Development of a Novel HCS Assay to Screen for Disruptors of AR-TIF2 Interactions

描述（由申请人提供）：累积证据表明，TIF2共激活因子表达水平升高与雄激素消融治疗（AAT）后前列腺癌（CaP）复发相关。过表达的TIF2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激活，这可能导致AAT后去势抵抗（CR） CaP的复发。AR-TIF2共激活物相互作用的干扰物将提供小分子探针，以研究这些相互作用在CR - CaP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测定方法，以测量和量化AR和TIF2之间的蛋白质-蛋白质相互作用（PPIs），并筛选阻止AR-TIF2 PPIs形成和/或破坏AR-TIF2复合物的探针化合物。AR-TIF2 PPI生物传感器（PPIB）检测利用蛋白质靶向细胞器、AR和TIF2功能域的特点，以及荧光报告产生定位生物传感器，测量和量化细胞中这些伙伴之间的相互作用，以及随后与TIF2的相互作用。该生物传感器可以通过几种形式进行筛选，以确定：1)ar激动剂，2)阻断AR-TIF2 PPIs诱导的化合物，以及3)破坏已建立的AR-TIF2复合物的化合物。我们将筛选格式#2和#3，因为存在许多检测格式来筛选AR激动剂。我们建议完成AR-TIF2 PPIB HCS试验的开发，并生成重组腺病毒（rAV）库，以针对30万种化合物进行MLPCN HCS试验。我们将在前列腺癌细胞系（PC-3、DU-45、LNCaP、LAPC4、C4-2和cwrr - r1）中进一步优化实验，选择最合适的细胞模型来代表CR-CaP进行HCS。然后，我们将调整和自动化分析，以筛选阻止或破坏AR-TIF2 ppi的分子，并在中试筛选中验证其性能。我们建议整合计数筛选和二级或三级分析来表征和确定AR-TIF2 PPI命中的作用机制。我们的计划是将完全优化和验证的AR-TIF2 PPIB HCS检测提交给MLPCN进行筛选。通过靶向AR信号传导的晚期，即AR- tif2相互作用的破坏，我们希望找到具有治疗潜力的抑制AR转激活的新化合物，以阻止aat耐药性的发展和CR-CaP的复发。

DESCRIPTION (provided by applicant): Cumulative evidence indicates that elevated TIF2 coactivator expression levels are associated with prostate cancer (CaP) recurrence after androgen ablation therapy (AAT). Overexpressed TIF2 leads to androgen receptor (AR) hypersensitivity and transactivation by lower affinity adrenal androgens or other steroids that may contribute to the recurrence of castration resistant (CR) CaP after AAT. Disruptors of AR-TIF2 coactivator interactions will provide small molecule probes to investigate the roles of these interactions in the progression to CR CaP, and may lead to development of new therapeutics for CaP. We are proposing to develop a novel high content image-based biosensor assay to measure and quantify the protein-protein interactions (PPIs) between AR and TIF2, and to screen for probe compounds that prevent the formation of AR-TIF2 PPIs and/or disrupt AR-TIF2 complexes. The AR-TIF2 PPI biosensor (PPIB) assay exploits features of protein targeting to organelles, AR and TIF2 functional domains, and fluorescent reporters to generate positional biosensors that measure and quantify the interactions between these partners in cell, and the subsequent interaction with TIF2. The biosensor can be screened in several formats to identify: 1) AR-agonists, 2) compounds that block induction of AR-TIF2 PPIs, and 3) compounds that disrupt established AR-TIF2 complexes. We will screen formats #2 and #3 because numerous assay formats exist to screen for AR agonists. We propose to complete the development of the AR-TIF2 PPIB HCS assay and generate recombinant adenovirus (rAV) banks to conduct an MLPCN HCS campaign against e 300,000 compounds. We will further optimize the assay in prostate cancer cell lines (PC-3, DU-45, LNCaP, LAPC4, C4-2, and CWR-R1) and select the most suitable cell model to represent CR-CaP for the HCS. We will then adapt and automate the assay to screen for molecules that prevent or disrupt AR-TIF2 PPIs, and validate its performance in pilot screens. We propose to integrate counter screens and secondary or tertiary assays to characterize and determine the mechanism of action of AR-TIF2 PPI hits. Our plan would then be to submit the fully optimized and validated AR-TIF2 PPIB HCS assay to the MLPCN for screening. By targeting a late stage in AR signaling, namely, disruption of AR-TIF2 interactions, we hope to identify novel compounds that inhibit AR transactivation with therapeutic potential to block the development of AAT-resistance and the recurrence of CR-CaP.