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Molecules (Basel, Switzerland) Aug 2022Cancer cells change their glucose and glutamine (GLU) metabolism to obtain the energy required to continue growing. Glutaminase (GLS) plays a crucial role in promoting...
Cancer cells change their glucose and glutamine (GLU) metabolism to obtain the energy required to continue growing. Glutaminase (GLS) plays a crucial role in promoting cell metabolism for cancer cell growth; targeting GLU metabolism by inhibiting GLS has attracted interest as a potential cancer management strategy. Herein, we employed a sequential screening of traditional Chinese medicine (TCM) database followed by drug-likeness and molecular dynamics simulations against the active site of GLS. We report 12 potent compounds after screening the TCM database against GLS, followed by a drug-likeness filter with Lipinski and Veber rule criteria. Among them, ZINC03978829 and ZINC32296657 were found to have higher binding energy (BE) values than the control compound 6-Diazo-5-Oxo-L-Norleucine, with BEs of -9.3 and -9.7 kcal/mol, respectively, compared to the BE of 6-Diazo-5-Oxo-L-Norleucine (-4.7 kcal/mol) with GLS. Molecular dynamics simulations were used to evaluate the results further, and a 100 ns MD simulation revealed that the hits form stable complexes with GLS and formed 2-5 hydrogen bond interactions. This study indicates that these hits might be employed as GLS inhibitors in the battle against cancer. However, more laboratory tests are a prerequisite to optimize them as GLS inhibitors.
Topics: Diazooxonorleucine; Early Detection of Cancer; Glutaminase; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Neoplasms; Neoplastic Processes
PubMed: 35956989
DOI: 10.3390/molecules27155042 -
Pharmacologically Inferred Glycolysis and Glutaminolysis Requirement of B Cells in Lupus-Prone Mice.Journal of Immunology (Baltimore, Md. :... May 2022Several studies have shown an enhanced metabolism in the CD4 T cells of lupus patients and lupus-prone mice. Little is known about the metabolism of B cells in lupus. In...
Several studies have shown an enhanced metabolism in the CD4 T cells of lupus patients and lupus-prone mice. Little is known about the metabolism of B cells in lupus. In this study, we compared the metabolism of B cells between lupus-prone B6. triple-congenic mice and C57BL/6 controls at steady state relative to autoantibody production, as well as during T cell-dependent (TD) and T cell-independent (TI) immunizations. Starting before the onset of autoimmunity, B cells from triple-congenic mice showed an elevated glycolysis and mitochondrial respiration, which were normalized in vivo by inhibiting glycolysis with a 2-deoxy-d-glucose (2DG) treatment. 2DG greatly reduced the production of TI-Ag-specific Abs, but showed minimal effect with TD-Ags. In contrast, the inhibition of glutaminolysis with 6-diazo-5-oxo-l-norleucine had a greater effect on TD than TI-Ag-specific Abs in both strains. Analysis of the TI and TD responses in purified B cells in vitro suggests, however, that the glutaminolysis requirement is not B cell-intrinsic. Thus, B cells have a greater requirement for glycolysis in TI than TD responses, as inferred from pharmacological interventions. B cells from lupus-prone and control mice have different intrinsic metabolic requirements or different responses toward 2DG and 6-diazo-5-oxo-l-norleucine, which mirrors our previous results obtained with follicular Th cells. Overall, these results predict that targeting glucose metabolism may provide an effective therapeutic approach for systemic autoimmunity by eliminating both autoreactive follicular Th and B cells, although it may also impair TI responses.
Topics: Animals; B-Lymphocytes; Diazooxonorleucine; Glycolysis; Humans; Mice; Mice, Congenic; Mice, Inbred C57BL; T-Lymphocytes, Helper-Inducer
PubMed: 35387839
DOI: 10.4049/jimmunol.2100356 -
Cellular Immunology Dec 2021Infection of the cornea with HSV results in an immune-inflammatory reaction orchestrated by proinflammatory T cells that is a major cause of human vision impairment. The...
Infection of the cornea with HSV results in an immune-inflammatory reaction orchestrated by proinflammatory T cells that is a major cause of human vision impairment. The severity of lesions can be reduced if the representation of inflammatory T cells is changed to increase the presence of T cells with regulatory function. This report shows that inhibiting glutamine metabolism using 6-Diazo-5-oxo-l-norleucine (DON) administered via intraperitoneal (IP) starting 6 days after ocular infection and continued until day 15 significantly reduced the severity of herpetic stromal keratitis lesions. The therapy resulted in reduced neutrophils, macrophages as well proinflammatory CD4 Th1 and Th17 T cells in the cornea, but had no effect on levels of regulatory T cells. A similar change in the representation of inflammatory and regulatory T cells occurred in the trigeminal ganglion (TG) the site where HSV infection establishes latency. Glutamine metabolism was shown to be required for the in-vitro optimal induction of both Th1 and Th17 T cells but not for the induction of Treg that were increased when glutamine metabolism was inhibited. Inhibiting glutamine metabolism also changed the ability of latently infected TG cells from animals previously infected with HSV to reactivate and produce infectious virus.
