Exploring the non-covalent ligand-binding mechanism on immunoproteasome by enhanced Molecular Dynamics

Selective inhibition of immunoproteasome is a valuable strategy to treat autoimmune and inflammatory diseases, and hematologic malignancies. In particular, non-covalent inhibition is strongly desirable because it is free of the drawbacks and side effects associated with covalent inhibition. Recently, a new series of amide derivatives with Ki values in the low/submicromolar ranges toward the β1i subunit have been identified as non-covalent inhibitors 1 . We investigated the binding mechanism of the most potent and selective inhibitor (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 400ns of MD-binding analysis, followed by 200ns of plain MD. The trajectories clustering allowed identifying three representative poses evidencing new key interactions with Phe31 and Lys33 together to a flipped orientation of a representative pose. Further, Binding pose metadynamics (BPMD) studies have been performed to evaluate the binding affinity, comparing (1) with other four inhibitors of β1i subunit (2, 3, 4, and 5). Results are consistent with 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