The Experts below are selected from a list of 1155 Experts worldwide ranked by ideXlab platform

Janusz Moryś - One of the best experts on this subject based on the ideXlab platform.

Craig M Crews - One of the best experts on this subject based on the ideXlab platform.

  • from Epoxomicin to carfilzomib chemistry biology and medical outcomes
    Natural Product Reports, 2013
    Co-Authors: Kyung Bo Kim, Craig M Crews
    Abstract:

    Covering: 1992 to 2012 The initial enthusiasm following the discovery of a pharmacologically active natural product is often fleeting due to the poor prospects for its ultimate clinical application. Despite this, the ever-changing landscape of modern biology has a constant need for molecular probes that can aid in our understanding of biological processes. After its initial discovery by Bristol-Myers Squibb as a microbial anti-tumor natural product, Epoxomicin was deemed unfit for development due to its peptide structure and potentially labile epoxyketone pharmacophore. Despite its drawbacks, Epoxomicin's pharmacophore was found to provide unprecedented selectivity for the proteasome. Epoxomicin also served as a scaffold for the generation of a synthetic tetrapeptide epoxyketone with improved activity, YU-101, which became the parent lead compound of carfilzomib (Kyprolis™), the recently approved therapeutic agent for multiple myeloma. In this era of rational drug design and high-throughput screening, the prospects for turning an active natural product into an approved therapy are often slim. However, by understanding the journey that began with the discovery of Epoxomicin and ended with the successful use of carfilzomib in the clinic, we may find new insights into the keys for success in natural product-based drug discovery.

  • targeted intracellular protein degradation induced by a small molecule en route to chemical proteomics
    Bioorganic & Medicinal Chemistry Letters, 2008
    Co-Authors: Ashley R Schneekloth, Mathieu Pucheault, Hyun Seop Tae, Craig M Crews
    Abstract:

    Abstract We have developed a heterobifunctional all-small molecule PROTAC (PROteolysis TArgeting Chimera) capable of inducing proteasomal degradation of the androgen receptor. This cell permeable PROTAC consists of a non-steroidal androgen receptor ligand (SARM) and the MDM2 ligand known as nutlin, connected by a PEG-based linker. The SARM–nutlin PROTAC recruits the androgen receptor to MDM2, which functions as an E3 ubiquitin ligase. This leads to the ubiquitination of the androgen receptor, and its subsequent degradation by the proteasome. Upon treatment of HeLa cells with 10 μM PROTAC for 7 h, we were able to observe a decrease in androgen receptor levels. This degradation is proteasome dependent, as it is mitigated in cells pre-treated with 10 μM Epoxomicin, a specific proteasome inhibitor. These results have implications for the potential study and treatment of various cancers with increased androgen receptor levels.

  • the selective proteasome inhibitors lactacystin and Epoxomicin can be used to either up or down regulate antigen presentation at nontoxic doses
    Journal of Immunology, 2000
    Co-Authors: Katrin Schwarz, Rita De Giuli, Gunter Schmidtke, Susanne Kostka, Maries Van Den Broek, Kyung Bo Kim, Craig M Crews, Regine Kraft, Marcus Groettrup
    Abstract:

    The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or Epoxomicin would affect Ag presentation. At 0.5-1 microM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89-168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or Epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5-1 microM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.

  • proteasome inhibition by the natural products Epoxomicin and dihydroeponemycin insights into specificity and potency
    Bioorganic & Medicinal Chemistry Letters, 1999
    Co-Authors: Kyung Bo Kim, Ny Sin, Jayhyuk Myung, Craig M Crews
    Abstract:

    While two structurally related epoxyketone-containing antitumor natural products, Epoxomicin and eponemycin, share the proteasome as a common intracellular target, they differ in their antiproliferative activity, proteasome subunit binding specificity, and rates of proteasome inhibition. As a first step towards understanding such differences and developing novel proteasome subunit-specific inhibitors, we report here the synthesis and characterization of Epoxomicin/dihydroeponemycin chimerae.

  • total synthesis of the potent proteasome inhibitor Epoxomicin a useful tool for understanding proteasome biology
    ChemInform, 1999
    Co-Authors: Ny Sin, Kyung Bo Kim, Mikael Elofsson, Lihao Meng, Hak Auth, Benjamin H B Kwok, Craig M Crews
    Abstract:

    Epoxomicin (1), a peptide alpha',beta'-epoxyketone isolated from the actinomycete strain No.Q996-17, possesses potent in vivo anti-tumor and anti-inflammatory activities. In this paper, we report the first syntheses of Epoxomicin, [3H]-Epoxomicin, and a biotinylated Epoxomicin analog as well as the absolute configuration of the epoxide stereocenter. The natural product and derivatives have permitted the first identification of the proteasome as the specific cellular target of Epoxomicin.

