Immunoproteasome-specific Inhibitors Research Articles

Targeting Immunoproteasome in Polarized Macrophages Ameliorates Experimental Emphysema Via Activating NRF1/2-P62 Axis and Suppressing IRF4 Transcription.

Chronic obstructive pulmonary disease (COPD) stands as the prevailing chronic airway ailment, characterized by chronic bronchitis and emphysema. Current medications fall short in treatment of these diseases, underscoring the urgent need for effective therapy. Prior research indicated immunoproteasome inhibition alleviated various inflammatory diseases by modulating immune cell functions. However, its therapeutic potential in COPD remains largely unexplored. Here, an elevated expression of immunoproteasome subunits LMP2 and LMP7 in the macrophages isolated from mouse with LPS/Elastase-induced emphysema and polarized macrophages in vitro is observed. Subsequently, intranasal administration of the immunoproteasome-specific inhibitor ONX-0914 significantly mitigated COPD-associated airway inflammation and improved lung function in mice by suppressing macrophage polarization. Additionally, ONX-0914 capsulated in PLGA nanoparticles exhibited more pronounced therapeutic effect on COPD than naked ONX-0914 by targeting immunoproteasome in polarized macrophages. Mechanistically, ONX-0914 activated autophagy and endoplasmic reticulum (ER) stress are not attribute to the ONX-0914 mediated suppression of macrophage polarization. Intriguingly, ONX-0914 inhibited M1 polarization through the nuclear factor erythroid 2-related factor-1 (NRF1) and NRF2-P62 axis, while the suppression of M2 polarization is regulated by inhibiting the transcription of interferon regulatory factor 4 (IRF4). In summary, the findings suggest that targeting immunoproteasome in macrophages holds promise as a therapeutic strategy for COPD.

Genomic and Biochemical Proteasomal Adaptations in Carfilzomib-Resistant Plasma Cells That Limit HLA-Desensitization in Transplantation Candidates

Bone marrow plasma cells (BMPCs) are the source of long-term humoral immunity including HLA antibodies, which promote organ rejection. End-stage renal disease (ESRD) affects more than 660,000 patients per year in the U.S. and is associated with significant morbidity and mortality. Renal transplantation remains the optimal treatment for a substantial proportion of ESRD patients and dramatically improves patient overall survival and quality-of-life. However, preformed human leukocyte antigen (HLA)-specific antibodies are associated with reduced transplantation rates, graft loss and poorer outcomes. Proteasome inhibitor (PI) therapies that target BMPCs to reduce HLA-antibody levels represent an effective means of enhancing transplantation outcomes but are limited by resistance mechanisms. Single antigen bead assays determined that the spectrum of antibody production by HLA-sensitized patient (BMPCs) was remarkably similar to that detected in matching serum samples, supporting the role of BMPCs as the primary source of circulating HLA antibodies. Desensitization therapy with the irreversible proteasome inhibitor carfilzomib reduced PCs in patient BM by ~70% and was associated with a profound reduction in HLA antibodies. We then used bulk RNA-seq transcriptomics to explore potential drug resistance mechanisms within carfilzomib-resistant BMPCs. Network-based pathway analysis revealed that genes encoding the immunoproteasome, a specialized proteasome variant with unique catalytic subunits, and the proteasome activator PA28 were upregulated in BMPCs that survived carfilzomib therapy. Native gel electrophoresis and in vitro functional studies confirmed that BMPCs isolated after carfilzomib therapy contained greater levels of immunoproteasomes as well as increased immunoproteasome-specific peptide-hydrolyzing activity relative to BMPCs from healthy donors or untreated HLA-sensitized patients. Supervised hierarchical clustering analysis (Fig. 1A) and western blotting (Fig. 1B) indicated that PSMB8, which encodes an immunoproteasome catalytic subunit, and PSME1 and PSME2 which encode the proteasome PA28 activator, were significantly upregulated in BMPCs isolated from patients following carfilzomib therapy. Assays then demonstrated that BMPCs that survived carfilzomib therapy were resistant to the FDA-approved PIs bortezomib, carfilzomib and ixazomib but were sensitive to an immunoproteasome-specific inhibitor KZR-616. Thus, we have uncovered proteasomal adaptations in BMPCs as a resistance mechanism underlying carfilzomib therapy and suggest novel strategies that could improve desensitization regimens and reduce antibody-mediated rejection in organ transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunoproteasome Activity and Content Determine Hematopoietic Cell Sensitivity to ONX-0914 and to the Infection of Cells with Lentiviruses.