Topics: Animals; Diazooxonorleucine; Glutamine; Keratitis, Herpetic; Latent Infection; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; T-Lymphocytes; Trigeminal Ganglion; Virus Activation; Virus Latency
PubMed: 34678554
DOI: 10.1016/j.cellimm.2021.104450 -
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 -
International Journal of Molecular... May 2021Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and...
Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and glutaminolysis are increased in rapidly proliferating VSMCs to support their increased energy requirements and biomass production. Thus, it is essential to develop new pharmacological tools that regulate metabolic reprogramming in VSMCs for treatment of atherosclerosis. The effects of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist, have been broadly investigated in highly proliferative cells; however, it is unclear whether DON inhibits proliferation of VSMCs and neointima formation. Here, we investigated the effects of DON on neointima formation in vivo as well as proliferation and migration of VSMCs in vitro. DON simultaneously inhibited FBS- or PDGF-stimulated glycolysis and glutaminolysis as well as mammalian target of rapamycin complex I activity in growth factor-stimulated VSMCs, and thereby suppressed their proliferation and migration. Furthermore, a DON-derived prodrug, named JHU-083, significantly attenuated carotid artery ligation-induced neointima formation in mice. Our results suggest that treatment with a glutamine antagonist is a promising approach to prevent progression of atherosclerosis and restenosis.
Topics: Animals; Antimetabolites, Antineoplastic; Cell Cycle; Cell Movement; Cell Proliferation; Cells, Cultured; Diazooxonorleucine; Glutamine; Glycolysis; Immunohistochemistry; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mitochondria; Muscle, Smooth, Vascular; Neointima; Oxidative Phosphorylation; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine
PubMed: 34070527
DOI: 10.3390/ijms22115602 -
Journal of Neuropathology and... Mar 2021Medulloblastoma is the most common malignant pediatric brain tumor. Amplification of c-MYC is a hallmark of a subset of poor-prognosis medulloblastoma. MYC upregulates...
Medulloblastoma is the most common malignant pediatric brain tumor. Amplification of c-MYC is a hallmark of a subset of poor-prognosis medulloblastoma. MYC upregulates glutamine metabolism across many types of cancer. We modified the naturally occurring glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) by adding 2 promoeities to increase its lipophilicity and brain penetration creating the prodrug isopropyl 6-diazo-5-oxo-2-(((phenyl (pivaloyloxy) methoxy) - carbonyl) amino) hexanoate, termed JHU395. This prodrug was shown to have a 10-fold improved CSF-to-plasma ratio and brain-to-plasma ratio relative to DON. We hypothesized that JHU395 would have superior cell penetration compared with DON and would effectively and more potently kill MYC-expressing medulloblastoma. JHU395 treatment caused decreased growth and increased apoptosis in multiple human high-MYC medulloblastoma cell lines at lower concentrations than DON. Parenteral administration of JHU395 in Nu/Nu mice led to the accumulation of micromolar concentrations of DON in brain. Treatment of mice bearing orthotopic xenografts of human MYC-amplified medulloblastoma with JHU395 increased median survival from 26 to 45 days compared with vehicle control mice (p < 0.001 by log-rank test). These data provide preclinical justification for the ongoing development and testing of brain-targeted DON prodrugs for use in medulloblastoma.
Topics: Animals; Apoptosis; Caproates; Cell Line, Tumor; Cerebellar Neoplasms; Diazooxonorleucine; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Female; Glutamine; Humans; Medulloblastoma; Mice; Mice, Nude
PubMed: 33712838
DOI: 10.1093/jnen/nlab018 -
The Laryngoscope Jul 2021Glutamine inhibition has been demonstrated an antifibrotic effect in iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts in vitro. We hypothesize that broadly...
OBJECTIVE/HYPOTHESIS
Glutamine inhibition has been demonstrated an antifibrotic effect in iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts in vitro. We hypothesize that broadly active glutamine antagonist, DON will reduce collagen formation and fibrosis-associated gene expression in iLTS mice.
STUDY DESIGN
Prospective controlled animal study.
