Immunoproteasome Inhibitors Research Articles

Immunoproteasome Inhibition Reduces the T Helper 2 Response in Mouse Models of Allergic Airway Inflammation.

BackgroundAllergic asthma is a chronic disease and medical treatment often fails to fully control the disease in the long term, leading to a great need for new therapeutic approaches. Immunoproteasome inhibition impairs T helper cell function and is effective in many (auto-) inflammatory settings but its effect on allergic airway inflammation is unknown.MethodsImmunoproteasome expression was analyzed in in vitro polarized T helper cell subsets. To study Th2 cells in vivo acute allergic airway inflammation was induced in GATIR (GATA-3-vYFP reporter) mice using ovalbumin and house dust mite extract. Mice were treated with the immunoproteasome inhibitor ONX 0914 or vehicle during the challenge phase and the induction of airway inflammation was analyzed.Results In vitro polarized T helper cell subsets (Th1, Th2, Th17, and Treg) express high levels of immunoproteasome subunits. GATIR mice proved to be a useful tool for identification of Th2 cells. Immunoproteasome inhibition reduced the Th2 response in both airway inflammation models. Furthermore, T cell activation and antigen-specific cytokine secretion was impaired and a reduced infiltration of eosinophils and professional antigen-presenting cells into the lung and the bronchoalveolar space was observed in the ovalbumin model.ConclusionThese results show the importance of the immunoproteasome in Th2 cells and airway inflammation. Our data provides first insight into the potential of using immunoproteasome inhibition to target the aberrant Th2 response, e.g. in allergic airway inflammation.

Identification of N, C-capped di- and tripeptides as selective immunoproteasome inhibitors

A series of N, C-capped di- and tripeptides were designed as selective immunoproteasome inhibitors based on the known inhibitor 4-CA. Forty-eight new compounds were synthesized and evaluated, and the structure-activity relationship (SAR) of this compound class as β5i selective inhibitors were explored. Most of these compounds showed significant inhibition against the β5i subunit of the immunoproteasome and the most potent β5i inhibitor (15) showed an IC50 of 0.94 nM. A selective β5i inhibitor (54) with over 500-fold β5i/β5c selectivity was identified. Three of the inhibitors were found to selectively inhibit β5i and β5c, and showed no noticeable inhibition against the other four subunits. Six inhibitors with significant inhibitory activity against the HCT-116 cells were recognized, and the most active inhibitors, 14 and 50, showed IC50 values of 0.46 μM and 0.16 μM, respectively. Some selective β5i inhibitors exhibited significant inhibitory effects on the release of the cytokines TNF-α and IL-6. The results not only afford effective chemical tools to elucidate the relationships between subunit selectivity and pharmacological profiles, but also offer useful clues for further optimization and development of selective immunoproteasome inhibitors.

Immunoproteasome Activity in Chronic Lymphocytic Leukemia as a Target of the Immunoproteasome-Selective Inhibitors.

Targeting proteasome with proteasome inhibitors (PIs) is an approved treatment strategy in multiple myeloma that has also been explored pre-clinically and clinically in other hematological malignancies. The approved PIs target both the constitutive and the immunoproteasome, the latter being present predominantly in cells of lymphoid origin. Therapeutic targeting of the immunoproteasome in cells with sole immunoproteasome activity may be selectively cytotoxic in malignant cells, while sparing the non-lymphoid tissues from the on-target PIs toxicity. Using activity-based probes to assess the proteasome activity profile and correlating it with the cytotoxicity assays, we identified B-cell chronic lymphocytic leukemia (B-CLL) to express predominantly immunoproteasome activity, which is associated with high sensitivity to approved proteasome inhibitors and, more importantly, to the immunoproteasome selective inhibitors LU005i and LU035i, targeting all immunoproteasome active subunits or only the immunoproteasome β5i, respectively. At the same time, LU102, a proteasome β2 inhibitor, sensitized B-CLL or immunoproteasome inhibitor-inherently resistant primary cells of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, multiple myeloma and plasma cell leukemia to low doses of LU035i. The immunoproteasome thus represents a novel therapeutic target, which warrants further testing with clinical stage immunoproteasome inhibitors in monotherapy or in combinations.

