Screening of FoxA1-ER-DNA disruptors for development of breast cancer therapeutic

筛选 FoxA1-ER-DNA 干扰物用于开发乳腺癌治疗药物

• 批准号: 8200699
• 负责人: Mary Szatkowski Ozers
• 金额: $ 21.82万
• 依托单位: PROTEOVISTA, LLC
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-16 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8200699
• 关键词: Affect; Affinity; Antineoplastic Agents; Area; Base Pairing; Binding; Binding Sites; Breast Cancer Cell; Cancer Patient; Cessation of life; DNA; DNA Binding; DNA Microarray Chip; DNA Probes; DNA Sequence; Data; Development; Drug Delivery Systems; Drug Design; Estrogen Receptor alpha; Estrogen Receptors; Estrogens; Factor X; Fluorescence; G-substrate; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Glucocorticoid Receptor; Human Genome; Incubated; Inflammation; Left; Ligands; Maps; Marketing; Mediating; Modeling; Molecular Target; NR4A1 gene; Patients; Peptide Nucleic Acids; Pharmacologic Substance; Phase; Physiological; Proteins; Regulation; Resistance; Response Elements; Screening procedure; Site; Slide; Specificity; Tamoxifen; Technology; Testing; Therapeutic; Time; Woman; base; design; drug discovery; genome wide association study; high throughput screening; improved; malignant breast neoplasm; new technology; novel; preference; protein complex; small molecule; stem; therapeutic target; tool; transcription factor

DESCRIPTION (provided by applicant): Estrogen receptor a (ERa) is the major therapeutic target for breast cancer, and ERa binding at target genes in breast cancer cells is guided by DNA binding pioneer factor proteins such as FoxA1. Although tamoxifen is a successful therapeutic for suppressing ER-mediated gene regulation, resistance to tamoxifen occurs in most breast cancer patients within five years and breast cancer remains one of the leading causes of women's death worldwide, highlighting the critical need for improved strategies. With an estimated annual breast cancer therapeutic market of $3-5 billion, the market is receptive to directed strategies. A major challenge in ERa drug design is the necessity to identify small-molecules that specifically target the molecular mechanisms of aberrant gene regulation in breast cancer, while leaving the physiological benefits of ERa action intact. The most critical interaction for ERa-mediated gene transcription occurs at its estrogen response element (ERE) DNA site, and FoxA1 enhances ERa binding at EREs in breast cancer cells. However, DNA-directed therapeutics focused on modulating the ERa-FoxA1-DNA interaction are under- studied areas of drug design because technologies to comprehensively examine this interaction are not yet amenable to high throughput drug discovery. We have developed Cognate Site Identifier (CSI) DNA microarrays, containing every permutation of a 10 to 12 base pair DNA sequence within duplex DNA, and Sequence Specificity Landscapes (SSLs) as a tool to analyze the million-plus data points resulting from the CSI array. This proposal will focus on understanding the comprehensive DNA binding specificity of FoxA1- ERa in breast cancer and specifically disrupting FoxA1 enhancement of ERa binding with DNA-directed peptide nucleic acids (PNAs). The specific aims of this Phase I proposal are: 1. The DNA binding preferences of FoxA1 will be characterized using FoxA1 from breast cancer cell lysates and as a purified protein, by CSI-SSL analysis. These studies will identify direct DNA binding sites of purified FoxA1 as well as potential protein-tethered FoxA1-DNA interactions in breast cancer cells. 2. DNA-binding PNAs will be designed to specifically target and disrupt the FoxA1-DNA interaction, as model therapeutics to down-regulate FoxA1-mediated transcription at genes instrumental in breast cancer regulation. 3. A high throughput screening platform will be developed using CSI DNA microarrays to examine the ERa- FoxA1-DNA interaction and screen test compounds as targeted disruptors of this interaction. Our findings will define a new arena for DNA-based therapeutics by expanding CSI-SSL technology to a DNA-bound heterodimeric protein complex, towards small-molecule screening, discovery of novel drug targets, and development of targeted DNA binding molecules as therapeutics for breast cancer. PUBLIC HEALTH RELEVANCE: Estrogen receptor alpha (ERa) is the major therapeutic target for breast cancer, and ERa binding at target genes in breast cancer cells is guided by DNA binding pioneer factor proteins such as FoxA1. While tamoxifen represents a first-line pharmaceutical defense for ERa-positive breast cancer, the field is in critical need of directed therapies because approximately 30% of patients are initially recalcitrant to tamoxifen and, of those who do respond, virtually all will become resistant to tamoxifen within five years. We are developing new technologies, the Cognate Site Identifier DNA microarray and Sequence Specificity Landscape, as a high throughput platform to rapidly identify compounds that specifically target and disrupt ERa-FoxA1-DNA interactions, for discovery of novel DNA-directed breast cancer drugs.

