Identification of Novel Inhibitors That Specifically Target the Immunoproteasome, and Selectively Induce Apoptosis in Multiple Myeloma and Other Immunoproteasome-Expressing Model Systems.

Abstract

Inhibition of the proteasome, a multi-catalytic proteinase complex responsible for regulated intracellular proteolysis, with bortezomib (VELCADE®) is a rational strategy against relapsed/refractory multiple myeloma, and studies are ongoing to further define the efficacy of this agent in other settings. However, bortezomib is associated with several toxicities, such as peripheral neuropathy, which can limit the ability of patients to receive maximal doses, possibly compromising anti-tumor efficacy. Novel proteasome inhibitors with an improved toxicity profile and improved anti-tumor activity are therefore needed. Most cells contain the constitutive version of the proteasome with three catalytic subunits, designated X, Y, and Z. Exposure of these cells to cytokines, such as γ-interferon, causes replacement of these subunits with three others, referred to as low molecular weight proteins (LMP)−2, −7, and −10. This LMP-containing proteasome, known as the immunoproteasome because it plays a role in generating antigens as part of the immune response, is also the major proteasome isoform in hematopoietic-derived cells even in the absence of exogenous cytokines. Using purified preparations of the XYZ and LMP proteasomes, we identified a panel of peptidyl-aldehydes that preferentially inhibit the immunoproteasome with relative sparing of the constitutive isoform. Among these, the most immunoproteasome specific inhibitors (IPSI) included compounds 001 and 006. IPSI-001 demonstrated a Ki of 1.03 μM against the chymotryptic activity of the LMP proteasome but only a 105 μM Ki against the XYZ proteasome, while IPSI-006 demonstrated Kis of 8.4 μM and 460 μM, respectively. In contrast, other commonly used peptidyl aldehydes, such as MG-132 and the aldehyde version of bortezomib, showed no ability to discriminate between these two proteasomes. Using radiolabeled 3,4-dichloroisocoumarin as a probe, IPSIs were shown to bind only to LMP-2 of the immunoproteasome, but not to any subunits of the constitutive proteasome in vitro. Studies of cell lines expressing immunoproteasome subunits showed IPSI-001 inhibited the chymotryptic activity of the LMP proteasome, with relative sparing of this activity in XYZ proteasome-expressing cell lines. IPSI-001 was also able to induce apoptosis in LMP-proteasome-expressing cells, including IM-9 lymphoblastoid cells and ANBL-6 interleukin-(IL)-6-dependent and RPMI-8226 IL-6-independent multiple myeloma cells, while relatively sparing XYZ-proteasome-expressing cell lines. Bortezomib, in contrast, induced apoptosis indiscriminately in both LMP- and XYZ-proteasome expressing cells. Taken together, these studies suggest that immunoproteasome specific inhibitors represent a novel class of drugs with activity against hematologic malignancies that may have less toxicity by virtue of their ability to spare the proteasome in most other tissues in the body, including in neural tissues. These properties could lead to an improved therapeutic index and anti-tumor efficacy, providing a rationale for development of these agents into clinically relevant drug candidates.