Syrbactin-class dual constitutive- and immuno-proteasome inhibitor TIR-199 impedes myeloma-mediated bone degeneration in vivo.

Proteasome-addicted neoplastic malignancies present a considerable refractory and relapsed phenotype with patients exhibiting drug resistance and high mortality rates. To counter this global problem, novel proteasome-based therapies are being developed. In the current study, we extensively characterize TIR-199, a syrbactin-class proteasome inhibitor derived from a plant virulence factor of bacterium Pseudomonas syringae pv syringae. We report that TIR-199 is a potent constitutive and immunoproteasome inhibitor, capable of inducing cell death in multiple myeloma, triple-negative breast cancer, (TNBC) and non-small cell lung cancer lines. TIR-199 also effectively inhibits the proteasome in primary myeloma cells of patients, and bypasses the PSMB5 A49T+A50V bortezomib-resistant mutant. TIR-199 treatment leads to accumulation of canonical proteasome substrates in cells, it is specific, and does not inhibit 50 other enzymes tested in vitro. The drug exhibits synergistic cytotoxicity in combination with proteasome-activating kinase DYRK2 inhibitor LDN192960. Furthermore, low-doses of TIR-199 exhibits in vivo activity by delaying myeloma-mediated bone degeneration in a mouse xenograft model. Together, our data indicates that proteasome inhibitor TIR-199 could indeed be a promising next-generation drug within the repertoire of proteasome-based therapeutics.

3112 – IMPACT OF IMMUNOPROTEASOME INHIBITION IN MOUSE MODEL OF HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS

Hemophagocytic lymphohistiocytosis (HLH) comprises an expanding group of disorders associated with overactive immune cells that mediate significant tissue damage. Prior studies reveal that persistence of activated antigen presenting cells drives hyperinflammation in HLH. The immunoproteasome is known to play a role in antigen presentation on class I major histocompatibility complex (MHC) and immune effector cell function. Accordingly, we hypothesized that immunoproteasome inhibition might decrease the cellular and molecular hyperinflammatory response in HLH. Supporting this notion, we find that immunoproteasome inhibition significantly reduces in vitro antigen-specific CD8 T cell proliferation and interferon-gamma (IFNg) production. To examine the effects of immunoproteasome inhibition in an in vivo model of primary (genetic) HLH, lymphocytic choriomeningitis virus (LCMV)-infected perforin-deficient (Prf1-/-) mice were treated with the selective immunoproteasome inhibitor zetomipzomib on varying days post-infection. On days 8-10, immune cell subsets, cytokine production, and lymphocytic tissue infiltration were assessed. Untreated LCMV-infected animals developed marked organomegaly with increased effector T cells, neutrophils, and elevated levels of serum IFNg. Each of these parameters was significantly diminished in zetomipzomib-treated animals. Upon ex vivo antigen stimulation, CD8 T cells from zetomipzomib-treated mice exhibited significantly decreased IFNg production. Together, these data highlight the role of persistent antigen presentation in disease development and suggest that immunoproteasome inhibition shows promise as a potential therapeutic strategy for HLH. Further studies are ongoing to optimize the effects and decipher underlying mechanisms. Hemophagocytic lymphohistiocytosis (HLH) comprises an expanding group of disorders associated with overactive immune cells that mediate significant tissue damage. Prior studies reveal that persistence of activated antigen presenting cells drives hyperinflammation in HLH. The immunoproteasome is known to play a role in antigen presentation on class I major histocompatibility complex (MHC) and immune effector cell function. Accordingly, we hypothesized that immunoproteasome inhibition might decrease the cellular and molecular hyperinflammatory response in HLH. Supporting this notion, we find that immunoproteasome inhibition significantly reduces in vitro antigen-specific CD8 T cell proliferation and interferon-gamma (IFNg) production. To examine the effects of immunoproteasome inhibition in an in vivo model of primary (genetic) HLH, lymphocytic choriomeningitis virus (LCMV)-infected perforin-deficient (Prf1-/-) mice were treated with the selective immunoproteasome inhibitor zetomipzomib on varying days post-infection. On days 8-10, immune cell subsets, cytokine production, and lymphocytic tissue infiltration were assessed. Untreated LCMV-infected animals developed marked organomegaly with increased effector T cells, neutrophils, and elevated levels of serum IFNg. Each of these parameters was significantly diminished in zetomipzomib-treated animals. Upon ex vivo antigen stimulation, CD8 T cells from zetomipzomib-treated mice exhibited significantly decreased IFNg production. Together, these data highlight the role of persistent antigen presentation in disease development and suggest that immunoproteasome inhibition shows promise as a potential therapeutic strategy for HLH. Further studies are ongoing to optimize the effects and decipher underlying mechanisms.

