6-Diazo-5-oxo-L-norleucine

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6-Diazo-5-oxo-L-norleucine
L-Diazooxonorleucine.svg
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Legal status
Identifiers
  • (5S)-5-Amino-1-diazonio-6-hydroxy-6-oxohex-1-en-2-olate [1]
CAS Number
PubChem CID
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ChEBI
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Chemical and physical data
Formula C6H9N3O3
Molar mass 171.156 g·mol−1
3D model (JSmol)
  • O=C(CC[C@H](N)C(O)=O)\C=[N+]=[N-]
  • InChI=1S/C6H9N3O3/c7-5(6(11)12)2-1-4(10)3-9-8/h3,5H,1-2,7H2,(H,11,12)/t5-/m0/s1 X mark.svgN
  • Key:YCWQAMGASJSUIP-YFKPBYRVSA-N X mark.svgN
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6-Diazo-5-oxo-L-norleucine (DON) is a glutamine antagonist, which was isolated originally from Streptomyces in a sample of Peruvian soil. This diazo compound is biosynthesized from lysine by three enzymes in bacteria. [2] It is one of the most famous non-proteinogenic amino acid and was characterized in 1956 by Henry W Dion et al., [3] who suggested a possible use in cancer therapy. This antitumoral efficacy was confirmed in different animal models. [4] DON was tested as chemotherapeutic agent in different clinical studies, but was never approved. In 2019, DON was shown to kill tumor cells while reversing disease symptoms and improve overall survival in late-stage experimental glioblastoma in mice, when combined with calorie-restricted ketogenic diet. [5]

Contents

Chemistry

DON is a water-soluble yellowish powder, which can be dissolved also in aqueous solutions of methanol, acetone or ethanol, but dissolution in absolute alcohols is difficult. Solutions of at least 50 μM DON in 0.9% NaCl are lightly yellowish. The crystalline form appears as yellowish greenish needles. The specific rotation is [α]26D +21° (c = 5.4% in H2O). In phosphate buffer, pH 7 are the ultraviolet absorption maxima at 274 nm (E1%1 cm. 683) and 244 nm (E1%1 cm 376). [3] [6]

Biochemistry

DON is used as inhibitor of different glutamine utilizing enzymes. Due to its similarity to glutamine, it can enter catalytic centres of these enzymes and inhibits them by covalent binding, or more precisely, by alkylation. [7] [8] The following table gives a survey of DON targets.

Selection of enzymes inhibited by DON
EnzymeMetabolic pathwayReferences
Carbamoyl phosphate synthase (CAD) Pyrimidine-De-Novo-Synthesis [7] [9]
CTP synthase (CTPS) Pyrimidine-De-Novo-Synthesis [7] [9]
FGAR amidotransferase Purine-De-Novo-Synthesis [7] [10]
Guanosine monophosphate synthetase (GMPS) Purine-De-Novo-Synthesis [7] [11]
PRPP amidotransferase Purine-De-Novo-Synthesis [7] [11]
Mitochondrial glutaminase First step of glutaminolysis [7] [11]
NAD synthase Coenzyme of the electron transport chain [7] [12]
Asparagine synthetaseAmino acid synthesis [7] [13]

Pharmacology

DON is a cytotoxic inhibitor of many enzymes of nucleotide synthesis. It could be shown in vitro that DON treatment led to apoptosis, or programmed cell death. Different pathways were investigated; it could be shown that the inner mitochondrial membrane was damaged, [14] and that single strand DNA breaks occurred. [15] The exact mode of action remains unclear and needs further research.

DON has not been approved as a pharmaceutical agent; however, it has been tested in combination with a recombinant glutaminase in clinical trials for the treatment of different solid tumors. [16]

See also

Related Research Articles

<span class="mw-page-title-main">Asparagine</span> Chemical compound

Asparagine is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group, an α-carboxylic acid group, and a side chain carboxamide, classifying it as a polar, aliphatic amino acid. It is non-essential in humans, meaning the body can synthesize it. It is encoded by the codons AAU and AAC.