Proteasomes are intracellular structures responsible for protein degradation. The 20S proteasome is a core catalytic element of the proteasome assembly. Variations of catalytic subunits generate different forms of 20S proteasomes including immunoproteasomes (iPs), which are present mostly in the immune cells. Certain cells of the immune system are primary targets of retroviruses. It has been shown that several viral proteins directly affect proteasome functionality, while inhibition of proteasome activity with broad specificity proteasome inhibitors stimulates viral transduction. Here we specifically addressed the role of the immunoproteasomes during early stages of viral transduction and investigated the effects of specific immunoproteasome inhibition and activation prior to infection using a panel of cell lines. Inhibition of iPs in hematopoietic cells with immunoproteasome-specific inhibitor ONX-0914 resulted in increased infection by VSV-G pseudotyped lentiviruses. Moreover, a tendency for increased infection of cloned cells with endogenously decreased proteasome activity was revealed. Conversely, activation of iPs by IFN-γ markedly reduced the viral infectivity, which was rescued upon simultaneous immunoproteasome inhibition. Our results indicate that immunoproteasome activity might be determinative for the cellular antiretroviral resistance at least for the cells with high iP content. Finally, therapeutic application of immunoproteasome inhibitors might promote retroviral infection of cells in vivo.

Expression of the immunoproteasome subunit β5i in non-small cell lung carcinomas

AimThe immunoproteasome is a specific proteasome isoform whose proteolytic activity enhances the generation of antigenic peptides to be presented by major histocompatibility complex class I molecules to CD8+ T cells....

ONX 0914 Lacks Selectivity for the Cardiac Immunoproteasome in CoxsackievirusB3 Myocarditis of NMRI Mice and Promotes Virus-Mediated Tissue Damage.

Inhibition of proteasome function by small molecules is highly efficacious in cancer treatment. Other than non-selective proteasome inhibitors, immunoproteasome-specific inhibitors allow for specific targeting of the proteasome in immune cells and the profound anti-inflammatory potential of such compounds revealed implications for inflammatory scenarios. For pathogen-triggered inflammation, however, the efficacy of immunoproteasome inhibitors is controversial. In this study, we investigated how ONX 0914, an immunoproteasome-selective inhibitor, influences CoxsackievirusB3 infection in NMRI mice, resulting in the development of acute and chronic myocarditis, which is accompanied by formation of the immunoproteasome in heart tissue. In groups in which ONX 0914 treatment was initiated once viral cytotoxicity had emerged in the heart, ONX 0914 had no anti-inflammatory effect in the acute or chronic stages. ONX 0914 treatment initiated prior to infection, however, increased viral cytotoxicity in cardiomyocytes, promoting infiltration of myeloid immune cells into the heart. At this stage, ONX 0914 completely inhibited the β5 subunit of the standard cardiac proteasome and less efficiently blocked its immunoproteasome counterpart LMP7. In conclusion, ONX 0914 unselectively perturbs cardiac proteasome function in viral myocarditis of NMRI mice, reduces the capacity of the host to control the viral burden and promotes cardiac inflammation.

Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells.

Donor-specific antibodies (DSAs) have a deleterious effect on allografts and remain a major immunologic barrier in transplantation. Current therapies to eliminate DSAs are ineffective in highly HLA-sensitized patients. Proteasome inhibitors have been employed as a strategy to target bone marrow plasma cells (BMPCs), the source of long-term antibody production; however, their efficacy has been limited by poorly defined drug-resistance mechanisms. Here, we performed transcriptomic profiling of CD138+ BMPCs that survived in vivo desensitization therapy with the proteasome inhibitor carfilzomib to identify mechanisms of drug resistance. The results revealed a genomic signature that included increased expression of the immunoproteasome, a highly specialized proteasomal variant. Western blotting and functional studies demonstrated that catalytically active immunoproteasomes and the immunoproteasome activator PA28 were upregulated in carfilzomib-resistant BMPCs. Carfilzomib-resistant BMPCs displayed reduced sensitivity to the proteasome inhibitors carfilzomib, bortezomib, and ixazomib, but enhanced sensitivity to an immunoproteasome-specific inhibitor ONX-0914. Finally, in vitro carfilzomib treatment of BMPCs from HLA-sensitized patients increased levels of the immunoproteasome β5i (PSMB8) catalytic subunit suggesting that carfilzomib therapy directly induces an adaptive immunoproteasome response. Taken together, our results indicate that carfilzomib induces structural changes in proteasomes and immunoproteasome formation.