METHODS
iLTS in mice were induced by chemomechanical injury of the trachea using a bleomycin-coated wire brush. PBS or DON (1.3 mg/kg) were administered by intraperitoneal injection (i.p.) every other day. Laryngotracheal complexes were harvested at days 7 and 14 after the initiation of DON treatment for the measurement of lamina propria thickness, trichrome stain, immunofluorescence staining of collagen 1, and fibrosis-associated gene expression.
RESULTS
The study demonstrated that DON treatment reduced lamina propria thickness (P = .025) and collagen formation in trichrome stain and immunofluorescence staining of collagen 1. In addition, DON decreased fibrosis-associated gene expression in iLTS mice. At day 7, DON inhibited Col1a1 (P < .0001), Col3a1 (P = .0046), Col5a1 (P < .0001), and Tgfβ (P = .023) expression. At day 14, DON reduced Co1a1 (P = .0076) and Tgfβ (P = .023) expression.
CONCLUSIONS
Broadly active glutamine antagonist, DON, significantly reduces fibrosis in iLTS mice. These results suggest that the concept of glutamine inhibition may be a therapeutic option to reduce fibrosis in the laryngotracheal stenosis.
LEVEL OF EVIDENCE
N/A Laryngoscope, 131:E2125-E2130, 2021.
Topics: Animals; Bleomycin; Collagen; Diazooxonorleucine; Disease Models, Animal; Fibroblasts; Fibrosis; Gene Expression; Glutamine; Iatrogenic Disease; Injections, Intraperitoneal; Laryngostenosis; Mice; Mucous Membrane; Prospective Studies; Trachea; Tracheal Stenosis
PubMed: 33433011
DOI: 10.1002/lary.29385 -
Frontiers in Immunology 2020Transplant tolerance in the absence of long-term immunosuppression has been an elusive goal for solid organ transplantation. Recently, it has become clear that metabolic...
Transplant tolerance in the absence of long-term immunosuppression has been an elusive goal for solid organ transplantation. Recently, it has become clear that metabolic reprogramming plays a critical role in promoting T cell activation, differentiation, and function. Targeting metabolism can preferentially inhibit T cell effector generation while simultaneously promoting the generation of T regulatory cells. We hypothesized that costimulatory blockade with CTLA4Ig in combination with targeting T cell metabolism might provide a novel platform to promote the induction of transplant tolerance.
Topics: Abatacept; Allografts; Animals; Deoxyglucose; Diazooxonorleucine; Glycolysis; Immunosuppression Therapy; Immunosuppressive Agents; Lymphocyte Activation; Metformin; Mice; T-Lymphocytes; Transplantation Tolerance
PubMed: 32328063
DOI: 10.3389/fimmu.2020.00572 -
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 -
Clinical Cancer Research : An Official... Oct 2019Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular...
PURPOSE
Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular heterogeneity in AT/RT with an aggressive subgroup featuring overexpression of the proto-oncogene. We perform the first comprehensive metabolic profiling of patient-derived AT/RT cell lines to identify therapeutic susceptibilities in high MYC-expressing AT/RT.
EXPERIMENTAL DESIGN
Metabolites were extracted from AT/RT cell lines and separated in ultra-high performance liquid chromatography mass spectrometry. Glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) was tested with growth and cell death assays and survival studies in orthotopic mouse models of AT/RT. Metabolic flux analysis was completed to identify combination therapies to act synergistically to improve survival in high MYC AT/RT.
RESULTS
Unbiased metabolic profiling of AT/RT cell models identified a unique dependence of high MYC AT/RT on glutamine for survival. The glutamine analogue, DON, selectively targeted high MYC cell lines, slowing cell growth, inducing apoptosis, and extending survival in orthotopic mouse models of AT/RT. Metabolic flux experiments with isotopically labeled glutamine revealed DON inhibition of glutathione (GSH) synthesis. DON combined with carboplatin further slowed cell growth, induced apoptosis, and extended survival in orthotopic mouse models of high MYC AT/RT.
CONCLUSIONS
Unbiased metabolic profiling of AT/RT identified susceptibility of high MYC AT/RT to glutamine metabolic inhibition with DON therapy. DON inhibited glutamine-dependent synthesis of GSH and synergized with carboplatin to extend survival in high MYC AT/RT. These findings can rapidly translate into new clinical trials to improve survival in high MYC AT/RT.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Cell Proliferation; Diazooxonorleucine; Female; Glutamine; Humans; Metabolome; Mice; Mice, Nude; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Rhabdoid Tumor; Teratoma; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 31300448
DOI: 10.1158/1078-0432.CCR-19-0189