Tony Muchamuel

A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis

The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low–molecular mass polypeptide-7 (LMP7, encoded by Psmb8), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I–restricted antigens in vitro and in vivo. Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-γ and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders.

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). Experimental Design: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors. Results: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure. Conclusion: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile. Clin Cancer Res; 17(9); 2734–43. ©2011 AACR.

Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (PR-047).

Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and non-Hodgkins lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents. The lead compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (PR-047), selectively inhibited CT-L activity of both the constitutive proteasome (beta5) and immunoproteasome (LMP7) and demonstrated an absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral administration at doses resulting in >80% proteasome inhibition in most tissues and elicited an antitumor response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse syngeneic models. The favorable pharmacologic profile supports its further development for the treatment of malignant diseases.

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

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSION 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.

Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG35–55 and PLP139–151‐induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine‐producing CD4+ cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing‐remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.

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 in vivo. Screening a focused library of epoxyketones revealed a series of potent dipeptides that were optimized to provide the highly selective inhibitor KZR-504 (12).

Abstract 2861: Validation of PERK as an oncology target: A role for the unfolded protein response in cancer

The Unfolded Protein Response (UPR) is a cellular stress response to stressors that induce accumulation of unfolded proteins in the endoplasmic reticulum (aka ER stress). The UPR protects cells from ER stress by increasing the capacity of the ER and attenuating bulk translation. Intense or unresolved ER stress induces apoptosis through pro-apoptotic factors like CHoP. The UPR is activated in tumors, especially those of hematological origin. PERK, a UPR sensor-kinase, is highly active in these settings and might be an attractive target in oncology. We have generated multiple potent, selective PERK inhibitor scaffolds. Low doses of PERK inhibitor ( Emerging data might provide a solution to these challenges. PERK IP-kinase assays demonstrate that compound binding at any dose activates PERK and this activity is retained after compound removal. Exposure modeling in vitro demonstrates that transient dosing followed by compound removal results in a conventional sigmoidal dose-response curve for viability. Intermittent dosing in vivo results in CHoP induction and tumor growth inhibition even at very high doses of PERK, consistent with PERK activation following compound clearance. These findings suggest that optimized scheduling might drive robust tumor growth inhibition with reduced risk of toxicity and facilitate a standard clinical dose escalation. Citation Format: Ken Dellamaggiore, Petia Mitchell, Ji-Rong Sun, Jeffrey Jones, Tony Muchamuel, David Hollenback, Seifu Tadesse, Shon Booker, Fang-Tsao Hong, Adrian Smith, Mark Rose, Pedro Beltran, James R. Lipford. Validation of PERK as an oncology target: A role for the unfolded protein response in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2861.

Abstract 4796: Carfilzomib and ONX 0912 reduce the level of STAT3 phosphorylation in human B-cell lymphoma cells in vitro and in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL BACKGROUND: Proteasome inhibitors, such as bortezomib and the tetrapeptide ketoepoxide carfilzomib (CFZ), have shown strong antitumor activity in patients with multiple myeloma and non-Hodgkins lymphoma (NHL). Anti-tumor responses are due to direct tumor cell cytotoxicity but may also involve effects on transcription factors such as signal transducers and activators of transcription (STATs). AIM: To evaluate the effect on STAT phosphorylation in NHL by proteasome inhibitors as a possible mechanism of antitumor activity. METHODS: The human lymphoma cell lines HS-Sultan and Mino were used for this study. In vitro studies involved overnight incubation of cells with DMSO, carfilzomib or ONX 0912 by flow cytometric analysis for STAT3 and STAT5 phosophorylation. In vivo studies, utilized NIH III immunocompromised mice implanted with HS-Sultan cells and single dose administration with carfilzomib or ONX 0912 when tumors reached 200 mm3. Twenty four hours after dosing, tumor cells were isolated by enzymatic digestion and analyzed by flow cytometry. RESULTS: Carfilzomib and ONX 0912 resulted in suppressed STAT3 tyrosine phosphorylation in both cell lines in vitro and in vivo. No effects on the level of STAT5 phosphorylation were detected. CONCLUSION: These findings suggest that carfilzomib and ONX 0912 exert an anti-tumor effect against human B-cell lymphoma in part through alterations in STAT3 phosphorylation levels. This is the first report that proteasome inhibitors can reduce STAT3 activation in human B-cell lymphomas in vivo. These data support the further evaluation of carfilzomib and ONX 0912 as novel therapeutic agents for the treatment of NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4796. doi:1538-7445.AM2012-4796

Abstract B104: The orally bioavailable proteasome inhibitor PR‐047 inhibits metastasis in a mouse mammary carcinoma model

Background: Proteasome inhibitors, such as bortezomib and the tetrapeptide epoxyketone carfilzomib, have shown strong antitumor activity in patients with multiple myeloma and mantle cell lymphoma. Anti‐tumor responses are due to direct tumor cell cytotoxicity but may also involve effects on growth factors, angiogenesis and stromal cell interactions. The effect of proteasome inhibitors on metastatic spread has not been well studied in pre‐clinical models. PR‐047 is a novel orally bioavailable inhibitor of the 20S proteasome, that is structurally related to carfilzomib. PR‐047 has demonstrated potent anti‐tumor activity in mice bearing subcutaneous tumors of multiple histotypes. Aim: To evaluate the effect of PR‐047 on metastatic spread in mice bearing tumors of the 4T1 mammary carcinoma, an invasive tumor cell line that can spontaneously metastasize from the primary tumor to multiple sites. Methods: 6–8 week old female BALB/c mice were challenged in the mammary fat pad (metastatic spread model) or via intravenous administration (micro‐metastasis model) with 4T1 cells (1 × 105/mouse). Treatment was initiated on Day 3 post tumor challenge in both models. PR‐047 was administered orally at 30 or 40 mg/kg on a weekly schedule of QDx2 or QDx5. The cytotoxic agent cyclophosphamide (CY) was used as a positive control. Primary tumors in the mammary fat pad and metastases in lungs were measured. Results: In the metastatic spread model, PR‐047 treatment reduced the number of metastases in the lung by ∼50% without an effect on the growth of the primary tumor. CY reduced the growth of the primary tumor and metastases in the lung. Both dose schedules and dose levels of PR‐047 were effective in blocking metastasis. To rule out a direct cytotoxic effect on tumor cells in the lung, we assessed the effect of PR‐047 treatment in mice intravenously challenged with 4T1 cells. Unlike CY, which inhibited the growth of micro‐metastases, PR‐047 treatment had no effect in this model, ruling out a direct cytotoxic effect. Conclusion: PR‐047 blocked the spread of 4T1 tumor cells from the mammary fat pad to the lung without an effect on primary tumor cell growth. This is the first report that an orally bioavailable proteasome inhibitor can block metastasis in vivo. These data support the further evaluation of PR‐047 as a novel therapeutic agent for the treatment of metastatic breast cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B104.