Nuclear Transcription Factor Translocation Assay

描述（由申请人提供）：转录因子（TF）是基因表达的关键调节因子。大多数TF存在于细胞质中，当被激活时，转移到细胞核中以刺激基因活性。治疗上重要的TF是NFkB，其刺激参与细胞周期、增殖和炎症反应的基因，并且是开发抗癌和抗炎药物的重要分子靶标。由于核转位是NFkB调节基因表达能力的关键事件，因此测量NFkB转位的技术可用于筛选阻断转位过程的新型抗肿瘤和抗炎剂。然而，很少有技术来测量TF从胞质溶胶到细胞核的移动，并且没有一种适合于高通量药物发现。DiscoveRx开发了一种酶片段互补（EFC）技术，可用于使用极其灵敏的β-半乳糖苷酶（B-gal）互补测定来检测TF的易位。 该方法利用E. coli Beta-gal.较大的片段称为酶受体（EA），在氨基末端附近含有缺失，而较小的片段称为酶供体（艾德），含有EA缺失的氨基末端序列。单独，EA是不活动的。 然而，它可以与艾德重组形成活性酶，该活性酶可以催化荧光产物的形成，通过光度法检测为视觉放大响应。我们建议使用EFC技术开发一种直接检测NF κ B易位的方法。我们将工程师艾德到NF κ B和目标EA的细胞核。当ED-NFkB易位到细胞核时，它将能够与EA重组以产生可以催化荧光产物形成的活性β-gal。作为测量NFkB易位的补充方法，我们将使用我们的EFC技术开发一种新的NFkB报告基因测定，其中NFkB反应元件驱动GST-ED融合蛋白的产生，其表达将在简化的基于细胞的荧光测定中用EA检测。这些研究将开发第一个用于药物发现的NF κ B易位。在未来，我们将适应这种检测作为一种通用的技术，用于药物发现对任何TF开辟一个全新的领域，转录因子药理学和治疗学。

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.