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Scientific Reports Aug 2017Pancreatic cancer is a lethal disease with poor prognosis. Gemcitabine has been the first line systemic treatment for pancreatic cancer. However, the rapid development...
Pancreatic cancer is a lethal disease with poor prognosis. Gemcitabine has been the first line systemic treatment for pancreatic cancer. However, the rapid development of drug resistance has been a major hurdle in gemcitabine therapy leading to unsatisfactory patient outcomes. With the recent renewed understanding of glutamine metabolism involvement in drug resistance and immuno-response, we investigated the anti-tumor effect of a glutamine analog (6-diazo-5-oxo-L-norleucine) as an adjuvant treatment to sensitize chemoresistant pancreatic cancer cells. We demonstrate that disruption of glutamine metabolic pathways improves the efficacy of gemcitabine treatment. Such a disruption induces a cascade of events which impacts glycan biosynthesis through Hexosamine Biosynthesis Pathway (HBP), as well as cellular redox homeostasis, resulting in global changes in protein glycosylation, expression and functional effects. The proteome alterations induced in the resistant cancer cells and the secreted exosomes are intricately associated with the reduction in cell proliferation and the enhancement of cancer cell chemosensitivity. Proteins associated with EGFR signaling, including downstream AKT-mTOR pathways, MAPK pathway, as well as redox enzymes were downregulated in response to disruption of glutamine metabolic pathways.
Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Deoxycytidine; Diazooxonorleucine; Drug Resistance, Neoplasm; Drug Synergism; Glutamine; Humans; Metabolic Networks and Pathways; Oxidation-Reduction; Pancreatic Neoplasms; Proteomics; Gemcitabine
PubMed: 28801576
DOI: 10.1038/s41598-017-08436-6 -
Bioorganic & Medicinal Chemistry Letters Oct 2021Two distinct diazo precursors, imidazotetrazine and nitrous amide, were explored as promoieties in designing prodrugs of 6-diazo-5-oxo-l-norleucine (DON), a glutamine...
Two distinct diazo precursors, imidazotetrazine and nitrous amide, were explored as promoieties in designing prodrugs of 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist. As a model for an imidazotetrazine-based prodrug, we synthesized (S)-2-acetamido-6-(8-carbamoyl-4-oxoimidazo[5,1-d][1,2,3,5]tetrazin-3(4H)-yl)-5-oxohexanoic acid (4) containing the entire scaffold of temozolomide, a precursor of the DNA-methylating agent clinically approved for the treatment of glioblastoma multiforme. For a nitrous amide-based prodrug, we synthesized 2-acetamido-6-(((benzyloxy)carbonyl)(nitroso)amino)-5-oxohexanoic acid (5) containing a N-nitrosocarbamate group, which can be converted to a diazo moiety via a mechanism similar to that of streptozotocin, a clinically approved diazomethane-releasing drug containing an N-nitrosourea group. Preliminary characterization confirmed formation of N-acetyl DON (6), also known as duazomycin A, from compound 4 in a pH-dependent manner while compound 5 did not exhibit sufficient stability to allow further characterization. Taken together, our model studies suggest that further improvements are needed to translate this prodrug approach into glutamine antagonist-based therapy.
Topics: Diazooxonorleucine; Drug Design; Drug Stability; Glutamine; Molecular Structure; Prodrugs
PubMed: 34400301
DOI: 10.1016/j.bmcl.2021.128321 -
Journal of Medicinal Chemistry Apr 20196-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy; however, its therapeutic potential was hampered by its biodistribution and...
6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy; however, its therapeutic potential was hampered by its biodistribution and toxicity to normal tissues, specifically gastrointestinal (GI) tissues. To circumvent DON's toxicity, we synthesized a series of tumor-targeted DON prodrugs designed to circulate inert in plasma and preferentially activate over DON in tumor. Our best prodrug 6 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate) showed stability in plasma, liver, and intestinal homogenates yet was readily cleaved to DON in P493B lymphoma cells, exhibiting a 55-fold enhanced tumor cell-to-plasma ratio versus that of DON and resulting in a dose-dependent inhibition of cell proliferation. Using carboxylesterase 1 knockout mice that were shown to mimic human prodrug metabolism, systemic administration of 6 delivered 11-fold higher DON exposure to tumor (target tissue; AUC = 5.1 nmol h/g) versus GI tissues (toxicity tissue; AUC = 0.45 nmol h/g). In summary, these studies describe the discovery of a glutamine antagonist prodrug that provides selective tumor exposure.
