Class Of Proteasome Inhibitors Research Articles

Synthesis and proteasome inhibition of lithocholic acid derivatives

A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of potency on proteasome inhibition. Among them, the phenylene diacetic acid hemiester derivative (13) displayed the most potent proteasome inhibition with IC(50) = 1.9 μM. Enzyme kinetic analysis indicates that these lithocholic acid derivatives are noncompetitive inhibitors of the proteasome.

Synthesis and biological evaluation of naphthoquinone analogs as a novel class of proteasome inhibitors

Screening of the NCI Diversity Set-1 identified PI-083 (NSC-45382) a proteasome inhibitor selective for cancer over normal cells. Focused libraries of novel compounds based on PI-083 chloronaphthoquinone and sulfonamide moieties were synthesized to gain a better understanding of the structure–activity relationship responsible for chymotrypsin-like proteasome inhibitory activity. This led to the demonstration that the chloronaphthoquinone and the sulfonamide moieties are critical for inhibitory activity. The pyridyl group in PI-083 can be replaced with other heterocyclic groups without significant loss of activity. Molecular modeling studies were also performed to explore the detailed interactions of PI-083 and its derivatives with the β5 and β6 subunits of the 20S proteasome. The refined model showed an H-bond interaction between the Asp-114 and the sulfonamide moiety of the PI-083 in the β6 subunit.

Neurotoxic and peripheral neuropathic effects in preclinical and clinical studies of carfilzomib (CFZ), a novel proteasome inhibitor (PI).

8135 Background: Treatment-induced peripheral neuropathy (TIPN) is a common, frequently debilitating, therapy-limiting complication in multiple myeloma (MM). Chemotherapies associated with TIPN in MM include thalidomide and bortezomib (BTZ). CFZ, a novel, highly selective PI, is structurally and mechanistically distinct from BTZ. In Ph I-II trials single-agent CFZ is active without dose-limiting PN in pts with relapsed and/or refractory (R/R) MM. Here we compare CFZ and BTZ in a neurotoxicity model and report clinical experience with CFZ in R/R MM. Methods: An in vitro neurotoxicity model established with differentiated SH-SY5Y neuroblastoma cells was used to investigate PI effects on neurite degeneration. A possible mechanism was pursued by bioinformatic and biochemical methods. Data were pooled from 2, ongoing Ph II trials of single-agent CFZ in pts with R/R MM, including neuropathy history, baseline neuropathic symptoms and neurological exam findings, prospective neurological exams, and FACT-GOG/NTx pt survey scores. PN adverse event data were also included. Results: Following 24 hrs of exposure to 10 nM BTZ, SH-SY5Y cells showed 40% reduction in neurite length/cell compared to CFZ- or vehicle-treated cells. Both agents induced equivalent proteasomal inhibition suggesting off-target effects induce neurodegeneration. HtrA2/Omi, a mitochondrial serine protease with a role in neuron survival was identified in databases and confirmed as a target of BTZ (IC50 = 3 nM), but not of CFZ (>10 μ M). At screening, 72% of 135 pts had active Grade (G) 1/2 PN; 81% had a history of TIPN. Pts received a mean of 30.4 CFZ doses (range 2–72) and 17% received >10 mo of therapy. New onset PN or PN worsening from baseline was reported in 16% of pts. There were no reports of G4 PN, missed doses, or treatment discontinuations. Only 1 pt was dose-reduced due to PN. Conclusions: In contrast to BTZ TIPN, CFZ-related PN is uncommon and not dose-limiting. In vitro, BTZ induces neurotoxicity in a proteasome-independent manner and potently inhibits a neuronal cell survival factor, HtrA2. These in vitro and clinical data suggest that severe PN is not a PI class effect. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Onyx Bristol-Myers Squibb, Celgene, Facet, Millennium, Novartis, Onyx Onyx Celgene, Johnson & Johnson, Merck, Millennium, Onyx, Ortho, Takeda Bristol-Myers Squibb, Celgene, Facet, Gloucester Pharmaceuticals, Millennium, Novartis, Onyx

Abstract LB-295: Identification of the first class of proteasome inhibitors that impair 26S proteasome assembly in vitro.

