Binding of Venezuelan Equine Encephalitis Virus Inhibitors to Importin-α Receptors Explored with All-Atom Replica Exchange Molecular Dynamics

Abstract

Although Venezuelan equine encephalitis virus (VEEV) is a life-threatening pathogen with a capacity for epidemic outbreaks, there are no FDA-approved VEEV antivirals for humans. VEEV cytotoxicity is partially attributed to the formation of a tetrameric complex between the VEEV capsid protein, the nuclear import proteins importin-α and importin-β, and the nuclear export protein CRM1, which together block trafficking through the nuclear pore complex. Experimental studies have identified small molecules from the CL6662 scaffold as potential inhibitors of the viral nuclear localization signal (NLS) sequence binding to importin-α. However, little is known about the molecular mechanism of CL6662 inhibition. To address this issue, we employed all-atom replica exchange molecular dynamics simulations to probe, in atomistic detail, the binding mechanism of CL6662 ligands to importin-α. Three ligands, including G281-1485 and two congeners with varying hydrophobicities, were considered. We investigated the distribution of ligand binding poses, their locations, and ligand specificities measured by the strength of binding interactions. We found that G281-1485 binds nonspecifically without forming well-defined binding poses throughout the NLS binding site. Binding of the less hydrophobic congener becomes strongly on-target with respect to the NLS binding site but remains nonspecific. However, a more hydrophobic congener is a strongly specific binder and the only ligand out of three to form a well-defined binding pose, while partially overlapping with the NLS binding site. On the basis of free energy estimates, we argue that all three ligands weakly compete with the viral NLS sequence for binding to importin-α in an apparent compromise to preserve host NLS binding. We further show that all-atom replica exchange binding simulations are a viable tool for studying ligands binding nonspecifically without forming well-defined binding poses.

Figure 1:

(a) Structure of inhibitors G281–1485 (I0), G281–1564 (I1), and I2. Locations, where the inhibitors are modified relative to I0, are highlighted in yellow. Ligand structural groups are marked. (b) All-atom REST simulations probe binding of G281–1485 (I0, in orange) inhibitor to impα. To enhance sampling of bound states, we impose a confining sphere with the radius R = 18 Å around VEEV minNLS binding site. Impα conformation is restrained to PDB structure 3VE6. (c) The complex formed by VEEV NLS peptide bound to impα (PDB code 3VE6). In (b) and (c) amino acids constituting the minNLS binding site are colored in ice blue. Three tryptophan amino acids are marked and presented in licorice. The hydrophobic cavity formed by non-minNLS amino acids Leu34, Pro40, Ile42 is in black. The rest of impα structure is in grey cartoon representation. NLS peptide in (c) is shown in cyan except for Lys7 and Lys9 side chains in yellow, which are intercalated between Trp side chains.

Figure 2:

I0 inhibitor centroid from the largest cluster CL0, which represents well the I0 interactions with impα. Amino acids constituting the minNLS binding site are in ice blue, except for the minNLS amino acids bound to I0 which are in purple. The non-minNLS amino acids binding I0 are in dark grey, of which three apolar amino acids, Leu34, Pro40, Ile42, forming a hydrophobic cavity are in black. Three tryptophan amino acids are presented in licorice and marked. The rest of impα structure is in grey cartoon representation. Ligand groups are colored as follows: L1 in red, L2 in orange, L3 in yellow, L4 in green, L5 in aqua, L6 in tan, and L7 in blue.

Figure 3:

Probability distributions P(γ₇₂₋₁₁₄) (in magenta) and P(γ_161−114) (in orange) map the orientations of Trp114 side chain with respect to the side chains of Trp72 and Trp161 upon binding of I0 (a), I1 (b), I2 (c) inhibitors and in the ligand-free impα (d). The probability distributions P(γ₇₂₋₁₆₁) between the side chains of Trp72 and Trp161 (in opal) indicate that these side chains always maintain approximately antiparallel orientation in all panels. The angles between the Trp side chains are defined in Models and Methods. The figure shows that the I0 and I2 probability distributions are qualitativey similar, whereas I1 distributions are distinct from them resembling those in the ligand-free impα.

Figure 4:

I1 inhibitor centroid from the largest cluster CL0, which represents well the I0 interactions with impα. The coloring and representation of impα amino acids and ligand groups follow Fig. 2. The gure reveals a notable shift of I1 binding pose toward minNLS amino acids, including three tryptophan residues.

Figure 5:

Superposition of I0 (in red), I1 (in blue), and I2 (in green) bound poses observed in the largest clusters CL0. The coloring and representation of impα amino acids follow Fig. 1b. The gure reveals distinct binding poses of the three ligands. Note that the CL0 clusters of I0 and I1 represent less than 10% of bound structures, whereas the I2 CL0 population is 27%.

Figure 6:

I2 inhibitor centroid from the largest cluster CL0, which represents well the I2 interactions with impα. The coloring and representation of impα amino acids and ligand groups follow Fig. 2. The gure reveals a notable shift of I2 binding away from the minNLS site and Trp cages toward the non-minNLS hydrophobic cleft.