This work reports
the development of highly potent and selective
inhibitors of the β5c catalytic activity of human constitutive
proteasomes. The work describes the design principles, large hydrophobic
P3 residue and small hydrophobic P1 residue, that led to the synthesis
of a panel of peptide epoxyketones; their evaluation and the selection
of the most promising compounds for further analyses. Structure–activity
relationships detail how in a logical order the β1c/i, β2c/i,
and β5i activities became resistant to inhibition as compounds
were diversified stepwise. The most effective compounds were obtained
as a mixture of <i>cis</i>- and <i>trans</i>-biscyclohexyl
isomers, and enantioselective synthesis resolved this issue. Studies
on yeast proteasome structures complexed with some of the compounds
provide a rationale for the potency and specificity. Substitution
of the N-terminus in the most potent compound for a more soluble equivalent
led to a cell-permeable molecule that selectively and efficiently
blocks β5c in cells expressing both constitutive proteasomes
and immunoproteasomes