Synthetic peptide-based activators of the proteasome

The development of small molecule peptide-based activators of the 20S proteasome or multicatalytic proteinase complex was initiated. The enhancement of antigen presentation by transfection of the protein activator PA28alpha into a mouse fibroblast cell line [10] supports the potential use of small molecule activators in stimulating the immune response. Four classes of peptide-based activators were synthesized, i.e. peptidyl alcohols, esters, p-nitroanilides and nitriles. These compounds markedly and reversibly stimulated the hydrolysis of suc-LLVY-MCA, Z-LLE-NA and Z-GPALG-p-aminobenzoate as well as hydrolysis of the decapeptide angiotensin I. Stimulation was due to a decrease in the Km and increase in the Vmax of the substrate. In general, the EC50 for activation ranged from 50-150 mM and maximal stimulation varied from 3 to 15 fold depending on the activity measured. Z-IE(Ot-Bu)AL-p-nitroanilide, a proteasome substrate, markedly stimulated the hydrolysis of Z-GPALG-pAB by binding to a saturable high affinity site distinct from its binding site as substrate. Since all effective activators contain hydrophobic groups in positions P1-P5, low aqueous solubility is a limitation of these compounds. Competition experiments suggest that these activators bind to the same site as PA28.