Inhibitors of the Immunoproteasome: Current Status and Future Directions

The ubiquitin-proteasome system (UPS) plays a vital role in maintaining protein homeostasis and regulating numerous cellular processes. The proteasome, a multi-protease complex, is the key component of the UPS and has been validated as a therapeutic target by the FDA's approval of bortezomib and carfilzomib. These proteasome inhibitor drugs have substantially improved outcomes in patients with hematological malignancies and are currently being investigated for other types of cancer as well as several other diseases. These approved proteasome inhibitors target the catalytic activity of both the constitutive proteasome and the immunoproteasome indiscriminately, and their inhibitory effects on the constitutive proteasome in normal cells are believed to contribute to unwanted side effects. In addition, selective immunoproteasome inhibition has been proposed to have unique effects on other diseases, including those involving aberrant immune function. Initially recognized for its role in the adaptive immune response, the immunoproteasome is often upregulated in disease states such as inflammatory diseases and cancer, suggesting functions beyond antigen presentation. In an effort to explore the immunoproteasome as a potential therapeutic target in these diseases, the development of immunoproteasome-specific inhibitors has become the focus of recent studies. Owing to considerable efforts by both academic and industry groups, immunoproteasome-selective inhibitors have now been identified and tested against several disease models. These inhibitors also provide a valuable set of chemical tools for investigating the biological function of the immunoproteasome. In this review, we will focus on the recent efforts towards the development of immunoproteasome-selective inhibitors.

Cytokine-induced oxidative stress in cardiac inflammation and heart failure—how the ubiquitin proteasome system targets this vicious cycle

The ubiquitin proteasome system (UPS) is critical for the regulation of many intracellular processes necessary for cell function and survival. The absolute requirement of the UPS for the maintenance of protein homeostasis and thereby for the regulation of protein quality control is reflected by the fact that deviation of proteasome function from the norm was reported in cardiovascular pathologies. Inflammation is a major factor contributing to cardiac pathology. Herein, cytokines induce protein translation and the production of free radicals, thereby challenging the cellular protein equilibrium. Here, we discuss current knowledge on the mechanisms of UPS-functional adaptation in response to oxidative stress in cardiac inflammation. The increasing pool of oxidant-damaged degradation-prone proteins in cardiac pathology accounts for the need for enhanced protein turnover by the UPS. This process is accomplished by an up-regulation of the ubiquitylation machinery and the induction of immunoproteasomes. Thereby, the inflamed heart muscle is cleared from accumulating misfolded proteins. Current advances on immunoproteasome-specific inhibitors in this field question the impact of the proteasome as a therapeutic target in heart failure.

The Immunoproteasome as a Target in Hematologic Malignancies

Suppression of proteasome function with the first-in-class small molecule inhibitor bortezomib is a rational therapeutic strategy against several hematologic malignancies, including multiple myeloma and mantle cell lymphoma. Second-generation inhibitors such as carfilzomib, ixazomib, and marizomib that, like bortezomib, target both the constitutive proteasome and the immunoproteasome, are also in clinical trials and showing encouraging activity. While the efficacy of these agents is well documented, toxicities associated with their use, such as peripheral neuropathy and gastrointestinal effects, can necessitate dose reductions or even discontinuations, possibly hampering their anti-neoplastic effects. These findings suggested that it could be possible to improve the therapeutic index of this class of drugs by specifically targeting only the immunoproteasome. Since the immunoproteasome is a unique target found in lymphoid-derived cells, immunoproteasome-specific inhibitors (IPSIs) could preserve efficacy while reducing treatment-emergent toxicities since they would spare other tissues with little to no immunoproteasome expression. This review discusses the current state of development of IPSIs, and the potential of using such agents for the treatment of hematologic malignancies.

