Nuclear Transcription Factor Translocation Assay

核转录因子易位测定

• 批准号: 6933630
• 负责人: PYARE L KHANNA
• 金额: $ 39.19万
• 依托单位: DISCOVERX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6933630
• 关键词: beta galactosidase; biotechnology; cell line; cell nucleus; drug discovery /isolation; drug screening /evaluation; enzyme activity; enzyme substrate; high throughput technology; nuclear factor kappa beta; protein transport; reporter genes; small molecule; technology /technique development; transcription factor

DESCRIPTION (provided by applicant): Transcription factors (TF) are key regulators of gene expression. Most TFs reside in the cytosol and when activated translocate to the nucleus to stimulate gene activity. A therapeutically important TF is NFkB which stimulates genes involved in cell cycling, proliferation and inflammatory responses and is an important molecular target for the development of anti-cancer and anti-inflammatory drugs. Since nuclear translocation is a critical event in NFkB's ability to regulate gene expression, technologies that measure NFkB translocation could be used to screen for novel anti-neoplastic and anti-inflammatory agents that block the translocation process. However, there are few technologies to measure the movement of TFs from the cytosol to the nucleus and none that is amenable for high throughput drug discovery. DiscoveRx has developed an enzyme fragment complementation (EFC) technology that could be used to detect the translocation of TFs using an extremely sensitive beta-galactosidase (B-gal) complementation assay. This method utilizes two genetically-engineered fragments of E. coli Beta-gal. The larger fragment, termed Enzyme Acceptor (EA), contains a deletion near the amino terminus, while the smaller fragment, termed Enzyme Donor (ED), contains the amino-terminal sequence missing from EA. Alone, EA is inactive. However, it can recombine with ED to form an active enzyme that can catalyze the formation of a fluorescent product detected photometrically as a visually amplified response. We propose to develop a direct assay for NFkB translocation using EFC technology. We will engineer ED into NFkB and target EA to the nucleus. When ED-NFkB translocates to the nucleus it will be able to recombine with EA to generate an active Beta-gal that can catalyze the formation of fluorescent products. As a complementary approach to measure NFkB translocation, we will develop a novel NFkB reporter gene assay using our EFC technology in which an NFkB response element drives the production of a GST-ED fusion protein whose expression will be detected with EA in a simplified cell based fluorescent assay. These studies will develop the first NFkB translocation for drug discovery. In the future, we will adapt this assay as a general technology for drug discovery against any TF to open up an entirely new field of transcription factor Pharmacology and therapeutics.

描述（由申请人提供）：转录因子（TF）是基因表达的关键调控因子。大多数tf驻留在细胞质中，当被激活时转移到细胞核以刺激基因活性。在治疗上重要的TF是NFkB，它刺激参与细胞循环、增殖和炎症反应的基因，是开发抗癌和抗炎药物的重要分子靶点。由于核易位是NFkB调节基因表达能力的关键事件，因此测量NFkB易位的技术可用于筛选阻断易位过程的新型抗肿瘤和抗炎药物。然而，很少有技术来测量tf从细胞质溶胶到细胞核的运动，而且没有一种技术适用于高通量药物发现。DiscoveRx开发了一种酶片段互补（EFC）技术，该技术可用于使用极其敏感的β -半乳糖苷酶（B-gal）互补测定来检测tf的易位。