| 1. |
- D'Arcy, Padraig, et al.
(författare)
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Deubiquitinase inhibition as a cancer therapeutic strategy
- 2015
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Ingår i: Pharmacology and Therapeutics. - : Elsevier. - 0163-7258 .- 1879-016X. ; 147, s. 32-54
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Forskningsöversikt (refereegranskat)abstract
- The ubiquitin proteasome system (UPS) is the main system for controlled protein degradation and a key regulator of fundamental cellular processes. The dependency of cancer cells on a functioning UPS has made this an attractive target for development of drugs that show selectivity for tumor cells. Deubiquitinases (DUBs, ubiquitin isopeptidases) are components of the UPS that catalyze the removal of ubiquitin moieties from target proteins or polyubiquitin chains, resulting in altered signaling or changes in protein stability. A number of DUBs regulate processes associated with cell proliferation and apoptosis, and as such represent candidate targets for cancer therapeutics. The majority of DUBs are cysteine proteases and are likely to be more "druggable" than E3 ligases. Cysteine residues in the active sites of DUBs are expected to be reactive to various electrophiles. Various compounds containing α,β-unsaturated ketones have indeed been demonstrated to inhibit cellular DUB activity. Inhibition of proteasomal cysteine DUB enzymes (i.e. USP14 and UCHL5) can be predicted to be particularly cytotoxic to cancer cells as it leads to blocking of proteasome function and accumulation of proteasomal substrates. We here provide an overall review of DUBs relevant to cancer and of various small molecules which have been demonstrated to inhibit DUB activity.
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| 2. |
- Fryknäs, Mårten, et al.
(författare)
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Screening for phenotype selective activity in multidrug resistant cells identifies a novel tubulin active agent insensitive to common forms of cancer drug resistance
- 2013
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 13, s. 374-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Drug resistance is a common cause of treatment failure in cancer patients and encompasses a multitude of different mechanisms. The aim of the present study was to identify drugs effective on multidrug resistant cells. Methods: The RPMI 8226 myeloma cell line and its multidrug resistant subline 8226/Dox40 was screened for cytotoxicity in response to 3,000 chemically diverse compounds using a fluorometric cytotoxicity assay (FMCA). Follow-up profiling was subsequently performed using various cellular and biochemical assays. Results: One compound, designated VLX40, demonstrated a higher activity against 8226/Dox40 cells compared to its parental counterpart. VLX40 induced delayed cell death with apoptotic features. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. Strong connections to tubulin inhibitors and microtubule cytoskeleton were retrieved. The mechanistic hypothesis of VLX40 acting as a tubulin inhibitor was confirmed by direct measurements of interaction with tubulin polymerization using a biochemical assay and supported by demonstration of G2/M cell cycle arrest. When tested against a broad panel of primary cultures of patient tumor cells (PCPTC) representing different forms of leukemia and solid tumors, VLX40 displayed high activity against both myeloid and lymphoid leukemias in contrast to the reference compound vincristine to which myeloid blast cells are often insensitive. Significant in vivo activity was confirmed in myeloid U-937 cells implanted subcutaneously in mice using the hollow fiber model. Conclusions: The results indicate that VLX40 may be a useful prototype for development of novel tubulin active agents that are insensitive to common mechanisms of cancer drug resistance.
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| 3. |
- Mazurkiewicz, Magdalena, et al.
