Virtual Screening Strategy and In Vitro Tests to Identify New Inhibitors of the Immunoproteasome

Abstract

Immunoproteasome inhibition is a promising strategy for the treatment of hematological malignancies, autoimmune diseases, and inflammatory diseases. The design of non-covalent inhibitors of the immunoproteasome β1i/β5i catalytic subunits could be a novel approach to avoid the drawbacks of the known covalent inhibitors, such as toxicity due to off-target binding. In this work, we report the biological evaluation of thirty-four compounds selected from a commercially available collection. These hit compounds are the outcomes of a virtual screening strategy including a dynamic pharmacophore modeling approach onto the β1i subunit and a pharmacophore/docking approach onto the β5i subunit. The computational studies were first followed by in vitro enzymatic assays at 100 μM. Only compounds capable of inhibiting the enzymatic activity by more than 50% were characterized in detail using Tian continuous assays, determining the dissociation constant (K_i) of the non-covalent complex where K_i is also the measure of the binding affinity. Seven out of thirty-four hits showed to inhibit β1i and/or β5i subunit. Compound 3 is the most active on the β1i subunit with K_i = 11.84 ± 1.63 µM, and compound 17 showed K_i = 12.50 ± 0.77 µM on the β5i subunit. Compound 2 showed inhibitory activity on both subunits (K_i = 12.53 ± 0.18 and K_i = 31.95 ± 0.81 on the β1i subunit and β5i subunit, respectively). The induced fit docking analysis revealed interactions with Thr1 and Phe31 of β1i subunit and that represent new key residues as reported in our previous work. Onto β5i subunit, it interacts with the key residues Thr1, Thr21, and Tyr169. This last hit compound identified represents an interesting starting point for further optimization of β1i/β5i dual inhibitors of the immunoproteasome.

Keywords: docking; immunoproteasome; in vitro enzymatic assay; induced fit docking; pharmacophore modeling; β1i subunit; β5i subunit.

Scheme 1

Structure of the selective β1i inhibitor 1.

Scheme 2

Most active compounds retrieved.

Figure 1

LigandScout pharmacophore model of (a) Pose1, (b) Pose2, (c) Pose3, and (d) Merged. Aromatic interactions are represented in yellow spheres, hydrogen bond donors in green spheres, and hydrogen bond acceptors in red spheres.

Figure 2

ROC curves of (a) LigandScout model and (b) PHASE model for β1i subunit.

Figure 3

PHASE pharmacophore models of (a) Pose1, (b) Pose2, (c) Pose3, and (d) Merged. The aromatic rings are represented in orange rings, hydrogen bond donors in blue spheres, and hydrogen bond acceptors in pink spheres.

Figure 4

(a) PR-957 covalent epoxyketone; (b) Modified PR-957, free of the epoxy ring portion (circled in red); (c) Superposition of the re-docked poses of PR-597: modified (green) and co-crystallized (white); (d) ROC curve for the retrospective analysis of β5i subunit.

Figure 5

(a) E-pharmacophore hypothesis for β5i, five features: two aromatic rings (orange), two hydrogen bond donors (blue), and a hydrogen bond acceptor (red). (b) ROC curves, zero feature omitted and (c) one feature omitted.

Figure 6

(a) LigandScout model for β5i, six features: an aromatic interaction (yellow), three hydrogen bond donors (green), and two hydrogen bond acceptors (red). (b) ROC curve.

Figure 7

Binding mode of 2 into the active site of (a) β1i and (b) β5i subunits. The H-bonds are represented in yellow dashes and the π-stacking in blue dashes.