Immunoproteasome and Non-Covalent Inhibition: Exploration by Advanced Molecular Dynamics and Docking Methods.

The selective inhibition of immunoproteasome is a valuable strategy to treat autoimmune, inflammatory diseases, and hematologic malignancies. Recently, a new series of amide derivatives as non-covalent inhibitors of the β1i subunit with Ki values in the low/submicromolar ranges have been identified. Here, we investigated the binding mechanism of the most potent and selective inhibitor, N-benzyl-2-(2-oxopyridin-1(2H)-yl)propanamide (1), to elucidate the steps from the ligand entrance into the binding pocket to the ligand-induced conformational changes. We carried out a total of 400 ns of MD-binding analyses, followed by 200 ns of plain MD. The trajectories clustering allowed identifying three representative poses evidencing new key interactions with Phe31 and Lys33 together in a flipped orientation of a representative pose. Further, Binding Pose MetaDynamics (BPMD) studies were performed to evaluate the binding stability, comparing 1 with four other inhibitors of the β1i subunit: N-benzyl-2-(2-oxopyridin-1(2H)-yl)acetamide (2), N-cyclohexyl-3-(2-oxopyridin-1(2H)-yl)propenamide (3), N-butyl-3-(2-oxopyridin-1(2H)-yl)propanamide (4), and (S)-2-(2-oxopyridin-1(2H)-yl)-N,4-diphenylbutanamide (5). The obtained results in terms of free binding energy were consistent with the experimental values of inhibition, confirming 1 as a lead compound of this series. The adopted methods provided a full dynamic description of the binding events, and the information obtained could be exploited for the rational design of new and more active inhibitors.