HCS Assay to Identify Disruptors of AR-TIF2 Protein-Protein Interactions

HCS 测定法鉴定 AR-TIF2 蛋白质-蛋白质相互作用的干扰物

• 批准号: 8098598
• 负责人: Paul A. Johnston
• 金额: $ 15.15万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8098598
• 关键词: AF2; Ablation; Adenoviruses; Adrenal Glands; Affinity; Androgen Receptor; Androgens; Anemia; Antisense Oligonucleotides; Apoptosis; Benign Prostatic Hypertrophy; Binding; Biological Assay; Biosensor; Cancer Etiology; Castration; Cell Nucleolus; Cell Nucleus; Cell Proliferation; Cells; Cessation of life; Chemotherapy-Oncologic Procedure; Chimeric Proteins; Clinical; Collection; Complex; Country; Cytoplasm; DNA Sequence; Development; Diffuse; Disease; Disease Progression; Down-Regulation; Estradiol; Exhibits; Exposure to; Family; Fatigue; Gene Expression; Gene Targeting; Genetic Transcription; Growth; Hormones; Hot flushes; Hypersensitivity; Image; Impaired cognition; Infection; Lead; Lesion; Libraries; Ligand Binding; Ligand Binding Domain; Ligands; Malignant neoplasm of prostate; Measures; Mental Depression; Messenger RNA; Muscular Atrophy; NCOA2 gene; Nuclear; Nuclear Export; Nuclear Hormone Receptors; Organelles; Osteoporosis; Patients; Performance; Phenotype; Production; Progesterone; Prostate; Prostate-Specific Antigen; Proteins; RNA Interference; Recombinants; Recurrence; Refractory; Relapse; Reporter; Resistance; Role; Sampling; Screening procedure; Second Primary Cancers; Series; Sexual Dysfunction; Signal Transduction; Steroid Receptors; Steroids; Structure; Surface; Technology; Tissues; Toxic effect; Transactivation; United States National Institutes of Health; Validation; base; glucocorticoid receptor-interacting protein 1; member; men; novel; novel therapeutics; promoter; protein protein interaction; receptor function; response; small molecule; transcription factor; tumor

DESCRIPTION (provided by applicant): There is a growing body of evidence that high TIF2 coactivator expression levels are associated with prostate cancer (CaP) recurrence after androgen ablation therapy (AAT). Over expressed TIF2 may lead to androgen receptor (AR) hypersensitivity and transactivation by lower affinity adrenal androgens or other steroids that could contribute to the growth of recurrent castration resistant (CR) CaP after AAT. Small molecule disruptors of the AR-TIF2 coactivator interaction will provide probes to investigate the role of this interaction in the development and progression to CR CaP, and may lead to the development of novel therapeutics for CaP. We are proposing to develop a novel high content image-based biosensor HCS assay to measure and quantify the protein-protein interactions (PPIs) between the AR and TIF2, and to screen for disruptors of these interactions. The AR-TIF2 protein-protein interaction biosensor (PPIB) assay exploits features of protein targeting to organelles, AR and TIF2 functional domains, and fluorescent reporters to generate positional biosensors to measure and quantify the interactions between these protein partners in cells. The AR-TIF2 PPIB proposed here recapitulates the ligand-induced translocation of AR from the cytoplasm to the nucleus and the subsequent recruitment and interaction with the TIF2 coactivator. The AR-TIF2 PPIB assay can be screened in two formats; to screen for compounds that block the DHT-induced formation of AR-TIF2 PPIs cells will be pre-exposed to compounds prior to DHT treatment, and to identify compounds capable of disrupting established AR-TIF2 PPI complexes cells will be pre-exposed to DHT prior to compound treatment. We recently described the development and characterization of p53-hDM2 PPIB assay utilizing this same technology that we successfully used to screen 220,000 compounds from the NIH library and to identify a novel chemotype series that disrupts p53-hDM2 PPIs in cells. We propose to optimize the AR-TIF2 biosensor HCS assay, adapt it to screen compound libraries for molecules that disrupt the AR-TIF2 PPIs, and to validate its performance by screening the LOPAC and NIH Clinical Collection compound libraries. After completing the development and validation of the AR-TIF2 PPIB HCS assay, our plan would be to submit the assay through the fast track mechanism into the MLPCN for screening. PUBLIC HEALTH RELEVANCE: Prostate cancer (CaP) remains incurable in the metastatic setting and despite the initial response to androgen ablation therapy (AATs) the disease transforms and progresses to the castration resistant (CR) state and is the most common cancer and second leading cause of cancer death among men in western countries,. To date however, no chemotherapy regimen has emerged as the standard therapy for metastatic CR CaP and current AATs are limited by toxicities including; muscle atrophy, osteoporosis, hot flashes, sexual dysfunction, fatigue, anemia, depression and cognitive dysfunction. We are proposing to develop and validate a novel AR-TIF2 protein-protein interaction biosensor assay that recapitulates the ligand-induced translocation of AR into the nucleus and the recruitment interactions with the TIF2 coactivator, and to use this assay to identify small molecule probes that disrupt AR-TIF2 protein-protein interactions and to use these probes to explore the role of such interactions in the development and progression of CR CaP.