Evaluation of Immunoproteasome-Specific Proteolytic Activity Using Fluorogenic Peptide Substrates

The 26S proteasome irreversibly hydrolyzes polyubiquitylated substrates to maintain protein homeostasis; it also regulates immune responses by generating antigenic peptides. An alternative form of the 26S proteasome is the immunoproteasome, which contains substituted catalytic subunits (β1i/PSMB9, β2i/PSMB10, and β5i/PSMB8) instead of constitutively expressed counterparts (β1/PSMB6, β2/PSMB7, and β5/PSMB5). The immunoproteasome expands the peptide repertoire presented on MHC class I molecules. However, how its activity changes in this context is largely elusive, possibly due to the lack of a standardized methodology to evaluate its specific activity. Here, we describe an assay protocol that measures the immunoproteasome activity of whole-cell lysates using commercially available fluorogenic peptide substrates. Our results showed that the most accurate assessment of immunoproteasome activity could be achieved by combining β5i-targeting substrate Ac-ANW-AMC and immunoproteasome inhibitor ONX-0914. This simple and reliable protocol may contribute to future studies of immunoproteasomes and their pathophysiological roles during viral infection, inflammation, and tumorigenesis.

Discovery and Early Clinical Development of Selective Immunoproteasome Inhibitors.

Inhibitors of the proteolytic activity of the 20S proteasome have transformed the treatment of multiple B-cell malignancies. These agents have also been employed with success in the treatment of patients with autoimmune diseases and immune-mediated disorders. However, new agents are needed to fully unlock the potential of proteasome inhibitors as immunomodulatory drugs. The discovery that selective inhibitors of the immunoproteasome possess broad anti-inflammatory activity in preclinical models has led to the progression of multiple compounds to clinical trials. This review focuses on the anti-inflammatory potential of immunoproteasome inhibition and the early development of KZR-616, the first selective inhibitor of the immunoproteasome to reach clinical testing.

Fragment-Sized and Bidentate (Immuno)Proteasome Inhibitors Derived from Cysteine and Threonine Targeting Warheads.

Constitutive- and immunoproteasomes are part of the ubiquitin–proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the β5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting (R)-boroleucine moieties. These compounds were designed to bind both the Thr1 and β5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the β5, β5i, β1, and β1i subunits with submicromolar to low-micromolar IC50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either β5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.

Improved Scalable Synthesis of Clinical Candidate KZR‐616, a Selective Immunoproteasome Inhibitor

AbstractKZR‐616 is a selective immunoproteasome inhibitor that is under clinical evaluation for the treatment of Systemic Lupus Erythematosus (SLE). KZR‐616 represents a first‐in‐class drug candidate for the treatment of autoimmune diseases targeting the immunoproteasome. Laboratory replication of the published synthetic route of KZR‐616 resulted in an inseparable mixture of enantiomers and diastereomers. Herein, we describe a stereoselective and scalable synthetic pathway for KZR‐616 that avoids laborious separation of enantiomeric mixtures of amino acids. Moreover, the new route provides KZR‐616 in higher overall yield (42.8 %), did not require a chiral chromatographic purification step, and the final product was obtained in high purity. The synthesized compound was evaluated for inhibition of human immunoproteasome and constitutive proteasome and found to target the β1 and β5 subunits of the immunoproteasome with higher potency than the equivalent subunits of the constitutive proteasome. Therefore, we have confirmed that KZR‐616, synthesized using this new method, is an immunoproteasome selective inhibitor.

