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Journal of Medicinal Chemistry Sep 2016The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due...
The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due to marked systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed. To enhance DON's therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Unexpectedly, simple alkyl ester-based prodrugs were ineffective due to chemical instability cyclizing to form a unique diazo-imine. However, masking both DON's amine and carboxylate functionalities imparted sufficient chemical stability for biological testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several provided excellent stability in monkey and human plasma. The most stable compound (5c, methyl-POM-DON-isopropyl-ester) was evaluated in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to DON utilization in glioblastoma multiforme patients.
Topics: Animals; Antimetabolites, Antineoplastic; Brain Neoplasms; Diazooxonorleucine; Female; Glioblastoma; Glutamine; Haplorhini; Humans; Mice; Mice, Nude; Prodrugs
PubMed: 27560860
DOI: 10.1021/acs.jmedchem.6b01069 -
Journal of Virology Oct 2016Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system...
UNLABELLED
Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (CNS). A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in adult C57BL/6 mice during clearance of infectious virus from the CNS, and the virus-specific immune response is implicated as a mediator of neuronal damage. Previous studies have shown that survival is improved in T-cell-deficient mice and in mice with pharmacological inhibition of the inflammatory response and glutamate excitotoxicity. Because glutamine metabolism is important in the CNS for the generation of glutamate and in the immune system for lymphocyte proliferation, we tested the effect of the glutamine antagonist DON (6-diazo-5-oxo-l-norleucine) on the outcome of NSV infection in mice. DON treatment for 7 days from the time of infection delayed the onset of paralysis and death. Protection was associated with reduced lymphocyte proliferation in the draining cervical lymph nodes, decreased leukocyte infiltration into the CNS, lower levels of inflammatory cytokines, and delayed viral clearance. In vitro studies showed that DON inhibited stimulus-induced proliferation of lymphocytes. When in vivo treatment with DON was stopped, paralytic disease developed along with the inflammatory response and viral clearance. These studies show that fatal NSV-induced encephalomyelitis is immune mediated and that antagonists of glutamine metabolism can modulate the immune response and protect against virus-induced neuroinflammatory disease.
IMPORTANCE
Encephalomyelitis due to infection with mosquito-borne alphaviruses is an important cause of death and of long-term neurological disability in those who survive infection. This study demonstrates the role of the virus-induced immune response in the generation of neurological disease. DON, a glutamine antagonist, inhibited the proliferation of lymphocytes in response to infection, prevented the development of brain inflammation, and protected mice from paralysis and death during treatment. However, because DON inhibited the immune response to infection, clearance of the virus from the brain was also prevented. When treatment was stopped, the immune response was generated, brain inflammation occurred, virus was cleared, and mice developed paralysis and died. Therefore, more definitive treatment for alphaviral encephalomyelitis should inhibit virus replication as well as neuroinflammatory damage.
Topics: Alphavirus; Alphavirus Infections; Animals; Cells, Cultured; Central Nervous System; Cytokines; Diazooxonorleucine; Encephalitis; Encephalitis, Viral; Encephalomyelitis; Female; Glutamine; Lymphocytes; Mice; Mice, Inbred C57BL; Paralysis; Sindbis Virus; Virus Replication
PubMed: 27489275
DOI: 10.1128/JVI.01045-16 -
Neurotherapeutics : the Journal of the... Jul 2016Mosquito-borne viruses are important causes of death and long-term neurologic disability due to encephalomyelitis. Studies of mice infected with the alphavirus Sindbis... (Review)
Review
Mosquito-borne viruses are important causes of death and long-term neurologic disability due to encephalomyelitis. Studies of mice infected with the alphavirus Sindbis virus have shown that outcome is dependent on the age and genetic background of the mouse and virulence of the infecting virus. Age-dependent susceptibility reflects the acquisition by neurons of resistance to virus replication and virus-induced cell death with maturation. In mature mice, the populations of neurons most susceptible to infection are in the hippocampus and anterior horn of the spinal cord. Hippocampal infection leads to long-term memory deficits in mice that survive, while motor neuron infection can lead to paralysis and death. Neuronal death is immune-mediated, rather than a direct consequence of virus infection, and associated with entry and differentiation of pathogenic T helper 17 cells in the nervous system. To modulate glutamate excitotoxicity, mice were treated with an N-methyl-D-aspartate receptor antagonist, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonists or a glutamine antagonist. The N-methyl-D-aspartate receptor antagonist MK-801 protected hippocampal neurons but not motor neurons, and mice still became paralyzed and died. α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonists GYKI-52466 and talampanel protected both hippocampal and motor neurons and prevented paralysis and death. Glutamine antagonist 6-diazo-5-l-norleucine protected hippocampal neurons and improved memory generation in mice surviving infection with an avirulent virus. Surprisingly, in all cases protection was associated with inhibition of the antiviral immune response, reduced entry of inflammatory cells into the central nervous system, and delayed virus clearance, emphasizing the importance of treatment approaches that include prevention of immunopathologic damage.
