A GFP Based HTS Assay for HIV-1 Tat Inhibitors (RMI)

基于 GFP 的 HIV-1 Tat 抑制剂 (RMI) HTS 测定

• 批准号: 6879840
• 负责人: OLAF KUTSCH
• 金额: $ 7.25万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879840
• 关键词: RNA binding protein; antiAIDS agent; biotechnology; cell line; chemical registry /resource; cytotoxicity; drug discovery /isolation; gene induction /repression; genetic transcription; green fluorescent proteins; high throughput technology; human immunodeficiency virus 1; protein protein interaction; technology /technique development; transcription factor

DESCRIPTION (provided by applicant): HIV-1 Tat transactivation, a key-step in HIV-1 transcription, is based on the unique interaction of Tat with the TAR element, a short nucleotide sequence on the nascent viral RNA, and has early on been recognized as an ideal target for HIV-1 inhibitory drugs. Although substantial efforts were made to develop HIV-1transcription inhibitors, promising in vitro results could thus far not be translated to the clinical situation. Nevertheless, with an increasing number of viral strains becoming resistant to the presently available anti-retrovirals, alternative anti-HIV-1 compounds would be urgently needed. To facilitate future screening for HIV-1 transcription inhibitors, we have developed a high throughput screening (HTS) system that uses enhanced green fluorescence protein expression (EGFP) as a direct and quantitative marker for HIV-1expression. The system is based on a T cell line that was stably transfected with a LTR-EGFP reporter plasmid and infected with a primary HIV-1 patient isolate. Clonal cell lines that expressed high levels of EGFP were derived from the cells that survived the initial infection. The cells, probably as a result of integration of the provirus into a site of high overall transcription activity, constitutively express all HIV-1genes, including HIV-1 Tat, which in turn activates the LTR-EGFP construct to express extremely high levels of EGFP. In contrast to other presently available assays that measure inhibition of HIV-1 expression, the resulting assay requires no cell manipulation during assay preparation or assay analysis, as EGFP, serves as a direct correlate of HIV-1 expression. The assay is thus extremely reliable, inexpensive and rapid. In a 96-well plate format the assay has a Z'-Factor of 0.95, and complete inhibition of HIV-1transcription results in a 35-fold decrease in EGFP fluorescence. In this application, we propose to transfer the reporter assay to a 384-weil plate format and to establish several counter-screen and verification assays that will allow for the rapid and reliable screen of large compound libraries.

描述（由申请人提供）：HIV-1达特反式激活是HIV-1转录的关键步骤，基于达特与TAR元件（新生病毒RNA上的短核苷酸序列）的独特相互作用，早期已被认为是HIV-1抑制药物的理想靶点。尽管人们在开发HIV-1转录抑制剂方面做了大量的努力，但到目前为止，有希望的体外结果还不能转化为临床情况。然而，随着越来越多的病毒株对目前可用的抗逆转录病毒药物产生耐药性，迫切需要替代的抗HIV-1化合物。为了便于将来筛选HIV-1转录抑制剂，我们开发了一种高通量筛选（HTS）系统，该系统使用增强型绿色荧光蛋白表达（EGFP）作为HIV-1表达的直接和定量标记。该系统基于用LTR-EGFP报告质粒稳定转染并用原代HIV-1患者分离株感染的T细胞系。表达高水平EGFP的克隆细胞系来自于在初始感染中存活的细胞。这些细胞，可能是由于前病毒整合到一个高转录活性位点的结果，组成型表达所有HIV-1基因，包括HIV-1达特，反过来激活LTR-EGFP构建体表达极高水平的EGFP。与其他目前可用的测量HIV-1表达抑制的测定相比，所得测定在测定制备或测定分析期间不需要细胞操作，因为EGFP用作HIV-1表达的直接相关物。因此，该测定法非常可靠、廉价和快速。在96孔板形式中，该测定具有0.95的Z '-因子，并且完全抑制HIV-1转录导致EGFP荧光降低35倍。在本申请中，我们建议将报告基因测定转移到384孔板格式，并建立几个计数器筛选和验证测定，这将允许快速和可靠的大型化合物文库的筛选。