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

描述（由申请方提供）：累积证据表明，TIF 2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。过度表达的TIF 2导致雄激素受体（AR）超敏反应和低亲和力肾上腺雄激素或其他类固醇的反式激活，这可能导致AAT后去势抵抗（CR）CaP的复发。AR-TIF 2辅激活因子相互作用的破坏剂将提供小分子探针来研究这些相互作用在CR CaP进展中的作用，并可能导致CaP新疗法的开发。我们建议开发一种新的高含量的基于图像的生物传感器测定来测量和量化AR和TIF 2之间的蛋白质-蛋白质相互作用（PPI），并筛选阻止AR-TIF 2 PPI形成和/或破坏AR-TIF 2复合物的探针化合物。AR-TIF 2 PPI生物传感器（PPIB）检测利用蛋白质靶向细胞器、AR和TIF 2功能结构域以及荧光报告分子的特征，生成位置生物传感器，测量和量化细胞中这些伴侣之间的相互作用，以及随后与TIF 2的相互作用。可以以几种形式筛选生物传感器以鉴定：1）AR激动剂，2）阻断AR-TIF 2 PPI诱导的化合物，和3）破坏已建立的AR-TIF 2复合物的化合物。我们将筛选形式#2和#3，因为存在许多用于筛选AR激动剂的测定形式。我们建议完成AR-TIF 2 PPIB HCS检测试剂盒的开发，并生成重组腺病毒（rAV）库，以针对约300，000种化合物进行MLPCN HCS活动。我们将进一步优化前列腺癌细胞系（PC-3、DU-45、LNCaP、LAPC 4、C4-2和CWR-R1）中的试验，并选择最合适的细胞模型来代表HCS的CR-CaP。然后，我们将调整和自动化检测方法，以筛选阻止或破坏AR-TIF 2 PPI的分子，并在中试筛选中验证其性能。我们建议整合计数器筛选和二级或三级检测，以表征和确定AR-TIF 2 PPI命中的作用机制。然后，我们的计划是将完全优化和验证的AR-TIF 2 PPIB HCS检测提交给MLPCN进行筛选。通过靶向AR信号传导的晚期阶段，即AR-TIF 2相互作用的破坏，我们希望鉴定出抑制AR反式激活的新型化合物，其具有阻断AAT抗性的发展和CR-CaP复发的治疗潜力。

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.