Biosynthesis, i.e., chemical synthesis occurring in biological contexts, is a term most often referring to multi-step, enzyme-catalyzed processes where chemical substances absorbed as nutrients serve as enzyme substrates, with conversion by the living organism either into simpler or more complex products. Examples of biosynthetic pathways include those for the production of amino acids, lipid membrane components, and nucleotides, but also for the production of all classes of biological macromolecules, and of acetyl-coenzyme A, adenosine triphosphate, nicotinamide adenine dinucleotide and other key intermediate and transactional molecules needed for metabolism. Thus, in biosynthesis, any of an array of compounds, from simple to complex, are converted into other compounds, and so it includes both the catabolism and anabolism of complex molecules. Biosynthetic processes are often represented via charts of metabolic pathways. A particular biosynthetic pathway may be located within a single cellular organelle, while others involve enzymes that are located across an array of cellular organelles and structures.

5α-Reductase Enzyme family

5α-Reductases, also known as 3-oxo-5α-steroid 4-dehydrogenases, are enzymes involved in steroid metabolism. They participate in three metabolic pathways: bile acid biosynthesis, androgen and estrogen metabolism. There are three isozymes of 5α-reductase encoded by the genes SRD5A1, SRD5A2, and SRD5A3.

<span class="mw-page-title-main">Tumor metabolome</span>

The study of the tumor metabolism, also known as tumor metabolome describes the different characteristic metabolic changes in tumor cells. The characteristic attributes of the tumor metabolome are high glycolytic enzyme activities, the expression of the pyruvate kinase isoenzyme type M2, increased channeling of glucose carbons into synthetic processes, such as nucleic acid, amino acid and phospholipid synthesis, a high rate of pyrimidine and purine de novo synthesis, a low ratio of Adenosine triphosphate and Guanosine triphosphate to Cytidine triphosphate and Uridine triphosphate, low Adenosine monophosphate levels, high glutaminolytic capacities, release of immunosuppressive substances and dependency on methionine.

<span class="mw-page-title-main">Amino acid synthesis</span> The set of biochemical processes by which amino acids are produced

Amino acid biosynthesis is the set of biochemical processes by which the amino acids are produced. The substrates for these processes are various compounds in the organism's diet or growth media. Not all organisms are able to synthesize all amino acids. For example, humans can synthesize 11 of the 20 standard amino acids. These 11 are called the non-essential amino acids.

<span class="mw-page-title-main">CAD protein</span> Protein-coding gene in the species Homo sapiens

CAD protein is a trifunctional multi-domain enzyme involved in the first three steps of pyrimidine biosynthesis. De-novo synthesis starts with cytosolic carbamoylphosphate synthetase II which uses glutamine, carbon dioxide and ATP. This enzyme is inhibited by uridine triphosphate.

<span class="mw-page-title-main">CTP synthetase</span> Enzyme

CTP synthase is an enzyme involved in pyrimidine biosynthesis that interconverts UTP and CTP.

<span class="mw-page-title-main">Azaserine</span> Chemical compound

Azaserine is a naturally occurring serine derivative diazo compound with antineoplastic and antibiotic properties deriving from its action as a purinergic antagonist and structural similarity to glutamine. Azaserine acts by competitively inhibiting glutamine amidotransferase, a key enzyme responsible for glutamine metabolism.

<span class="mw-page-title-main">Glutaminase</span>

Glutaminase is an amidohydrolase enzyme that generates glutamate from glutamine. Glutaminase has tissue-specific isoenzymes. Glutaminase has an important role in glial cells.

Barbiturase is a zinc-containing amidohydrolase. Its systemic name is barbiturate amidohydrolase (3-oxo-3-ureidopropanoate-forming). Barbiturase acts as a catalyst in the second step of oxidative pyrimidine degradation, promoting the ring-opening hydrolysis of barbituric acid to ureidomalonic acid. Although grouped into the naturally existing amidohydrolases, it demonstrates more homology with cyanuric acid amidohydrolase. Therefore, it has been proposed that barbiturase, along with cyanuric acid, should be grouped into a new family. KEGG

<span class="mw-page-title-main">Carbamoyl phosphate synthetase</span> Class of enzymes

Carbamoyl phosphate synthetase catalyzes the ATP-dependent synthesis of carbamoyl phosphate from glutamine or ammonia and bicarbonate. This enzyme catalyzes the reaction of ATP and bicarbonate to produce carboxy phosphate and ADP. Carboxy phosphate reacts with ammonia to give carbamic acid. In turn, carbamic acid reacts with a second ATP to give carbamoyl phosphate plus ADP.