Sławomir Wójcik - One of the best experts on this subject based on the ideXlab platform.

Maria Angeles Mena - One of the best experts on this subject based on the ideXlab platform.

  • Epoxomicin and trehalose differential effects in cellular viability on control and HD human skin fibroblasts.
    2014
    Co-Authors: Maria Angeles Fernandez-estevez, Maria Jose Casarejos, Jose López Sendon, Juan Garcia Caldentey, Carolina Ruiz, Ana Gomez, Juan Perucho, Justo García De Yebenes, Maria Angeles Mena
    Abstract:

    (A) Dose-dependent effects of Epoxomicin in caspase-3 activation, an indicator of apoptosis. (B) Photomicrographs of activated caspase-3+ cells (green) and total nuclei stained with bis-benzimide (blue) after Epoxomicin and trehalose treatments. (Scale bar  = 20 µm). (C) Percent of activated caspase-3+ cells in control and HD fibroblasts after Epoxomicin and trehalose treatments. Values are expressed as the mean ± SD, n = 4 patients. Control cell number (mean per field) 31.87±1.124, n = 4. HD cell number (mean per field) 44.75±2.456, n = 4. The data of each patient was obtained using 4 replicates. Statistical analysis was performed by one-way ANOVA with repeated measures followed by Bonferroni multiple comparison test: *p

  • Proliferation and apoptosis of human fibroblasts with compound heterozygous CHIP mutations.
    2014
    Co-Authors: Maria Jose Casarejos, Carolina Ruiz, Ana Gomez, Juan Perucho, Justo García De Yebenes, Jose Luis López-sendón, Conceição Bettencourt, Peter Heutink, Patrizia Rizzu, Maria Angeles Mena
    Abstract:

    Trehalose protects from apoptosis and Epoxomicin-induced cell death in CHIP-mutant human fibroblasts. A) Photomicrographs of dose-dependent effects of Epoxomicin and trehalose pre-treatment 15 min before Epoxomicin for 24 h on the expression of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts (scale bar  = 30 µm). B) Percentage of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts treated with 0, 2 and 10 nM Epoxomicin for 24 h and trehalose 50 mM pre-treatment after 5 days in vitro. One hundred fifty to two hundred cells per coverslip cells were counted. C) Photomicrographs of dividing BrdU+ cells and total nuclei stained with bis-benzimide. (Scale bar  = 30 µm). D) Percentage of BrdU+ cells with respect to the total number of fibroblasts with 7 and 12 passages, respectively. Sixty to eighty cells were counted per coverslip to obtain the percentages of BrdU+ cells. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM. Values are the mean of two experiments of 6 independent cell dishes (pseudo-replicates, n = 6) of the patient group and in control group: the mean of the 3 different controls with 3 dishes of cells each one (n = 3). Statistical analysis was performed by Student's t-test. **p

  • CHIP, HSP70 expression and HDAC6 accumulation in control and CHIP-mutant fibroblasts.
    2014
    Co-Authors: Maria Jose Casarejos, Carolina Ruiz, Ana Gomez, Juan Perucho, Justo García De Yebenes, Jose Luis López-sendón, Conceição Bettencourt, Peter Heutink, Patrizia Rizzu, Maria Angeles Mena
    Abstract:

    Effects of Epoxomicin and trehalose on chaperone proteins. After 3 days in vitro, the cells were pre-treated with trehalose (50 mM) for 15 minutes followed by addition of Epoxomicin (10 nM) or solvent for another 24 h. A) Representative image of control and CHIP-mutant fibroblasts immunostained with antibody to HDAC6 (a substrate of CHIP). B) Representative bands and quantification of CHIP and C) HSP70 Western blot. β-actin was used as an equal loading of proteins. In 1A, a total of 120 cells in 5 coverslips were analyzed in both groups. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM values. Values of 1B and 1C correspond to the mean of two experiments with 4 independent cell dishes of 3 different controls (n = 3). In the CHIP-mutant case six independent dishes of one patient (pseudo-replicates, n = 6). Statistical analysis was performed by Student's t-test. *p

  • Epoxomicin increases ROS levels, which are reduced by trehalose in HD firboblasts.
    2014
    Co-Authors: Maria Angeles Fernandez-estevez, Maria Jose Casarejos, Jose López Sendon, Juan Garcia Caldentey, Carolina Ruiz, Ana Gomez, Juan Perucho, Justo García De Yebenes, Maria Angeles Mena
    Abstract:

