Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance.

Zhanglong Liu,Trang T Tran,Gail E Fanucci,Daniel A Savin,Kyle Bentz,Lingna Hu,Linh Pham

doi:10.3390/v12111275

Abstract

Multidrug resistance continues to be a barrier to the effectiveness of highly active antiretroviral therapy in the treatment of human immunodeficiency virus 1 (HIV-1) infection. Darunavir (DRV) is a highly potent protease inhibitor (PI) that is oftentimes effective when drug resistance has emerged against first-generation inhibitors. Resistance to darunavir does evolve and requires 10–20 amino acid substitutions. The conformational landscapes of six highly characterized HIV-1 protease (PR) constructs that harbor up to 19 DRV-associated mutations were characterized by distance measurements with pulsed electron double resonance (PELDOR) paramagnetic resonance spectroscopy, namely double electron–electron resonance (DEER). The results show that the accumulated substitutions alter the conformational landscape compared to PI-naïve protease where the semi-open conformation is destabilized as the dominant population with open-like states becoming prevalent in many cases. A linear correlation is found between values of the DRV inhibition parameter Ki and the open-like to closed-state population ratio determined from DEER. The nearly 50% decrease in occupancy of the semi-open conformation is associated with reduced enzymatic activity, characterized previously in the literature.

Highlights

Human immunodeficiency virus 1 (HIV-1) protease (PR) is a potent target in the treatment of HIV-1 infection because its inhibition leads to non-infectious immature virus particles [1,2,3,4,5]
DRV-resistance oftentimes results in >18 amino acid changes, and these constructs represent the most highly mutated PR sequences we have investigated by double electron–electron resonance (DEER) spectroscopy to date
We have demonstrated that this single spin-labeled site reports changes in distances and distance distributions between the major conformations detected in numerous X-ray structures of closed (33 Å) and open (36 Å), and we find additional distance populations reflective of two other conformational states described as wide-open (>40 Å) and curled/tucked (25–30 Å) [16,50,51]; these results have been substantiated by molecular dynamic (MD) simulations [15,52] and crystallographic investigations [16,51]

Summary

Introduction

Human immunodeficiency virus 1 (HIV-1) protease (PR) is a potent target in the treatment of HIV-1 infection because its inhibition leads to non-infectious immature virus particles [1,2,3,4,5]. FOR PEER REVIEW wide-open orx curled/tucked) and destabilize closed-like conformations [12,13,14,15,16,17,18,19]. This conformational sampling scheme encompasses four conformational ensembles described as curled/tucked, wide-open, conformational sampling scheme encompasses four conformational ensembles described as semi-open, and closed (Figure 1). These conformations are proposed from a combination of X-ray curled/tucked, wide-open, semi-open, and closed (Figure 1) These conformations are proposed from structures, molecular dynamic (MD) simulations, and our DEER data [19,20]. Our prior work a combination of X-ray structures, molecular dynamic (MD) simulations, and our DEER data [19,20]

Methods

Results

Conclusion