Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Benzopyran-Based Inhibitors

Abstract

Inhibition of the insulin-regulated aminopeptidase (IRAP) improves memory and cognition in animal models. The enzyme has recently been crystallized and several series of inhibitors reported. We herein focused on one series of benzopyran-based inhibitors of IRAP known as the HFI series, with unresolved binding mode to IRAP, and developed a robust computational model to explain the structure-activity relationship (SAR) and potentially guide their further optimization. The binding model here proposed places the benzopyran ring in the catalytic binding site, coordinating the Zn²⁺ ion through the oxygen in position 3, in contrast to previous hypothesis. The whole series of HFI compounds was then systematically simulated, starting from this binding mode, using molecular dynamics and binding affinity estimated with the linear interaction energy (LIE) method. The agreement with experimental affinities supports the binding mode proposed, which was further challenged by rigorous free energy perturbation (FEP) calculations. Here, we found excellent correlation between experimental and calculated binding affinity differences, both between selected compound pairs and also for recently reported experimental data concerning the site directed mutagenesis of residue Phe544. The computationally derived structure-activity relationship of the HFI series and the understanding of the involvement of Phe544 in the binding of this scaffold provide valuable information for further lead optimization of novel IRAP inhibitors.

Keywords: Insulin regulated aminopeptidase (IRAP); benzopyran; free energy perturbation (FEP); linear interaction energy (LIE); molecular dynamics (MD).

FIGURE 1

Structure of IRAP inhibitors and substrates.

FIGURE 2

Binding pose of compound 7 (orange sticks) in IRAP active site obtained from the constrained docking calculations. Key residues in the active site are shown as gray lines, and the Zn²⁺ as light blue sphere.

FIGURE 3

Binding mode of compound 9 (panel A, green sticks), and 6 (panel B, yellow sticks) in IRAP as determined from the MD simulations used for LIE calculations. Zn²⁺ and water molecules are shown as gray and red spheres, respectively. All of the figures shown are extracted from representative snapshots of 2 ns MD simulations. H-bonds are shown as black dotted line, and π – stacking interactions in the purple color dashed line.

FIGURE 4

Scattered plot of LIE calculated (Y axis) and experimental (X-axis) binding affinities of all HFI compounds that have experimentally determined K_i values for WT (triangles) and F544I/V mutant version (stars) of IRAP. Main line denotes perfect agreement with experiments, while the two dashed lines are +/– 1 kcal⋅mol^{– 1}.

FIGURE 5

Closed thermodynamic cycle depicting the FEP pair transformations that connect the four compounds 6–9.

FIGURE 6

Binding modes of compound 8 (A, orange sticks) and 7 (D, cyan sticks) in wt-IRAP active site determined by FEP simulations. Binding mode of compound 8 in F544I (B) and F544V (C). Binding mode of compound 7 in F544I (E) and F544V (F). All of the figures shown are extracted from representative snapshot simulations. Residues playing a role in ligand binding are explicitly shown, while residues F544I and F544V are represented in magenta color. The remaining atom representations and protein-ligand interactions following the scheme as in Figure 3.

FIGURE 7

The GAMEN loop in the closed conformation (red color cartoon, PDB ID: 5MJ6) is compared with the GAMEN loop of open conformation (cyan color cartoon, PDB ID: 4PJ6) while compound 9 (green sticks) is docked in the active site. Zn²⁺ ion shown as gray sphere.