    (A) 2′, 7′–dichlorofluorescin (DCF) immunocytochemistry (green) and total nuclei stained with bis-benzimide (blue) in control and HD fibroblasts and (B) percentage of DCF positive cells respect to the total number. (Scale bar  = 20 µm). Values are expressed as the mean ± SD, n = 4 patients. Control cell number (mean per field) 28.83±0.9280, n = 4. HD cell number (mean per field) 46.40±5.247, n = 4. The data of each patient was obtained using 4 replicates. Statistical analysis was performed by one-way ANOVA with repeated measures followed by Bonferroni multiple comparison test: ***p

  • UPS activity in control and CHIP-mutant fibroblasts.
    2014
    Co-Authors: Maria Jose Casarejos, Carolina Ruiz, Ana Gomez, Juan Perucho, Justo García De Yebenes, Jose Luis López-sendón, Conceição Bettencourt, Peter Heutink, Patrizia Rizzu, Maria Angeles Mena
    Abstract:

    Trehalose protects from ubiquitin protein accumulation by Epoxomicin in CHIP-mutant and control human fibroblasts. A) Differential UPS activity in control and CHIP mutant fibroblasts. B) Accumulation of poly-ubiquitinated proteins and C) their corresponding densitometry analysis after 24 h of treatment corresponds to the whole smear. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM. Every value in 4A corresponds to the mean of two experiments: the mean of 4 independent dishes of cells of 3 different controls (n = 3) and for the CHIP-mutant group six independent dishes of one only patient (pseudo-replicates, n = 6). In 4C values are the mean of two independent experiments, with 6 cell dishes (pseudo-replicates, n = 6) of the patient group and, in the control group, the mean of 3 different dishes of cells for each one of the 3 controls (n = 3). Statistical analysis was performed by Student's t-test. *p

Maria Jose Casarejos - One of the best experts on this subject based on the ideXlab platform.

  • Trehalose Improves Human Fibroblast Deficits in a New CHIP-Mutation Related Ataxia
    2016
    Co-Authors: Maria Jose Casarejos, Carolina Ruiz, Juan Perucho, Peter Heutink, Jose Luis López-sendón, Justo Garcı́a De Yébenes, Patrizia Rizzu
    Abstract:

    In this work we investigate the role of CHIP in a new CHIP-mutation related ataxia and the therapeutic potential of trehalose. The patient’s fibroblasts with a new form of hereditary ataxia, related to STUB1 gene (CHIP) mutations, and three age and sex-matched controls were treated with Epoxomicin and trehalose. The effects on cell death, protein misfolding and proteostasis were evaluated. Recent studies have revealed that mutations in STUB-1 gene lead to a growing list of molecular defects as deregulation of protein quality, inhibition of proteasome, cell death, decreased autophagy and alteration in CHIP and HSP70 levels. In this CHIP-mutant patient fibroblasts the inhibition of proteasome with Epoxomicin induced severe pathophysiological age-associated changes, cell death and protein ubiquitination. Additionally, treatment with Epoxomicin produced a dose-dependent increase in the number of cleaved caspase-3 positive cells. However, co-treatment with trehalose, a disaccharide of glucose present in a wide variety of organisms and known as a autophagy enhancer, reduced these pathological events. Trehalose application also increased CHIP and HSP70 expression and GSH free radical levels. Furthermore, trehalose augmented macro and chaperone mediated autophagy (CMA), rising the levels of LC3, LAMP2, CD63 and increasing the expression of Beclin-1 and Atg5-Atg12. Trehalose treatment in addition increased the percentage of immunoreactive cells to HSC70 and LAMP2 and reduced the autophagic substrate, p62. Although this is a

  • Epoxomicin and trehalose differential effects in cellular viability on control and HD human skin fibroblasts.
    2014
    Co-Authors: Maria Angeles Fernandez-estevez, Maria Jose Casarejos, Jose López Sendon, Juan Garcia Caldentey, Carolina Ruiz, Ana Gomez, Juan Perucho, Justo García De Yebenes, Maria Angeles Mena
    Abstract:

    (A) Dose-dependent effects of Epoxomicin in caspase-3 activation, an indicator of apoptosis. (B) Photomicrographs of activated caspase-3+ cells (green) and total nuclei stained with bis-benzimide (blue) after Epoxomicin and trehalose treatments. (Scale bar  = 20 µm). (C) Percent of activated caspase-3+ cells in control and HD fibroblasts after Epoxomicin and trehalose treatments. Values are expressed as the mean ± SD, n = 4 patients. Control cell number (mean per field) 31.87±1.124, n = 4. HD cell number (mean per field) 44.75±2.456, n = 4. The data of each patient was obtained using 4 replicates. Statistical analysis was performed by one-way ANOVA with repeated measures followed by Bonferroni multiple comparison test: *p