Crystal structures to guide the design of immunoproteasome-specific inhibitors

Crystal structures to guide the design of immunoproteasome-specific inhibitors

Beneficial effect of novel proteasome inhibitors in murine lupus via dual inhibition of type I interferon and autoantibody‐secreting cells

To investigate the hypothesis that proteasome inhibition may have potential in the treatment of SLE, by targeting plasmacytoid dendritic cells (PDCs) and plasma cells, both of which are critical in disease pathogenesis. Lupus-prone mice were treated with the nonselective proteasome inhibitors carfilzomib and bortezomib, the immunoproteasome inhibitor ONX 0914, or vehicle control. Tissue was harvested and analyzed by flow cytometry using standard markers. Nephritis was monitored by evaluation for proteinuria and by histologic analysis of kidneys. Serum anti-double-stranded DNA (anti-dsDNA) levels were measured by enzyme-linked immunosorbent assay (ELISA), and total IgG and dsDNA antibody-secreting cells (ASCs) by enzyme-linked immunospot assay. Human peripheral blood mononuclear cells or mouse bone marrow cells were incubated with Toll-like receptor (TLR) agonists and proteasome inhibitors, and interferon-α (IFNα) levels were measured by ELISA and flow cytometry. Early treatment of lupus-prone mice with the dual-targeting proteasome inhibitors carfilzomib or bortezomib or the immunoproteasome-specific inhibitor ONX 0914 prevented disease progression, and treatment of mice with established disease dramatically abrogated nephritis. Treatment had profound effects on plasma cells, with greater reductions in autoreactive than in total IgG ASCs, an effect that became more pronounced with prolonged treatment and was reflected in decreasing serum autoantibody levels. Notably, proteasome inhibition efficiently suppressed production of IFNα by TLR-activated PDCs in vitro and in vivo, an effect mediated by inhibition of both PDC survival and PDC function. Inhibition of the immunoproteasome is equally efficacious as dual targeting agents in preventing lupus disease progression by targeting 2 critical pathways in disease pathogenesis, type I IFN activation and autoantibody production by plasma cells.

The Immunoproteasome: An Emerging Therapeutic Target

The proteasome, a large multisubunit protease complex, has been extensively investigated over the years, greatly enhancing our understanding of critical roles that the proteasome plays in cells. The FDA approval of bortezomib for the treatment of multiple myeloma and mantle cell lymphoma has validated the proteasome as an anticancer target. However, the undesirable toxicities of these agents limit their broad utility. The immunoproteasome, an alternative form of the constitutive proteasome, has recently been explored as a therapeutic target. While the immunoproteasome, normally expressed in cells of hematopoietic origin, has been shown to be associated with various types of cancer and inflammatory diseases, its multifaceted function is not fully understood due to the lack of appropriate molecular probes. In this review, recent advances in the immunoproteasome field are covered, including potential implications in disease states. In particular, recent developments in immunoproteasome-specific inhibitors are emphasized.

Second Generation Proteasome Inhibitors: Carfilzomib and Immunoproteasome-Specific Inhibitors (IPSIs)

The ubiquitin-proteasome pathway (UPP) is an attractive chemotherapeutic target due to its intrinsically stringent regulation of cell cycle, pro-survival, and anti-apoptotic regulators that disproportionately favor survival and proliferation in malignant cells. A reversible first-in-class proteasome inhibitor, bortezomib, is Food and Drug Administration approved for multiple myeloma and relapsed/refractory mantle cell lymphoma and has proven to be extremely effective, both as a single agent and in combination. An irreversible second generation proteasome inhibitor, carfilzomib, has shown preclinical effectiveness against hematological and solid malignancies both in vitro and in vivo. Carfilzomib, a peptidyl-epoxyketone functions similarly to bortezomib through primary inhibition of chymotrypsin-like (ChT-L) activity at the b5 subunits of the core 20S proteasome. Carfilzomib is also currently achieving successful response rates within the clinical setting. In addition to conventional proteasome inhibitors, a novel approach may be to specifically target the hematological-specific immunoproteasome, thereby increasing overall effectiveness and reducing negative off-target effects. The immunoproteasome-specific inhibitor, IPSI-001, was shown to have inhibitory preference over the constitutive proteasome, and display enhanced efficiency of apoptotic induction of tumor cells from a hematologic origin. Herein, we discuss the preclinical and clinical development of carfilzomib and explore the potential of immunoproteasome-specific inhibitors, like IPSI-001, as a rational approach to exclusively target hematological malignancies.

