New insights into the in silico prediction of HIV protease resistance to nelfinavir.

The Human Immunodeficiency Virus type 1 protease enzyme (HIV-1 PR) is one of the most important targets of antiretroviral therapy used in the treatment of AIDS patients. The success of protease-inhibitors (PIs), however, is often limited by the emergence of protease mutations that can confer resistance to a specific drug, or even to multiple PIs. In the present study, we used bioinformatics tools to evaluate the impact of the unusual mutations D30V and V32E over the dynamics of the PR-Nelfinavir complex, considering that codons involved in these mutations were previously related to major drug resistance to Nelfinavir. Both studied mutations presented structural features that indicate resistance to Nelfinavir, each one with a different impact over the interaction with the drug. The D30V mutation triggered a subtle change in the PR structure, which was also observed for the well-known Nelfinavir resistance mutation D30N, while the V32E exchange presented a much more dramatic impact over the PR flap dynamics. Moreover, our in silico approach was also able to describe different binding modes of the drug when bound to different proteases, identifying specific features of HIV-1 subtype B and subtype C proteases

Structural analysis of sB-D30V.

<p>(A) Superposition of the structures of sB-WT PR at 2,500 ps of simulation (grey) and at 50,000 ps (black). (B–C) Superposition of sB-D30N (red) and sB-D30V (green) at 50,000 ps over the respective structures at 2,500 ps (grey). (D–F) Measure of the deviation of ILE50 residue from PR Chain A considering the same structures from A, B and C, indicating the extent of Chain A flap movement. (G) Plot of the variation of the ASP25-ILE50 distance (Chain A) along the simulation. The stipulated threshold for semiopen conformation (1.58 nm) is indicated in blue.</p

Open conformation of the sB-V32E protease.

<p>Comparison between two frames of a molecular dynamics of the sB-V32E PR complexed with NF. Protease structure at 2,500 ps (25 ns) is represented in white (<i>cartoon</i>) with Nelfinavir depicted in purple (<i>sticks</i>). Protease structure at 50,000 ps (50 ns), in an open conformation, is depicted in blue (<i>cartoon</i>).</p

Conformational variability of Nelfinavir.

<p>Structural analysis of Nelfinavir in solution along 100(FES) of this simulation (top) indicates three “islands” of low energy conformations (i1, i2 and i3), from which different structures were recovered (NF-i1, NF-i2, NF-i3). The crystal structure of Nelfinavir (1OHR) was the input conformation, and Root Mean Square Deviation (RMSD) indicates that all conformations sampled during the simulation differ from original structure by at least 0.2 nm (down).</p

Molecular dynamics of sC-PRs bound to NF.

<p>Root Mean Square Deviation (RMSD) of subtype C (sC) proteases bound to Nelfinavir (NF) along 50 ns of molecular dynamics simulation. The colors are given in black, red and green for the wild-type (sC-WT), D30V (sC-D30V) and V32E (sC-V32E), respectively. Note that the sC-V32E RMSD behavior points to an open conformation state of the protease within the first 20 ns, alternating between open and closed conformation along the simulated period. The sC-WT and sC-D30V PRs remained in a closed conformation along the entire simulation.</p

Short replicated simulations of sB-PRs bound to NF.

<p>Average and Standard Deviation of the Root Mean Square Deviation (RMSD) for five independent 10 ns simulations of four different subtype B proteases (sB-PRs) bound to Nelfinavir (NF). Greater divergence is observed for sB-V32E, since two of its replicates presented a change to an open conformation of the flaps. Equilibration stages (before 2,500 ps) are not represented. Independent trajectories of each simulation can be observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087520#pone.0087520.s002" target="_blank">Figure S2</a>.</p

Hydrogen bonds between NF and residue 30 of sB-PRs

<p>. Number (above) and average (below) of hydrogen bonds performed between the ligand Nelfinavir and the residue 30 of each subtype B (sB) protease (Chain A) along 50 ns of molecular dynamics simulation. The colors are given in black, red and green for the wild-type (sB-WT), D30N (sB-D30N) and D30V (sB-D30V), respectively.</p

Rapid and Slow Progressors Show Increased IL-6 and IL-10 Levels in the Pre-AIDS Stage of HIV Infection

<div><p>Cytokines are intrinsically related to disease progression in HIV infection. We evaluated the plasma levels of Th1/Th2/Th17 cytokines in extreme progressors, including slow (SPs) and rapid (RPs) progressors, who were thus classified based on clinical and laboratory follow-up covering a period of time before the initiation of HAART, ranging from 93–136.5 months for SPs and 7.5–16.5 months for RPs. Analyses were also performed based on the different stages of HIV infection (chronic, pre-HAART individuals—subjects sampled before initiating HAART but who initiated therapy from 12 to 24 months—and those receiving HAART). The plasma cytokine levels of 16 HIV-infected rapid progressors and 25 slow progressors were measured using a Human Th1/Th2/Th17 CBA kit. The IL-6 and IL-10 plasma levels differed significantly between the stages of HIV infection. The IL-6 levels were higher in slow progressors pre-HAART than in chronically infected SPs and HIV-seronegative individuals. The IL-10 levels were higher in slow progressors pre-HAART than in slow progressors receiving HAART and HIV-seronegative controls, and in rapid progressors, the IL-10 levels were higher in pre-HAART subjects than in HIV-seronegative controls. The results reflect the changes in the cytokine profile occurring during different clinical stages in HIV+ subjects. Our results suggest an association between increased IL-6 and IL-10 levels and pre-HAART stages independent of the slow or rapid progression status of the subjects. Thus, increased IL-6 and IL-10 levels could indicate a global inflammatory status and could be used as markers of the disease course in HIV-infected individuals.</p></div

Flowchart of the sampling procedure.

<p>Flowchart of the sampling procedure.</p

Clinical baselines and demographic characteristics of the 41 HIV-infected subjects enrolled in this study as rapid or slow disease progressors.

<p>Clinical baselines and demographic characteristics of the 41 HIV-infected subjects enrolled in this study as rapid or slow disease progressors.</p