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

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 beta 1i/beta 5i 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 beta 1i subunit and a pharmacophore/docking approach onto the beta 5i subunit. The computational studies were first followed by in vitro enzymatic assays at 100 mu M. 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 beta 1i and/or beta 5i subunit. Compound 3 is the most active on the beta 1i subunit with K-i = 11.84 +/- 1.63 mu M, and compound 17 showed K-i = 12.50 +/- 0.77 mu M on the beta 5i subunit. Compound 2 showed inhibitory activity on both subunits (K-i = 12.53 +/- 0.18 and K-i = 31.95 +/- 0.81 on the beta 1i subunit and beta 5i subunit, respectively). The induced fit docking analysis revealed interactions with Thr1 and Phe31 of beta 1i subunit and that represent new key residues as reported in our previous work. Onto beta 5i 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 beta 1i/beta 5i dual inhibitors of the immunoproteasome.