Development of isoquinolinone derivatives as immunoproteasome inhibitors

The inhibition of immunoproteasome is considered nowadays a promising strategy for the treatment of hematologic malignancies. In this paper we report the design, synthesis, and biological evaluation as immunoproteasome inhibitors of a new series of isoquinolinone derivatives characterized by a (E)-prop-1-ene fragment that connects the heterocycle to a distal amide functionality. Among all the synthesized compounds, we identified an inhibitor with Ki values in the low micromolar or submicromolar range towards the chymotrypsin-like activities of both proteasome and immunoproteasome (β5c, β5i and β1i subunits). Molecular modeling studies suggest that the most potent compound of the series may act a single-site binder. In particular, through its isopentyl group, it might dock into P1 site in the case of the β1i catalytic subunit, while in the case of β5c and β5i subunits, the P3 site might be the preferred binding site.

Proteasome Inhibition Attenuates Self-Renewal in Human Acute Myeloid Leukemia By Targeting NF-Kappa B in Leukemia Stem Cells

In acute myeloid leukemia (AML), relapse following standard chemotherapy is common, leading to 2-year survival rates of less than 30%. Relapse is caused by leukemia stem cells (LSCs), a rare population of mostly quiescent cells that are chemo-refractory and can recapitulate the disease. Our overall goal is to define mechanisms of self-renewal that could be targeted to cure AML and prevent relapse. Functionally, self-renewal is defined by the ability to propagate leukemia in vivo.We use transcriptional and protein profiling to define the functional states of LSCs. We previously demonstrated that NRAS G12V facilitates self-renewal in a mouse model of AML (Mll-AF9/NRAS G12V). Using single-cell RNA sequencing and in vivo leukemia assays, we showed that the stem cell compartment in this model has two distinct subpopulations which differ in their self-renewal and proliferative abilities. The subset of LSCs marked by CD36 LowCD69 High (CD69 High) expression can self-renew and are poorly proliferative. The CD36 HighCD69 Low (CD36 High) subset is unable to self-renew and is highly proliferative. These data demonstrated that self-renewal and rapid proliferation are mutually exclusive functions among LSCs. We demonstrated that the gene expression profiles associated with these functionally distinct LSC subsets are also similarly differentially expressed in primary human LSCs, at the single-cell level (Sachs Cancer Research 2020). Given the functional differences between the CD36 LowCD69 High and CD36 HighCD69 Low subsets in murine AML, we tested whether CD69 and CD36 likewise discriminate self-renewal and proliferation in human AML. We sorted primary human AML samples according to CD69 and CD36 expression and found that Lin -CD69 High cells formed more colonies than Lin -CD36 High cells in every sample tested (n=6). Notably, in three of the six samples, the Lin -CD36 High population was unable to form any colonies (Panel A). These data suggest that CD69 may identify a self-renewing subset of human AML as it does in murine AML. Next, we asked whether Lin -CD69 High and Lin -CD36 High subsets harbor unique signaling protein activation profiles that could potentially be targeted therapeutically. We used CyTOF (mass cytometry) to compare the levels of 12 intracellular signaling proteins between these subpopulations in 14 human AML samples with intermediate and poor risk genetics. CyTOF quantitatively measures a panel of proteins at the single-cell level and allows us to profile activated signaling pathways within well-defined immunophenotypic subpopulations. We found upregulation of both total and phosphorylated (t- and p-) NF-kappa B, pERK, p4EBP1, pMAPKAPKII, and its upstream activator, pP38 in the Lin -CD69 High subset relative to the Lin -CD36 High. Notably, CD69 is a NF-kappa B target gene. Indeed, our single-cell transcriptional data showed that NF-kappa B target genes were upregulated in the self-renewing, CD69 High subset of murine and human LSCs (Sachs Cancer Research 2020). NF-kappa B is a well-known mediator of AML-self-renewal. Next, we asked whether targeting NF-kappa B with proteasome inhibitors might reduce self-renewal capacity in human AML. We plated six primary human samples in colony-forming assays with pan-proteasome inhibitor, carfilzomib, immunoproteasome inhibitor, PR957, or vehicle and found that both inhibitors reduced colony formation in every sample tested. We harvested colonies from each of the inhibitor and vehicle groups and plated them in secondary colony-forming assays. Colonies harvested from vehicle-treated samples formed colonies but those harvested from either carfilzomib or PR957-treated samples were unable to form any secondary colonies (Panel B). Using CyTOF, we validated that both inhibitors reduce NF-kappa B levels in these samples.These data demonstrate that CD69 marks a self-renewing subset of human AML, as it does in our murine AML model, and suggest that the transcriptional mechanisms of self-renewal that we defined in our murine model at the single-cell level may be shared with human AML. Additionally, these data demonstrate enhanced NF-kappa B levels in this self-renewing subset and that targeting the NF-kappa B pathway with proteasome inhibitors attenuates self-renewal in primary human AML samples (Panel C). These findings suggest that proteasome inhibition may be an effective approach to treating and preventing relapse in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proteasome Inhibitors and Their Pharmacokinetics, Pharmacodynamics, and Metabolism.

