Targeting HIV-1 Protease Autoprocessing for High-throughput Drug Discovery and Drug Resistance Assessment

Liangqun Huang,Chihfeng Tien,Linfeng Li,Daniel V Labarbera,Chaoping Chen

doi:10.1038/s41598-018-36730-4

Liangqun Huang, Chihfeng Tien + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-36730-4

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Abstract

HIV-1 protease autoprocessing liberates the free mature protease from its Gag-Pol polyprotein precursor through a series of highly regulated autoproteolysis reactions. Herein, we report the development and validation (Z’ ≥ 0.50) of a cell-based functional assay for high-throughput screening (HTS) of autoprocessing inhibitors using fusion precursors in combination with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Through pilot screening of a collection of 130 known protease inhibitors, the AlphaLISA assay confirmed all 11 HIV protease inhibitors in the library capable of suppressing precursor autoprocessing at low micromolar concentrations. Meanwhile, other protease inhibitors had no impact on precursor autoprocessing. We next conducted HTS of ~23,000 compounds but found no positive hits. Such high selectivity is advantageous for large-scale HTS campaigns and as anticipated based on assay design because a positive hit needs simultaneously to be nontoxic, cell permeable, and inhibiting precursor autoprocessing. Furthermore, AlphaLISA quantification of fusion precursors carrying mutations known to cause resistance to HIV protease inhibitors faithfully recapitulated the reported resistance, suggesting that precursor autoprocessing is a critical step contributing to drug resistance. Taken together, this reported AlphaLISA platform will provide a useful tool for drug discovery targeting HIV-1 protease autoprocessing and for quantification of PI resistance.

Highlights

From the Gag-Pol precursor to the free mature protease, HIV-1 protease autoprocessing is a complicated process in which the Gag-Pol precursor must function as both the catalyst and substrate before any mature PR becomes available
We have demonstrated that the currently available HIV- 1 protease inhibitors (PIs) are much less effective at suppressing precursor-mediated autoprocessing than inhibiting mature protease activity[33,34,38], which is consistent with other reports[29,30,31,39], confirming that the precursor is enzymatically different from the mature PR
We recently reported that precursor autoprocessing is context-dependent and the maltose binding protein signal peptide at the N-terminus leads to autoprocessing outcomes similar to those observed with viral particles[34]

Summary

Introduction

From the Gag-Pol precursor to the free mature protease, HIV-1 protease autoprocessing is a complicated process in which the Gag-Pol precursor must function as both the catalyst and substrate before any mature PR becomes available. We first examined AlphaLISA detection sensitivity using purified proteins at serial dilutions spiked into the lysates made from mock transfected cells (Fig. 1C). We compared AlphaLISA and western blotting detection by examining precursor autoprocessing in response to HIV PI treatment (Fig. 2).

Results

Conclusion