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Immunoproteasomes are a special class of proteasomes, which can be induced with IFN-γ in an inflammatory environment. In recent years, it became evident that certain immune cell types constitutively express high levels of immunoproteasomes. However, information regarding the basal expression of proteolytically active immunoproteasome subunits in different types of immune cells is still rare.

Signal peptide mimicry primes Sec61 for client-selective inhibition.

Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis.

Zetomipzomib (KZR-616) attenuates lupus in mice via modulation of innate and adaptive immune responses.

Zetomipzomib (KZR-616) is a selective inhibitor of the immunoproteasome currently undergoing clinical investigation in autoimmune disorders. Here, we characterized KZR-616 and using multiplexed cytokine analysis, lymphocyte activation and differentiation, and differential gene expression analysis. KZR-616 blocked production of >30 pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs), polarization of T helper (Th) cells, and formation of plasmablasts.

Role of Epoxide Hydrolases and Cytochrome P450s on Metabolism of KZR-616, a First-in-Class Selective Inhibitor of the Immunoproteasome.

KZR-616 is an irreversible tripeptide epoxyketone-based selective inhibitor of the human immunoproteasome. Inhibition of the immunoproteasome results in anti-inflammatory activity in vitro and based on promising therapeutic activity in animal models of rheumatoid arthritis and systemic lupus erythematosus KZR-616 is being developed for potential treatment of multiple autoimmune and inflammatory diseases. The presence of a ketoepoxide pharmacophore presents unique challenges in the study of drug metabolism during lead optimization and clinical candidate profiling.

Required Immunoproteasome Subunit Inhibition Profile for Anti-Inflammatory Efficacy and Clinical Candidate KZR-616 ((2 S,3 R)- N-(( S)-3-(Cyclopent-1-en-1-yl)-1-(( R)-2-methyloxiran-2-yl)-1-oxopropan-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-(( S)-2-(2-morpholinoacetamido)propanamido)propenamide).

Selective immunoproteasome inhibition is a promising approach for treating autoimmune disorders, but optimal proteolytic active site subunit inhibition profiles remain unknown. We reveal here our design of peptide epoxyketone-based selective low molecular mass polypeptide-7 (LMP7) and multicatalytic endopeptidase complex subunit-1 (MECL-1) subunit inhibitors. Utilizing these and our previously disclosed low molecular mass polypeptide-2 (LMP2) inhibitor, we demonstrate a requirement of dual LMP7/LMP2 or LMP7/MECL-1 subunit inhibition profiles for potent cytokine expression inhibition and in vivo efficacy in an inflammatory disease model.

Immunoproteasome functions explained by divergence in cleavage specificity and regulation.

The immunoproteasome (iP) has been proposed to perform specialized roles in MHC class I antigen presentation, cytokine modulation, and T cell differentiation and has emerged as a promising therapeutic target for autoimmune disorders and cancer. However, divergence in function between the iP and the constitutive proteasome (cP) has been unclear. A global peptide library-based screening strategy revealed that the proteasomes have overlapping but distinct substrate specificities.

Discovery of Highly Selective Inhibitors of the Immunoproteasome Low Molecular Mass Polypeptide 2 (LMP2) Subunit.

Building upon the success of bortezomib (VELCADE) and carfilzomib (KYPROLIS), the design of a next generation of inhibitors targeting specific subunits within the immunoproteasome is of interest for the treatment of autoimmune disease. There are three catalytic subunits within the immunoproteasome (low molecular mass polypeptide-7, -2, and multicatalytic endopeptidase complex subunit-1; LMP7, LMP2, and MECL-1), and a campaign was undertaken to design a potent and selective LMP2 inhibitor with sufficient properties to allow for sustained inhibition . Screening a focused library of epoxyketones revealed a series of potent dipeptides that were optimized to provide the highly selective inhibitor ().

CHAMPION-1: a phase 1/2 study of once-weekly carfilzomib and dexamethasone for relapsed or refractory multiple myeloma.

Carfilzomib, a proteasome inhibitor, is approved in the United States as a single agent, and in combination with dexamethasone or lenalidomide/dexamethasone (KRd) for relapsed or refractory multiple myeloma (MM). Under the single-agent and KRd approvals, carfilzomib is administered as a 10-minute IV infusion on days 1, 2, 8, 9, 15, and 16 of 28-day cycles (20 mg/m(2) [cycle 1, days 1-2]; 27 mg/m(2) thereafter). This multicenter, single-arm, phase 1/2 study, Community Harmonized Assessment of Myeloma Patients via an Integrated Oncology Network-1 (CHAMPION-1), evaluated once-weekly carfilzomib with dexamethasone in relapsed, or relapsed and refractory MM (1-3 prior therapies).

