Identification of Peptide Epoxy-Ketone Proteasome Inhibitors with Differential Active Site Selectivity.

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The proteasome is a multi-subunit structure responsible for degrading the majority of proteins in the cell. The three catalytic subunits in the 20S core of the proteasome are defined by their preference for amino acid side chains at the P1 position: the beta5 subunit (chymotrypsin-like), the beta2 subunit (trypsin-like), and the beta1 subunit (caspase-like) hydrolyze substrates after hydrophobic, basic, and acidic residues, respectively. Bortezomib (Velcade®), currently an approved drug for the treatment of multiple myeloma, is a potent inhibitor of the beta5 subunit but also has activity against the beta1 subunit (9-fold less active) and the beta2 subunit (250-fold less active). PR-171, a peptide epoxy-ketone proteasome inhibitor currently in Phase I clinical trials in multiple myeloma and NHL patients, has a distinct profile of inhibition. Similar to bortezomib, PR-171 is most potent against the beta5 subunit, but differs in its activity against the beta2 and beta1 subunits (45-fold less active and 140-fold less active, respectively). Although both bortezomib and PR-171 are cytotoxic to a variety of tumor cell types, the connection between inhibition of specific proteasomal subunits and cellular consequences, including induction of apoptosis, is currently unclear. To investigate this relationship, we synthesized a series of PR-171 analogs and profiled them in our proteasome-specific active site ELISA binding assay to identify compounds with differential binding profiles for the three catalytic subunits. Using this approach, we have identified compounds that bind all three catalytic sites with approximately equivalent activity as well as compounds that have increased specificity for the beta5 subunit. We have extended this approach to search for compounds with differential binding profiles for the immunoproteasome catalytic subunits. The immunoproteasome is the predominant form of the proteasome expressed in cells of hematopoetic origin and in other cell types after interferon-g stimulation. In the 20S core of the immunoproteasome the beta5, beta2 and beta1 subunits of the “constitutive” proteasome are replaced by the homologous subunits LMP7, MECL1, and LMP2. We have successfully identified inhibitors with distinct active site profiles for the immunoproteasome as well as the constitutive proteasome. The impact of differential active site inhibition on tumor cell viability is under investigation in a variety of solid and hematological tumor cell lines expressing the constitutive proteasome or the immunoproteasome.