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

开发一种新的 HCS 测定法来筛选 AR-TIF2 相互作用的干扰物

• 批准号: 8721728
• 负责人: Paul A. Johnston
• 金额: $ 31万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-16 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8721728
• 关键词: Ablation; Adenoviruses; Adrenal Glands; Affinity; Agonist; Androgen Receptor; Androgens; Antisense Oligonucleotides; Apoptosis; Benign Prostatic Hypertrophy; Binding; Biological Assay; Biosensor; Cancer Etiology; Castration; Cell Nucleus; Cell Proliferation; Cell model; Cells; Cessation of life; Chemicals; Chemotherapy-Oncologic Procedure; Clinical; Collection; Complex; Country; Cytoplasm; DNA Sequence; DU145; Development; Disease Progression; Down-Regulation; Estradiol; Family; Gene Expression; Gene Targeting; Genetic Transcription; Growth; Hypersensitivity; Image; LAPC4; LNCaP; Lead; Lesion; Ligands; Localized Disease; Malignant neoplasm of prostate; Measures; Messenger RNA; Methods; NCOA2 gene; Nuclear Hormone Receptors; Organelles; PC3 cell line; Patients; Performance; Pharmaceutical Preparations; Production; Progesterone; Property; Prostate; Prostate Cancer therapy; Prostate-Specific Antigen; Proteins; RNA Interference; Receptor Signaling; Recombinants; Recurrence; Relapse; Reporter; Resistance; Role; Sampling; Signal Transduction; Solid Neoplasm; Staging; Steroid Receptors; Steroids; Therapeutic; Tissues; Toxic effect; Transactivation; United States National Institutes of Health; assay development; base; glucocorticoid receptor-interacting protein 1; high risk; member; men; novel; novel therapeutics; overexpression; prevent; promoter; protein protein interaction; receptor function; response; screening; small molecule; transcription factor; tumor

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.

描述（由申请人提供）：累积证据表明，TIF2 共激活剂表达水平升高与雄激素消融疗法（AAT）后前列腺癌（CaP）复发相关。过度表达的 TIF2 会导致雄激素受体 (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，这可能导致 AAT 后去势抵抗 (CR) CaP 的复发。 AR-TIF2 共激活剂相互作用的干扰物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型高内涵基于图像的生物传感器测定法，以测量和量化 AR 和 TIF2 之间的蛋白质-蛋白质相互作用 (PPI)，并筛选可防止 AR-TIF2 PPI 形成和/或破坏 AR-TIF2 PPI 的探针化合物。 AR-TIF2 复合物。 AR-TIF2 PPI 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，用于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。生物传感器可以通过多种形式进行筛选，以识别：1) AR 激动剂，2) 阻断 AR-TIF2 PPI 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，因为存在多种筛选 AR 激动剂的测定格式。我们建议完成 AR-TIF2 PPIB HCS 检测的开发，并生成重组腺病毒 (rAV) 库，以针对 300,000 种化合物开展 MLPCN HCS 活动。我们将进一步优化前列腺癌细胞系（PC-3、DU-45、LNCaP、LAPC4、C4-2 和 CWR-R1）中的测定，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该测定，以筛选可预防或破坏 AR-TIF2 PPI 的分子，并在试点筛选中验证其性能。我们建议整合计数器筛选和二级或三级测定来表征和确定 AR-TIF2 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-TIF2 PPIB HCS 检测提交给 MLPCN 进行筛选。通过针对 AR 信号传导的后期，即 AR-TIF2 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，并具有阻止 AAT 耐药性发展和 CR-CaP 复发的治疗潜力。