Studies On The Screening Method Of HIV-1 Reverse Transcriptase Inhibitors

Reverse transcriptase (RT) plays a vital role in the early part of the life cycle of human immunodeficiency virus (HIV). RT has been one of the most successful targets for acquired immunodeficiency syndrome (AIDS) therapy over 20 years. Inhibitors of HIV RT are indispensable to the highly active antiretroviral therapy (HAART) of AIDS. The side effects and drug resistances of the existing drugs prompt an urgent research on searching for new inhibitors of HIV RT. Natural products are important sources of new drugs. To efficiently discover new HIV RT inhibitors, it will be very valuable to develop a method that can screen the inhibitory activity of crude natural product extracts. Most of the existing methods cannot be used for this purpose; it is therefore the aim of this project to develop a new screening method of HIV-1 RT inhibitors that can be used for screening crude natural product extracts in a bioactivity-guided manner.In principle, the method combines heterogeneous immunoassay and time-resolved fluorescence detection with reverse transcription reaction catalyzed by HIV-1 RT to detect HIV-1 RT activity. In this method, the capture is achieved via digoxigenin-anti-digoxigenin system, which effectively avoids the interference of endogenous biotin from crude natural product extracts. The detection is achieved via time-resolved fluorescence, which effectively avoids the interference of crude natural product extracts on the accuracy of assay. The feasibility of this method was firstly verified.