(författare)
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Acute lymphoblastic leukemia cells are sensitive to disturbances in protein homeostasis induced by proteasome deubiquitinase inhibition
- 2017
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Ingår i: Oncotarget. - : IMPACT JOURNALS LLC. - 1949-2553. ; 8:13, s. 21115-21127
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Tidskriftsartikel (refereegranskat)abstract
- The non-genotoxic nature of proteasome inhibition makes it an attractive therapeutic option for the treatment of pediatric malignancies. We recently described the small molecule VLX1570 as an inhibitor of proteasome deubiquitinase (DUB) activity that induces proteotoxic stress and apoptosis in cancer cells. Here we show that acute lymphoblastic leukemia (ALL) cells are highly sensitive to treatment with VLX1570, resulting in the accumulation of polyubiquitinated proteasome substrates and loss of cell viability. VLX1570 treatment increased the levels of a number of proteins, including the chaperone HSP70B , the oxidative stress marker heme oxygenase-1 (HO-1) and the cell cycle regulator p21(Cip1). Unexpectedly, polybiquitin accumulation was found to be uncoupled from ER stress in ALL cells. Thus, increased phosphorylation of eIF2a occurred only at supra-pharmacological VLX1570 concentrations and did not correlate with polybiquitin accumulation. Total cellular protein synthesis was found to decrease in the absence of eIF2a phosphorylation. Furthermore, ISRIB (Integrated Stress Response inhibitor) did not overcome the inhibition of protein synthesis. We finally show that VLX1570 can be combined with L-asparaginase for additive or synergistic antiproliferative effects on ALL cells. We conclude that ALL cells are highly sensitive to the proteasome DUB inhibitor VLX1570 suggesting a novel therapeutic option for this disease.
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| 4. |
- Selvaraju, Karthik, et al.
(författare)
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Inhibition of proteasome deubiquitinase activity : a strategy to overcome resistance to conventional proteasome inhibitors?
- 2015
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Ingår i: Drug resistance updates. - : Elsevier BV. - 1368-7646 .- 1532-2084. ; 21-22, s. 20-29
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Forskningsöversikt (refereegranskat)abstract
- Although more traditionally associated with degradation and maintenance of protein homeostasis, the ubiquitin-proteasome system (UPS) has emerged as a critical component in the regulation of cancer cell growth and survival. The development of inhibitors that block the proteolytic activities of the proteasome have highlighted its suitability as a bona fide anti-cancer drug target. However, key determinants including the development of drug resistance and dose-limiting toxicity call for the identification of alternative components of the UPS for novel drug targeting. Recently the deubiquitinases (DUBS), a diverse family of enzymes that catalyze ubiquitin removal, have attracted significant interest as targets for the development of next generation UPS inhibitors. In particular, pharmacological inhibition of the proteasomal cysteine DUBs (i.e., USP14 and UCHL5) has been shown to be particularly cytotoxic to cancer cells and inhibit tumour growth in several in vivo models. In the current review we focus on the modes of action of proteasome DUB inhibitors and discus the potential of DUB inhibitors to circumvent acquired drug resistance and provide a therapeutic option for the treatment of cancer.
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| 5. |
- Shukla, Neerav, et al.
(författare)
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Proteasome Addiction Defined in Ewing Sarcoma Is Effectively Targeted by a Novel Class of 19S Proteasome Inhibitors
- 2016
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Ingår i: Cancer Research. - : AMER ASSOC CANCER RESEARCH. - 0008-5472 .- 1538-7445. ; 76:15, s. 4525-4534
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Tidskriftsartikel (refereegranskat)abstract
- Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. (C) 2016 AACR.
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| 6. |
- Sun, Chao, et al.
(författare)
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Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:10, s. e108839-
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Tidskriftsartikel (refereegranskat)abstract
- Inhibitors of the catalytic activity of the 20S proteasome are cytotoxic to tumor cells and are currently in clinical use for treatment of multiple myeloma, whilst the deubiquitinase activity associated with the 19S regulatory subunit of the proteasome is also a valid target for anti-cancer drugs. The mechanisms underlying the therapeutic efficacy of these drugs and their selective toxicity towards cancer cells are not known. Here, we show that increasing the cellular levels of proteasome substrates using an inhibitor of Sec61-mediated protein translocation significantly increases the extent of apoptosis that is induced by inhibition of proteasomal deubiquitinase activity in both cancer derived and non-transformed cell lines. Our results suggest that increased generation of misfolded proteasome substrates may contribute to the mechanism(s) underlying the increased sensitivity of tumor cells to inhibitors of the ubiquitin-proteasome system.
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| 7. |
- Wang, Xin, et al.
