6HWE

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the ??1c, ??1i, ??5c, and ??5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting ??2c or ??2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the ??2c- and ??2i-selective compounds LU-002c (4; IC 50 ??2c: 8 nM, IC 50 ??2i/??2c: 40-fold) and LU-002i (5; IC 50 ??2i: 220 nM, IC 50 ??2c/??2i: 45-fold), respectively. Co-crystal structures with ??2 humanized yeast proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of ??2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.