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Abstract
Grant Number: 1R03NS050810-01 Project Title: A GFP Based HTS Assay for HIV-1 Tat Inhibitors (RMI)
PI Information: Name Title KUTSCH, OLAF okutsch@uab.edu Abstract: 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.
Public Health Relevance:
This Public Health Relevance is not available.Thesaurus Terms:
antiAIDS agent, drug discovery /isolation, gene induction /repression, genetic transcription, green fluorescent protein, high throughput technology, human immunodeficiency virus 1, technology /technique development, transcription factor
RNA binding protein, cytotoxicity, protein protein interaction
biotechnology, cell line, chemical registry /resource
Institution: UNIVERSITY OF ALABAMA AT BIRMINGHAM 1530 3rd Avenue South BIRMINGHAM, AL 35294 Fiscal Year: 2004 Department: MEDICINE Project Start: 30-SEP-2004 Project End: 31-AUG-2005 ICD: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE IRG: ZNS1