(författare)
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Synthesis and Evaluation of Derivatives of the Proteasome Deubiquitinase Inhibitor b-AP15
- 2015
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Ingår i: Chemical Biology and Drug Design. - : Wiley. - 1747-0277 .- 1747-0285. ; 86:5, s. 1036-1048
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Tidskriftsartikel (refereegranskat)abstract
- The ubiquitin-proteasome system (UPS) is increasingly recognized as a therapeutic target for the development of anticancer therapies. The success of the 20S proteasome core particle (20S CP) inhibitor bortezomib in the clinical management of multiple myeloma has raised the possibility of identifying other UPS components for therapeutic intervention. We previously identified the small molecule b-AP15 as an inhibitor of 19S proteasome deubiquitinase (DUB) activity. Building upon our previous data, we performed a structure-activity relationship (SAR) study on b-AP15 and identified VLX1570 as an analog with promising properties, including enhanced potency and improved solubility in aqueous solution. In silico modeling was consistent with interaction of VLX1570 with key cysteine residues located at the active sites of the proteasome DUBs USP14 and UCHL5. VLX1570 was found to inhibit proteasome deubiquitinase activity in vitro in a manner consistent with competitive inhibition. Furthermore, using active-site-directed probes, VLX1570 also inhibited proteasome DUB activity in exposed cells. Importantly, VLX1570 did not show inhibitory activity on a panel of recombinant non-proteasome DUBs, on recombinant kinases, or on caspase-3 activity, suggesting that VLX1570 is not an overtly reactive general enzyme inhibitor. Taken together, our data shows the chemical and biological properties of VLX1570 as an optimized proteasome DUB inhibitor.
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| 8. |
- Wang, Xin, et al.
(författare)
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The 19S Deubiquitinase Inhibitor b-AP15 Is Enriched in Cells and Elicits Rapid Commitment to Cell Death
- 2014
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Ingår i: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0026-895X .- 1521-0111. ; 85:6, s. 932-945
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Tidskriftsartikel (refereegranskat)abstract
- b-AP15 [(3E, 5E)-3,5-bis[(4-nitrophenyl) methylidene]-1-(prop-2enoyl) piperidin-4-one] is a small molecule inhibitor of the ubiquitin specific peptidase (USP) 14/ubiquitin carboxyl-terminal hydrolase (UCH) L5 deubiquitinases of the 19S proteasome that shows antitumor activity in a number of tumor models, including multiple myeloma. b-AP15 contains an alpha,beta-unsaturated carbonyl unit that is likely to react with intracellular nucleophiles such as cysteine thiolates by Michael addition. We found that binding of b-AP15 to USP14 is partially reversible, and that inhibition of proteasome function is reversible in cells. Despite reversible binding, tumor cells are rapidly committed to apoptosis/cell death after exposure to b-AP15. We show that b-AP15 is rapidly taken up from the medium and enriched in cells. Enrichment provides an explanation of the stronger potency of the compound in cellular assays compared with in vitro biochemical assays. Cellular uptake was impaired by 30-minute pretreatment of cells with low concentrations of N-ethylmaleimide (10 mu M), suggesting that enrichment was thiol dependent. We report that in addition to inhibition of deubiquitinases, b-AP15 inhibits the selenoprotein thioredoxin reductase (TrxR). Whereas proteasome inhibition was closely associated with cell death induction, inhibition of TrxR was not. TrxR inhibition is, however, likely to contribute to triggering of oxidative stress observed with b-AP15. Furthermore, we present structure-activity, in vivo pharmacokinetic, and hepatocyte metabolism data for b-AP15. We conclude that the strong enrichment of b-AP15 in cells and a rapid commitment to apoptosis/cell death are factors that likely contribute to the strong antitumor activity of this compound.
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| 9. |
- Wang, Xin, et al.
(författare)
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The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells
- 2016
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 6, s. 1-14
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Tidskriftsartikel (refereegranskat)abstract
- Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity currently in clinical trials for relapsed multiple myeloma. Here we show that VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Exposure of multiple myeloma cells to VLX1570 resulted in thermostabilization of USP14 at therapeutically relevant concentrations. Transient knockdown of USP14 or UCHL5 expression by electroporation of siRNA reduced the viability of multiple myeloma cells. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response. Sensitivity to VLX1570 was moderately affected by altered drug uptake, but was unaffected by overexpression of BCL2-family proteins or inhibitors of caspase activity. Finally, treatment with VLX1570 was found to lead to extended survival in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.
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