Topics: Alphavirus Infections; Animals; Benzodiazepines; Cell Death; Diazooxonorleucine; Dizocilpine Maleate; Encephalomyelitis; Hippocampus; Humans; Inflammation; Memory; Mice; Neurons; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Sindbis Virus; Survival Analysis; Th17 Cells
PubMed: 27114366
DOI: 10.1007/s13311-016-0434-6 -
Aging Cell Jun 2016Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)-CREB signaling plays a critical role in learning and...
Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)-CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O-GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O-GlcNAcylation in metabolically active rat brain slices by O-(2-acetamido-2-deoxy-d-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc), an inhibitor of N-acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non-PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O-GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O-GlcNAcylation through intracerebroventricular injection of 6-diazo-5-oxo-l-norleucine (DON), the inhibitor of glutamine fructose-6-phosphate amidotransferase, suppressed PKA-CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O-GlcNAcylation is a novel mechanism that regulates PKA-CREB signaling. Downregulation of O-GlcNAcylation suppresses PKA-CREB signaling and consequently causes learning and memory deficits in AD.
Topics: Acetylglucosamine; Alzheimer Disease; Animals; Brain; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Diazooxonorleucine; Down-Regulation; Glycosylation; HEK293 Cells; HeLa Cells; Humans; Learning; Male; Memory; Memory Disorders; Mice, Inbred C57BL; Phosphorylation; Protein Transport; Rats, Sprague-Dawley; Subcellular Fractions; tau Proteins
PubMed: 26840030
DOI: 10.1111/acel.12449 -
Nature Reviews. Drug Discovery Dec 2015
Topics: Animals; Antimalarials; Diazooxonorleucine; Glutamine; Malaria, Cerebral; Malaria, Falciparum
PubMed: 26585535
DOI: 10.1038/nrd4786 -
Cell Reports Oct 2015Upon antigen recognition and co-stimulation, T lymphocytes upregulate the metabolic machinery necessary to proliferate and sustain effector function. This metabolic...
Upon antigen recognition and co-stimulation, T lymphocytes upregulate the metabolic machinery necessary to proliferate and sustain effector function. This metabolic reprogramming in T cells regulates T cell activation and differentiation but is not just a consequence of antigen recognition. Although such metabolic reprogramming promotes the differentiation and function of T effector cells, the differentiation of regulatory T cells employs different metabolic reprogramming. Therefore, we hypothesized that inhibition of glycolysis and glutamine metabolism might prevent graft rejection by inhibiting effector generation and function and promoting regulatory T cell generation. We devised an anti-rejection regimen involving the glycolytic inhibitor 2-deoxyglucose (2-DG), the anti-type II diabetes drug metformin, and the inhibitor of glutamine metabolism 6-diazo-5-oxo-L-norleucine (DON). Using this triple-drug regimen, we were able to prevent or delay graft rejection in fully mismatched skin and heart allograft transplantation models.
Topics: Allografts; Animals; CD8-Positive T-Lymphocytes; Cells, Cultured; Deoxyglucose; Diazooxonorleucine; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glutamine; Glycolysis; Graft Rejection; Heart Transplantation; Metformin; Mice; Mice, Inbred BALB C; Mice, Transgenic; Phosphorylation; T-Lymphocytes, Regulatory
PubMed: 26489460
DOI: 10.1016/j.celrep.2015.09.036 -
Proceedings of the National Academy of... Oct 2015The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective...
