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

描述(由申请人提供)：越来越多的证据表明，高水平的TIF2辅活化子表达与前列腺癌(CAP)雄激素消融治疗(AAT)后的复发有关。TIF2的过度表达可能导致雄激素受体(AR)超敏和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后复发的去势抵抗(CR)帽的生长。AR-TIF2共激活子相互作用的小分子干扰物将提供探针，以研究这种相互作用在CR CAP的发生和进展中的作用，并可能导致CAP的新疗法的开发。我们建议开发一种新的高含量基于图像的生物传感器HCS分析来测量和量化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因素。AR-TIF2蛋白质相互作用生物传感器(PPIB)利用蛋白质靶向细胞器、AR和TIF2功能区以及荧光报告的特点来产生位置生物传感器来测量和量化细胞中这些蛋白质伙伴之间的相互作用。本文提出的AR-TIF2 PPIB概括了配体诱导的AR从胞浆到细胞核的移位以及随后的募集和与TIF2共激活子的相互作用。AR-TIF2 PPIB分析可通过两种形式进行筛选；为了筛选阻止DHT诱导的AR-TIF2 PPI形成的化合物，细胞将在DHT处理之前预先暴露于化合物，以及识别能够破坏已建立的AR-TIF2 PPI复合体的化合物；细胞在复合处理之前将预先暴露于DHT。我们最近描述了P53-Hdm2 PPIB试验的发展和特征，利用我们成功地从NIH文库中筛选220,000种化合物和鉴定一系列新的化学类型系列来破坏细胞中的P53-Hdm2 PPIB的技术。我们建议优化AR-TIF2生物传感器HCS分析，使其适用于筛选破坏AR-TIF2 PPI的分子的化合物文库，并通过筛选LOPAC和NIH临床收集化合物文库来验证其性能。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：前列腺癌(CAP)在转移性环境中仍然是无法治愈的，尽管对雄激素消融治疗(AATS)有初步反应，但这种疾病转变并进展到去势抵抗(CR)状态，是西方国家男性最常见的癌症和第二大癌症死亡原因。然而，到目前为止，还没有出现化疗方案作为转移性CR CAP的标准治疗方案，目前的AAT受到毒副作用的限制，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。我们建议建立和验证一种新的AR-TIF2蛋白-蛋白质相互作用生物传感器实验，该方法概括了配体诱导的AR移位到细胞核中以及与TIF2共激活因子的招募相互作用，并利用该方法识别破坏AR-TIF2蛋白质-蛋白质相互作用的小分子探针，并使用这些探针来探索这种相互作用在CR CAP的发生和发展中的作用。

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.