Dual High-Throughput Imaging Screen for Nucleolar Integrity and RNA Transcription

描述（由申请人提供）：癌症改变了转录程序，导致正常细胞功能失调并支持癌细胞特征。癌细胞的先决条件是增加其核糖合成活性，以支持增加的蛋白质合成需求。这些过程由 RNA 聚合酶 I (Pol I) 控制，该酶对外部刺激高度敏感，并且在癌症中通常过度激活。然而，它尚未被用作临床相关靶点。 Pol I 转录和核糖体生物合成是核仁的精细过程。 Pol I 转录的畸变会导致核仁应激和核仁解体，从而产生独特的、可识别的细胞表型。这个 该项目提出了一种基于双细胞的高内涵成像筛选，适用于高通量筛选（HTS）平台，用于核仁应激和 Pol I 的调节剂。该筛选将利用稳定整合在癌细胞中的核仁荧光融合蛋白的易位以及 Pol I 催化亚基的表达作为可识别标记。此处提供的数据表明，Pol I 催化亚基可以选择性地靶向降解，并且其靶向与癌细胞活力降低有关。该项目旨在确定 HTS 平台读数的可行性，并证明该测定法识别新型调节剂的适用性和可重复性。拟议工作的理由是它将为 Pol I 活动的监管提供根本性的新颖筛选工具。目标是利用筛选首先识别核仁活性抑制剂，其次识别 Pol I 催化亚基的特定调节剂。为了实现这些目标，在目标 1 中，将建立、测试双重筛选测定，并为命中阈值设置参数。 Aim 2 使用当前治疗中使用的已定义的药物化学库，旨在系统地筛选影响核仁完整性和 Pol I 转录的药物和化合物，并为高通量平台提供组装设置和可重复性的验证。 Aim 3 旨在通过二次筛选和测定来验证筛选命中，以确定哪些 Pol I 复合因子和核仁过程受到影响。这些方法将定义当前影响 Pol I 的药物空间，识别有利于治疗结果的潜在新效应，并支持筛选应用于各种大规模化学库收藏。该研究将显着增进人们对 rRNA 转录抑制如何有助于抑制癌细胞生长的理解，并在此过程中鉴定新药和小分子先导分子。

DESCRIPTION (provided by applicant): Cancers have altered transcriptional programs that lead to deregulation of normal cellular functions and support the cancer cell characteristics. A prerequisite for the cancer cell is an increase in its ribosynthetic activity to support the increaed protein synthetic needs. These processes are governed by RNA polymerase I (Pol I), which is highly responsive to external stimuli and commonly hyperactivated in cancer. Yet, it has not been exploited as a clinically relevant target. Pol I transcription and ribosome biosynthesis are exquisite processes of the nucleolus. Aberration of Pol I transcription leads to nucleolar stress and disintegration of the nucleolus leading to a distinctive, identifiable cellular phenotype. This project sets forth a dual cell-based high-content imaging screen, adaptable to high-throughput screening (HTS) platforms, for regulators of nucleolar stress and of Pol I. The screen will utilize as identifiable marks, the translocation of a nucleolar fluorescent fusion protein stably integrate in cancer cells and the expression of the catalytic subunit of Pol I. Data presented here show that the Pol I catalytic subunit can be selectively targeted for degradation and that its targetingis associated with decreased cancer cell viability. This project aims to define the feasibility of thee readouts for HTS platforms, and to demonstrate the suitability and reproducibility of the assay to identify novel regulators. The rationale for the proposed work is that it will provide fundamentall novel screening tools for regulation of Pol I activity. The goal is to employ the screen to identif, firstly, inhibitors of nucleolar activities and secondly, specific regulators of the Pol I catalyti subunit. Towards these goals, in Aim 1, the dual-screening assay will be established, tested and parameters will be set for hit thresholds. Using a defined chemical library of pharmaceuticals in current therapy use, Aim 2 has been devised to systematically screen for drugs and compounds that affect the nucleolar integrity and Pol I transcription, and to provide verification of the assy set-up and reproducibility for high-throughput platforms. Aim 3 has been designed to validate the screen hits through secondary screens and assays that determine which Pol I complex factors and nucleolar processes are affected. These approaches will define the current drug space affecting Pol I, identify potential new effects beneficial for therapeutic outcome, and support application of the screen to diverse large-scale chemical library collections. The study will significantly increase the understanding how inhibition of rRNA transcription contributes to curb growth of cancer cells, and identify new drugs and small-molecule lead molecules in this process.