A Novel HTS Cell-Based Assay For Cyclic GMP

描述（由申请人提供）：环鸟苷单磷酸（cGMP）在信号转导中发挥重要作用，调节多种细胞过程，包括肿瘤细胞存活。细胞内cGMP水平受到磷酸二酯酶（PDE）同工酶家族的严格调节，因此抑制剂可以放大信号的强度和/或持续时间。我们以往的研究表明，cGMP PDE抑制是一种重要的脱靶效应，是某些强效非甾体类抗炎药如舒林酸抑制肿瘤细胞生长活性的原因。这些研究使我们假设，通过询问大型化合物文库的cGMP PDE抑制活性，可以发现更安全，更有选择性的癌症药物。然而，尚不清楚哪一种PDE同工酶负责舒林酸的活性，并且很少有抑制剂可用于确定参与致癌作用的特定PDE同工酶。为了实现我们确定具有抗癌活性的选择性cGMP PDE抑制剂的长期目标，我们建议使用一种新型cGMP生物传感器开发一种HTS检测方法，该传感器涉及由青色和黄色荧光蛋白（CFP/YFP）组成的嵌合蛋白与PDE2或PDE5的cGMP结合域融合。荧光共振能量转移（FRET）是由于蛋白质在cGMP结合时发生构象变化，导致YFP荧光的极化（各向异性）降低，这为使用激光扫描荧光仪在活细胞群体中成像细胞内cGMP提供了高度特异性的分析物。作为原理的证明，我们已经证明了已知的cGMP PDE抑制剂可以增加瞬时转染细胞中的cGMP水平。该提案的具体目标是：1)生成表达cGMP生物传感器的稳定转染结肠肿瘤细胞系，2)开发基于图像的检测方法来测量转染细胞群中的cGMP水平，以及3)配置用于HTS的检测方法并开发二级检测方法来评估命中特异性。这种基于细胞的检测方法的一个关键创新之处在于，它能够识别药理学上相关的cGMP PDE抑制剂，这些抑制剂可以抑制肿瘤细胞的生长，而无需事先了解致癌过程中涉及的特定同工酶。结直肠癌是一个重大的公共卫生问题，尽管先前的研究表明某些非甾体抗炎药（NSAIDs）可以提供显著的保护作用。机制研究表明，环鸟苷单磷酸（cGMP）磷酸二酯酶（PDE）抑制是某些高效非甾体抗炎药（如舒林酸）对结肠肿瘤细胞生长抑制活性的重要脱靶作用。这些研究使我们假设，通过对cGMP PDE抑制活性的大型化合物文库进行查询，可以发现安全且高选择性的抗癌药物。为了实现这一目标，我们建议开发一种创新的基于细胞的检测方法，包括用于高通量筛选的荧光cGMP生物传感器。

DESCRIPTION (provided by applicant): Cyclic guanosine monophosphate (cGMP) plays an essential role in signal transduction to regulate a diverse range of cellular processes, including tumor cell survival. Intracellular cGMP levels are tightly regulated by a family of phosphodiesterase (PDE) isozymes, whereby inhibitors can amplify the magnitude and/or duration of the signal. Previous studies that we have conducted suggest that cGMP PDE inhibition is an important off-target effect, which is responsible for the tumor cell growth inhibitory activity of certain highly potent nonsteroidal anti-inflammatory drugs such as sulindac. These studies led us to hypothesize that safer and more selective cancer drugs can be discovered by interrogating a large compound library for cGMP PDE inhibitory activity. However, it is unclear which PDE isozyme is responsible for the activity of sulindac and few inhibitors are available to identify the specific PDE isozyme(s) involved in carcinogenesis. Towards our long term goal of identifying selective cGMP PDE inhibitors with anti-cancer activity, we propose to develop a HTS assay using a novel cGMP biosensor involving a chimeric protein composed of cyan- and yellow fluorescent proteins (CFP/YFP) fused with the cGMP binding domain of PDE2 or PDE5. Fluorescence resonance energy transfer (FRET) results from a conformational change of the protein upon cGMP binding to cause decreased polarization (anisotropy) of YFP fluorescence, which provides a highly specific analyte to image intracellular cGMP in live cell populations using a laser scanning fluorimeter. As proof of principle, we have shown that known cGMP PDE inhibitors can increase cGMP levels in transiently transfected cells. The specific aims of this proposal are to: 1) generate a stable transfected colon tumor cell line that expresses the cGMP biosensor, 2) develop an image-based assay to measure cGMP levels in transfected cell populations, and 3) configure the assay for HTS and develop secondary assays to assess hit specificity. A key innovation of this cell-based assay is its ability to identify pharmacologically relevant cGMP PDE inhibitors that suppress tumor cell growth without prior knowledge of the specific isozymes involved in carcinogenesis. Colorectal cancer is a major public health problem, although previous studies have shown that certain nonsteroidal anti-inflammatory drugs (NSAIDs) can provide a significant protective benefit. Mechanistic studies suggest that cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) inhibition is an important off- target responsible for the growth inhibitory activity of certain highly potent NSAIDs such as sulindac against colon tumor cells. These studies led us to hypothesize that safe and highly selective anticancer drugs can be discovered by interrogating a large compound library for cGMP PDE inhibitory activity. Towards this goal, we propose to develop an innovative cell-based assay involving a fluorescence cGMP biosensor for high throughput screening.