6-Diazo-5-oxo-L-norleucine explained

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.[1] It is one of the most famous non-proteinogenic amino acid and was characterized in 1956 by Henry W Dion et al.,[2] who suggested a possible use in cancer therapy. This antitumoral efficacy was confirmed in different animal models.[3] 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.[4]

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).[2] [5]

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.[6] The following table gives a survey of DON targets.

Metabolic pathwayReferences
Carbamoyl phosphate synthase (CAD) [7] [8]
CTP synthase (CTPS)
FGAR amidotransferase [9]
Guanosine monophosphate synthetase (GMPS) [10]
PRPP amidotransferase
NAD synthase [11]
Asparagine synthetase Amino acid synthesis [12]

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,[13] and that single strand DNA breaks occurred.[14] 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.[15]

See also

Notes and References

  1. Kawai S, Sugaya Y, Hagihara R, Tomita H, Katsuyama Y, Ohnishi Y . 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 . April 2021 . 33624374 . 10.1002/anie.202100462 . 232039107 .
  2. Book: Dion HW, Fusari SA, Jakubowski ZL, Zora JG, Bartz QR . 6-diazo-5-oxo-L-norleucine, A new tumor inhibitory substance. II: Isolation and Characterization . . 78 . 3075–7 . 1954. etal.
  3. Yoshioka K, Takehara H, Okada A, Komi N . Glutamine antagonist with diet deficient in glutamine and aspartate reduce tumor growth . The Tokushima Journal of Experimental Medicine . 39 . 1–2 . 69–76 . June 1992 . 1412455 .
  4. Mukherjee P, Augur ZM, Li M, Hill C, Greenwood B, Domin MA, Kondakci G, Narain NR, Kiebish MA, Bronson RT, Arismendi-Morillo G, Chinopoulos C, Seyfried TN . 6 . Therapeutic benefit of combining calorie-restricted ketogenic diet and glutamine targeting in late-stage experimental glioblastoma . Communications Biology . 2 . 1 . 200 . 29 May 2019 . 31149644 . 6541653 . 10.1038/s42003-019-0455-x .
  5. DeWald HA, Moore AM . 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 . August 1958 . 80 . 15 . 3941–3945 . 10.1021/ja01548a036 .
  6. Ortlund E, Lacount MW, Lewinski K, Lebioda L . 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 . February 2000 . 10684596 . 10.1021/bi991797d .
  7. Book: Pinkus LM . Affinity labeling . Glutamine binding sites . Methods in Enzymology . 46 . 414–27 . 1977 . 909432 . 10.1016/S0076-6879(77)46049-X . 978-0-12-181946-0 .
  8. Eidinoff ML, Knoll JE, Marano B, Cheong L . Pyrimidine Studies: I. Effect of DON (6-Diazo-5-oxo-l-norleucine) on Incorporation of Precursors into Nucleic Acid Pyrimidines . Cancer Research . 1 January 1958 . 18 . 1 . 105–109 .
  9. Levenberg B, Melnick I, Buchanan JM . 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 . March 1957 . 13416227 . 10.1016/S0021-9258(18)64919-1 . free .
  10. Ahluwalia GS, Grem JL, Hao Z, Cooney DA . Metabolism and action of amino acid analog anti-cancer agents . Pharmacology & Therapeutics . 46 . 2 . 243–271 . 1990 . 2108451 . 10.1016/0163-7258(90)90094-I .
  11. Barclay RK, Phillipps MA . 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 . February 1966 . 4285554 .
  12. Rosenbluth RJ, Cooney DA, Jayaram HN, Milman HA, Homan ER . DON, CONV and DONV-II. Inhibition of L-'asparagine synthetase in vivo . Biochemical Pharmacology . 25 . 16 . 1851–1858 . August 1976 . 9091 . 10.1016/0006-2952(76)90189-1 .
  13. Wu F, Lukinius A, Bergström M, Eriksson B, Watanabe Y, Långström B . 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 . July 1999 . 10533463 . 10.1016/S0959-8049(99)00099-4 .
  14. Hiramoto K, Fujino T, Kikugawa K . DNA strand cleavage by tumor-inhibiting antibiotic 6-diazo-5-oxo-L-norleucine . Mutation Research . 360 . 2 . 95–100 . June 1996 . 8649470 . 10.1016/0165-1161(95)00073-9 .
  15. Mueller C, Al-Batran S, Jaeger E, Schmidt B, Bausch M, Unger C, Sethuraman N . 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 . 2008 . 10.1200/jco.2008.26.15_suppl.2533 .

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