  • Trehalose Reverses Cell Malfunction in Fibroblasts from Normal and Huntington’s Disease Patients Caused by Proteosome Inhibition
    2014
    Co-Authors: Maria Angeles Fern, Maria Jose Casarejos, Juan Garcia Caldentey, Carolina Ruiz, Ana Gomez, Juan Perucho, Jose López Sendon, Justo Garcı́a De Yebenes
    Abstract:

    Huntington’s disease (HD) is a neurodegenerative disorder characterized by progressive motor, cognitive and psychiatric deficits, associated with predominant loss of striatal neurons and is caused by polyglutamine expansion in the huntingtin protein. Mutant huntingtin protein and its fragments are resistant to protein degradation and produce a blockade of the ubiquitin proteasome system (UPS). In HD models, the proteasome inhibitor Epoxomicin aggravates protein accumulation and the inductor of autophagy, trehalose, diminishes it. We have investigated the effects of Epoxomicin and trehalose in skin fibroblasts of control and HD patients. Untreated HD fibroblasts have increased the levels of ubiquitinized proteins and higher levels of reactive oxygen species (ROS), huntingtin and the autophagy marker LAMP2A. Baseline replication rates were higher in HD than in controls fibroblasts but that was reverted after 12 passages. Epoxomicin increases the activated caspase-3, HSP70, huntingtin, ubiquitinated proteins and ROS levels in both HD and controls. Treatment with trehalose counteracts the increase in ROS, ubiquitinated proteins, huntingtin and activated caspase-3 levels induced by Epoxomicin, and also increases the LC3 levels more in HD fibroblast than controls. These results suggest that trehalose could revert protein processing abnormalities in patients with Huntington’s Disease

  • Proliferation and apoptosis of human fibroblasts with compound heterozygous CHIP mutations.
    2014
    Co-Authors: Maria Jose Casarejos, Carolina Ruiz, Ana Gomez, Juan Perucho, Justo García De Yebenes, Jose Luis López-sendón, Conceição Bettencourt, Peter Heutink, Patrizia Rizzu, Maria Angeles Mena
    Abstract:

    Trehalose protects from apoptosis and Epoxomicin-induced cell death in CHIP-mutant human fibroblasts. A) Photomicrographs of dose-dependent effects of Epoxomicin and trehalose pre-treatment 15 min before Epoxomicin for 24 h on the expression of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts (scale bar  = 30 µm). B) Percentage of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts treated with 0, 2 and 10 nM Epoxomicin for 24 h and trehalose 50 mM pre-treatment after 5 days in vitro. One hundred fifty to two hundred cells per coverslip cells were counted. C) Photomicrographs of dividing BrdU+ cells and total nuclei stained with bis-benzimide. (Scale bar  = 30 µm). D) Percentage of BrdU+ cells with respect to the total number of fibroblasts with 7 and 12 passages, respectively. Sixty to eighty cells were counted per coverslip to obtain the percentages of BrdU+ cells. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM. Values are the mean of two experiments of 6 independent cell dishes (pseudo-replicates, n = 6) of the patient group and in control group: the mean of the 3 different controls with 3 dishes of cells each one (n = 3). Statistical analysis was performed by Student's t-test. **p

  • CHIP, HSP70 expression and HDAC6 accumulation in control and CHIP-mutant fibroblasts.
    2014
    Co-Authors: Maria Jose Casarejos, Carolina Ruiz, Ana Gomez, Juan Perucho, Justo García De Yebenes, Jose Luis López-sendón, Conceição Bettencourt, Peter Heutink, Patrizia Rizzu, Maria Angeles Mena
    Abstract:

    Effects of Epoxomicin and trehalose on chaperone proteins. After 3 days in vitro, the cells were pre-treated with trehalose (50 mM) for 15 minutes followed by addition of Epoxomicin (10 nM) or solvent for another 24 h. A) Representative image of control and CHIP-mutant fibroblasts immunostained with antibody to HDAC6 (a substrate of CHIP). B) Representative bands and quantification of CHIP and C) HSP70 Western blot. β-actin was used as an equal loading of proteins. In 1A, a total of 120 cells in 5 coverslips were analyzed in both groups. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM values. Values of 1B and 1C correspond to the mean of two experiments with 4 independent cell dishes of 3 different controls (n = 3). In the CHIP-mutant case six independent dishes of one patient (pseudo-replicates, n = 6). Statistical analysis was performed by Student's t-test. *p