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        Screening of Novel Inhibitors of HIV-1 Reverse Transcriptase with a Reporter Ribozyme Assay

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        “Highly active anti-retroviral therapy (HAART)” is currently the standard treatment for human immunodeficiency virus (HIV). This treatment consists of a cocktail of two reverse transcriptase (RT) inhibitors and a protease inhibitor. Despite the success of this regimen, there is a continuing need for innovative drug to overcome problems with tolerability and the emergence of viral resistance. The present protocol describes a novel strategy to rapidly screening a new class of small molecule HIV-1 RT inhibitors, which bind to the primer/template binding site of RT, as yet an unexplored site for small molecule interference on this target. The assay is based on aptamer-displacement which is visualized by applying a rationally designed HIV-1 RT responsive ribozyme. The handiness of the assay procedure permits automation, compatible with high-throughput screening (HTS). Subsequently, the identified hit compounds have been evaluated by an in vitro enzymatic assay to test the inhibitory potential. The strategy provides a powerful and efficient screening format for site-directed inhibitors with biological activity.
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