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

描述（申请人提供）：雌激素受体a （Estrogen receptor a, ERa）是乳腺癌的主要治疗靶点，在乳腺癌细胞中，雌激素受体a结合靶基因是由FoxA1等DNA结合先锋因子蛋白引导的。尽管他莫昔芬是一种成功的抑制内质网介导的基因调控的治疗方法，但大多数乳腺癌患者在五年内都会出现对他莫昔芬的耐药性，乳腺癌仍然是全球妇女死亡的主要原因之一，这凸显了改进策略的迫切需要。据估计，每年乳腺癌治疗市场的规模为30亿至50亿美元，市场接受定向策略。ERa药物设计的一个主要挑战是必须确定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，FoxA1增强了乳腺癌细胞中ERa与雌激素反应元件（ERE）的结合。然而，专注于调节ERa-FoxA1-DNA相互作用的dna定向疗法是药物设计的研究领域，因为全面检查这种相互作用的技术还不适合高通量药物发现。我们开发了同源位点标识（CSI） DNA微阵列，包含双工DNA中10到12个碱基对DNA序列的每个排列，以及序列特异性景观（SSLs）作为分析CSI阵列产生的百万多个数据点的工具。本研究将重点了解FoxA1- ERa在乳腺癌中的综合DNA结合特异性，并特异性地破坏FoxA1增强ERa与DNA导向肽核酸（DNA-directed peptide nucleic acids, PNAs）的结合。第一阶段建议的具体目标是：FoxA1的DNA结合偏好将使用乳腺癌细胞裂解物中的FoxA1和纯化蛋白，通过CSI-SSL分析来表征。这些研究将确定纯化FoxA1的直接DNA结合位点以及乳腺癌细胞中潜在的蛋白拴系FoxA1-DNA相互作用。2. dna结合PNAs将被设计为特异性靶向和破坏FoxA1-DNA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. 一个高通量筛选平台将使用CSI DNA微阵列来检测ERa- FoxA1-DNA相互作用，并筛选测试化合物作为这种相互作用的靶向干扰物。通过将CSI-SSL技术扩展到DNA结合的异二聚体蛋白复合物、小分子筛选、新药物靶点的发现以及靶向DNA结合分子作为乳腺癌治疗方法的开发，我们的发现将为基于DNA的治疗开辟一个新的领域。

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.