A Novel HTS Cell-Based Assay For Cyclic GMP

一种新型 HTS 细胞循环 GMP 检测方法

• 批准号: 7292261
• 负责人: Gary A Piazza
• 金额: $ 25.64万
• 依托单位: SOUTHERN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-05 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7292261
• 关键词: Affect; Anisotropy; Antineoplastic Agents; Binding; Biological Assay; Biosensor; Cell Line; Cell Survival; Cell physiology; Cells; Chimeric Proteins; Colonic Neoplasms; Colorectal Cancer; Cyclic GMP; Disease; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Growth; Image; Isoenzymes; Knowledge; Lasers; Libraries; Life; Malignant Neoplasms; Measurement; Measures; Neurons; Non-Steroidal Anti-Inflammatory Agents; PDE2 phosphodiesterase; Phosphodiesterase Inhibitors; Physiological; Play; Population; Process; Property; Proteins; Public Health; Range; Regulation; Role; Scanning; Screening procedure; Signal Transduction; Smooth Muscle; Specificity; Sulindac; anticancer activity; base; carcinogenesis; cell growth; high throughput screening; inhibitor/antagonist; innovation; neoplastic cell; novel; phosphoric diester hydrolase; tool; tumor

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.

描述（申请人提供）：环磷酸鸟苷（cGMP）在信号转导中起重要作用，可调节多种细胞过程，包括肿瘤细胞存活。细胞内cGMP水平受磷酸二酯酶（PDE）同工酶家族的严格调节，抑制剂可以放大信号的幅度和/或持续时间。我们之前进行的研究表明，环鸟苷酸偏二酸抑制是一种重要的脱靶效应，它是某些高效非甾体抗炎药（例如舒林酸）的肿瘤细胞生长抑制活性的原因。这些研究使我们假设，可以通过询问大型化合物库的cGMP PDE抑制活性来发现更安全和更有选择性的癌症药物。然而，尚不清楚哪种PDE同工酶负责舒林酸的活性，并且很少有抑制剂可用于鉴定参与致癌作用的特异性PDE同工酶。为了实现我们的长期目标，确定选择性cGMP PDE抑制剂的抗癌活性，我们建议开发一种HTS测定使用一种新的cGMP生物传感器，涉及的嵌合蛋白组成的青色和黄色荧光蛋白（CFP/YFP）融合cGMP结合结构域的PDE 2或PDE 5。荧光共振能量转移（FRET）是由于cGMP结合后蛋白质的构象变化导致YFP荧光的偏振（各向异性）降低，这提供了高度特异性的分析物，以使用激光扫描荧光计对活细胞群中的细胞内cGMP进行成像。作为原理的证明，我们已经表明已知的cGMP PDE抑制剂可以增加瞬时转染细胞中的cGMP水平。本提案的具体目的是：1）生成表达cGMP生物传感器的稳定转染结肠肿瘤细胞系，2）开发基于图像的测定以测量转染细胞群中的cGMP水平，以及3）配置HTS测定并开发二次测定以评估命中特异性。这种基于细胞的测定的一个关键创新是其能够鉴定与肿瘤相关的cGMP PDE抑制剂，该抑制剂抑制肿瘤细胞生长，而无需预先了解致癌作用中涉及的特异性同工酶。结直肠癌是一个主要的公共卫生问题，虽然以前的研究表明，某些非甾体抗炎药（NSAID）可以提供显着的保护作用。机制研究表明，环鸟苷一磷酸（cGMP）磷酸二酯酶（PDE）抑制是某些高效NSAID（如舒林酸）对结肠肿瘤细胞的生长抑制活性的重要脱靶原因。这些研究使我们假设，可以通过询问大型化合物库的cGMP PDE抑制活性来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，涉及荧光cGMP生物传感器的高通量筛选。

项目成果

Colon tumor cell growth-inhibitory activity of sulindac sulfide and other nonsteroidal anti-inflammatory drugs is associated with phosphodiesterase 5 inhibition.

• DOI: 10.1158/1940-6207.capr-10-0030
• 发表时间: 2010-10
• 期刊: Cancer prevention research (Philadelphia, Pa.)
• 作者: Tinsley HN;Gary BD;Thaiparambil J;Li N;Lu W;Li Y;Maxuitenko YY;Keeton AB;Piazza GA
• 通讯作者: Piazza GA

Sulindac sulfide selectively inhibits growth and induces apoptosis of human breast tumor cells by phosphodiesterase 5 inhibition, elevation of cyclic GMP, and activation of protein kinase G.

• DOI: 10.1158/1535-7163.mct-09-0758
• 发表时间: 2009-12
• 期刊: Molecular cancer therapeutics
• 影响因子: 5.7
• 作者: Tinsley HN;Gary BD;Keeton AB;Zhang W;Abadi AH;Reynolds RC;Piazza GA
• 通讯作者: Piazza GA