ABSTRACT
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.
Subject(s)
Diazooxonorleucine/analogs & derivatives , Diazooxonorleucine/pharmacology , Glutamine/antagonists & inhibitors , Prodrugs/chemistry , Prodrugs/pharmacology , Diazooxonorleucine/chemistry , Drug Design , Drug Stability , Molecular StructureABSTRACT
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.
Subject(s)
Cell Movement/drug effects , Cell Proliferation/drug effects , Diazooxonorleucine/pharmacology , Glutamine/antagonists & inhibitors , Glycolysis/drug effects , Muscle, Smooth, Vascular/drug effects , Neointima/drug therapy , Oxidative Phosphorylation/drug effects , Animals , Antimetabolites, Antineoplastic/pharmacology , Cell Cycle/drug effects , Cells, Cultured , Diazooxonorleucine/analogs & derivatives , Glutamine/metabolism , Immunohistochemistry , Male , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Inbred C57BL , Mitochondria/drug effects , Mitochondria/metabolism , Muscle, Smooth, Vascular/metabolism , Neointima/metabolism , Platelet-Derived Growth Factor/pharmacology , Rats , Rats, Sprague-Dawley , Serum Albumin, Bovine/pharmacologyABSTRACT
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.
Subject(s)
Apoptosis/drug effects , Caproates/pharmacology , Cerebellar Neoplasms/drug therapy , Diazooxonorleucine/analogs & derivatives , Diazooxonorleucine/pharmacology , Glutamine/antagonists & inhibitors , Medulloblastoma/drug therapy , Animals , Apoptosis/physiology , Caproates/chemistry , Caproates/therapeutic use , Cell Line, Tumor , Cerebellar Neoplasms/pathology , Diazooxonorleucine/therapeutic use , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/chemistry , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Amino Acid Antagonists/therapeutic use , Female , Glutamine/metabolism , Humans , Medulloblastoma/pathology , Mice , Mice, NudeABSTRACT
CTP synthase is compartmentalized within a subcellular structure, termed the cytoophidium, in a range of organisms including bacteria, yeast, fruit fly and rat. Here we show that CTP synthase is also compartmentalized into cytoophidia in human cells. Surprisingly, the occurrence of cytoophidia in human cells increases upon treatment with a glutamine analog 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of glutamine-dependent enzymes including CTP synthase. Experiments in flies confirmed that DON globally promotes cytoophidium assembly. Clonal analysis via CTP synthase RNA interference in somatic cells indicates that CTP synthase expression level is critical for the formation of cytoophidia. Moreover, DON facilitates cytoophidium assembly even when CTP synthase level is low. A second glutamine analog azaserine also promotes cytoophidum formation. Our data demonstrate that glutamine analogs serve as useful tools in the study of cytoophidia.
Subject(s)
Cell Compartmentation/drug effects , Diazooxonorleucine/pharmacology , Drosophila melanogaster/cytology , Drosophila melanogaster/drug effects , Enzyme Inhibitors/pharmacology , Glutamine/analogs & derivatives , Intracellular Space/drug effects , Intracellular Space/metabolism , Animals , Apoptosis/drug effects , Azaserine/analogs & derivatives , Azaserine/pharmacology , Carbon-Nitrogen Ligases/antagonists & inhibitors , Carbon-Nitrogen Ligases/deficiency , Carbon-Nitrogen Ligases/genetics , Carbon-Nitrogen Ligases/metabolism , Cell Cycle/drug effects , Diazooxonorleucine/analogs & derivatives , Drosophila melanogaster/enzymology , Drosophila melanogaster/metabolism , Enzyme Inhibitors/chemistry , Female , HeLa Cells , Humans , Intracellular Space/enzymology , Male , Oogenesis/drug effects , RNA InterferenceABSTRACT
The substitution of a glutamine residue with 6-diazo-5-oxo-norleucine (DON) transforms an immunodominant gluten peptide into a potent inhibitor of tissue transglutaminase. DON-modified peptides could be useful for the study and therapy of celiac sprue.