Topics: Acetylation; Animals; Antineoplastic Agents; Area Under Curve; Carboxylic Ester Hydrolases; Cell Line, Tumor; Diazooxonorleucine; Drug Delivery Systems; Humans; Lysine; Mice; Mice, Knockout; Prodrugs; Swine
PubMed: 30892035
DOI: 10.1021/acs.jmedchem.8b02009 -
American Journal of Physiology. Cell... Jun 2003Nuclear, mitochondrial, and plasma membrane events associated with apoptosis were investigated in rat neutrophils cultivated for 3, 24, and 48 h in the absence or...
Nuclear, mitochondrial, and plasma membrane events associated with apoptosis were investigated in rat neutrophils cultivated for 3, 24, and 48 h in the absence or presence of glutamine (0.5, 1.0, and 2.0 mM). Condensation of chromatin was reduced after 24 or 48 h of culture in the presence of glutamine compared with its absence as assessed by Hoechst 33342 staining. The level of Escherichia coli phagocytosis in the presence of glutamine was markedly increased compared with the level achieved by cells cultured in the absence of glutamine. Annexin V binding to externalized phosphatidylserine was reduced in the presence of glutamine. Sensitive fluorochrome rhodamine 123, as determined by fluorescence-activated cell sorting and confocal microscopy, was used to monitor loss of the mitochondrial transmembrane potential. In the absence of glutamine, neutrophils exhibited a marked reduction in the uptake of rhodamine 123. In the presence of 1.0 or 2.0 mM glutamine, the uptake of rhodamine was 20 or 38% higher, respectively. Similar effect was found in human neutrophils by measuring DNA fragmentation and mitochondrial transmembrane potential. Therefore, glutamine protects from events associated with triggering and executing apoptosis in both rat and human neutrophils.
Topics: Animals; Apoptosis; Cells, Cultured; Chromatin; Diazooxonorleucine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutamine; Humans; Male; Membrane Potentials; Mitochondria; Neutrophils; Phagocytosis; Phosphatidylserines; Rats; Rats, Wistar
PubMed: 12529242
DOI: 10.1152/ajpcell.00224.2002 -
Chembiochem : a European Journal of... Apr 2022During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6-diazo-5-oxo-L-norleucine (DON) and one of alanine, the α/β hydrolase AzpM...
During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6-diazo-5-oxo-L-norleucine (DON) and one of alanine, the α/β hydrolase AzpM synthesizes the DON-DON dipeptide using DON tethered to the carrier protein AzpF (DON-AzpF). However, whether AzpM catalyzes the condensation of DON-AzpF with DON or DON-AzpF remains unclear. Here, to distinguish between these two condensation possibilities, the reaction catalyzed by AzpM was examined in vitro using a DON analogue, azaserine (AZS). We found that AzpM catalyzed the condensation between AZS-AzpF and DON-AzpF, but not between AZS-AzpF and DON. Possible reaction intermediates, DON-DON-AzpF and AZS-AZS-AzpF, were also detected during AzpM-catalyzed dipeptide formation from DON-AzpF and AZS-AzpF, respectively. From these results, we concluded that AzpM catalyzed the condensation of the two molecules of DON-AzpF and subsequent hydrolysis to produce DON-DON. Thus, AzpM is an unprecedented α/β hydrolase that catalyzes dipeptide synthesis from two molecules of a carrier protein-tethered amino acid.
Topics: Carrier Proteins; Diazooxonorleucine; Dipeptides; Hydrolases
PubMed: 35132756
DOI: 10.1002/cbic.202100700 -
Lloydia 1977
Review
Topics: Amines; Animals; Anti-Bacterial Agents; Azaserine; Azo Compounds; Carcinogens; Chemical Phenomena; Chemistry; Cycasin; Diazooxonorleucine; Hydrazines; Hydroxylamines; Nitrosamines; Pteridines; Pyrazoles; Streptozocin; Toxins, Biological
PubMed: 142890
DOI: No ID Found -
ELife Jun 2018Energy metabolism is essential for T cell function. However, how persistent antigenic stimulation affects T cell metabolism is unknown. Here, we report that long-term in...
