Solid-state NMR and MD simulations of the antiviral drug amantadine solubilized in DMPC bilayers

Abstract

The interactions of (15)N-labeled amantadine, an antiinfluenza A drug, with DMPC bilayers were investigated by solid-state NMR and by a 12.6-ns molecular dynamics (MD) simulation. The drug was found to assume a single preferred orientation and location when incorporated in these bilayers. The experimental and MD computational results demonstrate that the long axis of amantadine is on average parallel to the bilayer normal, and the amine group is oriented toward the headgroups of the lipid bilayers. The localization of amantadine was determined by paramagnetic relaxation and by the MD simulation showing that amantadine is within the interfacial region and that the amine interacts with the lipid headgroup and glycerol backbone, while the hydrocarbon portion of amantadine interacts with the glycerol backbone and much of the fatty acyl chain as it wraps underneath the drug. The lipid headgroup orientation changes on drug binding as characterized by the anisotropy of (31)P chemical shielding and (14)N quadrupolar interactions and by the MD simulation.

FIGURE 1

Static ¹⁵N spectra of amantadine in hydrated randomly oriented DMPC bilayers at pH 2.0 (A) and pH 8.0 (B) at 30°C in a 400-MHz spectrometer.

FIGURE 2

Static ³¹P spectra (left column) of DMPC using 300-MHz spectrometer and ¹⁵N spectra (right column) of amantadine in hydrated DMPC bilayers using 400-MHz spectrometer: (A and D) no alignment, (B and E) perpendicular, and (C and F) parallel with respect to the static magnetic field (B_o). The experiments were performed at 30°C and pH 8.0. The molar ratio of amantadine to lipid is 1:20.

FIGURE 3

Static ³¹P spectra (left column) from a 300-MHz spectrometer and ¹⁴N spectra from an 830-MHz spectrometer (right column) of randomly oriented hydrated DMPC in the presence of amantadine in mole percent: 0% (A and D), 5% (B and E), and 10% (C and F) amantadine. The experiments were performed at 30°C and pH 8.0.

FIGURE 4

Static ³¹P spectra (left column) from a 300-MHz spectrometer of DMPC and ¹⁵N spectra (right column) from a 400-MHz spectrometer of amantadine (AMT/lipid, 1:20 molar ratio) in aligned hydrated DMPC bilayers (parallel with B₀) in the presence of Mn²⁺ in mole percent: 0% (A and C) and 1.5% Mn²⁺ (B and D). The experiments were performed at 30°C and pH 8.0.

FIGURE 5

¹³C CP-MAS spectra of (A) dry powder sample of amantadine (the free base form, the molecule is shown to the left and the resonance assignments are labeled in the spectrum). (B) Amantadine in hydrated DMPC lipid bilayers: the signals from amantadine are labeled with asteriks. (C) The same sample as in B in the presence of 1.5% mol % MnCl₂. (D) The same sample as in B in the presence of 5 mol % MnCl₂. The experiments were performed at 30°C and pH 8.0. The molar ratio of amantadine to lipid is 1:5.

FIGURE 6

The membrane-amantadine-water system in MD simulation. DMPC lipid molecules, amantadine, and water box are represented by line, sphere, and surface, respectively.

FIGURE 7

The angle of amantadine C_α-N vector to the membrane normal along the 10-ns trajectory (black) and its distribution with the peak at 30° (red).

FIGURE 8

The relative depth of the amantadine (red, nitrogen atom) with respect to the DMPC lipid molecules in the upper leaflet. Distances from the bilayer central plane (0 Å) along the 10-ns trajectory are shown. For comparison, the average distance of nitrogen (black), phosphorus (green), and oxygen (blue) atoms in choline, phosphate, and carbonyl groups from the membrane center are shown as well. The broken black line shows the distribution of the amantadine amine location with a peak at 15 Å.

FIGURE 9

The average angle of DMPC P-N vectors in the upper leaflet with respect to the membrane normal, within 12 Å of amantadine (green) and that of the outside lipid molecules (blue) along the 10-ns trajectory. Amantadine location (red) and P-N angle (green) show an anti-correlation.

FIGURE 10

A snapshot of the MD simulation at 3.8 ns from the beginning of 10-ns trajectory. Lipids around and away from amantadine in the upper leaflet are shown by stick and surface representations, respectively. Amantadine is represented by spheres. Carbon, nitrogen, oxygen, hydrogen, and phosphorus atoms are colored in cyan, blue, red, white, and brown, respectively.