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，它在调节涉及炎症和免疫抑制的基因中起着重要作用，代表了哮喘，关节炎，卢普斯和其他疾病的主要治疗靶标。 GR药物设计的主要挑战是需要增强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介导的基因转录的最关键相互作用发生在其糖皮质激素反应元件（GRE）DNA位点。 GRE的变化决定了差异基因转录，但是尚未完成对GR相互作用的全面分析。我们已经开发了同源位点标识符（CSI）DNA微阵列，其中包含双链DNA中12个碱基对DNA序列的每一个置换。我们的建议将集中于了解与配体和异二聚体蛋白的相互作用如何影响对DNA的GR识别，扩大CSI技术，以发现用于药物设计，小分子筛查以及人工转录因子的新目标。该阶段I建议的具体目的是：1。响应于受体相互作用与配体和异二聚体伴侣AP-1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物靶标和DNA结合特异性的关键变化。 2。由N-甲基吡咯和N-甲基咪唑对组成的DNA结合小分子聚酰胺，可以精确靶向DNA位点的N-甲基咪唑对，以破坏GR-GRE和GR-AP1-DNA相互作用。这些分子将有助于在涉及负副作用的皮质类固醇调节基因上下调GR介导的转录的治疗剂。 3.使用CSI微阵列将测试化合物区分为GR-GRE相互作用的破坏者或增强子的高吞吐量筛选平台将开发出复合位点的GR-GRE相互作用和GR-AP1结合。在此提案中，我们开发了一个微阵列平台，以检查重要药物靶标的DNA结合特异性用于抗炎治疗性发育。蛋白-DNA相互作用和DNA介导的变构作用对蛋白质 - 配体相互作用的靶向效应因素是具有巨大治疗潜力的药物设计领域。来自GR-DNA相互作用的CSI信息将用于有意义地注释人基因组，以确定患者人群中关键的单核苷酸多态性引起的差异，并预测药物靶标是否会影响不良基因或途径。 CSI方法可以应用于任何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）药物设计的主要挑战是必须确定“解离的配体”，从而增强抗炎和免疫抑制作用，以治疗哮喘，关节炎，狼疮和其他疾病，同时下调了糖皮质激素介导的副作用的副作用，例如，抑制了疾病，疾病伴随着疾病的疾病。 GR介导的基因转录的最关键相互作用发生在其糖皮质激素反应元件（GRE）DNA位点。 GRE的变化决定了差异基因转录，但是尚未完成对GR相互作用的全面分析。我们的建议使用表现出12个碱基对DNA序列的每一个排列的Cognate位点标识符（CSI）DNA微阵列，我们的建议将重点介绍如何了解与配体的相互作用和异二聚体蛋白会影响DNA的GR识别DNA的识别，以发现针对药物设计，小分子筛选和开发有指导的转录因子的新型目标。