The proteasome is responsible for mediating intracellular protein degradation and regulating cellular function with impact on tumor and immune effector cell biology. The proteasome is found predominantly in two forms, the constitutive proteasome and the immunoproteasome. It has been validated as a therapeutic drug target through regulatory approval with 2 distinct chemical classes of small molecular inhibitors (boronic acid derivatives and peptide epoxyketones), including 3 compounds, bortezomib (VELCADE), carfilzomib (KYPROLIS), and ixazomib (NINLARO), for use in the treatment of the plasma cell neoplasm, multiple myeloma. Additionally, a selective inhibitor of immunoproteasome (KZR-616) is being developed for the treatment of autoimmune diseases. Here, we compare and contrast the pharmacokinetics (PK), pharmacodynamics (PD), and metabolism of these 2 classes of compounds in preclinical models and clinical studies. The distinct metabolism of peptide epoxyketones, which is primarily mediated by microsomal epoxide hydrolase, is highlighted and postulated as a favorable property for the development of this class of compound in chronic conditions.

Macrocyclic Immunoproteasome Inhibitors as a Potential Therapy for Alzheimer's Disease.

Previously, we reported that immunoproteasome (iP)-targeting linear peptide epoxyketones improve cognitive function in mouse models of Alzheimer's disease (AD) in a manner independent of amyloid β. However, these compounds' clinical prospect for AD is limited due to potential issues, such as poor brain penetration and metabolic instability. Here, we report the development of iP-selective macrocyclic peptide epoxyketones prepared by a ring-closing metathesis reaction between two terminal alkenes attached at the P2 and P3/P4 positions of linear counterparts. We show that a lead macrocyclic compound DB-60 (20) effectively inhibits the catalytic activity of iP in ABCB1-overexpressing cells (IC50: 105 nM) and has metabolic stability superior to its linear counterpart. DB-60 (20) also lowered the serum levels of IL-1α and ameliorated cognitive deficits in Tg2576 mice. The results collectively suggest that macrocyclic peptide epoxyketones have improved CNS drug properties than their linear counterparts and offer promising potential as an AD drug candidate.