Prevention of Experimental Colitis by a Selective Inhibitor of the Immunoproteasome

The proteasome, a multicatalytic protease, is responsible for the degradation of intracellular proteins. Stimulation of cells with inflammatory cytokines, such as IFN-gamma, leads to the replacement of the constitutive catalytic proteasome subunits by the inducible subunits low molecular mass polypeptide (LMP)2 (beta1i), multicatalytic endopeptidase complex-like-1 (beta2i), and LMP7 (beta5i), which are required for the production of certain MHC class I-restricted T cell epitopes. In this study, we investigated the effect of immunoproteasomes on the development of dextran sulfate sodium-induced colitis. Colitis induction in LMP2-, LMP7-, and multicatalytic endopeptidase complex-like-1-deficient mice caused reduced weight loss compared with wild-type mice. Although colon lengths were shortened in wild-type mice, no reduction was observed in immunoproteasome-deficient mice. In accordance with this, proinflammatory cytokines, such as TNF-alpha and IL-1beta, were not upregulated in these mice. Blockage of LMP7 by a novel LMP7-selective inhibitor (PR-957) strongly reduced pathological symptoms of dextran sulfate sodium-induced colitis. Production of numerous cytokines in PR-957-treated mice was suppressed, resulting in reduced inflammation and tissue destruction. Taken together, these results demonstrate that an immunoproteasome-specific inhibitor can be used to attenuate autoimmune diseases like colitis.

Targeted inhibition of the immunoproteasome is a potent strategy against models of multiple myeloma that overcomes resistance to conventional drugs and nonspecific proteasome inhibitors

AbstractProteasome inhibition is a validated strategy for therapy of multiple myeloma, but this disease remains challenging as relapses are common, and often associated with increasing chemoresistance. Moreover, nonspecific proteasome inhibitors such as bortezomib can induce peripheral neuropathy and other toxicities that may compromise the ability to deliver therapy at full doses, thereby decreasing efficacy. One novel approach may be to target the immunoproteasome, a proteasomal variant found predominantly in cells of hematopoietic origin that differs from the constitutive proteasome found in most other cell types. Using purified preparations of constitutive and immunoproteasomes, we screened a rationally designed series of peptidyl-aldehydes and identified several with relative specificity for the immunoproteasome. The most potent immunoproteasome-specific inhibitor, IPSI-001, preferentially targeted the β1i subunit of the immunoproteasome in vitro and in cellulo in a dose-dependent manner. This agent induced accumulation of ubiquitin-protein conjugates, proapoptotic proteins, and activated caspase-mediated apoptosis. IPSI-001 potently inhibited proliferation in myeloma patient samples and other hematologic malignancies. Importantly, IPSI-001 was able to overcome conventional and novel drug resistance, including resistance to bortezomib. These findings provide a rationale for the translation of IPSIs to the clinic, where they may provide antimyeloma activity with greater specificity and less toxicity than current inhibitors.

Development of Immunoproteasome-Specific Inhibitors as Novel Anti-Cancer Drugs.

