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 β5i and β1i sites of the immunoproteasome over the β5c and β1c sites of the constitutive proteasome with nearly 20‐fold selective inhibition of β1i over the homologous β1c. Molecular modelling attributes the β1i over β1c selectivity to the small hydrophobic S1 pocket of β1i and β5i over β5c 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.

Proteasomes play an important role in protein degradation and regulation of many cellular pathways by maintaining protein balance. Inhibitors of the proteasome disrupt this balance affecting proteins that are key...