Purine metabolism refers to the metabolic pathways to synthesize and break down purines that are present in many organisms.

<span class="mw-page-title-main">Amidophosphoribosyltransferase</span> Mammalian protein found in Homo sapiens

Amidophosphoribosyltransferase (ATase), also known as glutamine phosphoribosylpyrophosphate amidotransferase (GPAT), is an enzyme responsible for catalyzing the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosyl-1-amine (PRA), using the amine group from a glutamine side-chain. This is the committing step in de novo purine synthesis. In humans it is encoded by the PPAT gene. ATase is a member of the purine/pyrimidine phosphoribosyltransferase family.

<span class="mw-page-title-main">Asparagine synthetase</span> Mammalian protein found in Homo sapiens

Asparagine synthetase is a chiefly cytoplasmic enzyme that generates asparagine from aspartate. This amidation reaction is similar to that promoted by glutamine synthetase. The enzyme is ubiquitous in its distribution in mammalian organs, but basal expression is relatively low in tissues other than the exocrine pancreas.

<span class="mw-page-title-main">Homoserine dehydrogenase</span> Enzyme

In enzymology, a homoserine dehydrogenase (EC 1.1.1.3) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Omega-amidase</span>

In enzymology, an omega-amidase (EC 3.5.1.3) is an enzyme that catalyzes the chemical reaction

In enzymology, a glutamine-pyruvate transaminase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">UCK2</span> Protein-coding gene in the species Homo sapiens

Uridine-cytidine kinase 2 (UCK2) is an enzyme that in humans is encoded by the UCK2 gene.

Glutaminolysis (glutamine + -lysis) is a series of biochemical reactions by which the amino acid glutamine is lysed to glutamate, aspartate, CO2, pyruvate, lactate, alanine and citrate.

<span class="mw-page-title-main">Alazopeptin</span> Chemical compound

Alazopeptin is an antibiotic, with moderate anti-trypanosomal and antitumor activity. It was originally isolated from Streptacidiphilus griseoplanus, sourced from soil near Williamsburg, Iowa. It is also isolated from Kitasatospora azatica It is still largely produced via fermentation broths of that organism. Structurally, alazopeptin is a tripeptide and contains 2 molecules of 6-diazo-5-oxo-L-norleucine and one molecule of L-alanine. In 2021 the biosynthetic pathway of alazopeptin was elucidated.