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. This study presents a thorough and systematic in vitro and cell-based enzymatic metabolism and kinetic investigation to identify the major enzymes involved in the metabolism and elimination of KZR-616. Upon exposure to liver microsomes in the absence of NADPH, KZR-616 and its analogs were converted to their inactive diol derivatives with varying degrees of stability. Diol formation was also shown to be the major metabolite in pharmacokinetic studies in monkeys and correlated with in vitro stability results for individual compounds. Further study in intact hepatocytes revealed that KZR-616 metabolism was sensitive to an inhibitor of microsomal epoxide hydrolase (mEH) but not inhibitors of cytochrome P450 (P450) or soluble epoxide hydrolase (sEH). Primary human hepatocytes were determined to be the most robust source of mEH activity for study in vitro. These findings also suggest that the exposure of KZR-616 in vivo is unlikely to be affected by coadministration of inhibitors or inducers of P450 and sEH. SIGNIFICANCE STATEMENT: This work presents a thorough and systematic investigation of metabolism and kinetics of KZR-616 and related analogs in in vitro and cell-based enzymatic systems. Information gained could be useful in assessing novel covalent proteasome inhibitors during lead compound optimization. These studies also demonstrate a robust source in vitro test system that correlated with in vivo pharmacokinetics for KZR-616 and two additional tripeptide epoxyketones.

Immunoproteasome Function in Normal and Malignant Hematopoiesis.

The ubiquitin–proteasome system (UPS) is a central part of protein homeostasis, degrading not only misfolded or oxidized proteins but also proteins with essential functions. The fact that a healthy hematopoietic system relies on the regulation of protein homeostasis and that alterations in the UPS can lead to malignant transformation makes the UPS an attractive therapeutic target for the treatment of hematologic malignancies. Herein, inhibitors of the proteasome, the last and most important component of the UPS enzymatic cascade, have been approved for the treatment of these malignancies. However, their use has been associated with side effects, drug resistance, and relapse. Inhibitors of the immunoproteasome, a proteasomal variant constitutively expressed in the cells of hematopoietic origin, could potentially overcome the encountered problems of non-selective proteasome inhibition. Immunoproteasome inhibitors have demonstrated their efficacy and safety against inflammatory and autoimmune diseases, even though their development for the treatment of hematologic malignancies is still in the early phases. Various immunoproteasome inhibitors have shown promising preliminary results in pre-clinical studies, and one inhibitor is currently being investigated in clinical trials for the treatment of multiple myeloma. Here, we will review data on immunoproteasome function and inhibition in hematopoietic cells and hematologic cancers.

Development of peptide epoxyketones as selective immunoproteasome inhibitors.

A series of epoxyketone analogues with varying N-caps and P3-configurations were designed, synthesized and evaluated. We found that D-Ala in P3 was crucial for β5i selectivity over β5c. Notably, compounds 20j (β5i IC50=26.0nM, 25-fold selectivity) and 20l (β5i IC50=25.1nM, 24-fold selectivity) with the D-configuration at P3 were the most selective inhibitors. Although 20j and 20l showed only moderate anti-proliferative activity against RPMI-8226 and MM.1S cell lines, based on our experiments, it indicates that the inhibition of β5i alone is not sufficient to exert anticancer effects and may rely on the complementary inhibition of β1i, β5c and β5i. These data further increase our understanding of immunoproteasome inhibitors in hematologic malignancies.

Activity of immunoproteasome inhibitor ONX-0914 in acute lymphoblastic leukemia expressing MLL\u2013AF4 fusion protein

