Bivalency as a principle for proteasome inhibition

The proteasome, a multicatalytic protease, is known to degrade unfolded pol ypeptides with low specificity in substrate selection and cleavage pattern. This lack of well-defined substrate specificities makes the design of pept ide-based highly selective inhibitors extremely difficult. However, the x-r ay structure of the proteasome from Saccharomyces cerevisiae reveals a uniq ue topography of the six active sites in the inner chamber of the protease, which lends itself to strategies of specific multivalent inhibition. Struc ture-derived active site separation distances were exploited for the design of homo- and heterobivalent inhibitors based on peptide aldehyde head grou ps and polyoxyethylene as spacer element. Polyoxyethylene was chosen as a f lexible, linear, and proteasome-resistant polymer to mimic unfolded polypep tide chains and thus to allow access to the proteolytic chamber. Spacer len gths were selected that satisfy the inter- and intra-ring distances for occ upation of the active sites from the S subsites, X-ray analysis of the prot easome/bivalent inhibitor complexes confirmed independent recognition and b inding of the inhibitory head groups, Their inhibitory potencies, which are by 2 orders of magnitude enhanced, compared with pegylated monovalent inhi bitors, result from the bivalent binding. The principle of multivaleney, ub iquitous in nature, has been successfully applied in the past to enhance af finity and avidity of ligands in molecular recognition processes. The prese nt study confirms its utility also for inhibition of multicatalytic proteas e complexes.