Introduction: The proteasome is a large (~2.5 MDa), ATP-dependent, intracellular protease responsible for degrading ubiquitinated proteins as part of the ubiquitin-proteasome pathway. The immunoproteasome is a unique proteasomal variant with distinct catalytic subunits termed low molecular mass proteins that functions predominately in cells derived from hematopoietic precursors, and differs from the constitutive proteasome found in most other cells. Bortezomib (VELCADE®; Millennium Pharmaceuticals, Inc.) is a first-in-class proteasome inhibitor, which is approved for treatment of multiple myeloma patients who have received at least one prior therapy. While the overall safety profile of bortezomib is manageable and predictable, some toxicities, such as peripheral neuropathy, associated with bortezomib treatment can be dose-limiting. These toxicities may be due to the inhibition of all isoforms of the proteasome. Development of immunoproteasome-specific inhibitors (IPSIs) would allow for targeted therapy against cancers arising from hematologic origins, thereby sparing normal tissues, such as gastrointestinal and neurological tissues.Methods: We have identified several novel IPSIs, most notably IPSI-001, with selective activity against the immunoproteasome, which we therefore sought to characterize.Results: Expression of proteins associated with the immunoproteasome (low molecular mass protein-2; 11S Reg-α) was found primarily in cell lines of hematopoietic origin, while solid tumor cell lines exhibited expression of constitutive proteasome proteins (β5; 19S S6′). IPSI-001 exposure induced preferential inhibition of the chymotrypsin-like activity, the rate-limiting step of proteolysis, in hematologic cell lines over solid tumors. This inhibition was associated with an increase in ubiquitinated substrates, activation of c-Jun N-terminal kinase, and accumulation of Bax. IPSI-001 treatment led to preferential induction of apoptosis as evidenced by DNA fragmentation assays and cleavage of β-actin by caspase-3 into an apoptotic marker, fractin. Furthermore, IPSI-001 had potent chymotrypsin-like inhibitory activity in patient samples of chronic lymphocytic leukemia and acute myeloid leukemia. A dose-dependent decrease in proliferation was observed in additional patient samples of acute myeloid leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma treated with IPSI-001. Also, IPSI-001 exposure induced apoptosis in multiple myeloma and chronic lymphocytic leukemia patient samples. In an effort to increase the efficacy of IPSIs, a series of boronic acid analogs were made of several IPSIs. Conversion into the boronate analogs increased the potency by up to 1000-fold against the chymotrypsin-like activity of the immunoproteasome in vitro and in cellulo. Dose-dependent inhibition of proliferation was observed in ANBL-6, KAS-6/1, and Ramos cell lines, which was associated with induction of apoptosis.Conclusions: Studies are ongoing to characterize the specificity and molecular effects associated with IPSI-boronic acid derivatives exposure in immunoproteasome- and constitutive proteasome-containing cell types.

Immunoproteasome-Selective Keto-Epoxide Inhibitors: Potential Therapeutic Agents for the Treatment of Hematological Disorders.

The clinical development of the dipeptide boronate bortezomib (Velcade™) has validated proteasome inhibition as a treatment for multiple myeloma and non-Hodgkin's lymphoma. In hematopoetically derived normal and malignant cells, the form of the proteasome expressed, termed the immunoproteasome, differs from that found in most tissues, referred to as the constitutive proteasome. Bortezomib inhibits the chymotrypsin-like activity of the immunoproteasome and the constitutive proteasome with equal potency and induces side effects such as peripheral neuropathy and gastrointestinal toxicity that may be related to proteasome inhibition in cells expressing the constitutive proteasome. This finding suggests that development of immunoproteasome-specific inhibitors would selectively target hematological malignances without affecting non-hematological cells. We have undertaken an effort to profile hematological tumor cell lines for immunoproteasome expression to determine which hematological cancers could be effectively targeted using an immunoproteasome selective inhibitor. We have observed that several cell lines derived from hematologic malignancies express predominantly the immunoproteasome while others express a mixture of both proteasome types. To develop selective inhibitors of the immunoproteasome, we generated analogs of PR-171, a peptide keto-epoxide that is currently being investigated in clinical trials for the treatment of myeloma and lymphoma. Purified 20S preparations were used to screen for compounds that selectively inhibit the chymotrypsin-like activity of the immunoproteasome (subunit LMP7) over the constitutive proteasome (subunit beta5). While the parental molecule PR-171 targets both forms of the proteasome with similar potency, several analogs have been identified with an IC50 below 50 nM on LMP7 and greater than 10-fold selectivity compared to beta5. To evaluate the immunoproteasome selectivity of our compounds in cells, an active site-specific ELISA was utilized. Cells expressing both constitutive and immunoproteasome were treated with the analogs and the occupancy of the LMP7 and beta5 active sites was determined. Several compounds retained their immunoproteasome selectivity in cells with an IC50 below 300 nM for LMP7 inhibition. In cell viability studies, some of these compounds demonstrated enhanced cytotoxicity in hematological tumor cell lines expressing the immunoproteasome as compared to solid tumor cell lines which express the constitutive proteasome. Finally, when pharmacodynamics was evaluated following intravenous administration to mice, LMP7 selectivity was retained in tissues expressing both the immunoproteasome and constitutive proteasome. The in vivo anti-tumor properties and toxicity profiles of the immunoproteasome-selective inhibitors are currently being evaluated.

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

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.