描述（由申请人提供）：越来越多的证据表明，高 TIF2 共激活剂表达水平与雄激素消融疗法 (AAT) 后前列腺癌 (CaP) 复发相关。过度表达的 TIF2 可能导致雄激素受体 (AR) 过敏，并通过亲和力较低的肾上腺雄激素或其他类固醇进行反式激活，从而导致 AAT 后复发性去势抵抗 (CR) CaP 的生长。 AR-TIF2 共激活剂相互作用的小分子干扰物将为研究这种相互作用在 CR CaP 的发生和进展中的作用提供探针，并可能导致新的 CaP 治疗方法的开发。我们建议开发一种新型高内涵基于图像的生物传感器 HCS 测定法，以测量和量化 AR 和 TIF2 之间的蛋白质-蛋白质相互作用 (PPI)，并筛选这些相互作用的干扰物。 AR-TIF2 蛋白质-蛋白质相互作用生物传感器 (PPIB) 测定利用蛋白质靶向细胞器、AR 和 TIF2 功能域以及荧光报告基因的特征来生成位置生物传感器，以测量和量化细胞中这些蛋白质伙伴之间的相互作用。这里提出的 AR-TIF2 PPIB 概括了配体诱导的 AR 从细胞质到细胞核的易位以及随后与 TIF2 共激活剂的募集和相互作用。 AR-TIF2 PPIB 检测可以通过两种形式进行筛选；筛选能够阻断 DHT 诱导的 AR-TIF2 PPI 形成的化合物 在 DHT 处理之前，细胞将预先暴露于化合物，并鉴定能够破坏已建立的 AR-TIF2 PPI 复合物的化合物 细胞将预先暴露于 DHT复合治疗前。我们最近描述了 p53-hDM2 PPIB 测定的开发和表征，该技术利用了与我们成功地从 NIH 库中筛选 220,000 种化合物并鉴定出破坏细胞中 p53-hDM2 PPI 的新型化学型系列相同的技术。我们建议优化 AR-TIF2 生物传感器 HCS 测定，使其适应于筛选破坏 AR-TIF2 PPI 分子的化合物库，并通过筛选 LOPAC 和 NIH 临床收集化合物库来验证其性能。在完成 AR-TIF2 PPIB HCS 检测的开发和验证后，我们的计划是通过快速通道机制将该检测提交到 MLPCN 进行筛选。 公共卫生相关性：转移性前列腺癌 (CaP) 仍然无法治愈，尽管雄激素消融疗法 (AAT) 最初有反应，但该疾病会转变并进展至去势抵抗 (CR) 状态，是最常见的癌症和第二大原因西方国家男性癌症死亡的情况。然而，迄今为止，还没有化疗方案成为转移性 CR CaP 的标准疗法，并且当前的 AAT 受到毒性的限制，包括：肌肉萎缩、骨质疏松、潮热、性功能障碍、疲劳、贫血、抑郁和认知功能障碍。我们提议开发和验证一种新型 AR-TIF2 蛋白质-蛋白质相互作用生物传感器测定法，该测定法概括了配体诱导的 AR 易位到细胞核中以及与 TIF2 共激活剂的募集相互作用，并使用该测定法来鉴定小分子探针，破坏 AR-TIF2 蛋白质-蛋白质相互作用，并使用这些探针探索这种相互作用在 CR CaP 发生和进展中的作用。