Membrane Partitioning of Anionic, Ligand-Coated Nanoparticles Is Accompanied by Ligand Snorkeling, Local Disordering, and Cholesterol Depletion

<div><p>Intracellular uptake of nanoparticles (NPs) may induce phase transitions, restructuring, stretching, or even complete disruption of the cell membrane. Therefore, NP cytotoxicity assessment requires a thorough understanding of the mechanisms by which these engineered nanostructures interact with the cell membrane. In this study, extensive Coarse-Grained Molecular Dynamics (MD) simulations are performed to investigate the partitioning of an anionic, ligand-decorated NP in model membranes containing dipalmitoylphosphatidylcholine (DPPC) phospholipids and different concentrations of cholesterol. Spontaneous fusion and translocation of the anionic NP is not observed in any of the 10-µs unbiased MD simulations, indicating that longer timescales may be required for such phenomena to occur. This picture is supported by the free energy analysis, revealing a considerable free energy barrier for NP translocation across the lipid bilayer. 5-µs unbiased MD simulations with the NP inserted in the bilayer core reveal that the hydrophobic and hydrophilic ligands of the NP surface rearrange to form optimal contacts with the lipid bilayer, leading to the so-called snorkeling effect. Inside cholesterol-containing bilayers, the NP induces rearrangement of the structure of the lipid bilayer in its vicinity from the liquid-ordered to the liquid phase spanning a distance almost twice its core radius (8–10 nm). Based on the physical insights obtained in this study, we propose a mechanism of cellular anionic NPpartitioning, which requires structural rearrangements of both the NP and the bilayer, and conclude that the translocation of anionic NPs through cholesterol-rich membranes must be accompanied by formation of cholesterol-lean regions in the proximity of NPs.</p></div

Bilayer thickness, defined as the distance between phosphate groups (PO4) of different bilayer leaflets, for different cholesterol concentrations as a function of the distance from the NP-center of mass.

<p>Bilayer thickness, defined as the distance between phosphate groups (PO4) of different bilayer leaflets, for different cholesterol concentrations as a function of the distance from the NP-center of mass.</p

PMF for NP partitioning in a cholesterol-free DPPC lipid bilayer.

<p>The error bars represent standard deviations from two independent sets of Umbrella sampling calculations using the bootstrapping technique. Detailed analysis of the PMF convergence is provided in Text S1.</p

Number density maps of the negatively charged end-terminal groups of the NP ligands.

<p>The calculation was performed over the last 500 ns of the simulation for the different systems. The snapshots correspond to the final frame of the simulation and are depicted to indicate the relative position of the NP with respect to the lipid bilayer at the end of the simulation. White corresponds to zero density of the negatively-charged moieties, indicating the snorkeling effect, where charged-end terminal groups orient themselves towards the lipid head groups and outside of the bilayer core. A typical RGB color scale is used to show increasing occupancy. Only the NP core is shown for clarity. The coloring is the same as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003917#pcbi-1003917-g001" target="_blank">Figure 1</a>.</p

2D Radial concentration of DPPC and CHOL from the NP center of mass.

<p>The concentration values as a function of the distance from the NP center of mass, c(d), are normalized with respect to the bulk values, c_bulk.</p

Coarse-grained models of the system components.

<p>DPPC molecule (top, left), cholesterol molecule (bottom, left), nanoparticle simulated in the water phase (center), and nanoparticle simulated in a membrane containing 30% cholesterol (right) (final snapshots). The molecules or CG beads are not shown to scale. Colors: Negative beads bearing -1e charge = purple; hydrophobic beads = cyan; positive beads = blue; cholesterol hydroxyl bead = gray; glycerol backbone beads = white; cholesterol sterol body beads = lime. The core of the NP is shown in gray and surface representation, whereas the hydrophobic parts of NP surface ligands are shown in a bead-spring representation. Arrows indicate the tendency of the surface charged ligands to associate with the DPPC head groups and their hydrophobic parts to interact with the hydrophobic core, inducing a snorkeling effect.</p

Free Energy Calculations Reveal the Origin of Binding Preference for Aminoadamantane Blockers of Influenza A/M2TM Pore

Aminoadamantane derivatives, such as amantadine and rimantadine, have been reported to block the M2 membrane protein of influenza A virus (A/M2TM), but their use has been discontinued due to reported resistance in humans. Understanding the mechanism of action of amantadine derivatives could assist the development of novel potent inhibitors that overcome A/M2TM resistance. Here, we use Free Energy Perturbation calculations coupled with Molecular Dynamics simulations (FEP/MD) to rationalize the thermodynamic origin of binding preference of several aminoadamantane derivatives inside the A/M2TM pore. Our results demonstrate that apart from crucial protein–ligand intermolecular interactions, the flexibility of the protein, the water network around the ligand, and the desolvation free energy penalty to transfer the ligand from the aqueous environment to the transmembrane region are key elements for the binding preference of these compounds and thus for lead optimization. The high correlation of the FEP/MD results with available experimental data (R² = 0.85) demonstrates that this methodology holds predictive value and can be used to guide the optimization of drug candidates binding to membrane proteins

Exploring a Non-ATP Pocket for Potential Allosteric Modulation of PI3Kα

Allosteric modulators offer a novel approach for kinase inhibition because they target less conserved binding sites compared to the active site; thus, higher selectivity may be obtained. PIK-108, a known pan phosphoinositide 3-kinase (PI3K) inhibitor, was recently detected to occupy a non-ATP binding site in the PI3Kα C-lobe. This newly identified pocket is located close to residue 1047, which is frequently mutated in human cancers (H1047R). In order to assess the interactions, stability, and any possible allosteric effects of this inhibitor on PI3Kα, extensive molecular dynamics (MD) simulations in aqueous solution were performed for the wild type (WT) human, WT murine, and H1047R human mutant PI3Kα proteins with PIK-108 placed in both catalytic and non-ATP sites. We verify the existence of the second binding site in the vicinity of the hotspot H1047R PI3Kα mutation through binding site identification and MD simulations. PIK-108 remains stable in both sites in all three variants throughout the course of the simulations. We demonstrate that the pose and interactions of PIK-108 in the catalytic site are similar in the murine WT and human mutant forms, while they are significantly different in the case of human WT PI3Kα protein. PIK-108 binding in the non-ATP pocket also differs significantly among the three variants. Finally, we examine whether the non-ATP binding site is implicated in PI3Kα allostery in terms of its communication with the active site using principal component analysis and perform in vitro experiments to verify our hypotheses

Normalized interaction energies of the NP with the different components of the system in KJ/mol.

<p>Normalized interaction energies of the NP with the different components of the system in KJ/mol.</p

Spatially averaged lipid order parameters for bilayer systems with different cholesterol concentrations.

<p>The characteristic values P₂ order parameter for various bilayer phases are demarcated in the color bar on the right <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003917#pcbi.1003917-Waheed1" target="_blank">[35]</a>. The different box sizes are due to the gradual condensing effect of cholesterol on the bilayer.</p