Inhibition of the precursor and mature forms of HIV-1 protease as a tool for drug evaluation

Jana Humpolíčková,Jana Starková,Jan Konvalinka,Eva Mašínová,Iva Flaisigová,Taťána Majerová,Jana Günterová,Jan Weber

doi:10.1038/s41598-018-28638-w

Abstract

HIV-1 protease (PR) is a homodimeric enzyme that is autocatalytically cleaved from the Gag-Pol precursor. Known PR inhibitors bind the mature enzyme several orders of magnitude more strongly than the PR precursor. Inhibition of PR at the precursor level, however, may stop the process at its rate-limiting step before the proteolytic cascade is initiated. Due to its structural heterogeneity, limited solubility and autoprocessing, the PR precursor is difficult to access by classical methods, and limited knowledge regarding precursor inhibition is available. Here, we describe a cell-based assay addressing precursor inhibition. We used a reporter molecule containing the transframe (TFP) and p6* peptides, PR, and N-terminal fragment of reverse transcriptase flanked by the fluorescent proteins mCherry and EGFP on its N- and C- termini, respectively. The level of FRET between EGFP and mCherry indicates the amount of unprocessed reporter, allowing specific monitoring of precursor inhibition. The inhibition can be quantified by flow cytometry. Additionally, two microscopy techniques confirmed that the reporter remains unprocessed within individual cells upon inhibition. We tested darunavir, atazanavir and nelfinavir and their combinations against wild-type PR. Shedding light on an inhibitor’s ability to act on non-mature forms of PR may aid novel strategies for next-generation drug design.

Highlights

Extensive studies of HIV-1 protease (PR) have expanded knowledge about the biological, chemical and structural aspects governing retroviral infections and led to successful development of antiretroviral drugs[1,2]
A PR precursor containing TFP-p6*, p6*-PR and PR-RT cleavage sites was inserted between the mCherry and EGFP fluorescent proteins to obtain mCherry-TFP-p6*-PR-RT′-EGFP
We identified weak synergy between darunavir and atazanavir (CI < 1) and antagonism between nelfinavir and atazanavir (CI > 1) in cells transiently transfected with the mCherry-PRprec-EGFP reporter, as well in the drug susceptibility assay with fully infectious virus (Table 2)

Summary

Introduction

Extensive studies of HIV-1 protease (PR) have expanded knowledge about the biological, chemical and structural aspects governing retroviral infections and led to successful development of antiretroviral drugs[1,2]. The design of the more recently approved PIs in clinical use ( tipranavir, atazanavir and darunavir) was inspired by the effort to target drug-resistant PR variants[3,4]. HIV-1 PR is produced as part of the Gag-Pol polyprotein. Each Gag-Pol polyprotein contains one HIV-1 PR monomer (Fig. 1A). HIV-1 PR autoproteolysis is a key step in viral maturation, which is critical for successful production of infectious viral progeny[1]. One site in the Gag region (p2-NC) and one site in the Pol region (TFP-p6*) are cleaved intramolecularly, followed by cis N-terminal cleavage of HIV-1 PR out of the precursor. A delay in HIV-1 autoprocessing leads to formation of viral particles with irregular morphology[9], while overactivation of HIV-1 PR blocks production of viral progeny[10,11]

Methods

Results

Conclusion