疗剂以下调参与负面副作用的皮质类固醇调节基因处的GR介导的转录。3.将开发使用CSI微阵列的高通量筛选平台，以区分测试化合物作为GR-GRE相互作用和GR-AP 1在复合位点结合的破坏剂或增强剂。在这个提议中，我们开发了一个微阵列平台来检查抗炎治疗开发的重要药物靶点的DNA结合特异性。蛋白质-DNA相互作用的靶向效应物和DNA介导的蛋白质-配体相互作用的变构效应是研究不足的药物设计领域，具有巨大的治疗潜力。来自GR-DNA相互作用的CSI信息将用于有意义地注释人类基因组，识别患者群体中关键单核苷酸多态性引起的差异，并预测药物靶点是否可能影响不良基因或途径。CSI方法可应用于任何DNA结合蛋白和分子，其中许多与癌症、糖尿病、肥胖和其他疾病有关。公共卫生相关性：糖皮质激素受体（GR）药物设计中的主要挑战是必须鉴定"解离配体"，其增强抗炎和免疫抑制作用以治疗哮喘、关节炎、狼疮和其它病症，同时下调皮质类固醇介导的有害副作用，如体重增加、糖尿病、高血压和骨质疏松症。GR介导的基因转录中最关键的相互作用发生在其糖皮质激素反应元件（GRE）DNA位点。GRE的变异决定了差异基因转录，但尚未对GR相互作用进行全面分析，探索GRE的每一种排列。使用同源位点标识符（CSI）DNA微阵列显示12个碱基对DNA序列的每一个排列，我们的建议将集中在了解如何与配体和异二聚体蛋白质的相互作用影响GR识别的DNA发现新的药物设计，小分子筛选和定向转录因子的发展目标。

DESCRIPTION (provided by applicant): Glucocorticoid receptor (GR) is a member of the nuclear receptor (NR) superfamily that is instrumental in regulating genes involved in inflammation and immunosuppression and represents a major therapeutic target for asthma, arthritis, lupus, and other chronic disorders. A major challenge in GR drug design is the necessity to enhance GR-mediated anti-inflammatory and immunosuppressive action, while down-regulating deleterious corticosteroid side effects including diabetes, weight gain, hypertension, and osteoporosis. The most critical interaction for GR-mediated gene transcription occurs at its glucocorticoid response element (GRE) DNA site. Variations of the GRE dictate differential gene transcription, yet a comprehensive analysis of GR interaction exploring every permutation of the GRE has not been done. We have developed Cognate Site Identifier (CSI) DNA microarrays containing every permutation of a 12 base pair DNA sequence within duplex DNA. Our proposal will focus on understanding how interactions with ligands and heterodimeric proteins affect GR recognition of DNA, expanding the CSI technology for discovery of novel targets for drug design, small-molecule screening, and development of artificial transcription factors. The specific aims of this Phase I proposal are: 1. DNA binding preferences of GR in response to receptor interactions with ligand and with a heterodimeric partner AP-1 will be characterized using CSI analysis. These studies will identify potential new drug targets and key changes in DNA binding specificity. 2. DNA-binding small-molecule polyamides, comprised of N-methylpyrrole and N-methylimidazole pairs that can precisely target DNA sites, will be designed to disrupt GR-GRE and GR-AP1-DNA interactions. These molecules will be useful as therapeutics to down-regulate GR-mediated transcription at corticosteroid-regulated genes involved in negative side effects. 3. A high throughput screening platform using CSI microarrays to differentiate test compounds as disruptors or enhancers of GR-GRE interactions and GR-AP1 binding at composite sites will be developed. In this proposal, we develop a microarray platform to examine the DNA binding specificity of important drug targets for anti-inflammatory therapeutic development. Targeted effectors of protein-DNA interactions and DNA-mediated allosteric effects on protein-ligand interactions are under-studied areas of drug design that have enormous therapeutic potential. CSI information from GR-DNA interactions will be used to meaningfully annotate the human genome, to identify differences due to critical single nucleotide polymorphisms in patient populations, and to predict whether a drug target may affect undesirable genes or pathways. The CSI methodology can be applied for any DNA-binding proteins and molecules, many of which are linked to cancer, diabetes, obesity and other disorders. PUBLIC HEALTH RELEVANCE: A major challenge in glucocorticoid receptor (GR) drug design is the necessity to identify "dissociated ligands" that enhance anti-inflammatory and immunosuppressive action for treatment of asthma, arthritis, lupus and other disorders, while down-regulating corticosteroid-mediated deleterious side effects such as weight gain, diabetes, hypertension, and osteoporosis. The most critical interaction for GR-mediated gene transcription occurs at its glucocorticoid response element (GRE) DNA site. Variations of the GRE dictate differential gene transcription, yet a comprehensive analysis of GR interaction exploring every permutation of the GRE has not been done. Using Cognate Site Identifier (CSI) DNA microarrays displaying every permutation of a 12 base pair DNA sequence, our proposal will focus on understanding how interactions with ligands and heterodimeric proteins affect GR recognition of DNA for discovery of novel targets for drug design, small-molecule screening, and development of directed transcription factors.