The Influence of 150-Cavity Binders on the Dynamics of Influenza A Neuraminidases as Revealed by Molecular Dynamics Simulations and Combined Clustering

Neuraminidase inhibitors are the main pharmaceutical agents employed for treatments of influenza infections. The neuraminidase structures typically exhibit a 150-cavity, an exposed pocket that is adjacent to the catalytic site. This site offers promising additional contact points for improving potency of existing pharmaceuticals, as well as generating entirely new candidate inhibitors. Several inhibitors based on known compounds and designed to interact with 150-cavity residues have been reported. However, the dynamics of any of these inhibitors remains unstudied and their viability remains unknown. This work reports the outcome of long-term, all-atom molecular dynamics simulations of four such inhibitors, along with three standard inhibitors for comparison. Each is studied in complex with four representative neuraminidase structures, which are also simulated in the absence of ligands for comparison, resulting in a total simulation time of 9.6µs. Our results demonstrate that standard inhibitors characteristically reduce the mobility of these dynamic proteins, while the 150-binders do not, instead giving rise to many unique conformations. We further describe an improved RMSD-based clustering technique that isolates these conformations – the structures of which are provided to facilitate future molecular docking studies – and reveals their interdependence. We find that this approach confers many advantages over previously described techniques, and the implications for rational drug design are discussed

N2-1 alternate enzyme poses.

<p>Shown are the C1, C3, C8 of N2 with L1 of <b>3</b>. The color scheme is identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059873#pone-0059873-g005" target="_blank">Figure 5</a>.</p

Simultaneous 150-loop closure and ligand sidechain ejection for N8_open-6.

<p>Shown is C1 and C3 of N8_open with L1 and L2 of <b>6</b>. The color scheme is identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059873#pone-0059873-g005" target="_blank">Figure 5</a>.</p

Independence of cavity-width and key residue conformations.

<p>Superposition of four MD snapshots of N8_open-<b>6</b> that exhibit a cavity-width of 1.25 nm but differ significantly in conformations of key active site residues, shown as sticks.</p

N2–5 facilitating R152 moving into the active site center.

<p>Shown is C1 and C5 of N2 and L1, L3, and L4 of <b>5</b>. The color scheme is identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059873#pone-0059873-g005" target="_blank">Figure 5</a>.</p

Compounds of interest.

<p>Compounds of interest.</p

Neuraminidase active site, N8_closed and N8_open.

<p>Active site of N8_closed (left) with standard binders <b>1–3</b> and N8_open (right) with 150-binders <b>4–7</b>. Compound <b>1</b> is shown in beige, <b>2</b> in yellow, <b>3</b> in purple, <b>4</b> in light blue, <b>5</b> in teal, <b>6</b> in grey, and <b>7</b> in brown. Key residues are labeled.</p

N2–6 demonstrating a recessed D151–R152.

<p>Shown is C1 and C6 of N2 and L1 of <b>6</b>. The color scheme is identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059873#pone-0059873-g005" target="_blank">Figure 5</a>.</p

Loop-closed populations based on cavity-width.

<p>Loop-closed populations based on cavity-width.</p

Averaged clustering results and standard deviations.

a<p>See materials and methods for criteria.</p>b<p>Percentage of simulation time that a loop-closed cluster is occupied while the ligand remains in the active site.</p