Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits

Background: The 26S proteasome is responsible for most cytosolic proteolysi s. and is an important protease in major histocompatibility complex class I -mediated antigen presentation. Constitutively expressed proteasomes from m ammalian sources possess three distinct catalytically active species, beta1 , beta2 and beta5, which are replaced in the gamma -interferon-inducible im munoproteasome by a different set of catalytic subunits, beta li, beta 2i a nd beta 5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to indi vidual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-lik e' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies wit h protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual act ivities and subunits. Results: A new class of proteasome inhibitors, considerably extended in com parison with the commonly used fluorescent substrates and peptide-based inh ibitors, has been prepared. Application of the safety catch resin allowed t he generation of the target compounds using a solid phase protocol, Evaluat ion of the new compounds revealed a set of highly potent proteasome inhibit ors that target all individual active subunits with comparable affinity, un like the other inhibitors described to date. Modification of the most activ e compound, adamantane-acetyl(6-aminohexanoyl)(3)-(leucinyl)(3)-vinyl-(meth yl)-sulfone (Ada-Ahx(3)L(3)VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels. Conclusions: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors . Such extensions greatly enhance inhibition and largely obliterate selecti vity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is norm ally assumed based on the use of small peptide-based substrates and inhibit ors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide viny l sulfone counterparts. (C) 2001 Elsevier Science Ltd. All rights reserved.