Proteasome inhibitors bortezomib and carfilzomib are approved for the treatment of multiple myeloma and mantle cell lymphoma and have demonstrated clinical efficacy for the treatment of acute lymphoblastic leukemia (ALL). The t(4;11)(q21;q23) chromosomal translocation that leads to the expression of MLL–AF4 fusion protein and confers a poor prognosis, is the major cause of infant ALL. This translocation sensitizes tumor cells to proteasome inhibitors, but toxicities of bortezomib and carfilzomib may limit their use in pediatric patients. Many of these toxicities are caused by on-target inhibition of proteasomes in non-lymphoid tissues (e.g., heart muscle, gut, testicles). We found that MLL–AF4 cells express high levels of lymphoid tissue-specific immunoproteasomes and are sensitive to pharmacologically relevant concentrations of specific immunoproteasome inhibitor ONX-0914, even in the presence of stromal cells. Inhibition of multiple active sites of the immunoproteasomes was required to achieve cytotoxicity against ALL. ONX-0914, an inhibitor of LMP7 (ß5i) and LMP2 (ß1i) sites of the immunoproteasome, and LU-102, inhibitor of proteasome ß2 sites, exhibited synergistic cytotoxicity. Treatment with ONX-0914 significantly delayed the growth of orthotopic ALL xenograft tumors in mice. T-cell ALL lines were also sensitive to pharmacologically relevant concentrations of ONX-0914. This study provides a strong rationale for testing clinical stage immunoproteasome inhibitors KZ-616 and M3258 in ALL.

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.

Discovery of selective fragment-sized immunoproteasome inhibitors.

Proteasomes contribute to maintaining protein homeostasis and their inhibition is beneficial in certain types of cancer and in autoimmune diseases. However, the inhibition of the proteasomes in healthy cells leads to unwanted side-effects and significant effort has been made to identify inhibitors specific for the immunoproteasome, especially to treat diseases which manifest increased levels and activity of this proteasome isoform. Here, we report our efforts to discover fragment-sized inhibitors of the human immunoproteasome. The screening of an in-house library of structurally diverse fragments resulted in the identification of benzo[d]oxazole-2(3H)-thiones, benzo[d]thiazole-2(3H)-thiones, benzo[d]imidazole-2(3H)-thiones, and 1-methylbenzo[d]imidazole-2(3H)-thiones (with a general term benzoXazole-2(3H)-thiones) as inhibitors of the chymotrypsin-like (β5i) subunit of the immunoproteasome. A subsequent structure-activity relationship study provided us with an insight regarding growing vectors. Binding to the β5i subunit was shown and selectivity against the β5 subunit of the constitutive proteasome was determined. Thorough characterization of these compounds suggested that they inhibit the immunoproteasome by forming a disulfide bond with the Cys48 available specifically in the β5i active site. To obtain fragments with biologically more tractable covalent interactions, we performed a warhead scan, which yielded benzoXazole-2-carbonitriles as promising starting points for the development of selective immunoproteasome inhibitors with non-peptidic scaffolds.

Mitigated viral myocarditis in A/J mice by the immunoproteasome inhibitor ONX 0914 depends on inhibition of systemic inflammatory responses in CoxsackievirusB3 infection

A preclinical model of troponin I-induced myocarditis (AM) revealed a prominent role of the immunoproteasome (ip), the main immune cell-resident proteasome isoform, in heart-directed autoimmunity. Viral infection of the heart is a known trigger of cardiac autoimmunity, with the ip enhancing systemic inflammatory responses after infection with a cardiotropic coxsackievirusB3 (CV). Here, we used ip-deficient A/J-LMP7−/− mice to investigate the role of ip-mediated effects on adaptive immunity in CV-triggered myocarditis and found no alteration of the inflammatory heart tissue damage or cardiac function in comparison to wild-type controls. Aiming to define the impact of the systemic inflammatory storm under the control of ip proteolysis during CV infection, we targeted the ip in A/J mice with the inhibitor ONX 0914 after the first cycle of infection, when systemic inflammation has set in, well before cardiac inflammation. During established acute myocarditis, the ONX 0914 treatment group had the same reduction in cardiac output as the controls, with inflammatory responses in heart tissue being unaffected by the compound. Based on these findings and with regard to the known anti-inflammatory role of ONX 0914 in CV infection, we conclude that the efficacy of ip inhibitors for CV-triggered myocarditis in A/J mice relies on their immunomodulatory effects on the systemic inflammatory reaction.