Abstract The Ubiquitin-Proteasome System (UPS) is the main non-lysosomial proteolytic machinery in the eukaryotic cells. Prior to their degradation proteins are adressed to the catalytic 26S proteasome complex composed of a 20S “core” that contains proteolytic activities, assembled with one or two 19S regulatory particles that bind proteins to be degraded. Due to its central role of in regulating cell cycle progression and signal transduction the UPS has been proposed as a suitable target for antineoplastic strategies. To date only catalytic inhibitors of proteasome, targeting proteolytic activities of the 20S core particle, have been characterized and/or have emerged as potent anticancer drugs (1). However, the capacity of the UPS to degrade proteins not only depends of the catalytic activities of the 20S core, but also of the 26S proteasome assembly, a dynamic and tightly controlled cellular process. Thus, compounds impairing 26S proteasome assembly could represent a novel class of proteasome inhibitors. With the aim of identifying such molecules we first set up a robust biochemical screening assay allowing us to follow 26S proteasome assembly in vitro. First, 26S proteasome was purified from Hela cells using affinity chromatography followed by gel filtration. Then, experimental conditions were determined for in vitro 26S disassembly into its constitutive sub-complexes (19S+20S) ands its re-assembly as an active and functional 26S entity. The differential CTL-activity between 26S and 20S proteasomes (2) was then used as the readout to follow the reassembly of 26S in vitro, prior to be confirmed using in gel overlay experiments and western-blotting. Using this biochemical assay: 1) we screened naturals compounds endowed with antiproliferative properties and described as inhibitors of the ubiquitine-proteasome pathway (3). 2) we identified structurally-related natural molecules, epidithiodioxopiperazine and diketodithiopiperazine derivatives, as the first class of molecules inhibiting proteasome assembly without significantly affecting neither 26S nor 20S catalytic activity in vitro. Our research efforts now concentrate in showing that: 1) these molecules inhibit proteasome assembly in cells, and that 2) this inhibition can be correlated with their antiproliferative effects. References Sterz J. et al. Extert Opin. Invest. Drugs, 2008, 17 Rivett AJ et al., Experimental Gerontology, 2002, 37 Vandenberghe I. et al., Biochem. Pharmacol., 2008, 76 Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-295.

Abstract 2632: Syrbactin, a novel proteasome inhibitor class, induces apoptosis and autophagy in neuroblastoma

Abstract Syrbactins belong to a new class of proteasome inhibitors which include syringolins and glidobactins. These small molecules are structurally distinct from other, well-established proteasome inhibitors, and bind the eukaryotic 20S proteasome by a novel mechanism. In this study, we examined the effects of syringolin A (SylA) and glidobactin A (GlbA) as well as two synthetic SylA-analogs (SylA-PEG and SylA-LIP) in human neuroblastoma (SK-N-SH), human multiple myeloma (MM1.S, MM1.RL, and U266), and human ovarian cancer (SKOV3) cells. While all four syrbactins inhibited cell proliferation in a dose-dependent manner, GlbA was most potent in both dexamethasone-sensitive MM1.S cells (IC50: 0.004 µM) and dexamethasone-resistant MM1.RL cells (IC50: 0.005 µM). Syrbactins also inhibited the chymotrypsin-like proteasome activity in a dose-dependent fashion, and GlbA was most effective in SK-N-SH cells (IC50: 0.015 µM). The GlbA-promoted inhibition of proteasomal activity in SK-N-SH cells resulted in the accumulation of ubiquitinated proteins and tumor suppressor protein p53, and led to apoptotic cell death. Strikingly, GlbA treatment also promoted the activation of Akt/PKB via phosphorylation at residue Ser473 and induced autophagy as judged by the presence of the lipidated form of microtubule-associated protein 1 light chain 3 (LC3) and autophagosomes. Collectively, our data suggest that syrbactins belong to a new and effective proteasome inhibitor class which promotes cell death. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2632.

Syrbactin class proteasome inhibitor-induced apoptosis and autophagy occurs in association with p53 accumulation and Akt/PKB activation in neuroblastoma

Syrbactins belong to a new class of proteasome inhibitors which include syringolins and glidobactins. These small molecules are structurally distinct from other, well-established proteasome inhibitors, and bind the eukaryotic 20S proteasome by a novel mechanism. In this study, we examined the effects of syringolin A (SylA) and glidobactin A (GlbA) as well as two synthetic SylA-analogs (SylA-PEG and SylA-LIP) in human neuroblastoma (SK-N-SH), human multiple myeloma (MM1.S, MM1.RL, and U266), and human ovarian cancer (SKOV-3) cells. While all four syrbactins inhibited cell proliferation in a dose-dependent manner, GlbA was most potent in both dexamethasone-sensitive MM1.S cells (IC 50: 0.004 μM) and dexamethasone-resistant MM1.RL cells (IC 50: 0.005 μM). Syrbactins also inhibited the chymotrypsin-like proteasome activity in a dose-dependent fashion, and GlbA was most effective in SK-N-SH cells (IC 50: 0.015 μM). The GlbA-promoted inhibition of proteasomal activity in SK-N-SH cells resulted in the accumulation of ubiquitinated proteins and tumor suppressor protein p53 and led to apoptotic cell death in a time-dependent manner. GlbA treatment also promoted the activation of Akt/PKB via phosphorylation at residue Ser 473 and induced autophagy as judged by the presence of the lipidated form of microtubule-associated protein 1 light chain 3 (LC3) and autophagosomes. Collectively, our data suggest that syrbactins belong to a new and effective proteasome inhibitor class which promotes cell death. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics.

Studies on terrein as a new class of proteasome inhibitors

The proteasome is an intracellular multicatalytic protease involved in the cell cycle regulation, signaling response, antigen presentation and apoptosis. Since proteasome inhibitors promote cell death by apoptosis, they have been proposed as new anti-tumoral drugs. Terrein, a secondary metabolite secreted by the fungus Aspergillus terreus, was firstly described in 1935. In the present work we report that terrein isolated through the screening for inhibitors of the 20S proteasome showed inhibitory effect upon both chymotrypsin- and trypsin-like activities of the multicatalytic core particle, the 20S proteasome. Despite of the high inhibitory concentration determined in vitro, that verified by incubating cells (fibroblasts and a pulmonary tumor cell line) in the presence of terrein was 4-fold lower indicating the proteasome as a selective intracellular target. Moreover, terrein promoted apoptotic cell death on both fibroblasts and pulmonary tumor cell line tested. Although terrein concentrations (mM range) necessary to elicit apoptosis in the cellular models herein tried were high when compared to those (μM and nM range) of other inhibitors recently described, its chemical structure is not correlated to any other inhibitor reported thus far. Therefore, the present results point out for the possibility of exploring terrein as a new molecular fragment for the development of synthetic proteasome inhibitors.

Alkaline Phosphatase (ALP) Variation During Carfilzomib Treatment Is Associated to Best Response in Multiple Myeloma.

Abstract 2865Poster Board II-841 BACKGROUND:The ubiquitin-proteasome pathway, which has been shown to be an essential cellular degradative system in myeloma cells, can also regulate bone formation through its effects on osteoblast differentiation. Retrospective analysis of variation of ALP during treatment with bortezomib indicates a close correlation between myeloma response and serum ALP levels which multiple studies have shown to be of bone origin. To determine if this effect is a class effect of proteasome inhibitors (PIs), this retrospective study analyzed variation of ALP in relationship to myeloma response during treatment with carfilzomib, the first in a new class of selective epoxyketone PIs that has demonstrated encouraging safety and efficacy in two phase 2 studies of relapsed or refractory myeloma patients. METHODS:Retrospective analysis of serum ALP was performed on relapsed or refractory myeloma patients enrolled on two phase 2 studies (PX-171-003 and PX- 171-004) evaluating the safety and efficacy of single agent carfilzomib. We analyzed data from 38 patients in the first cohort of the PX-171-003 study, a relapsed and refractory myeloma trial for patients who have received ≥ 3 prior therapies including bortezomib and an IMiD and 29 patients in PX-171-004, a relapsed or refractory myeloma trial that included bortezomib naïve patients. All patients received 20 mg/m2 of carfilzomib on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle. RESULTS:Seventy-seven patients were enrolled. The median age was 63 years with a median time since diagnosis of 4.6 years, 52% were male, 84.% had relapsed after autologous transplants, 82.% were previously exposed to bortezomib, and 92% were previously exposed to an IMiD. Sixty seven patients with ALP data were evaluable for response. In PX-171-003 the ORR (≥PR) was 18% and the clinical benefit response (CBR; ≥MR) was 26%, while in PX-171-004 the ORR was 35.5% overall and 57% in bortezomib naive patients. ALP increment from baseline, which was most evident during the second cycle of treatment, was statistically different in patients who achieved ≥VGPR compared to all others on Days 1 (P=0.0049) and 8 (P=0.006) of Cycle 2. In all patients achieving a VGPR or better, ALP increased more than 15 units per liter at Cycle 2 Day 1 over baseline An ALP increase over the same period of time was seen in 26 %, 13%, and 11% of patients achieving PR, MR, and SD, respectively. None of the patients with progressive disease exhibited a similar increase. Our study indicates that response first assessed on Day 15 of Cycle 1 parallels the ALP elevation which returned to baseline levels at the end of Cycle 3. CONCLUSIONS:This retrospective analysis on a subset of patients in these ongoing phase 2 studies of single agent carfilzomib in relapsed or refractory multiple myeloma suggests that elevation in ALP may be associated with best response. Taken with previous publications describing bortezomib treatment, these results suggest that this specific anabolic bone phenomenon could be a class effect of proteasome inhibitors. These phase 2 studies are ongoing with a higher dose of carfilzomib (27 mg/m2) being evaluated. The data from this small subset analysis suggests that further exploration of this relationship is warranted. Disclosures:Zangari:Milllennium: Honoraria, Research Funding; Novartis: Research Funding; Celgene: Honoraria; OrthoBiotech: Honoraria; Optum Health: Honoraria; Educational Concepts Group, LLC: Membership on an entity's Board of Directors or advisory committees. Vij:Proteolix: Consultancy, Research Funding. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Merck: Honoraria. Siegel:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Stewart:Millennium: Consultancy, Research Funding; Proteolix: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Honoraria. Wang:Proteolix: Honoraria, Research Funding. Belch:Ortho Biotech: Honoraria, Research Funding. Jakubowiak:Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Centocor Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Trudel:Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Bahlis:Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria, Speakers Bureau. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Singhal:Celgene: Speakers Bureau; Millennium: Speakers Bureau.

Carfilzomib (CFZ), a Novel Proteasome Inhibitor for Relapsed or Refractory Multiple Myeloma, Is Associated with Minimal Peripheral Neuropathic Effects.

Carfilzomib (CFZ), a Novel Proteasome Inhibitor for Relapsed or Refractory Multiple Myeloma, Is Associated with Minimal Peripheral Neuropathic Effects.

Bortezomib induces canonical nuclear factor-κB activation in multiple myeloma cells

Bortezomib is a proteasome inhibitor with remarkable preclinical and clinical antitumor activity in multiple myeloma (MM) patients. The initial rationale for its use in MM was inhibition of nuclear factor (NF)-κB activity by blocking proteasomal degradation of inhibitor of κBα (IκBα). Bortezomib inhibits inducible NF-κB activity; however, its impact on constitutive NF-κB activity in MM cells has not yet been defined. In this study, we demonstrate that bortezomib significantly down-regulated IκBα expression and triggered NF-κB activation in MM cell lines and primary tumor cells from MM patients. Importantly, no inhibition of p65 (RelA) nuclear translocation was recognized after bortezomib treatment in a murine xenograft model bearing human MM cells. Bortezomib-induced NF-κB activation was mediated via the canonical pathway. Moreover, other classes of proteasome inhibitors also induced IκBα down-regulation associated with NF-κB activation. Molecular mechanisms whereby bortezomib induced IκBα down-regulation were further examined. Bortezomib triggered phosphorylation of IκB kinase (IKKβ) and its upstream receptor-interacting protein 2, whereas IKKβ inhibitor MLN120B blocked bortezomib-induced IκBα down-regulation and NF-κB activation, indicating receptor-interacting protein 2/IKKβ signaling plays crucial role in bortezomib-induced NF-κB activation. Moreover, IKKβ inhibitors enhanced bortezomib-induced cytotoxicity. Our studies therefore suggest that bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NF-κB activity in MM cells.

The peptide‐semicarbazone S‐2209, a representative of a new class of proteasome inhibitors, induces apoptosis and cell growth arrest in multiple myeloma cells

Bortezomib is the first approved member of a new class of anti-myeloma agents, the proteasome inhibitors. Further proteasome inhibitors are needed to optimise this promising treatment option. S-2209 [1-[1-{1-[(2,4-Dioxo-imidazolidin-1-ylimino)-methyl]-2-phenyl-ethylcarbamoyl}-2-(1H-indol-3-yl)-ethylcarbamoyl]-2-(1H-indol)] inhibits the chymotryptic activity of the human 20S proteasome (half maximal effective concentration, IC(50) approximately 220 nmol/l) which was determined by a proteasome inhibition assay. A nuclear factor kappaB inhibition assay revealed a half maximal effective concentration (EC(50)) of 0.9 micromol/l. The WST-1 growth assay showed inhibition of cell growth of all tested multiple myeloma (MM) cell lines with an IC(50) between 100 nmol/l and 600 nmol/l. Strong induction of apoptosis was seen in MM cells at nanomolar concentrations (IC(50) approximately 300 nm) as well as in primary myeloma cells. No induction of apoptosis was detected in peripheral blood mononuclear cells from healthy humans. Upregulation of p53, activation of JNK protein, and downregulation of Mcl-1 was revealed. Despite the administration of 15 mg S-2209/kg/d in wistar rats, no toxicity with respect to body weight, hepatic enzymes, creatinine or haemoglobin was seen. Proteasome inhibition in white blood cells isolated from the treated rats was higher in the S-2209 treated animals in comparison with the control animals treated with 0.1 mg/kg/d bortezomib. S-2209 is active in myeloma cells and shows a favourable toxicity profile in first in-vivo studies. S-2209 is a promising agent for further clinical development.

Dietary Supplement Vitamin C Significantly Abrogates Bortezomib-Induced Multiple Myeloma (MM) Cell Growth Inhibition

Vitamin C (ascorbic acid, AA) has antioxydative effects and is widely taken as a food supplement. Previous studies have shown that AA inhibits bortezomib-induced cytotoxicity in human cancer cell lines in vitro thought a direct interaction of both agents. Since bortezomib is a standard treatment option in multiple myeloma (MM), we have determined whether daily oral intake of AA could alter anti-MM activities of bortezomib treatment in MM patients. We first examined plasma levels of vitamin C after oral intake of AA 1000 mg (about 15mg/kg). The mean peak plasma value was 135 μmol/l, and the median concentration at steady state in healthy volunteers ranged from 60 to 100 μmol/l. We next determined direct cytoxicity of AA in several MM cell lines, assessed by [3H]-thymidine uptake assay at 24h culture. The growth inhibition of AA at physiological concentration of 125 μM was 20% in MM1S, 15% in OPM1, 0% in RPMI8226, 17% in U266, 13% in OPM2 and 1% in H9299 cells, indicating marginal growth inhibitory effect. Higher concentration of AA (1mM), induced remarkable cytotoxicity (20–99%) in the majority of MM cell lines. To confirm inhibitory effect of AA on bortezomib-induced cell death, we treated RPMI 8226 cells with therapeutic doses of bortezomib (5nM), in the absence or presence of AA. Importantly, AA (125 μM and 250 μM) significantly inhibited bortezomib (5 ηM)-triggered growth inhibition in a dose-dependent fashion in RPMI8226 cells. We also examined the inhibition of proteasome activity by bortezomib in the presence of AA (125 μM), assessed by poly-ubiquitinated protein level in RPMI cells treated with bortezomib (20 ηM, 8h). Consistent with growth inhibition assays, anti-ubiquitin immunoblotting showed significant reduction of ubiquinated protein level in the presence of AA, confirming that AA blocked inhibition of proteasomal degradation of ubiquitineted proteins by bortezomib. Similar results were observed with other antioxidant agents (ie, LNAC). We further examined whether AA blocks other classes of proteasome inhibitors. Although AA strongly inhibited peptide boronate (MG262 and bortezomib), it did not block lactacystin or MG132-triggered proteasomal inhibition. Importantly, AA could not overcome proteasome inhibition by NPI0052, currently under evaluation in clinical trials in MM. These results confirmed that AA only blocked inhibition of proteasome activity and growth of MM cells triggered by peptide boronate proteasome bortezomib. To investigate whether a vitamin C-rich diet affects bortezomib-induced cytotoxicity we used plasma collected from healthy volunteers (n=4) taking 1000 mg AA for 4 consequtive days The mean baseline AA level was 35μmol/l, with peak concentration (120 μmol/l) at 4h after taking AA. Bortezomib treatment (10 ηM, 24h) triggered 67% growth inhibition in RPMI8226 cells, which was significantly abrogated (30%) by vitamin-C enriched plasma. We confirmed that inhibition of 20S proteasome activity by bortezomib was also markedly blocked (52% vs 7%) by those plasma samples used in growth inhibition assay. Specifically accumulation of polyubiquitinated proteins triggered by bortezomib was also reduced by the plasma. Ongoing studies in a human MM cell xenograft mouse model will delineate the inhibitory effect of AA against anti-tumor activities of bortezomib in vivo.

Non-Proteasomal Targets of Proteasome Inhibitors Bortezomib and Carfilzomib

The proteasome is a multicatalytic protease complex that has been validated as a therapeutic target in oncology with the approval of bortezomib for the treatment of multiple myeloma and mantle cell lymphoma. Carfilzomib is a next generation proteasome inhibitor that is structurally and mechanistically distinct from bortezomib and has entered clinical development in oncology. Both inhibitors target the chymotrypsin-like activity of the proteasome, but their mechanism of action differs due to their unique chemical pharmacores (or “warheads”): bortezomib is a boronate while carfilzomib is an epoxyketone. Phase 1 studies with carfilzomib suggest a clinical safety profile that has both commonalities and distinctions from bortezomib. Transient thrombocytopenia is observed with both molecules suggesting that this event is a proteasome inhibitor class effect. In contrast, the painful peripheral neuropathy that is commonly observed with bortezomib appears to be less severe and possibly less frequent with carfilzomib, raising the possibility that non-proteasome mechanisms may underlie this toxicity. To gain potential insight into the common and unique clinical toxicities of bortezomib and carfilzomib, we evaluated their propensity to act as inhibitors of non-proteasomal enzymes. Bortezomib and carfilzomib were initially screened in a panel of candidate cysteine, aspartyl, metallo-, and serine proteases. Bortezomib significantly inhibited the serine proteases cathepsin G (IC50= 0.3μM) and chymase (IC50= 1.1μM), while carfilzomib did not inhibit these enzymes (IC50>10μM). These effects were further validated in cell extracts prepared from the Thp1 monocyte cell line and peripheral blood mononuclear cells (PBMC), where the inhibition of cathepsin G and chymase by bortezomib was detected using FP-biotin, a serine hydrolase-specific fluorophosphonate activity-based probe. These results suggest that the boronic acid warhead of bortezomib has greater off-target activity than the epoxyketone warhead of carfilzomib. To further investigate this hypothesis, off-target binding of proteasome inhibitors with either the boronate (Cbz-LLL-boronate) or epoxyketone (Cbz-LLL-epoxyketone) warheads were tested in cell extracts and intact cells using FP-biotin detection. Several serine hydrolases in liver (HepG2), lung (A549), kidney (786-O), and leukemia (Thp1) tumor cell lines were found to bind the boronate inhibitor but not the epoxyketone inhibitor. This result suggests that the boronate warhead is more “promiscuous” than the expoxyketone warhead. To obtain a global inhibition profile of the serine hydrolases that are targeted by bortezomib, multidimensional protein identification technology (MudPIT) analysis was performed on FP-biotin-reactive proteins from HepG2 cell extracts. Bortezomib was found to bind several serine hydrolases, including cathepsin A and dipeptidyl peptidase IV (DPP IV) and we have further validated these targets using specific antibodies. Taken together, these data demonstrate that non-proteasomal enzymes can be targeted by bortezomib (cathepsin G, cathepsin A, DPP IV and chymase), whereas carfilzomib does not appear to inhibit non-proteasomal targets in our assays. Work in progress includes biochemical and cell-based characterization for non-proteasomal targets, further global proteomic inhibition profiling using MudPIT analyses, and assessing the potential contribution of non-proteasomal targets to the differential toxicity profiles of the two proteasome inhibitor classes.

231 POSTER Profiling of cancer cell signaling pathways activated by a novel proteasome inhibitor class (syrbactins) in human neuroblastoma

The objective of this study was to analyze the expression and clinical role of 13 signaling molecules in a large cohort of breast carcinoma patients with long follow-up period. Breast carcinomas (n = 410) were analyzed for protein expression of phosphorylated mitogen-activated protein kinases (p-ERK, p-JNK, p-p38) and phosphoinositide 3-kinase signaling pathway proteins (p-AKT, p-mTOR, p-p70S6K); the BAG family proteins BAG-1 and BAG-4/SODD; the antiapoptotic protein Bcl-2; the inhibitor of apoptosis family member Survivin; and the heat shock protein family members HSP27, HSP70, and HSP90. Protein expression was studied for association with clinicopathological parameters and survival. Significantly higher expression of p-AKT (P < .001), p-mTOR (P < .001), p-p70S6K (P < .001), Bcl-2 (P < .001), BAG-4/SODD (P < .001), HSP27 (P < .001), HSP70 (P = .012), HSP90 (P < .001), and Survivin (P = .004) was found in infiltrating ductal and lobular carcinomas compared to mucinous carcinomas. Bcl-2 expression was significantly higher in grades 1 and 2 compared to grade 3 carcinomas (P < .001). p-AKT expression was higher in tumors more than 2 cm (P = .027), whereas p-mTOR expression was lowest in tumors more than 5 cm (P = .019). Higher BAG-4/SODD, HSP70, and HSP90 expression was associated with poor overall survival (P = .016, P = .039, and P = .023, respectively) in univariate analysis, whereas the only independent prognosticator in Cox multivariate survival analysis was tumor diameter (P = .003). In conclusion, BAG-4/SODD, HSP70, and HSP90 are potential prognostic markers in node-negative breast carcinoma that merit further research.

TROSY-Based NMR Evidence for a Novel Class of 20S Proteasome Inhibitors

The proteasome plays a central role in maintaining cellular homeostasis, in controlling the cell cycle, in removing misfolded proteins that can be toxic, and in regulating the immune system. It is also an important target for novel anticancer drugs, such as bortezomib, a potent inhibitor that has been used successfully in the treatment of multiple myeloma. Here, we show that the antimalaria drug chloroquine inhibits proteasome function in eukaryotic cell extracts and in preparations of purified 20S archaeal proteasome from Thermoplasma acidophilium. Methyl-TROSY-based NMR spectroscopy experiments conducted with the 670 kDa 20S proteasome localize chloroquine binding to regions between the alpha and beta subunits of the alpha-beta-beta-alpha barrel-like structure, approximately 20 A from the proteolytic active sites in this 7-fold symmetric molecule. Complementary amide TROSY experiments that provide further probes of proteasome-inhibitor interactions were performed on a novel 180 kDa single-ring construct containing only alpha subunits, the proper assembly of which was confirmed by electron microscopy. In contrast to the chloroquine-proteasome interaction described here, all previously reported inhibitors of the proteasome, including MG132, bind the catalytic region directly. Consistent with the NMR chemical shift perturbation data reported here that place chloroquine binding distal from sites of proteolysis, we show that MG132 and chloroquine can bind the proteasome simultaneously, further establishing that they exploit two completely separate binding pockets. Our data thus establish a novel class of proteasome inhibitor that functions via a mechanism distinct from binding to active sites.

A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism

Pathogenic bacteria often use effector molecules to increase virulence. In most cases, the mode of action of effectors remains unknown. Strains of Pseudomonas syringae pv. syringae (Pss) secrete syringolin A (SylA), a product of a mixed non-ribosomal peptide/polyketide synthetase, in planta. Here we identify SylA as a virulence factor because a SylA-negative mutant in Pss strain B728a obtained by gene disruption was markedly less virulent on its host, Phaseolus vulgaris (bean). We show that SylA irreversibly inhibits all three catalytic activities of eukaryotic proteasomes, thus adding proteasome inhibition to the repertoire of modes of action of virulence factors. The crystal structure of the yeast proteasome in complex with SylA revealed a novel mechanism of covalent binding to the catalytic subunits. Thus, SylA defines a new class of proteasome inhibitors that includes glidobactin A (GlbA), a structurally related compound from an unknown species of the order Burkholderiales, for which we demonstrate a similar proteasome inhibition mechanism. As proteasome inhibitors are a promising class of anti-tumour agents, the discovery of a novel family of inhibitory natural products, which we refer to as syrbactins, may also have implications for the development of anti-cancer drugs. Homologues of SylA and GlbA synthetase genes are found in some other pathogenic bacteria, including the human pathogen Burkholderia pseudomallei, the causative agent of melioidosis. It is thus possible that these bacteria are capable of producing proteasome inhibitors of the syrbactin class.

Safety and Antitumor Efficacy of the Proteasome Inhibitor Carfilzomib (PR-171) Dosed for Five Consecutive Days in Hematologic Malignancies: Phase 1 Results.

Introduction: Carfilzomib (CFZ) is a structurally- and mechanistically-novel proteasome inhibitor (PI) of the peptide epoxyketone class that exhibits a high level of selectivity for the unique N-terminal threonine active sites within the proteasome. CFZ is similar to bortezomib (BTZ; VELCADE®), in that it is a potent inhibitor of the chymotrypsin-like activity, but unlike BTZ, CFZ has shown minimal cross-reactivity with the other catalytic sites within the proteasome or across other protease classes. Preclinical human tumor xenograft models indicate that consecutive day (D1, D2) dosing resulting in 48 hours of proteasome suppression was superior to split dosing (D1, D4).Methods: This phase 1 sequential dose escalation trial tested an intensive proteasome inhibition schedule with CFZ administered as an intravenous push (IVP) daily (QD) for 5 consecutive days on a 14 day cycle. The doses administered ranged from 1.2 mg/m2 to 20 mg/m2. Multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) that included mantle cell lymphoma (MCL), Hodgkin's disease (HD), or Waldenström's macroglobulinemia (WM) were eligible if they had relapsed after 2 or more prior therapies.Results: A total of 29 subjects have been treated. The minimal effective dose (MED) was 11 mg/m2. A maximum of 80% proteasome inhibition was achieved in peripheral blood and mononuclear cells at ≥15 mg/m2. Five responses have occurred in 14 patients (6 MM, 3 MCL, 1 WM, 4 NHL) enrolled at ≥ the MED: 1 MM partial response (PR), 2 MM minimal responses (MR), 1 WM MR and one macroscopic CR in a patient with gastrointestinal MCL. Responses occurred in patients whose disease had failed therapy with BTZ, immunomodulatory agents, and stem cell transplant. Four NHL patients have had stable disease and remained on treatment for up to 7 months. Dose limiting toxicity reported at 20 mg/m2 included febrile neutropenia (reversible) and grade 4 thrombocytopenia. Grade 3 (25%) or grade 4 events (8%) were primarily hematologic events. Most withdrawals occurred due to the inconvenience of frequent visits, or progressive disease.Conclusions: This study demonstrates that intensive CFZ dosing leading to sustained proteasome inhibition is well tolerated. The selectivity of the epoxyketone class of PI may promote a favorable safety profile. Furthermore, antitumor responses in heavily pretreated patients suggest non-cross resistant activity. Phase 2 trials in MM and solid tumors are proceeding with an alternate IV schedule. An oral agent suitable for frequent dosing schedules is under development.

Phase 1 Single Agent Antitumor Activity of Twice Weekly Consecutive Day Dosing of the Proteasome Inhibitor Carfilzomib (PR-171) in Hematologic Malignancies.

