Assay Optimization for Identification of Novel HIV-1 Protease Autoprocessing Inhibitors Project Summary This study aims to optimize a cell-based functional assay for high-throughput screens of small molecules that selectively suppress HIV-1 protease autoprocessing, an essential viral-specific process that has not been exploited for anti-HIV drug development. In the infected cell, HIV protease is initially synthesized as part of the Gag-Pol polyprotein precursor. During the late stage of virion production, the precursor catalyzes the cleavage reactions that lead to the liberation of the free, fully active, mature protease. The currently available FDA- approved protease inhibitors (PIs) primarily target the mature protease at its catalytic site. These PIs, however, are significantly less effective at suppressing precursor autoprocessing, suggesting that these two forms of HIV-1 protease are not enzymatically identical. We have recently developed an assay for quantification of precursor autoprocessing. This assay also has the potential to be used for high-throughput screen (HTS) of novel autoprocessing inhibitors with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Our pilot screen demonstrated a z'factor ranging from 0.5 to 0.7 and S/N ratios greater than 100. Built upon this platform, we here propose to establish/optimize the primary assay conditions that can be used for HTS of novel autoprocessing inhibitors (Aim 1);and to evaluate the primary assay via a small-scale screen (Aim 2). Results of these developments will lay the foundation for identification of novel autoprocessing inhibitors through large scale screens. Further characterization of the identified compounds will aid in the development of a much-needed new class of therapeutic drugs that target the HIV-1 protease at regions/stages different from those targeted by the current PIs. A cocktail combining this next generation of autoprocessing inhibitors with the current regimen may significantly improve the treatment outcomes of those living with HIV. Biochemical and structural examination of these new drugs may also shed light on the mechanism of precursor autoprocessing.
HIV-1 is the causative agent of the tragic AIDS epidemic that has claimed more than 25 million lives worldwide since 1981. The existing anti-HIV drugs are effective but often unable to completely suppress HIV-1 infection and the emergence of drug resistance is a persistent problem;therefore, innovative compounds with novel inhibition mechanisms are in constant need in order to achieve an AIDS-free generation in the near future. This project seeks to establish and optimize a cell-based functional assay that will enable identification of new anti-HIV medicines targeting protease precursor autoprocessing, an essential virus-specific process to which there is no specific inhibitor.