Bivalency as a principle for proteasome inhibition

Abstract

The proteasome, a multicatalytic protease, is known to degrade unfolded polypeptides with low specificity in substrate selection and cleavage pattern. This lack of well-defined substrate specificities makes the design of peptide-based highly selective inhibitors extremely difficult. However, the x-ray structure of the proteasome from Saccharomyces cerevisiae reveals a unique topography of the six active sites in the inner chamber of the protease, which lends itself to strategies of specific multivalent inhibition. Structure-derived active site separation distances were exploited for the design of homo- and heterobivalent inhibitors based on peptide aldehyde head groups and polyoxyethylene as spacer element. Polyoxyethylene was chosen as a flexible, linear, and proteasome-resistant polymer to mimic unfolded polypeptide chains and thus to allow access to the proteolytic chamber. Spacer lengths were selected that satisfy the inter- and intra-ring distances for occupation of the active sites from the S subsites. X-ray analysis of the proteasome/bivalent inhibitor complexes confirmed independent recognition and binding of the inhibitory head groups. Their inhibitory potencies, which are by 2 orders of magnitude enhanced, compared with pegylated monovalent inhibitors, result from the bivalent binding. The principle of multivalency, ubiquitous in nature, has been successfully applied in the past to enhance affinity and avidity of ligands in molecular recognition processes. The present study confirms its utility also for inhibition of multicatalytic protease complexes.

Figure 1

Schematic representation of the central β-rings of the yeast proteasome with selected distances between active sites as derived from the x-ray structure (13).

Scheme 1

Synthesis of Ac-Arg-Val-Arg-H. (a) i) Z-Arg(Adoc)₂-OH, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)/1-hydroxybenzotriazole/diisopropylethylamine (DIEA), dimethylformamide (DMF); ii) Pd/C (10%), EtOH. (b) i) Ac₂O, DIEA, DMF; ii) 95% trifluoroacetic acid.

Scheme 2

Synthesis of the PEG/peptide aldehyde conjugates. (a) 1 equ. H-Leu-Leu-Nle-Sc, TBTU/1-hydroxybenzotriazole (HOBt)/DIEA, DMF. (b) 1 equ. H-Arg(Adoc)₂-Val-Arg(Adoc)₂-diethyl acetal, TBTU/HOBt/DIEA, DMF. (c) 2 equ. H-Leu-Leu-Nle-Sc, TBTU/HOBt/DIEA, DMF. (d) AcOH, 37% HCHO, MeOH. (e) 95% trifluoroacetic acid.

Figure 2

Part of the x-ray structure of the yeast 20S proteasome/OHC-CO-LGPGGLLnL-H (3) adduct. Protein subunits are marked with different colors: blue for β1, red for β2, yellow for β7, and the inhibitor is shown in white (drawn with main; ref. 27).

Figure 3

Part of the electron density map with 2F_o-F_c coefficients after 2-fold averaging of the yeast 20S proteasome/(PEG)_19–25-[NH-CO-(CH₂)₂-CO-Leu-Leu-Nle-H]₂ (8) complex. The electron density map was calculated with phases of the free enzyme structure and contoured around the inhibitor molecule at 2ó cutoff. The carbon atoms are marked with different colors: green for β5, red for β6, and white for the inhibitor.