Structure-based identification of inhibitors targeting obstruction of the HIVgp41 N-heptad repeat trimer

Abstract

The viral protein HIVgp41 is an attractive and validated drug target that proceeds through a sequence of conformational changes crucial for membrane fusion, which facilitates viral entry. Prior work has identified inhibitors that interfere with the formation of a required six-helix bundle, composed of trimeric C-heptad (CHR) and N-heptad (NHR) repeat elements, through blocking association of an outer CHR helix or obstructing formation of the inner NHR trimer itself. In this work, we employed similarity-based scoring to identify and experimentally characterize 113 compounds, related to 2 small-molecule inhibitors recently reported by Allen et al. (Bioorg. Med. Chem Lett.2015, 25 2853-59), proposed to act via the NHR trimer obstruction mechanism. The compounds were first tested in an HIV cell-cell fusion assay with the most promising evaluated in a second, more biologically relevant viral entry assay. Of the candidates, compound #11 emerged as the most promising hit (IC₅₀=37.81µM), as a result of exhibiting activity in both assays with low cytotoxicity, as was similarly seen with the known control peptide inhibitor C34. The compound also showed no inhibition of VSV-G pseudotyped HIV entry compared to a control inhibitor suggesting it was specific for HIVgp41. Molecular dynamics simulations showed the predicted DOCK pose of #11 interacts with HIVgp41 in an energetic fashion (per-residue footprints) similar to the four native NHR residues (IQLT) which candidate inhibitors were intended to mimic.

Keywords: Computer-aided drug design; DOCK; Docking; Footprint similarity; HIV; Hungarian similarity; Structure-based drug design; Viral entry; Virtual screening; gp41.

Figure 1

(A) Top, ribbon representation of the HIVgp41 NHR-trimer model, based on prior work reported by McGillick et al, with highlighted spheres (orange) showing the location of the four residues that correspond to the IQLT pocket. (Bottom) Close-up view of the four native (IQLT) residues that interpolate the pocket. (B) Structures of A2 and D9.

Figure 2

(A) The 3D predicted binding geometries for A2 (purple surface) and D9 (light blue surface) with each group of analogs (colored sticks). (B) Comparison of van der Waals and electrostatic footprints of A2 (purple) and D9 (light blue) with the top 5 analogs (green) showing the best overlap in each case. A distant-dependent-dielectric cutoff (ddd=4r) was used to compute the electrostatic footprint score.

Figure 3

Experimental cell-cell fusion (A) and cell viability (B) for the top 35 out of 113 compounds when ranked by activity using a combined luciferase reporter and cell viability assay. Colored blue are compounds with a luciferase signal ≤ 0.5 and outlined in gray are compounds with a luciferase signal >0.5. Also shown are DMSO only (black), control peptide inhibitor C34 (red), and the two previously identified inhibitors A2 (magenta) and D9 (light blue). Compounds labeled in alphanumeric code along the x-axis.

Figure 4

Experimental viral entry (A) and cell viability (B) results for the top 35 out of 113 compounds when ranked by activity using a combined luciferase reporter and cell viability assay. Colored blue are compounds with a luciferase signal ≤ 0.5 and outlined in gray are compounds with a luciferase signal >0.5. Also shown are DMSO only (black), control peptide inhibitor C34 (red), and the two previously identified inhibitors A2 (magenta) and D9 (light blue). Compounds labeled in alphanumeric code along the x-axis.

Figure 5

VSV-G mediated viral entry results of compounds #11 and #73, colored blue. Also depicted are DMSO (black) and the control Bafilomycin A1 (BFA1).

Figure 6

Dose–response viral entry curve (black), cytotoxicity curve (red), of compounds #11 (left), #73 (middle) and the control peptide inhibitor C34 (right). The 2-dimensional structures of #11 and #73.

Figure 7

(A) The DOCK pose of #11 was the reference for all RMSD calculations. Left, RMSDs vs. time for #11 from six different MD simulations (colored lines); Middle, RMSDs vs. time for #11 with the more flexible 1-methylbenzimidazole ring excluded. Right, composite histogram of internally-fit RMSDs for compound #11 from all simulations; (B) Twenty evenly spaced MD snapshots of #11 (line representation) extracted every 1000 ps from the most stable (cyan) trajectory compared to the initial DOCK pose (surface representation) in the IQLT pocket formed by two gp41 NHR peptides (purple ribbon).

Figure 8

Footprint comparison for the original DOCK pose of #11 (blue), six MD-averaged trajectories of #11 (black) and time-averaged trajectory of the IQLT peptide reference (orange) segmented into van der Waals (top) and electrostatic components (bottom). For consistency with Allen et al electrostatics were computed without a distant-dependent-dielectric cutoff.