References

  1. ↑ "CID 5359375". PubChem. United States National Library of Medicine.
  2. ↑ Kawai S, Sugaya Y, Hagihara R, Tomita H, Katsuyama Y, Ohnishi Y (April 2021). "Complete Biosynthetic Pathway of Alazopeptin, a Tripeptide Consisting of Two Molecules of 6-Diazo-5-oxo-l-norleucine and One Molecule of Alanine". Angewandte Chemie. 60 (18): 10319–10325. doi:10.1002/anie.202100462. PMID   33624374. S2CID   232039107.
  3. 1 2 Dion HW, Fusari SA, Jakubowski ZL, Zora JG, Bartz QR, et al. (1954). 6-diazo-5-oxo-L-norleucine, A new tumor inhibitory substance. II: Isolation and Characterization. Antibiotics and Chemotherapy. Vol. 78. pp. 3075–7.
  4. ↑ Yoshioka K, Takehara H, Okada A, Komi N (June 1992). "Glutamine antagonist with diet deficient in glutamine and aspartate reduce tumor growth". The Tokushima Journal of Experimental Medicine. 39 (1–2): 69–76. PMID   1412455.
  5. ↑ Mukherjee P, Augur ZM, Li M, Hill C, Greenwood B, Domin MA, et al. (29 May 2019). "Therapeutic benefit of combining calorie-restricted ketogenic diet and glutamine targeting in late-stage experimental glioblastoma". Communications Biology. 2 (1): 200. doi:10.1038/s42003-019-0455-x. PMC   6541653 . PMID   31149644.
  6. ↑ DeWald HA, Moore AM (August 1958). "6-Diazo-5-oxo-L-norleucine, a New Tumor-inhibitory Substance.1a Preparation of L-, D- and DL-Forms1b". Journal of the American Chemical Society. 80 (15): 3941–3945. doi:10.1021/ja01548a036.
  7. 1 2 3 4 5 6 7 8 9 Pinkus LM (1977). "Glutamine binding sites". Affinity labeling. Methods in Enzymology. Vol. 46. pp. 414–27. doi:10.1016/S0076-6879(77)46049-X. ISBN   978-0-12-181946-0. PMID   909432.
  8. ↑ Ortlund E, Lacount MW, Lewinski K, Lebioda L (February 2000). "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". Biochemistry. 39 (6): 1199–1204. doi:10.1021/bi991797d. PMID   10684596.
  9. 1 2 Eidinoff ML, Knoll JE, Marano B, Cheong L (1 January 1958). "Pyrimidine Studies: I. Effect of DON (6-Diazo-5-oxo-l-norleucine) on Incorporation of Precursors into Nucleic Acid Pyrimidines". Cancer Research. 18 (1): 105–109.
  10. ↑ Levenberg B, Melnick I, Buchanan JM (March 1957). "Biosynthesis of the purines. XV. The effect of aza-L-serine and 6-diazo-5-oxo-L-norleucine on inosinic acid biosynthesis de novo". The Journal of Biological Chemistry. 225 (1): 163–176. doi: 10.1016/S0021-9258(18)64919-1 . PMID   13416227.
  11. 1 2 3 Ahluwalia GS, Grem JL, Hao Z, Cooney DA (1990). "Metabolism and action of amino acid analog anti-cancer agents". Pharmacology & Therapeutics. 46 (2): 243–271. doi:10.1016/0163-7258(90)90094-I. PMID   2108451.
  12. ↑ Barclay RK, Phillipps MA (February 1966). "Effects of 6-diazo-5-oxol-norleucine and other tumor inhibitors on the biosynthesis of nicotinamide adenine dinucleotide in mice". Cancer Research. 26 (2): 282–286. PMID   4285554.
  13. ↑ Rosenbluth RJ, Cooney DA, Jayaram HN, Milman HA, Homan ER (August 1976). "DON, CONV and DONV-II. Inhibition of L-'asparagine synthetase in vivo". Biochemical Pharmacology. 25 (16): 1851–1858. doi:10.1016/0006-2952(76)90189-1. PMID   9091.
  14. ↑ Wu F, Lukinius A, Bergström M, Eriksson B, Watanabe Y, Långström B (July 1999). "A mechanism behind the antitumour effect of 6-diazo-5-oxo-L-norleucine (DON): disruption of mitochondria". European Journal of Cancer. 35 (7): 1155–1161. doi:10.1016/S0959-8049(99)00099-4. PMID   10533463.
  15. ↑ Hiramoto K, Fujino T, Kikugawa K (June 1996). "DNA strand cleavage by tumor-inhibiting antibiotic 6-diazo-5-oxo-L-norleucine". Mutation Research. 360 (2): 95–100. doi:10.1016/0165-1161(95)00073-9. PMID   8649470.
  16. ↑ Mueller C, Al-Batran S, Jaeger E, Schmidt B, Bausch M, Unger C, Sethuraman N (2008). "A phase IIa study of PEGylated glutaminase (PEG-PGA) plus 6-diazo-5-oxo-L-norleucine (DON) in patients with advanced refractory solid tumors". J Clin Oncol. 26 (May 20 Suppl): abstr 2533. doi:10.1200/jco.2008.26.15_suppl.2533.
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