Subject(s)
Celiac Disease/drug therapy , Diazooxonorleucine/analogs & derivatives , Diazooxonorleucine/chemical synthesis , GTP-Binding Proteins/antagonists & inhibitors , Glutens/chemistry , Peptides/chemical synthesis , Transglutaminases/antagonists & inhibitors , Celiac Disease/enzymology , Diazooxonorleucine/pharmacology , Glutamine/chemistry , Humans , Peptides/chemistry , Peptides/pharmacology , Protein Glutamine gamma Glutamyltransferase 2ABSTRACT
Recent studies have implicated a crucial role for tissue transglutaminase (TG2) in the pathogenesis of Celiac Sprue, a disorder of the small intestine triggered in genetically susceptible individuals by dietary exposure to gluten. Proteolytically stable peptide inhibitors of human TG2 were designed containing acivicin or alternatively 6-diazo-5-oxo-norleucine (DON) as warheads. In biochemical and cell-based assays, the best of these inhibitors, Ac-PQP-(DON)-LPF-NH(2), was considerably more potent and selective than other TG2 inhibitors reported to date. Selective pharmacological inhibition of extracellular TG2 should be useful in exploring the mechanistic implications of TG2-catalyzed modification of dietary gluten, a phenomenon of considerable relevance in Celiac Sprue.
Subject(s)
Diazooxonorleucine/analogs & derivatives , Diazooxonorleucine/chemical synthesis , Drug Design , Enzyme Inhibitors/chemical synthesis , GTP-Binding Proteins/antagonists & inhibitors , Glutens/chemistry , Peptides/chemical synthesis , Transglutaminases/antagonists & inhibitors , Animals , Cells, Cultured , Diazooxonorleucine/chemistry , Diazooxonorleucine/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , GTP-Binding Proteins/chemistry , Glutamic Acid/chemistry , Humans , Isoxazoles/chemistry , Peptides/chemistry , Peptides/pharmacology , Protein Glutamine gamma Glutamyltransferase 2 , Structure-Activity Relationship , Transglutaminases/chemistryABSTRACT
In vitro experiments showed that 6-diazo-5-oxo-L-norleucylglycine, a dipeptide analog of L-glutaminylglycine, inactivates gamma-glutamyl transferase bound to renal brush border membrane vesicles but does not inactivate the purified transferase. The rate of inactivation of the membrane-bound enzyme decreased markedly in the presence of dipeptides, such as L-leucylglycine and L-alanylglycine, or in the presence of o-phenanthroline, an inhibitor of renal peptidases. The presence of L-cysteinylglycine S-acetyldextran polymer (Mr 500,000), which does not permeate membranes, protected the membrane-bound transferase from inactivation by 6-diazo-5-oxo-L-norleucyglycine. This and other findings suggest that the norleucylglycine derivative was hydrolyzed by peptidase(s) bound to the outer surface of the brush border membranes and that the 6-diazo-5-oxo-L-norleucine thus released acts as an affinity-labeling reagent for the membrane-bound transferase. Similar effects were observed in vivo. Intravenous administration of 6-diazo-5-oxo-L-norleucylglycine to mice resulted in a marked decrease in renal transferase activity. Mice thus pretreated with 6-diazo-5-oxo-L-norleucylglycine, but not an untreated group, excreted significant amounts of S-carbamido[14C]methylglutathione in their urine within 30 min of intravenous administration of this compound. This finding suggests that the renal transferase was involved in the hydrolysis of the glutathione S-conjugate in the glomerular filtrate in vivo and that the administered 6-diazo-5-oxo-L-norleucylglycine underwent hydrolysis peptidase(s)-catalyzed to liberate 6-diazo-5-oxo-L-norleucine that reacted with the membrane-bound gamma-glutamyl transferase.