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

描述（由申请人提供）：雌激素受体a（ER a）是乳腺癌的主要治疗靶点，ER a与乳腺癌细胞中靶基因的结合由DNA结合先锋因子蛋白（如FoxA 1）指导。虽然他莫昔芬是一种成功的治疗抑制ER介导的基因调控，耐药发生在大多数乳腺癌患者在五年内，乳腺癌仍然是全球妇女死亡的主要原因之一，突出了改善策略的迫切需要。估计每年的乳腺癌治疗市场为30亿至50亿美元，市场接受定向策略。ER α药物设计的一个主要挑战是需要鉴定特异性靶向乳腺癌中异常基因调控的分子机制的小分子，同时保持ER α作用的生理益处不变。ER α介导的基因转录的最关键的相互作用发生在其雌激素反应元件（ERE）DNA位点，并且FoxA 1增强乳腺癌细胞中ER α与ERE的结合。然而，专注于调节ERa-FoxAl-DNA相互作用的DNA导向治疗是药物设计的研究不足的领域，因为全面检查这种相互作用的技术还不适合于高通量药物发现。我们已经开发了同源位点标识符（CSI）DNA微阵列，包含双链DNA中10至12个碱基对DNA序列的每一种排列，以及序列特异性景观（SSL）作为分析CSI阵列产生的数百万个数据点的工具。该提案将集中于了解乳腺癌中FoxA 1- ERa的全面DNA结合特异性，并特别破坏FoxA 1增强ERa与DNA指导的肽核酸（PNA）结合。第一阶段提案的具体目标是：1. FoxA 1的DNA结合偏好将使用来自乳腺癌细胞裂解物的FoxA 1和作为纯化蛋白通过CSI-SSL分析来表征。这些研究将确定纯化FoxA 1的直接DNA结合位点以及乳腺癌细胞中潜在的蛋白质束缚FoxA 1-DNA相互作用。2. DNA结合PNA将被设计为特异性靶向和破坏FoxA 1-DNA相互作用，作为模型疗法下调FoxA 1介导的乳腺癌调控基因转录。3.将使用CSI DNA微阵列开发高通量筛选平台以检查ERa-FoxA 1-DNA相互作用并筛选作为该相互作用的靶向破坏剂的测试化合物。我们的研究结果将定义一个新的竞技场的DNA为基础的治疗方法，扩大CSI-SSL技术的DNA结合异二聚体蛋白复合物，对小分子筛选，发现新的药物靶点，并开发有针对性的DNA结合分子作为乳腺癌的治疗。 公共卫生相关性：雌激素受体α（ER α）是乳腺癌的主要治疗靶点，ER α与乳腺癌细胞中靶基因的结合由DNA结合先锋因子蛋白如FoxA 1指导。虽然他莫昔芬代表了ER α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产生了抗药性，并且在那些确实有反应的患者中，几乎所有人都会在五年内对他莫昔芬产生抗药性。我们正在开发新的技术，同源位点标识符DNA微阵列和序列特异性景观，作为一个高通量平台，快速识别化合物，特异性靶向和破坏ERa-FoxA 1-DNA相互作用，发现新的DNA导向的乳腺癌药物。

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.