-
Communications Biology 2019Glioblastoma (GBM) is an aggressive primary human brain tumour that has resisted effective therapy for decades. Although glucose and glutamine are the major fuels that...
Glioblastoma (GBM) is an aggressive primary human brain tumour that has resisted effective therapy for decades. Although glucose and glutamine are the major fuels that drive GBM growth and invasion, few studies have targeted these fuels for therapeutic management. The glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), was administered together with a calorically restricted ketogenic diet (KD-R) to treat late-stage orthotopic growth in two syngeneic GBM mouse models: VM-M3 and CT-2A. DON targets glutaminolysis, while the KD-R reduces glucose and, simultaneously, elevates neuroprotective and non-fermentable ketone bodies. The diet/drug therapeutic strategy killed tumour cells while reversing disease symptoms, and improving overall mouse survival. The therapeutic strategy also reduces edema, hemorrhage, and inflammation. Moreover, the KD-R diet facilitated DON delivery to the brain and allowed a lower dosage to achieve therapeutic effect. The findings support the importance of glucose and glutamine in driving GBM growth and provide a therapeutic strategy for non-toxic metabolic management.
Topics: Animals; Body Weight; Brain; Brain Neoplasms; Caloric Restriction; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Diet, Ketogenic; Disease Models, Animal; Female; Fermentation; Glioblastoma; Glucose; Glutamine; Humans; Immunohistochemistry; Ketone Bodies; Ketones; Male; Mice; Mice, Inbred C57BL; Neoplasm Transplantation
PubMed: 31149644
DOI: 10.1038/s42003-019-0455-x -
Scientific Reports Apr 2019Glutamine metabolism and the mechanistic target of rapamycin (mTOR) pathway are activated cooperatively in the differentiation and activation of inflammatory immune...
Glutamine metabolism and the mechanistic target of rapamycin (mTOR) pathway are activated cooperatively in the differentiation and activation of inflammatory immune cells. But the combined inhibition of both pathways was rarely investigated. This study investigated how inhibiting both glutamine metabolism with 6-diazo-5-oxo-L-norleucine (DON) and mTOR with rapamycin affects immune cells and the arthritis in a mouse model. We revealed that rapamycin and DON additively suppressed CD4 T cell proliferation, and both of them inhibited Th17 cell differentiation. While DON inhibited the differentiation of dendritic cells and macrophages and facilitated that of Ly6G granulocytic (G)-MDSCs more strongly than did rapamycin, G-MDSCs treated with rapamycin but not DON suppressed CD4 T cell proliferation in vitro. The combination of rapamycin and DON significantly suppressed the arthritis in SKG mice more strongly than did each monotherapy in vivo. The numbers of CD4 T and Th17 cells in the spleen were lowest in mice treated with the combination therapy. Thus, combined treatment with rapamycin and DON additively ameliorated the arthritis in SKG mice, possibly by suppressing CD4 T cell proliferation and Th17 differentiation. These results suggest the combination of rapamycin and DON may be a potential novel therapy for arthritis.
Topics: Animals; Arthritis; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Dendritic Cells; Diazooxonorleucine; Female; Glutamine; Immunosuppression Therapy; Macrophages; Mice, Inbred BALB C; Myeloid-Derived Suppressor Cells; Sirolimus; TOR Serine-Threonine Kinases; Th17 Cells
PubMed: 31011190
DOI: 10.1038/s41598-019-42932-1 -
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 -
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 -
Molecular Carcinogenesis Jun 2019Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy....
Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy. However, platinum-based regimens have reached a plateau of therapeutic efficacy; therefore, it is critical to implement novel approaches for improvement. The hexosamine biosynthesis pathway (HBP), which produces amino-sugar N-acetyl-glucosamine for protein glycosylation, is important for protein function and cell survival. Here we show a beneficial effect by the combination of cisplatin with HBP inhibition. Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of HBP, was increased in NSCLC cell lines and tissues. Pharmacological inhibition of GFAT activity or knockdown of GFATimpaired cell proliferation and exerted synergistic or additive cytotoxicity to the cells treated with cisplatin. Mechanistically, GFAT positively regulated the expression of binding immunoglobulin protein (BiP; also known as glucose-regulated protein 78, GRP78), an endoplasmic reticulum chaperone involved in unfolded protein response (UPR). Suppressing GFAT activity resulted in downregulation of BiP that activated inositol-requiring enzyme 1α, a sensor protein of UPR, and exacerbated cisplatin-induced cell apoptosis. These data identify GFAT-mediated HBP as a target for improving platinum-based chemotherapy for NSCLC.
Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Diazooxonorleucine; Down-Regulation; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Heat-Shock Proteins; Hexosamines; Humans; Lung Neoplasms
PubMed: 30790354
DOI: 10.1002/mc.22992 -
Proceedings of the National Academy of... Dec 2018The deadliest complication of infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial...
The deadliest complication of infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood-brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.
Topics: Adult; Animals; Antimalarials; Biomarkers; Blood-Brain Barrier; Brain; Brain Edema; Child; Diazooxonorleucine; Disease Models, Animal; Disease Progression; Female; Glutamine; Humans; Magnetic Resonance Imaging; Malaria, Cerebral; Malaria, Falciparum; Mice; Mice, Inbred C57BL; Plasmodium falciparum
PubMed: 30514812
DOI: 10.1073/pnas.1812909115 -
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