Energy metabolism is essential for T cell function. However, how persistent antigenic stimulation affects T cell metabolism is unknown. Here, we report that long-term in vivo antigenic exposure induced a specific deficit in numerous metabolic enzymes. Accordingly, T cells exhibited low basal glycolytic flux and limited respiratory capacity. Strikingly, blockade of inhibitory receptor PD-1 stimulated the production of IFNγ in chronic T cells, but failed to shift their metabolism towards aerobic glycolysis, as observed in effector T cells. Instead, chronic T cells appeared to rely on oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) to produce ATP for IFNγ synthesis. Check-point blockade, however, increased mitochondrial production of superoxide and reduced viability and effector function. Thus, in the absence of a glycolytic switch, PD-1-mediated inhibition appears essential for limiting oxidative metabolism linked to effector function in chronic T cells, thereby promoting survival and functional fitness.
Topics: Adenosine Triphosphate; Animals; Antibodies, Monoclonal; Antimetabolites, Antineoplastic; B7-H1 Antigen; Cell Lineage; DNA-Binding Proteins; Diazooxonorleucine; Epoxy Compounds; Gene Expression Profiling; Gene Expression Regulation; Glycolysis; Interferon-gamma; Interleukin Receptor Common gamma Subunit; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Oligomycins; Oxidative Phosphorylation; Programmed Cell Death 1 Receptor; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes; Transplantation, Homologous
PubMed: 29911570
DOI: 10.7554/eLife.30938 -
The Journal of Clinical Investigation Jan 2020Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic...
Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA4, PD1, PDL1) has not been very successful against PDAC. The robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on the one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase 1 (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). In the current manuscript, we targeted this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-l-norleucine [DON]). Our results showed that DON decreased the self-renewal potential and metastatic ability of tumor cells. Further, treatment with DON decreased hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM and an increased infiltration of CD8+ T cells. Additionally, treatment with DON sensitized pancreatic tumors to anti-PD1 therapy, resulting in tumor regression and prolonged survival.
Topics: Animals; CD8-Positive T-Lymphocytes; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Diazooxonorleucine; Female; Hexosamines; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Proteins; Pancreatic Neoplasms; Programmed Cell Death 1 Receptor; Xenograft Model Antitumor Assays
PubMed: 31613799
DOI: 10.1172/JCI127515 -
Biochemical Society Transactions Nov 1992
Review
Topics: Animals; Biological Transport, Active; Diazooxonorleucine; Erythrocytes; Glutamine; Humans; Malaria, Falciparum; Plasmodium falciparum; Surface Properties
PubMed: 1487065
DOI: 10.1042/bst0200790 -
Physiological Reports Mar 2019Despite recent advances, acute respiratory distress syndrome (ARDS) remains a severe and often fatal disease for which there is no therapy able to reduce the underlying...
Despite recent advances, acute respiratory distress syndrome (ARDS) remains a severe and often fatal disease for which there is no therapy able to reduce the underlying excessive lung inflammation or enhance resolution of injury. Metabolic programming plays a critical role in regulating inflammatory responses. Due to their high metabolic needs, neutrophils, macrophages, and lymphocytes rely upon glutamine metabolism to support activation and function. Additionally, during times of physiologic stress, nearly all cells, including fibroblasts and epithelial cells, require glutamine metabolism. We hypothesized that inhibiting glutamine metabolism reduces lung inflammation and promotes resolution of acute lung injury. Lung injury was induced by instilling lipopolysaccharide (LPS) intratracheally. To inhibit glutamine metabolism, we administered a glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON) that binds to glutamine-utilizing enzymes and transporters, after injury was well established. Treatment with DON led to less lung injury, fewer lung neutrophils, lung inflammatory and interstitial macrophages, and lower levels of proinflammatory cytokines and chemokines at 5 and/or 7 days after injury. Additionally, DON led to earlier expression of the growth factor amphiregulin and more rapid recovery of LPS-induced weight loss. Thus, DON reduced lung inflammation and promoted resolution of injury. These data contribute to our understanding of how glutamine metabolism regulates lung inflammation and repair, and identifies a novel target for future therapies for ARDS and other inflammatory lung diseases.
Topics: Acute Lung Injury; Amphiregulin; Animals; Anti-Inflammatory Agents; Antimetabolites; Cytokines; Diazooxonorleucine; Disease Models, Animal; Glutamine; Inflammation Mediators; Lipopolysaccharides; Lung; Macrophages; Mice, Inbred C57BL; Neutrophil Infiltration; Pneumonia; Time Factors
PubMed: 30821123
DOI: 10.14814/phy2.14019