BACKGROUND: The 26S
proteasome is responsible for most
cytosolic proteolysis, and is an important
protease in
major histocompatibility complex class I-mediated
antigen presentation. Constitutively expressed
proteasomes from mammalian sources possess three distinct catalytically
active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible
immunoproteasome by a different set of
catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic
peptide substrates, activities of the
proteasome have been assigned to individual
subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl
peptide hydrolyzing' (beta1). Studies with 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 activities and
subunits. RESULTS: A new class of
proteasome inhibitors, considerably extended in comparison with the commonly used
fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch
resin allowed the generation of the target compounds using a
solid phase protocol. Evaluation of the new compounds revealed a set of highly potent
proteasome inhibitors that target all individual active
subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)(3)-(leucinyl)(3)-vinyl-(
methyl)-
sulfone (AdaAhx(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 selectivity 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 normally assumed based on the use of small peptide-based
substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in
living cells than their shorter
peptide vinyl sulfone counterparts.
