RESUMEN
Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the ß1c, ß1i, ß5c, and ß5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting ß2c or ß2i 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 ß2c- and ß2i-selective compounds LU-002c (4; IC50 ß2c: 8 nM, IC50 ß2i/ß2c: 40-fold) and LU-002i (5; IC50 ß2i: 220 nM, IC50 ß2c/ß2i: 45-fold), respectively. Co-crystal structures with ß2 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 ß2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.