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

描述(由申请人提供)：HIV-1Tat反式激活，HIV-1转录的关键步骤，基于Tat与TAR元件的独特相互作用，TAR元件是新生病毒RNA上的短核苷酸序列，很早就被认为是HIV-1抑制药物的理想靶点。尽管在开发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现有的抗逆转录病毒药物产生抗药性，迫切需要替代的抗艾滋病毒-1化合物。为了便于将来筛选HIV-1转录抑制物，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T细胞系，该T细胞系稳定地转染了LTR-EGFP报告质粒，并感染了HIV-1患者的主要分离株。表达高水平绿色荧光蛋白的克隆细胞系来自最初感染后存活的细胞。这些细胞可能是由于前病毒整合到一个总转录活性较高的部位而结构性地表达所有HIV-1基因，包括HIV-1 Tat，这反过来又激活LTR-EGFP结构以表达极高水平的EGFP。与目前测量HIV-1表达抑制的其他检测方法不同，所得到的检测方法在检测准备或检测分析过程中不需要细胞操作，因为EGFP与HIV-1表达直接相关。因此，该检测方法极其可靠、廉价和快速。在96孔板格式中，该检测的Z‘因子为0.95，完全抑制HIV-1转录导致EGFP荧光减少35倍。在这项应用中，我们建议将报告分析转换为384-Weil平板格式，并建立几种反筛选和验证分析，以允许快速和可靠地筛选大型化合物文库。

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.