Identification of a new class of proteasome inhibitors based on a naphthyl-azotricyclic-urea-phenyl scaffold.

Proteasomes play an important role in protein degradation and regulation of many cellular pathways by maintaining protein balance. Inhibitors of proteasomes disrupt this balance affecting proteins that are key in malignancies and as such have found applications in the treatment of multiple myeloma and mantle cell lymphoma. However, resistance mechanisms have been reported for these proteasome inhibitors including mutations at the ß5 site which necessitates the constant development of new inhibitors. In this work, we report the identification of a new class of proteasome inhibitors, polycyclic molecules bearing a naphthyl-azotricyclic-urea-phenyl scaffold, from screening of the ZINC library of natural products. The most potent of these compounds showed evidence of dose dependency through proteasome assays with IC50 values in the low micromolar range, and kinetic analysis revealed competitive binding at the ß5c site with an estimated inhibition constant, K i, of 1.15 µM. Inhibition was also shown for the ß5i site of the immunoproteasome at levels similar to those of the constitutive proteasome. Structure-activity relationship studies identified the naphthyl substituent to be crucial for activity and this was attributed to enhanced hydrophobic interactions within ß5c. Further to this, halogen substitution within the naphthyl ring enhanced the activity and allowed for π-π interactions with Y169 in ß5c and Y130 and F124 in ß5i. The combined data highlight the importance of hydrophobic and halogen interactions in ß5 binding and assist in the design of next generation inhibitors of proteasomes.

Argyrin B, a non-competitive inhibitor of the human immunoproteasome exhibiting preference for ß1i.

Inhibitors of the proteasome have found broad therapeutic applications; however, they show severe toxicity due to the abundance of proteasomes in healthy cells. In contrast, inhibitors of the immunoproteasome, which is upregulated during disease states, are less toxic and have increased therapeutic potential including against autoimmune disorders. In this project, we report argyrin B, a natural product cyclic peptide to be a reversible, non-competitive inhibitor of the immunoproteasome. Argyrin B showed selective inhibition of the ß5i and ß1i sites of the immunoproteasome over the ß5c and ß1c sites of the constitutive proteasome with nearly 20-fold selective inhibition of ß1i over the homologous ß1c. Molecular modelling attributes the ß1i over ß1c selectivity to the small hydrophobic S1 pocket of ß1i and ß5i over ß5c to site-specific amino acid variations that enable additional bonding interactions and stabilization of the binding conformation. These findings facilitate the design of immunoproteasome selective and reversible inhibitors that may have a greater therapeutic potential and lower toxicity.