Screening of glucocorticoid receptor small-molecule regulators using cognate site

使用同源位点筛选糖皮质激素受体小分子调节剂

• 批准号: 7671718
• 负责人: Mary Szatkowski Ozers
• 金额: $ 18.19万
• 依托单位: VISTAMOTIF LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7671718
• 关键词: Adrenal Cortex Hormones; Adverse effects; Affect; Anti-Inflammatory Agents; Anti-inflammatory; Area; Arthritis; Asthma; Base Pairing; Binding; Binding Proteins; Binding Sites; Chronic Disease; DNA; DNA Binding; DNA Microarray Chip; DNA Sequence; DNA-Binding Proteins; DNA-Protein Interaction; Development; Diabetes Mellitus; Disease; Drug Delivery Systems; Drug Design; Engineering; Enhancers; Figs - dietary; Fluorescence; Gene Expression; Genes; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Human Genome; Hypertension; Immunosuppression; Immunosuppressive Agents; Incubated; Inflammation; Ligands; Link; Lupus; Malignant Neoplasms; Mediating; Methodology; Nuclear Receptors; Nylons; Obesity; Osteoporosis; Pathway interactions; Phase; Physiology; Proteins; Response Elements; Screening procedure; Single Nucleotide Polymorphism; Site; Specificity; Technology; Testing; Therapeutic; Transcription Factor AP-1; Variant; Weight Gain; design; high throughput screening; improved; member; novel; patient population; preference; public health relevance; receptor; response; small molecule; stem; therapeutic development; therapeutic target; transcription factor

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.

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，有助于调节与炎症和免疫抑制有关的基因，并且是哮喘、关节炎、狼疮和其他慢性疾病的主要治疗靶点。 GR 药物设计的一个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 的变异决定了基因转录的差异，但尚未完成探索 GRE 每种排列的 GR 相互作用的全面分析。我们开发了同源位点标识符 (CSI) DNA 微阵列，其中包含双链 DNA 内 12 碱基对 DNA 序列的每种排列。我们的提案将侧重于了解配体和异二聚体蛋白的相互作用如何影响 DNA 的 GR 识别，扩展 CSI 技术以发现药物设计、小分子筛选和人工转录因子开发的新靶点。该第一阶段提案的具体目标是： 1. 将使用 CSI 分析来表征 GR 响应受体与配体和异二聚体伙伴 AP-1 相互作用的 DNA 结合偏好。这些研究将确定潜在的新药物靶点和 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，旨在破坏GR-GRE和GR-AP1-DNA相互作用。这些分子将可用作治疗剂，下调与负面副作用相关的皮质类固醇调节基因的 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 相互作用和复合位点 GR-AP1 结合的破坏剂或增强剂。在本提案中，我们开发了一个微阵列平台来检查抗炎治疗开发重要药物靶点的 DNA 结合特异性。蛋白质-DNA 相互作用的靶向效应器和 DNA 介导的蛋白质-配体相互作用的变构效应是药物设计中尚未得到充分研究的领域，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造成的差异，并预测药物靶标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。公共健康相关性：糖皮质激素受体（GR）药物设计的一个主要挑战是需要识别“解离配体”，以增强治疗哮喘、关节炎、狼疮和其他疾病的抗炎和免疫抑制作用，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 的变异决定了基因转录的差异，但尚未完成探索 GRE 每种排列的 GR 相互作用的全面分析。使用同源位点标识符 (CSI) DNA 微阵列显示 12 碱基对 DNA 序列的每个排列，我们的提案将重点了解配体和异二聚体蛋白的相互作用如何影响 DNA 的 GR 识别，以发现药物设计、小分子筛选和定向转录因子开发的新靶点。