Carfilzomib demonstrates broad anti-tumor activity in pre-clinical non-small cell and small cell lung cancer models.

Background: Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1B studies of CFZ reported signals of clinical activity in solid tumors, including small cell lung cancer (SCLC). The aim of this study was to investigate the activity of CFZ in lung cancer models.

Analysis of autophagosome formation using lentiviral biosensors for live fluorescent cellular imaging.

Autophagy, a highly regulated homeostatic degradative process, allows cells to reallocate nutrients from less important to more essential processes under extreme conditions of starvation. Autophagy also prevents the buildup of damaged proteins and organelles that cause chronic tissue damage and disease. Although a topic of great interest with involvement of multiple signaling pathways, there are limitations in real-time detection of the autophagic process.

Antileukemic activity and mechanism of drug resistance to the marine Salinispora tropica proteasome inhibitor salinosporamide A (Marizomib).

Salinosporamide A (NPI-0052, marizomib) is a naturally occurring proteasome inhibitor derived from the marine actinobacterium Salinispora tropica, and represents a promising clinical agent in the treatment of hematologic malignancies. Recently, these actinobacteria were shown to harbor self-resistance properties to salinosporamide A by expressing redundant catalytically active mutants of the 20S proteasome β-subunit, reminiscent of PSMB5 mutations identified in cancer cells with acquired resistance to the founding proteasome inhibitor bortezomib (BTZ). Here, we assessed the growth inhibitory potential of salinosporamide A in human acute lymphocytic leukemia CCRF-CEM cells, and its 10-fold (CEM/BTZ7) and 123-fold (CEM/BTZ200) bortezomib-resistant sublines harboring PSMB5 mutations.

Anti-leukemic activity and mechanisms underlying resistance to the novel immunoproteasome inhibitor PR-924.

PR-924 is a novel prototypic immunoproteasome inhibitor bearing markedly enhanced specificity for the β5i immunoproteasome subunit, compared to the classical proteasome inhibitor bortezomib. Here, we assessed the growth inhibitory potential of PR-924 in three human hematologic malignancy cell lines (CCRF-CEM, THP1, and 8226) and their bortezomib-resistant sublines. Parental cells displayed equal sensitivity to PR-924 (IC₅₀: 1.

Interferon-γ-induced upregulation of immunoproteasome subunit assembly overcomes bortezomib resistance in human hematological cell lines.

Background: Despite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, hence calling for novel strategies to overcome bortezomib-resistance. We previously showed that bortezomib-resistant human leukemia cell lines expressed significantly lower levels of immunoproteasome at the expense of constitutive proteasomes, which harbored point mutations in exon 2 of the PSMB5 gene encoding the β5 subunit. Here we investigated whether up-regulation of immunoproteasomes by exposure to interferon-γ restores sensitivity to bortezomib in myeloma and leukemia cell lines with acquired resistance to bortezomib.

Higher ratio immune versus constitutive proteasome level as novel indicator of sensitivity of pediatric acute leukemia cells to proteasome inhibitors.

The ex vivo sensitivity of pediatric leukemia cells to the proteasome inhibitor bortezomib was compared to 3 next generation proteasome inhibitors: the epoxyketone-based irreversible proteasome inhibitors carfilzomib, its orally bio-available analog ONX 0912, and the immunoproteasome inhibitor ONX 0914. LC50 values were determined by MTT cytotoxicity assays for 29 childhood acute lymphoblastic leukemia and 12 acute myeloid leukemia patient samples and correlated with protein expression levels of the constitutive proteasome subunits (β5, β1, β2) and their immunoproteasome counterparts (β5i, β1i, β2i). Acute lymphoblastic leukemia cells were up to 5.

Correction of cystathionine β-synthase deficiency in mice by treatment with proteasome inhibitors.

Cystathionine beta-synthase (CBS) deficiency is an inborn error of metabolism characterized by extremely elevated levels of plasma total homocysteine. The vast majority of CBS-deficient patients have missense mutations located in the CBS gene that result in the production of either misfolded or unstable protein. Here, we examine the effect of proteasome inhibitors on mutant CBS function using two different mouse models of CBS deficiency.

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors.

Background: In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence.

Methods: Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC).

Nonproteasomal targets of the proteasome inhibitors bortezomib and carfilzomib: a link to clinical adverse events.

Purpose: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR).

A neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts.