Proteasome Inhibitors Do Not Inhibit the Serine Protease HtrA2/Omi

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5023-5023
Author(s):  
Joseph Senn ◽  
Vilmos Csizmadia ◽  
Paul Hales ◽  
Larry Dick ◽  
Vivek J. Kadambi
Keyword(s):  
Peripheral Neuropathy ◽  
Serine Protease ◽  
Proteasome Inhibitor ◽  
Cultured Cells ◽  
Proteasome Inhibitors ◽  
Specific Inhibitor ◽  
Positive Control ◽  
Recombinant Enzyme ◽  
Enzyme Assays

Abstract Abstract 5023 Based on unprecedented efficacy, the proteasome inhibitor (PI) bortezomib has become the cornerstone of multiple myeloma treatment. Nevertheless, in a subset of patients bortezomib causes painful peripheral neuropathy and this side effect can limit its potential benefit for those patients. Although the mechanism of bortezomib associated neuropathy is unknown, we have previously suggested that it is related to the mechanism of action (Csizmadia at. al. Vet Pathol 2010; 47:358–367.). Recently Arastu-Kapur et al. (Clin Cancer Res 2011;17:2734–2743.) have reported that the serine protease HtrA2/Omi was inhibited by bortezomib (a peptide boronate proteasome inhibitor) and not by carfilzomib (an epoxyketone proteasome inhibitor). Further, since HtrA2/Omi is involved in neuronal survival (Martins et al. Mol Cell Biol 2004; 24: 9848–9862.) they suggested that this off target inhibition by bortezomib could be the mechanism underlying bortezomib associated peripheral neuropathy. To confirm and extend these published results, we investigated the effects of these two PIs on HtrA2/Omi activity in recombinant enzyme assays, in SH-SY5Y neuroblastoma-, and wild type and HtrA2/Omi double negative mouse embryonic fibroblast cells (MEF). In contrast to the results of Arastu-Kapur et al., our results clearly demonstrated that neither bortezomib nor carfilzomib inhibits HtrA2/Omi in recombinant enzyme assays at concentrations up to 100μM. As a positive control we used Ucf-101 an HtrA2/Omi specific inhibitor (Cilenti et al. J Biol Chem 2003; 278:11489–11494.) which in our assay behaved in a manner consistent with the published literature. Similarly, in MEF cells, only Ucf-101 prevented the degradation of validated HtrA2/Omi substrates eIF4G1 and UCH-L1, while neither bortezomib nor carfilzomib prevented the degradation of these two substrates. In conclusion, we have assessed the protease activity of HtrA2/Omi both in vitro with purified enzyme and in cultured cells and we find that neither PI inhibits this protease. Therefore we think it is unlikely that PI associated peripheral neuropathy is caused by off target inhibition of HtrA2/Omi. Further research is needed to understand the side effects of PIs. Disclosures: Senn: Millennium Pharmaceuticals, Inc.: Employment. Csizmadia:Millennium Pharmaceuticals, Inc.: Employment. Hales:Millennium Pharmaceuticals, Inc.: Employment. Dick:Millennium Pharmaceuticals, Inc.: Employment. Kadambi:Millennium Pharmaceuticals, Inc.: Employment.

scholarly journals Proteasome inhibitors bortezomib and carfilzomib used for the treatment of multiple myeloma do not inhibit the serine protease HtrA2/Omi

2016 ◽  
Vol 5 (6) ◽  
pp. 1619-1628 ◽  
Author(s):  
Vilmos Csizmadia ◽  
Paul Hales ◽  
Christopher Tsu ◽  
Jingya Ma ◽  
Jiejin Chen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Peripheral Neuropathy ◽  
Serine Protease ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors

The proteasome inhibitor bortezomib is associated with the development of peripheral neuropathy in patients, but the mechanism is not fully understood.

Neurodegeneration Induced by Bortezomib Exposure in Vitro Occurs Via Proteasome Independent Mechanisms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2859-2859
Author(s):  
Shirin Arastu-Kapur ◽  
Andrew J. Ball ◽  
Janet L. Anderl ◽  
Mark K Bennett ◽  
Christopher J Kirk
Keyword(s):  
Peripheral Nerve ◽  
Proteasome Inhibitor ◽  
Serine Proteases ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Nerve Degeneration ◽  
Differentiated Cells ◽  
Favorable Safety ◽  
Neurite Degeneration

Abstract Abstract 2859 Poster Board II-835 BACKGROUND: The dipeptide boronate proteasome inhibitor bortezomib (BTZ; Velcade®) is approved for the treatment of multiple myeloma and non-Hodgkin's lymphoma. Bortezomib-induced peripheral neuropathy (BIPN, Blood (2008)112:1593-1599) is seen in ∼30% of BTZ-treated patients and can result in dose reductions and discontinuations that may result in suboptimal levels of proteasome inhibition. Carfilzomib (CFZ), a tetrapeptide epoxyketone, is a selective and irreversible proteasome inhibitor that is structurally and mechanistically distinct from bortezomib. Single agent treatment with CFZ has demonstrated strong activity in relapsed and refractory myeloma and a favorable safety profile in Phase 2 trials (ASH2008:864 & 865). Importantly treatment-emergent PN was seen at low levels and did not result in dose modifications or discontinuations. The disparate safety data for these proteasome inhibitors suggest that non-proteasomal mechanisms may underlie BIPN. Using activity-based probes in peripheral blood mononuclear cell (PBMC) lysates, we previously demonstrated inhibition of non-proteasomal proteases by BTZ and other proteasome inhibitors with a boronate pharmacophore (EHA2009:0939). However, the involvement of the proteasome in the peripheral nerve degeneration and BIPN in BTZ-treated myeloma patients remains to be established. AIMS: To establish an in vitro model of peripheral nerve degeneration and to determine the effects of proteasome inhibition by BTZ and CFZ on neurite outgrowth and cell survival. METHODS: SH-SY5Y neuroblastoma cells were differentiated by long term culture in retinoic acid and brain derived nerve growth factor to induce neurite outgrowth. The effects of proteasome inhibitors were measured by high content image analysis of fluorescent images for cell survival (Hoechst nuclear counterstain) and neurite degeneration (FITC-mouse anti-beta-III-tubulin). Phase contrast images were also collected to observe morphological effects and gross cell death. Cell viability and proteasome inhibition was measured in undifferentiated and differentiated cells. The MEROPS (peptidase) database was mined for candidate serine proteases with a P1 selectivity of Leu/Phe/Tyr to identify candidate off-targets CFZ and BTZ and candidate proteases were validated by standard biochemical and cell biology techniques. RESULTS: In differentiated SH-SY5Y cells, the average neurite length decreased by 33% following 24 hr exposure to 10nM BTZ but was unaffected by the same concentration of CFZ. Proteasome inhibition as determined by a fluorescent substrate for the chymotrypsin-like activity was equivalent (∼70%) after a 24 hr exposure for both compounds in differentiated cells, suggesting that neurodegeneration involves non-proteasomal pathways. With 72 hrs continuous exposure, BTZ was 10-fold more potent than CFZ at inducing neurodegeneration. Furthermore, in both undifferentiated and differentiated SH-SY5Y cells, BTZ was 5-fold more cytotoxic than CFZ. Database mining for serine proteases with a selectivity for Leu/Phe/Tyr at P1 was used to identify other potential BTZ targets that might underlie neurotoxicity. One candidate is HtrA2 (also called Omi), an inducible mitochondrial serine protease whose activity protects neurons from stress induced apoptosis (Hum Mol Genet (2005) 14(5):2099-2111). HtrA2 levels increased 2-fold in SH-SY5Y cells treated with either BTZ or CFZ for 6 hrs at 40 nM. Using a gel based assay and purified enzyme preparations, BTZ inhibited HtrA2 activity with an IC50 ∼ 4 nM, equivalent to its activity against the proteasome. In contrast, Carfilzomib did not inhibit HtrA2 at the highest concentration tested (10 mM). CONCLUSIONS: These data demonstrate that BTZ induces neuronal cell death and neurite degeneration in vitro by proteasome-independent mechanisms. We propose that combined inhibition of the proteasome and HtrA2 by BTZ may underlie peripheral nerve toxicities in vitro and may be involved in BIPN in myeloma patients. In this model, CFZ, which mediates equivalent proteasome inhibition to BTZ in neurons, does not induce neurodegeneration due to inactivity against HtrA2. Future profiling of non-proteasomal targets of BTZ, including HtrA2 activity, in patient samples is merited. These results suggest that the favorable safety profile of CFZ in myeloma patients may be a result of its high selectivity for proteasomal proteases. Disclosures: Arastu-Kapur: Proteolix, Inc: Employment. Ball:Millipore Corp: Employment. Anderl:Millipore Corp: Employment. Bennett:Proteolix: Employment. Kirk:Proteolix, Inc: Employment.

Investigational Agent MLN2238/MLN9708, a Specific, Orally Available, Small Molecule Proteasome Inhibitor, Shows Promising In Vitro Activity Against Multiple Myeloma Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3014-3014
Author(s):  
Giada Bianchi ◽  
Vijay G. Ramakrishnan ◽  
Teresa Kimlinger ◽  
Jessica Haug ◽  
S. Vincent Rajkumar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Cell Lines ◽  
Small Molecule ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Proteasome Inhibitors ◽  
Biologically Active ◽  
Investigational Agent

Abstract Abstract 3014 Background: Proteasome inhibitors have proven particularly effective in treatment of multiple myeloma, the second most frequent hematologic malignancy in the western world. Bortezomib, the first in class proteasome inhibitor in clinical use, was first approved in 2003 via fast FDA track, given the remarkable activity shown during phase II clinical trials. Nevertheless, more than 50% of multiple myeloma patients did not respond to single agent bortezomib when administered as second line agent. Moreover, bortezomib is only available for intravenous administration, representing a cumbersome therapy for patients, and its use is limited by significant toxicities (especially peripheral neuropathy). MLN9708 (Millennium Pharmaceuticals, Inc.), an investigational orally available, small molecule, is a potent, specific and reversible inhibitor of the 20S proteasome. It is currently under clinical investigation for the treatment of hematologic and non-hematologic malignancies. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form, and MLN2238 was used for all of the preclinical studies reported here. In vitro biochemistry studies have shown that MLN2238 has a faster dissociation rate from the proteasome compared to bortezomib, and in vivo studies of MLN2238 have shown antitumor activity in a broader range of tumor xenografts when compared to bortezomib. Given these encouraging preclinical results, we set to investigate the anti-myeloma activity of MLN2238 in vitro. Results: MLN2238 proved to have anti-proliferative and pro-apoptotic activity against a broad range of MM cell lines with EC50 at 24 hours ranging between 10 and 50 nM, even in relatively resistant MM cell lines (OPM2, DOX6, RPMI, etc.). In MM.1S cells, induction of apoptosis was time and dose dependent and related to activation of both caspase 8 and 9. When compared to MM.1S treated for 24 hours with EC50 dose of bortezomib, treatment with EC50 dose of MLN2238 resulted in the same extent of caspases cleavage occurring at an earlier time point (8-12 hours), possibly suggesting more rapid onset and/or irreversibility of apoptosis in cells treated with MLN2238. Treatment with MLN2238 was associated with early, but persistent induction of endoplasmic reticulum (ER) stress with BiP being induced 2–4 hours after treatment with EC50 dose and gradually increasing over time. While bortezomib has been associated with early induction and late decrease in proteins involved in ER stress, MLN2238 appears to induce a persistent rise in these factors, suggesting either more sustained proteasome blockade with stabilization of proteasome substrates or de-novo induction of unfolded protein response (UPR) genes. MLN2238 also proved effective in reducing phosphorylation of ERK1-2 with no overall alteration in the total ERK level, thus accounting for the observed reduction in proliferation upon treatment. Preliminary data indicate potential for additive and synergistic combination with widely used drugs, including doxorubicin and dexamethasone. Conclusion: While further clinical data are needed to establish the effectiveness of MLN2238 in the treatment of multiple myeloma, these preliminary nonclinical data, together with the favorable biochemical and pharmacokinetic properties, including oral bioavailability, make the investigational agent MLN9708 an appealing candidate for treatment of multiple myeloma. Further in vitro data could help establish whether a difference in the apoptotic mechanisms exist between MLN2238 and other proteasome inhibitors, primarily bortezomib, and could also help inform combination treatment approaches aimed at increasing effectiveness, overcoming bortezomib resistance and decreasing toxicity. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

scholarly journals Mutations in a Conserved Residue in the Influenza Virus Neuraminidase Active Site Decreases Sensitivity to Neu5Ac2en-Derived Inhibitors

1998 ◽  
Vol 72 (3) ◽  
pp. 2456-2462 ◽  
Author(s):  
Jennifer L. McKimm-Breschkin ◽  
Anjali Sahasrabudhe ◽  
Tony J. Blick ◽  
Mandy McDonald ◽  
Peter M. Colman ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Active Site ◽  
Liquid Culture ◽  
Specific Inhibitor ◽  
Side Chain ◽  
Enzyme Assays ◽  
Hydrophobic Group ◽  
Influenza Virus Neuraminidase ◽  
Conserved Residue

ABSTRACT The influenza virus neuraminidase (NA)-specific inhibitor zanamivir (4-guanidino-Neu5Ac2en) is effective in humans when administered topically within the respiratory tract. The search for compounds with altered pharmacological properties has led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide). NWS/G70C variants generated in vitro, with decreased sensitivity to 6-carboxamide, contained hemagglutinin (HA) and/or NA mutations. HA mutants bound with a decreased efficiency to the cellular receptor and were cross-resistant to all the NA inhibitors tested. The NA mutation, an Arg-to-Lys mutation, was in a previously conserved site, Arg292, which forms part of a triarginyl cluster in the catalytic site. In enzyme assays, the NA was equally resistant to zanamivir and 4-amino-Neu5Ac2en but showed greater resistance to 6-carboxamide and was most resistant to a new carbocyclic NA inhibitor, GS4071, which also has a hydrophobic side chain at the 6 position. Consistent with enzyme assays, the lowest resistance in cell culture was seen to zanamivir, more resistance was seen to 6-carboxamide, and the greatest resistance was seen to GS4071. Substrate binding and enzyme activity were also decreased in the mutant, and consequently, virus replication in both plaque assays and liquid culture was compromised. Altered binding of the hydrophobic side chain at the 6 position or the triol group could account for the decreased binding of both the NA inhibitors and substrate.

scholarly journals Parkin facilitates proteasome inhibitor-induced apoptosis via suppression of NF-κB activity in hepatocellular carcinoma

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Xiaolan Zhang ◽  
Chun Lin ◽  
Junwei Song ◽  
Han Chen ◽  
Xuhong Chen ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Tumor Growth ◽  
Protein Degradation ◽  
Anticancer Agents ◽  
Proteasome Inhibitor ◽  
Primary Liver Cancer ◽  
Proteasome Inhibitors ◽  
Apoptosis Resistance ◽  
Induced Apoptosis

Abstract The ubiquitin–proteasome system (UPS) is a tight homeostatic control mechanism of intracellular protein degradation and turnover involved in many human diseases. Proteasome inhibitors were initially developed as anticancer agents with potential benefits in the suppression of tumor growth. However, clinical trials of patients with solid tumors fail to demonstrate the same efficacy of these proteasome inhibitors. Here, we show that Parkin, an E3 ubiquitin ligase, is implicated in tumorigenesis and therapy resistance of hepatocellular carcinoma (HCC), the most common type of primary liver cancer in adults. Lower Parkin expression correlates with poor survival in patients with HCC. Ectopic Parkin expression enhances proteasome inhibitor-induced apoptosis and tumor suppression in HCC cells in vitro and in vivo. In contrast, knockdown of Parkin expression promotes apoptosis resistance and tumor growth. Mechanistically, Parkin promotes TNF receptor-associated factor (TRAF) 2 and TRAF6 degradation and thus facilitates nuclear factor-kappa-B (NF-κB) inhibition, which finally results in apoptosis. These findings reveal a direct molecular link between Parkin and protein degradation in the control of the NF-κB pathway and may provide a novel UPS-dependent strategy for the treatment of HCC by induction of apoptosis.

In Vitro Growth Inhibition, Target Modulation and Drug Synergy In Pediatric Leukemia By The Novel Proteasome Inhibitor Carfilzomib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2673-2673
Author(s):  
Aarthi Jayanthan ◽  
Yibing Ruan ◽  
Meaghan Hagerty ◽  
Ravi Shah ◽  
Tony Truong ◽  
...  
Keyword(s):  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Leukemia Cells ◽  
Toxicity Profile ◽  
Pediatric Leukemia ◽  
Over Expression ◽  
Drug Synergy ◽  
Gm Csf ◽  
Induction Of Apoptosis

Abstract Introduction Despite the considerable progress that has been made in past two decades, relapsed or refractory leukemia still remains a leading cause of death in children. Acute and long term toxicities prevent significant further intensification of current chemotherapeutic regimens. Hence, effective pre-clinical data on new agents and novel therapeutic approaches are urgently needed. Recent studies have established proteasome inhibition as a distinctive and effective way to induce cytotoxicity in tumor cells that have acquired resistance to conventional chemotherapy. Although they represent a major advancement in the treatment of various hematological malignancies, the first generation proteasome inhibitors exhibit measurable off-target toxicities and the eventual development of resistance. Carfilzomib (CFZ) is a selective proteasome inhibitor that is structurally distinct from bortezomib and has shown efficacy and favorable toxicity profile in multiple myeloma patients. Mechanistically, carfilzomib has been shown to irreversibly bind and inhibit the chymotrypsin-like activity of the 20S proteasome and to cause the accumulation of polyubiquinated proteins resulting in cell cycle arrest, apoptosis, and suppression of tumor growth. Methods A diverse panel of pediatric leukemia derived cell lines and primary leukemia specimens (n = 12) were used to evaluate CFZ induced in vitro cytotoxicity using the Alamar blue assay. These cell lines include leukemia with abnormal FLT3 (FLT3-ITD and over-expression), Bcr-Abl fusion, extremely high white blood cell count variants, mixed lineage leukemia and Juvenile Myelomonocytic Leukemia (JMML) cells that are hyper-stimulated with exogenous GM-CSF. A luminescent based technique that individually measures protease activities associated with the proteasome complex in cultured cells (Promega, Cell-Based Proteasome-Glo Assay) was used to evaluate the mechanism of CFZ activity in these cells. Drug combination studies were carried out with etoposide, cytarabine, sorafenib and mefloquine using Chou and Talalay methodology. Target modulation, induction of apoptosis and the modulation of autophagy were evaluated by Western Blot analysis of cells treated with CFZ at defined time periods. Results Carfilzomib induced effective cytotoxicity in all leukemia cells tested (IC50 mean = 7 nM, range = 0.2 – 10 nM). Cell based proteasome assays confirmed the targeted and specific activity of CFZ in these cells. Infant leukemia cells with FLT3 over-expression were highly sensitive to CFZ followed by cells with ITD. Primary JMML cells that showed high growth stimulation with GM-CSF were also significantly affected by CFZ (IC50 = 0.2 nM). Although the extent of drug synergy varied between AML and ALL cells, CFZ synergized with all four agents (Combination Index (CI) mean = 0.47, range = 0.2 – 0.9). Induction of apoptosis by CFZ was evidenced by the increase in the active fragments of caspase 7 and 8 and PARP cleavage. CFZ also modulated autophagy by showing concentration regulated changes in p62 and LC3B. However, this effect appears to be restricted to AML cells. In vitro clonogenic assays using normal human CD34+ cells showed that even at 10 nM concentration, CFZ has no detectable inhibition on erythroid or myeloid colony formation. Discussion Carfilzomib is a potent, selective and irreversible inhibitor of the ubiquitin-proteasome pathway in cancer cells and has shown an acceptable toxicity profile in adult clinical trials. Our current data substantiates its potential as an active anti-leukemic agent in currently difficult to cure pediatric leukemia subtypes. Furthermore, we provide evidence on useful drug combinations and target modulation data to characterize the molecular mechanisms and biological correlates of distinct proteasome inhibitors in pediatric leukemia. This information provides key primary data for further in vivo studies and to design effective early phase clinical trials in the near future. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of the small molecule ARC39, a direct and specific inhibitor of acid sphingomyelinase in vitro

2020 ◽  
Vol 61 (6) ◽  
pp. 896-910 ◽  
Author(s):  
Eyad Naser ◽  
Stephanie Kadow ◽  
Fabian Schumacher ◽  
Zainelabdeen H. Mohamed ◽  
Christian Kappe ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Specific Inhibitor ◽  
Acid Sphingomyelinase ◽  
Intact Cells ◽  
Protein Levels ◽  
High Doses ◽  
Hydrolysis Of

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM’s catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

scholarly journals The inositol-requiring enzyme 1 (IRE1α) RNAse inhibitor, 4µ8C, is also a potent cellular antioxidant

2018 ◽  
Vol 475 (5) ◽  
pp. 923-929 ◽  
Author(s):  
Stanley M.H. Chan ◽  
Mark P. Lowe ◽  
Ashton Bernard ◽  
Alyson A. Miller ◽  
Terence P. Herbert
Keyword(s):  
Er Stress ◽  
Cultured Cells ◽  
Specific Inhibitor ◽  
Unfolded Protein ◽  
Ic50 Values ◽  
A Cell ◽  
Target Effect ◽  
The Unfolded Protein Response ◽  
Protein Response

Inositol-requiring enzyme 1 alpha (IRE1α) is an endoplasmic reticulum (ER)-transmembrane endonuclease that is activated in response to ER stress as part of the unfolded protein response (UPR). Chronic activation of the UPR has been implicated in the pathogenesis of many common diseases including diabetes, cancer, and neurological pathologies such as Huntington's and Alzheimer's disease. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (4µ8C) is widely used as a specific inhibitor of IRE1α ribonuclease activity (IC50 of 6.89 µM in cultured cells). However, in this paper, we demonstrate that 4µ8C acts as a potent reactive oxygen species (ROS) scavenger, both in a cell-free assay and in cultured cells, at concentrations lower than that widely used to inhibit IRE1α activity. In vitro we show that, 4µ8C effectively decreases xanthine/xanthine oxidase catalysed superoxide production with an IC50 of 0.2 µM whereas in cultured endothelial and clonal pancreatic β-cells, 4µ8C inhibits angiotensin II-induced ROS production with IC50 values of 1.92 and 0.29 µM, respectively. In light of this discovery, conclusions reached using 4µ8C as an inhibitor of IRE1α should be carefully evaluated. However, this unexpected off-target effect of 4µ8C may prove therapeutically advantageous for the treatment of pathologies that are thought to be caused by, or exacerbated by, both oxidative and ER stress such as endothelial dysfunction and/or diabetes.

scholarly journals The Proteasome Inhibitor Velcade Enhances rather than Reduces Disease in Mouse Hepatitis Coronavirus-Infected Mice

2010 ◽  
Vol 84 (15) ◽  
pp. 7880-7885 ◽  
Author(s):  
Matthijs Raaben ◽  
Guy C. M. Grinwis ◽  
Peter J. M. Rottier ◽  
Cornelis A. M. de Haan
Keyword(s):  
Anticancer Drug ◽  
Proteasome Inhibitor ◽  
Cultured Cells ◽  
Lethal Outcome ◽  
Attractive Candidate

ABSTRACT Many viruses, including coronaviruses (CoVs), depend on a functional cellular proteasome for efficient infection in vitro. Hence, the proteasome inhibitor Velcade (bortezomib), a clinically approved anticancer drug, shown in an accompanying study (M. Raaben et al., J. Virol. 84:7869-7879, 2010) to strongly inhibit mouse hepatitis CoV (MHV) infection in cultured cells, seemed an attractive candidate for testing its antiviral properties in vivo. Surprisingly, however, the drug did not reduce replication of the virus in mice. Rather, inhibition of the proteasome caused enhanced infection with lethal outcome, calling for caution when using this type of drug during infection.

scholarly journals Inhibition of p-glycoprotein does not increase the efficacy of proteasome inhibitors in multiple myeloma cells

2020 ◽  
Author(s):  
Rachel L. Mynott ◽  
Craig T. Wallington-Beddoe
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Specific Inhibitor ◽  
Myeloma Cell ◽  
Drug Transporter ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell ◽  
P Glycoprotein

AbstractThe aim of this study is to determine whether manipulation of the drug transporter P-glycoprotein improves the efficacy of proteasome inhibitors in multiple myeloma cells. P-glycoprotein is a well-known drug transporter that is associated with chemotherapy resistance in a number of cancers but its role in modulating the efficacy of proteasome inhibitors in multiple myeloma is not well understood. Research has shown that the second generation proteasome inhibitor carfilzomib is a substrate of P-glycoprotein and as such its efficacy may correlate with P-glycoprotein activity. In contrast to carfilzomib, research concerning the first-in-class proteasome inhibitor bortezomib is inconsistent with some reports suggesting that inhibition of P-glycoprotein increases bortezomib cytotoxicity in multiple myeloma cells whereas others have shown no effect. Through the mining of publicly available gene expression microarrays of patient bone marrow, we show that P-glycoprotein gene expression increases with the disease stages leading to multiple myeloma. However, RNA-seq on LP-1 cells treated with bortezomib or carfilzomib demonstrated minimal basal P-glycoprotein expression which did not increase with treatment. Moreover, only one (KMS-18) of nine multiple myeloma cell lines expressed P-glycoprotein, including RPMI-8226 cells that are resistant to bortezomib or carfilzomib. We hypothesised that by inhibiting P-glycoprotein, multiple myeloma cell sensitivity to proteasome inhibitors would increase, thus providing a potential approach to improving responses and reversing resistance to these agents. However, the sensitivity of multiple myeloma cells lines to proteasome inhibition was not enhanced by inhibition of P-glycoprotein with the specific inhibitor tariquidar. In addition, targeting glucosylceramide synthase with eliglustat did not inhibit P-glycoprotein activity and also did not improve proteasome inhibitor efficacy except at a high concentration. We conclude that P-glycoprotein is poorly expressed in multiple myeloma cells, its inhibition does not enhance the efficacy of proteasome inhibitors, and it is unlikely to be a useful avenue for further translational research.

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