Model studies towards prodrugs of the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) containing a diazo precursor.

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.

Complete Biosynthetic Pathway of Alazopeptin, a Tripeptide Consisting of Two Molecules of 6-Diazo-5-oxo-l-norleucine and One Molecule of Alanine.

DON (6-diazo-5-oxo-l-norleucine), a diazo-containing amino acid, has been studied for more than 60 years as a potent antitumor agent, but its biosynthesis has not been elucidated. Here we reveal the complete biosynthetic pathway of alazopeptin, the tripeptide Ala-DON-DON, which has antitumor activity, by gene inactivation and in vitro analysis of recombinant enzymes. We also established heterologous production of N-acetyl-DON in Streptomyces albus. DON is synthesized from lysine by three enzymes and converted to alazopeptin by five enzymes and one carrier protein. Most interestingly, transmembrane protein AzpL was indicated to catalyze diazotization using 5-oxolysine and nitrous acid as substrates. Site-directed mutagenesis of AzpL indicated that the hydroxy group of Tyr-93 is important for the diazotization. These findings expand our knowledge of the enzymology of N-N bond formation.

We're Not "DON" Yet: Optimal Dosing and Prodrug Delivery of 6-Diazo-5-oxo-L-norleucine.

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. Mol Cancer Ther; 17(9); 1824-32. ©2018 AACR.

Structure of 6-diazo-5-oxo-norleucine-bound human gamma-glutamyl transpeptidase 1, a novel mechanism of inactivation.

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-1 min-1 and the Ki 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.

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

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.

Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-deconjugating enzyme is an unusual aspartate amidase.

Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.

Synthesis of potent water-soluble tissue transglutaminase inhibitors.

Dipeptide-based sulfonium peptidylmethylketones derived from 6-diazo-5-oxo-L-norleucine (DON) have been investigated as potential water-soluble inhibitors of extracellular transglutaminase. The lead compounds were prepared in four steps and exhibited potent activity against tissue transglutaminase.

Glutamine stimulates argininosuccinate synthetase gene expression through cytosolic O-glycosylation of Sp1 in Caco-2 cells.

Glutamine stimulates the expression of the argininosuccinate synthetase (ASS) gene at both the level of enzyme activity and mRNA in Caco-2 cells. Searching to identify the pathway involved, we observed that (i) the stimulating effect of glutamine was totally mimicked by glucosamine addition, and (ii) its effect but not that of glucosamine was totally blocked by 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of amidotransferases, suggesting that the metabolism of glutamine to glucosamine 6-phosphate was required. Moreover, run-on assays revealed that glucosamine was acting at a transcriptional level. Because three functional GC boxes were identified on the ASS gene promoter (Anderson, G. M., and Freytag, S. O. (1991) Mol. Cell Biol. 11, 1935-1943), the potential involvement of Sp1 family members was studied. Electrophoretic mobility shift assays using either the Sp1 consensus sequence or an appropriate fragment of the ASS promoter sequence as a probe demonstrated that both glutamine and glucosamine increased Sp1 DNA binding. Immunoprecipitation-Western blot experiments demonstrated that both compounds increased O-glycosylation of Sp1 leading to its translocation into nucleus. Again, the effect of glutamine on Sp1 was inhibited by the addition of DON but not of glucosamine. Taken together, the results clearly demonstrate that the metabolism of glutamine through the hexosamine pathway leads to the cytosolic O-glycosylation of Sp1, which, in turn, translocates into nucleus and stimulates the ASS gene transcription. Collectively, the results constitute the first demonstration of a functional relationship between a regulating signal (glutamine), a transcription factor (Sp1), and the transcription of the ASS gene.

The active conformation of glutamate synthase and its binding to ferredoxin.

Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases. The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain. Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form. The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex. These structures represent the enzyme in the active conformation. By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites. X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd). Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd. A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis.

Design, synthesis, and evaluation of gluten peptide analogs as selective inhibitors of human tissue transglutaminase.

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.

Mechanism for acivicin inactivation of triad glutamine amidotransferases.

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.

Do bacterial L-asparaginases utilize a catalytic triad Thr-Tyr-Glu?

The structures of Erwinia chrysanthemi L-asparaginase (ErA) complexed with the L- and D-stereoisomers of the suicide inhibitor, 6-diazo-5-oxy-norleucine, have been solved using X-ray crystallography and refined with data extending to 1.7 A. The distances between the Calpha atoms of the inhibitor molecules and the hydroxyl oxygen atoms of Thr-15 and Tyr-29 (1.20 and 1.60 A, respectively) clearly indicate the presence of covalent bonds between these moieties, confirming the nucleophilic role of Thr-15 during the first stage of enzymatic reactions and also indicating direct involvement of Tyr-29. The factors responsible for activating Tyr-29 remain unclear, although some structural changes around Ser-254', Asp-96, and Glu-63, common to both complexes, suggest that those residues play a function. The role of Glu-289' as the activator of Tyr-29, previously postulated for the closely related Pseudomonas 7A L-glutaminase-asparaginase, is not confirmed in this study, due to the lack of interactions between these residues in these complexes and in holoenzymes. The results reported here are consistent with previous reports that mutants of Escherichia coli L-asparaginase lacking Glu-289 remain catalytically active and prove the catalytic roles of both Thr-15 and Tyr-29, while still leaving open the question of the exact mechanism resulting in the unusual chemical properties of these residues.

Reactions of Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibitions and suggests an unusual catalytic triad Thr-Tyr-Glu.

Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D and L isomers of glutamine and asparagine. Crystals of PGA were reacted with diazo analogues of glutamine (6-diazo-5-oxo-L-norleucine, DON) and asparagine (5-diazo-4-oxo-L-norvaline, DONV), which are known inhibitors of the enzyme. The derivatized crystals remained isomorphous to native PGA crystals. Their structures were refined to crystallographic R = 0.20 and R(free) = 0.24 for PGA-DON and R = 0.19 and R = 0.23 for PGA-DONV. Difference Fourier electron density maps clearly showed that both DON and DONV inactivate PGA through covalent inhibition. Continuous electron density connecting the inhibitor to both Thr20 and Tyr34 of the flexible loop was observed providing strong evidence that Thr20 is the primary catalytic nucleophile and that Tyr34 plays an important role in catalysis as well. The unexpected covalent binding observed in the PGA-DON and PGA-DONV complexes shows that a secondary reaction involving the formation of a Tyr34-inhibitor bond takes place with concomitant inactivation of PGA. The predicted covalent linkage is not seen, however, suggesting an alternative method of inhibition not yet seen for these diazo analogues. These surprising results give insight as to the role of the flexible loop Thr and Tyr in the catalytic mechanism.