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）后的前列腺癌（CAP）复发有关。过度表达的TIF2可能会导致雄激素受体（AR）过敏性和通过较低亲和力肾上腺雄激素或其他类固醇的反式激活，这可能有助于AAT后复发性castatration（CR）CAP的生长。 AR-TIF2共激活因子相互作用的小分子破坏者将提供探针，以研究这种相互作用在开发和发展为CR CAP中的作用，并可能导致CAP的新型治疗剂的开发。我们建议开发一种新型的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破坏者。 AR-TIF2蛋白 - 蛋白质相互作用生物传感器（PPIB）分析利用蛋白质靶向细胞器，AR和TIF2功能结构域的特征，以及荧光报告基因生成位置生物传感器，以测量和量化细胞中这些蛋白质伴侣之间的相互作用。此处提出的AR-TIF2 PPIB概括了配体诱导的AR从细胞质到细胞核的转运以及随后的募集以及与TIF2共激活因子的相互作用。 AR-TIF2 PPIB测定可以以两种格式筛选。为了筛选阻断DHT诱导的AR-TIF2 PPI细胞形成的化合物，将在DHT治疗之前预先暴露于化合物，并鉴定能够破坏已建立的AR-TIF2 PPI复合物细胞的化合物，将在化合物处理之前预先暴露于DHT。最近，我们使用了相同的技术来成功地使用我们成功地从NIH文库筛选220,000种化合物，并确定一个新颖的化学型系列，并确定了破坏细胞中p53-HDM2 PPI的新型化学型系列。我们建议优化AR-TIF2生物传感器HCS分析，使其适应筛选化合物库的分子，以破坏AR-TIF2 PPI，并通过筛选LOPAC和NIH临床收集复合文库来验证其性能。在完成AR-TIF2 PPIB HCS分析的开发和验证后，我们的计划是通过快速轨道机制将测定法提交给MLPCN进行筛查。 公共卫生相关性：前列腺癌（CAP）在转移性环境中仍然无法治愈，尽管对雄激素消融疗法（AATS）的最初反应（AATS）疾病转变并发展为耐castration（CR）状态，并且是西方国家男性中癌症死亡的最常见癌症，是第二个主要原因。然而，迄今为止，尚无化学疗法方案作为转移CR帽的标准疗法，并且电流AAT受到毒性的限制，包括：肌肉萎缩，骨质疏松症，热闪光，性功能障碍，疲劳，贫血，抑郁和认知功能障碍。 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帽子。