Carfilzomib (CFZ) is a structurally- and mechanistically-novel proteasome inhibitor of peptide epoxyketone class that exhibits a high level of selectivity for the unique N-terminal threonine active sites within the proteasome. CFZ is similar to bortezomib (BTZ,Velcade®), in that it is a potent inhibitor of the proteasome chymotrypsin-like activity, but unlike BTZ, CFZ has shown minimal cross-reactivity with the other catalytic sites within the proteasome or across other protease classes. Preclinical human tumor xenograft models indicated that consecutive day (D1, D2) dosing resulting in 48 hours of proteasome suppression was superior to split dosing (D1, D4). This phase 1 sequential dose escalation trial tested twice weekly consecutive day dosing (48 hour proteasome inhibition) with carfilzomib. Carfilzomib was administered as an IVP on a 28 day cycle (D1, 2, 8, 9, 15 and 16 with 12 days rest) at doses from 1.2 mg/m2 to 27 mg/m2. Patients with Multiple myeloma (MM), non-Hodgkin's Lymphoma (NHL) that included mantle cell lymphoma (MCL), Hodgkin's disease (HD), or Waldenström's macroglobulinemia (WM) were eligible if they had relapsed after at least 2 prior therapies. A total of 37 subjects have been treated. The minimal effective dose (MED) was 15 mg/m2, and maximum (80%) proteasome inhibition was achieved in peripheral blood and mononuclear cells at this dose. Five objective responses have occurred in 16 patients (13 MM, 3 MCL) enrolled at ≥ the MED: 4 MM partial response (PR), 1 MM minimal responses (MR). The responses have been durable (134 to 392 days) and occurred in patients who had failed bortezomib, immunomodulatory agents and stem cell transplant. Six additional patients have had stable disease (SD): 2 MM, 1 T NHL, 2 MCL, 1 B NHL. Dose limiting toxicity reported at 27 mg/m2 included a hypoxic event and Grade 2 thrombocytopenia increasing to Grade 4. A reversible Grade 2 creatinine D2 was reported in 3/5 MM patients treated at 27 mg/m2 and was associated with a rapid decline in M-protein without evidence of tumor lysis syndrome and the event did not occur on rechallenge. Cyclic thrombocytopenia was rapidly reversible and painful peripheral neuropathy was not reported. This study demonstrates that consecutive CFZ dosing leading to sustained proteasome inhibition is well tolerated on a weekly basis. The selectivity of epoxyketone class of proteasome inhibitors may provide a wide therapeutic index. Patients have remained on therapy for over a year, and furthermore, antitumor responses in heavily pretreated MM patients suggest non-cross resistant activity. Phase 2 trials in relapse and refractory MM and relapse solid tumors are proceeding with this schedule.

Inhibitors of Ubiquitin-Activating Enzyme (E1), a New Class of Potential Cancer Therapeutics

The conjugation of proteins with ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous malignancies. For this reason, there is great interest in targeting the ubiquitin-proteasome system in cancer. Several classes of proteasome inhibitors, which block degradation of ubiquitylated proteins, are widely used in research, and one, Bortezomib, is now in clinical use. Despite the well-defined and central role of the ubiquitin-activating enzyme (E1), no cell permeable inhibitors of E1 have been identified. Such inhibitors should, in principle, block all functions of ubiquitylation. We now report 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41) as the first such inhibitor. Unexpectedly, in addition to blocking ubiquitylation, PYR-41 increased total sumoylation in cells. The molecular basis for this is unknown; however, increased sumoylation was also observed in cells harboring temperature-sensitive E1. Functionally, PYR-41 attenuates cytokine-mediated nuclear factor-kappaB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of TRAF6, which is essential to IkappaB kinase activation. PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of IkappaBalpha. Furthermore, PYR-41 inhibits degradation of p53 and activates the transcriptional activity of this tumor suppressor. Consistent with this, it differentially kills transformed p53-expressing cells. Thus, PYR-41 and related pyrazones provide proof of principle for the capacity to differentially kill transformed cells, suggesting the potential for E1 inhibitors as therapeutics in cancer. These inhibitors can also be valuable tools for studying ubiquitylation.

Mantle cell lymphoma: Evolving novel options

Mantle cell lymphoma (MCL) represents only 6% of non-Hodgkin's lymphoma but is one of the most active fields of clinical investigation. Front-line therapy appears to benefit from intensification either through high-dose therapy with stem cell transplant consolidation or dose-intense chemotherapy with hyper-CVAD (fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) and rituximab. Unfortunately, no standard therapy has been defined, and most patients eventually relapse. An impressive number of novel agents are currently being tested, the bulk of which are biologic agents or targeted therapies. Bortezomib is the first in class of proteasome inhibitors and the first new agent to be approved in relapsed/refractory MCL. Other small molecules have shown encouraging activity, including mTOR and Bcl-2 inhibitors, novel antibodies, and new cytotoxic agents. Future trials will also benefit from new molecular approaches through pharmacogenomics.