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

Abstract

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.

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

“…The previous MD study [ 24 ] allowed identifying three representative poses (pose 1, pose 2, and pose 3) that elucidated the dynamic behavior and stability of compound 1 within the binding cavity. The interactions pattern of inhibition included two H-bonds between the amide group and Ser21 and Gly47, a π-stacking between the benzyl group and Phe31, and a cation-pi stacking interaction between the 2-pyridone moiety and the epsilon amino group of Lys33.…”

“…Induced Fit Docking (IFD) developed by Schrödinger [ 40 ] was performed using the standard protocol, as reported in the different studies [ 24 , 29 , 41 , 42 , 43 , 44 , 45 ]. The receptor box for β5i was set on the PR-957 pose with no constraints applied.…”

“…Both pharmacophore models were used for a virtual screening campaign on commercially available compound collection of 2 million compounds (hereby called The mechanism of the non-covalent inhibition of this compound has been investigated using advanced molecular dynamics methods such as MD-binding (MDB) and Binding Pose MetaDynamics (BPMD). Moreover, an advanced docking method, Induced Fit Docking (IFD), was used for further investigation [24]. These approaches allowed for analyzing the binding mechanism, gaining insights into the ligand entrance pathway, and elucidating the dynamic behavior of the ligand within the binding cavity, providing a dynamic point of view for the definition of the pharmacophore features.…”

Investigating the Inhibition of FTSJ1, a Tryptophan tRNA-Specific 2′-O-Methyltransferase by NV TRIDs, as a Mechanism of Readthrough in Nonsense Mutated CFTR

“…Three MD simulation replicas of 100 ns each were carried out using a Desmond 6.5 [29] using the OPLS4 force field for each complex FTSJ1/DAP, FTSJ1/SAM, and FTSJ1/NV848, NV914, and NV930. The system setup and the simulation options are the same as reported in previous manuscripts [30][31][32][33]. Initial velocities were determined with random seeds.…”

Investigating the Inhibition of FTSJ1 a Tryptophan tRNA-Specific 2′-O-Methyltransferase by NV TRIDs, as a Mechanism of Readthrough in Nonsense Mutated CFTR

“…Three MD simulation replicas of 100 ns each were carried out using a Desmond 6.5 [24] using the OPLS4 force field for each complex FTSJ1/DAP, FTSJ1/SAM, and FTSJ1/NV848, NV914, and NV930. The system setup and the simulation options are the same as reported in previous manuscripts [25][26][27][28]. Initial velocities were determined with random seeds.…”