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Molecular Cancer Therapeutics Sep 2018The broadly active glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) has been studied for 60 years as a potential anticancer therapeutic. Clinical studies of DON in... (Review)
Review
The broadly active glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) has been studied for 60 years as a potential anticancer therapeutic. Clinical studies of DON in the 1950s using low daily doses suggested antitumor activity, but later phase I and II trials of DON given intermittently at high doses were hampered by dose-limiting nausea and vomiting. Further clinical development of DON was abandoned. Recently, the recognition that multiple tumor types are glutamine-dependent has renewed interest in metabolic inhibitors such as DON. Here, we describe the prior experience with DON in humans. Evaluation of past studies suggests that the major impediments to successful clinical use included unacceptable gastrointestinal (GI) toxicities, inappropriate dosing schedules for a metabolic inhibitor, and lack of targeted patient selection. To circumvent GI toxicity, prodrug strategies for DON have been developed to enhance delivery of active compound to tumor tissues, including the CNS. When these prodrugs are administered in a low daily dosing regimen, appropriate for metabolic inhibition, they are robustly effective without significant toxicity. Patients whose tumors have genetic, metabolic, or imaging biomarker evidence of glutamine dependence should be prioritized as candidates for future clinical evaluations of novel DON prodrugs, given either as monotherapy or in rationally directed pharmacologic combinations. .
Topics: Animals; Antimetabolites, Antineoplastic; Diazooxonorleucine; Glutamine; Humans; Molecular Structure; Nausea; Neoplasms; Prodrugs; Vomiting
PubMed: 30181331
DOI: 10.1158/1535-7163.MCT-17-1148 -
Biochemical Pharmacology Oct 2018Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates...
Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates the function of human umbilical vein ECs, it is not known whether these findings extend to human ECs beyond the fetal circulation. Furthermore, the molecular mechanism by which GLS1 regulates EC function is not defined. In this study, we show that the absence of glutamine in the culture media or the inhibition of GLS1 activity or expression blocked the proliferation and migration of ECs derived from the human umbilical vein, the human aorta, and the human microvasculature. GLS1 inhibition arrested ECs in the G/G phase of the cell cycle and this was associated with a significant decline in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer improved the proliferative, but not the migratory, response of GLS1-inhibited ECs. Glutamine deprivation or GLS1 inhibition also stimulated the production of reactive oxygen species and this was associated with a marked decline in heme oxygenase-1 (HO-1) expression. GLS1 inhibition also sensitized ECs to the cytotoxic effect of hydrogen peroxide and this was prevented by the overexpression of HO-1. In conclusion, the metabolism of glutamine by GLS1 promotes human EC proliferation, migration, and survival irrespective of the vascular source. While cyclin A contributes to the proliferative action of GLS1, HO-1 mediates its pro-survival effect. These results identify GLS1 as a promising therapeutic target in treating diseases associated with aberrant EC proliferation, migration, and viability.
Topics: Aorta; Benzeneacetamides; Cell Movement; Cell Proliferation; Cell Survival; Cyclin A; Diazooxonorleucine; Endothelial Cells; Gene Expression Regulation, Enzymologic; Glutaminase; Glutamine; Heme Oxygenase-1; Humans; RNA Interference; Thiadiazoles; Veins
PubMed: 30144404
DOI: 10.1016/j.bcp.2018.08.032 -
Neuropsychopharmacology : Official... Mar 2019There are a number of clinically effective treatments for stress-associated psychiatric diseases, including major depressive disorder (MDD). Nonetheless, many patients...
There are a number of clinically effective treatments for stress-associated psychiatric diseases, including major depressive disorder (MDD). Nonetheless, many patients exhibit resistance to first-line interventions calling for novel interventions based on pathological mechanisms. Accumulating evidence implicates altered glutamate signaling in MDD pathophysiology, suggesting that modulation of glutamate signaling cascades may offer novel therapeutic potential. Here we report that JHU-083, our recently developed prodrug of the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) ameliorates social avoidance and anhedonia-like behaviors in mice subjected to chronic social defeat stress (CSDS). JHU-083 normalized CSDS-induced increases in glutaminase activity specifically in microglia-enriched CD11b cells isolated from the prefrontal cortex and hippocampus. JHU-083 treatment also reverses the CSDS-induced inflammatory activation of CD11b cells. These results support the importance of altered glutamate signaling in the behavioral abnormalities observed in the CSDS model, and identify glutaminase in microglia-enriched CD11b cells as a pharmacotherapeutic target implicated in the pathophysiology of stress-associated psychiatric conditions such as MDD.
Topics: Animals; Behavior, Animal; CD11b Antigen; Depression; Diazooxonorleucine; Disease Models, Animal; Glutaminase; Hippocampus; Inflammation; Male; Mice; Mice, Inbred C57BL; Prefrontal Cortex; Prodrugs; Signal Transduction; Stress, Psychological
PubMed: 30127344
DOI: 10.1038/s41386-018-0177-7 -
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 -
Scientific Reports May 2018Bacteria in nature often reside in differentiated communities termed biofilms, which are an active interphase between uni-cellular and multicellular life states for...
Bacteria in nature often reside in differentiated communities termed biofilms, which are an active interphase between uni-cellular and multicellular life states for bacteria. Here we demonstrate that the development of B. subtilis biofilms is dependent on the use of glutamine or glutamate as a nitrogen source. We show a differential metabolic requirement within the biofilm; while glutamine is necessary for the dividing cells at the edges, the inner cell mass utilizes lactic acid. Our results indicate that biofilm cells preserve a short-term memory of glutamate metabolism. Finally, we establish that drugs that target glutamine and glutamate utilization restrict biofilm development. Overall, our work reveals a spatial regulation of nitrogen and carbon metabolism within the biofilm, which contributes to the fitness of bacterial complex communities. This acquired metabolic division of labor within biofilm can serve as a target for novel anti-biofilm chemotherapies.