The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective antimalarial chemotherapy. There are no adjunctive treatments for CM, so there is an urgent need to identify new targets for therapy. Here we show that the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) rescues mice from CM when administered late in the infection a time at which mice already are suffering blood-brain barrier dysfunction, brain swelling, and hemorrhaging accompanied by accumulation of parasite-specific CD8(+) effector T cells and infected red blood cells in the brain. Remarkably, within hours of DON treatment mice showed blood-brain barrier integrity, reduced brain swelling, decreased function of activated effector CD8(+) T cells in the brain, and levels of brain metabolites that resembled those in uninfected mice. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM in African children.
Topics: Animals; Antimalarials; Blood-Brain Barrier; Diazooxonorleucine; Glutamine; Malaria, Cerebral; Malaria, Falciparum; Mice
PubMed: 26438846
DOI: 10.1073/pnas.1516544112 -
BioMed Research International 2015Abnormal metabolism is another cancer hallmark. The two most characterized altered metabolic pathways are high rates of glycolysis and glutaminolysis, which are natural... (Review)
Review
Abnormal metabolism is another cancer hallmark. The two most characterized altered metabolic pathways are high rates of glycolysis and glutaminolysis, which are natural targets for cancer therapy. Currently, a number of newer compounds to block glycolysis and glutaminolysis are being developed; nevertheless, lonidamine and 6-diazo-5-oxo-L-norleucine (DON) are two old drugs well characterized as inhibitors of glycolysis and glutaminolysis, respectively, whose clinical development was abandoned years ago when the importance of cancer metabolism was not fully appreciated and clinical trial methodology was less developed. In this review, a PubMed search using the words lonidamine and 6-diazo-5-oxo-L-norleucine (DON) was undertaken to analyse existing information on the preclinical and clinical studies of these drugs for cancer treatment. Data show that they exhibit antitumor effects; besides there is also the suggestion that they are synergistic. We conclude that lonidamine and DON are safe and potentially effective drugs that need to be reevaluated in combination as metabolic therapy of cancer.
Topics: Antineoplastic Combined Chemotherapy Protocols; Clinical Trials as Topic; Diazooxonorleucine; Humans; Indazoles; Neoplasms
PubMed: 26425550
DOI: 10.1155/2015/690492 -
Oncotarget Sep 2015Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We...
Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.
Topics: Adenocarcinoma; Aged; Amidophosphoribosyltransferase; Aneuploidy; Animals; Biomarkers, Tumor; Carboxy-Lyases; Cell Line, Tumor; Cell Proliferation; Chickens; Diazooxonorleucine; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glutamine; Humans; Lung Neoplasms; Male; Mice; Middle Aged; Neoplasm Invasiveness; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Peptide Synthases; Prognosis; Purines
PubMed: 26140362
DOI: 10.18632/oncotarget.4352 -
Oncology Reports Sep 2015There is a strong rationale for targeting the metabolic alterations of cancer cells. The most studied of these are the higher rates of glycolysis, glutaminolysis and...
There is a strong rationale for targeting the metabolic alterations of cancer cells. The most studied of these are the higher rates of glycolysis, glutaminolysis and de novo synthesis of fatty acids (FAs). Despite the availability of pharmacological inhibitors of these pathways, no preclinical studies targeting them simultaneously have been performed. In the present study it was determined whether three key enzymes for glycolysis, glutaminolysis and de novo synthesis of FAs, hexokinase-2, glutaminase and fatty acid synthase, respectively, were overexpressed as compared to primary fibroblasts. In addition, we showed that at clinically relevant concentrations lonidamine, 6-diazo-5-oxo-L-norleucine and orlistat, known inhibitors of the mentioned enzymes, exerted a cell viability inhibitory effect. Genetic downregulation of the three enzymes also reduced cell viability. The three drugs were highly synergistic when administered as a triple combination. Of note, the cytotoxicity of the triple combination was low in primary fibroblasts and was well tolerated when administered into healthy BALB/c mice. The results suggest the feasibility and potential clinical utility of the triple metabolic targeting which merits to be further studied by using either repositioned old drugs or newer, more selective inhibitors.
Topics: Animals; Apoptosis; Cell Survival; Diazooxonorleucine; Drug Synergism; Fatty Acid Synthases; Fatty Acids; Fibroblasts; Gene Expression Regulation, Neoplastic; Glutaminase; Glycolysis; Hexokinase; Humans; Indazoles; Lactones; Metabolic Networks and Pathways; Mice; Neoplasms; Orlistat
PubMed: 26134042
DOI: 10.3892/or.2015.4077