说明(申请人提供)：雌激素受体a(ERA)是乳腺癌的主要治疗靶点，ERA与乳腺癌细胞靶基因的结合是由DNA结合先锋因子蛋白如FoxA1引导的。尽管他莫昔芬是一种成功的抑制ER介导的基因调控的疗法，但在五年内大多数乳腺癌患者对他莫昔芬产生抗药性，乳腺癌仍然是全球妇女死亡的主要原因之一，这突显了改进战略的迫切需要。据估计，乳腺癌治疗市场的年销售额为30-50亿美元，该市场愿意接受有针对性的策略。Era药物设计中的一个主要挑战是有必要识别专门针对乳腺癌异常基因调控分子机制的小分子，同时保持Era作用的生理益处不变。Era介导的基因转录最关键的相互作用发生在其雌激素反应元件(ERE)DNA位点，而FoxA1增强了乳腺癌细胞中ERE的Era结合。然而，专注于调节Era-FoxA1-DNA相互作用的DNA导向疗法是药物设计中研究不足的领域，因为全面检查这种相互作用的技术还不适用于高通量的药物发现。我们已经开发出包含双链DNA中10到12个碱基对DNA序列的每一种排列的Cognate位点识别符(CSI)DNA微阵列，以及序列特异性景观(SSLS)，作为分析CSI阵列产生的100多万个数据点的工具。这项提案将侧重于了解FoxA1-era在乳腺癌中的全面DNA结合特异性，并特异性地干扰FoxA1增强Era与DNA导向的肽核酸(PNA)的结合。这一阶段建议的具体目标是：1.FoxA1的DNA结合偏好将使用乳腺癌细胞裂解物中的FoxA1作为纯化蛋白，通过CSI-SSL分析来表征。这些研究将确定纯化的FoxA1的直接DNA结合部位，以及乳腺癌细胞中潜在的蛋白质拴系的FoxA1-DNA相互作用。2.DNA结合的PNA将被设计成特异性地靶向和干扰FoxA1-DNA的相互作用，作为下调FoxA1介导的乳腺癌调控基因转录的模型疗法。3.将开发一个高通量的筛选平台，利用CSI DNA微阵列研究Era-FoxA1-DNA相互作用，并筛选化合物作为这种相互作用的目标干扰物。我们的发现将通过将CSI-SSL技术扩展到DNA结合的异二聚体蛋白质复合体，朝着小分子筛选、发现新的药物靶点以及开发靶向DNA结合分子作为乳腺癌的治疗方法，从而定义基于DNA的治疗的新领域。 公共卫生相关性：雌激素受体α(ERA)是乳腺癌的主要治疗靶点，ERA与乳腺癌细胞中靶基因的结合是由DNA结合先锋因子蛋白如FoxA1引导的。虽然他莫昔芬代表了治疗Era阳性乳腺癌的一线药物防御，但该领域迫切需要定向治疗，因为大约30%的患者最初对他莫昔芬有抵抗力，而在那些确实有反应的患者中，几乎所有人都将在5年内对他莫昔芬产生抗药性。我们正在开发新技术，Cognate Site IdentifierDNA微阵列和序列特异性景观，作为一个高通量平台，快速识别特定靶向并破坏Era-FoxA1-DNA相互作用的化合物，以发现新的DNA导向的乳腺癌药物。