Topics: Aminooxyacetic Acid; Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Biofilms; Carbon; Diazooxonorleucine; Gene Expression Regulation, Bacterial; Glutamic Acid; Glutamine; Mutant Proteins; Nitrogen; Repressor Proteins
PubMed: 29740028
DOI: 10.1038/s41598-018-25401-z -
The Laryngoscope Feb 2018Management of laryngotracheal stenosis (LTS) remains primarily surgical, with a critical need to identify targets for adjuvant therapy. Laryngotracheal stenosis scar...
OBJECTIVE
Management of laryngotracheal stenosis (LTS) remains primarily surgical, with a critical need to identify targets for adjuvant therapy. Laryngotracheal stenosis scar fibroblasts exhibit a profibrotic phenotype with distinct metabolic shifts, including an increased glycolysis/oxidative phosphorylation ratio. This study examines the effects of the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) on collagen production, gene expression, proliferation, and metabolism of human LTS-derived fibroblasts in vitro.
METHOD
Paired normal and scar-derived fibroblasts isolated from subglottic and proximal tracheal tissue in patients with iatrogenic laryngotracheal stenosis (iLTS) were cultured. Proliferation rate, gene expression, protein production, and cellular metabolism were assessed in two conditions: 1) fibroblast growth medium, and 2) fibroblast growth medium with 1 × 10 M DON.
RESULTS
DON treatment reduced cellular proliferation rate (n = 7, P = 0.0150). Expression of genes collagen 1 and collagen 3 both were reduced (n = 7, P = 0.0102, 0.0143, respectively). Soluble collagen production decreased (n = 7, P = 0.0056). As measured by the rate of extracellular acidification, glycolysis and glycolytic capacity decreased (n = 7, P = 0.0082, 0.0003, respectively). adenosine triphosphate (ATP) production and basal respiration decreased (n = 7, P = 0.0045, 0.0258, respectively), determined by measuring the cellular rate of oxygen consumption.
CONCLUSION
The glutamine antagonist DON reverses profibrotic changes by inhibiting both glycolysis and oxidative phosphorylation in iLTS scar fibroblasts. In contrast to untreated iLTS scar fibroblasts, collagen gene expression, protein production, metabolic rate, and proliferation were significantly reduced. These results suggest DON and/or its derivatives as strong candidates for adjuvant therapy in the management of iatrogenic laryngotracheal stenosis. Enzymes involved in glutamine metabolism inhibited by DON offer targets for future investigation.
LEVEL OF EVIDENCE
NA. Laryngoscope, 128:E59-E67, 2018.
Topics: Adult; Aged; Antimetabolites, Antineoplastic; Cell Culture Techniques; Cell Proliferation; Cicatrix; Collagen; Diazooxonorleucine; Female; Fibroblasts; Fibrosis; Gene Expression; Glycolysis; Humans; Iatrogenic Disease; Laryngostenosis; Male; Middle Aged; Oxygen Consumption; Real-Time Polymerase Chain Reaction; Tracheal Stenosis; Young Adult
PubMed: 28940431
DOI: 10.1002/lary.26893 -
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 -
Journal of Medicinal Chemistry Aug 2017Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders...
Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.
Topics: Aminocaproates; Animals; Azo Compounds; Blood; Brain; Diazooxonorleucine; Drug Stability; Female; Glutamic Acid; Glutaminase; HIV Infections; Humans; Male; Mice, Inbred C57BL; Neurocognitive Disorders; Nootropic Agents; Prodrugs; Swine; Viral Load
PubMed: 28759224
DOI: 10.1021/acs.jmedchem.7b00966 -
Virology Aug 2017Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that...
Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that is partially prevented by treatment with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist (Potter et al., J Neurovirol 21:159, 2015). To determine the mechanism(s) of protection, lymph node and central nervous system (CNS) tissues from SINV-infected mice treated daily for 1 week with low (0.3mg/kg) or high (0.6mg/kg) dose DON were examined. DON treatment suppressed lymphocyte proliferation in cervical lymph nodes resulting in reduced CNS immune cell infiltration, inflammation, and cell death compared to untreated SINV-infected mice. Production of SINV-specific antibody and interferon-gamma were also impaired by DON treatment with a delay in virus clearance. Cessation of treatment allowed activation of the antiviral immune response and viral clearance, but revived CNS pathology, demonstrating the ability of the immune response to mediate both CNS damage and virus clearance.
Topics: Alphavirus Infections; Animals; Antiviral Agents; Diazooxonorleucine; Encephalomyelitis; Glutamine; Humans; Interferon-gamma; Male; Mice; Mice, Inbred C57BL; Sindbis Virus
PubMed: 28531865
DOI: 10.1016/j.virol.2017.05.013 -
Protein Science : a Publication of the... Jun 2017Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and...
Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6-diazo-5-oxo-norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM min and the K was 2.7 ± 0.7 mM. The crystal structure of DON-inactivated hGGT1 contained a molecule of DON without the diazo-nitrogen atoms in the active site. The overall structure of the hGGT1-DON complex resembled the structure of the apo-enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1-DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α-amine of Thr381. The structure of DON-bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.
Topics: Catalytic Domain; Crystallography, X-Ray; Diazooxonorleucine; Enzyme Inhibitors; Humans; Protein Structure, Secondary; gamma-Glutamyltransferase
PubMed: 28378915
DOI: 10.1002/pro.3172