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Journal of the American Chemical Society Aug 2023l-Alanosine is a diazeniumdiolate (-nitrosohydroxylamine) antibiotic that inhibits MTAP-deficient tumor cells by blocking adenine biosynthesis. Previous work revealed...
l-Alanosine is a diazeniumdiolate (-nitrosohydroxylamine) antibiotic that inhibits MTAP-deficient tumor cells by blocking adenine biosynthesis. Previous work revealed the early steps in the biosynthesis of l-alanosine. In the present study, we used genome mining to discover two new l-alanosine-producing strains that lack the spartate-itrouccinate pathway genes found in the original l-alanosine producer. Instead, nitrate is reduced with a unique set of nitrate-nitrite reductases. These enzymes are typically used as part of the nitrogen cycle for denitrification or assimilation, and our report here shows how enzymes from the nitrogen cycle can be repurposed for the biosynthesis of specialized metabolites. The widespread distribution of nitric-oxide-producing reductases also indicates a potential for the discovery of new nitric-oxide-derived natural products.
Topics: Nitric Oxide; Nitrates; Oxidoreductases; Nitrite Reductases; Nitrate Reductases
PubMed: 37478476
DOI: 10.1021/jacs.3c04447 -
Angewandte Chemie (International Ed. in... Mar 2020The formation of a N-N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among...
The formation of a N-N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N-N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate-containing amino acid l-alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l-alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl-carrier-protein-based mechanism for activation of the precursor l-diaminopropionate, and we also show that nitric oxide can participate in non-enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N-terminal cupin domain and a C-terminal AraC-like DNA-binding domain, is required for alanosine biosynthesis.
Topics: Alanine; Aspartic Acid; Glutamic Acid; Molecular Structure; Multigene Family; Streptomyces
PubMed: 31823464
DOI: 10.1002/anie.201913458 -
Oncotarget Mar 2023Homozygous deletion of upregulates synthesis of purine (DNSP) and increases the proliferation of neoplastic cells. This increases the sensitivity of breast cancer...
INTRODUCTION
Homozygous deletion of upregulates synthesis of purine (DNSP) and increases the proliferation of neoplastic cells. This increases the sensitivity of breast cancer cells to DNSP inhibitors such as methotrexate, L-alanosine and pemetrexed.
MATERIALS AND METHODS
7,301 cases of MBC underwent hybrid-capture based comprehensive genomic profiling (CGP). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. Tumor cell PD-L1 expression was determined by IHC (Dako 22C3).
RESULTS
208 (2.84%) of MBC featured loss. loss patients were younger ( = 0.002) and were more frequently ER- (30% vs. 50%; < 0.0001), triple negative (TNBC) (47% vs. 27%; < 0.0001) and less frequently HER2+ (2% vs. 8%; = 0.0001) than intact MBC. Lobular histology and mutations were more frequent in intact (14%) than loss MBC ( < 0.0001). (100%) and (97%) loss (9p21 co-deletion) were significantly associated with loss ( < 0.0001). Likely associated with the increased TNBC cases, BRCA1 mutation was also more frequent in loss MBC (10% vs. 4%; < 0.0001). As for immune checkpoint inhibitors biomarkers, higher TMB >20 mut/Mb levels in the intact MBC ( < 0.0001) and higher PD-L1 low expression (1-49% TPS) in the loss ( = 0.002) were observed.
CONCLUSIONS
loss in MBC has distinct clinical features with genomic alterations (GA) affecting both targeted and immunotherapies. Further efforts are necessary to identify alternative means of targeting PRMT5 and MTA2 in -ve cancers to benefit from the high-MTA environment of -deficient cancers.
Topics: Humans; B7-H1 Antigen; Homozygote; Triple Negative Breast Neoplasms; Sequence Deletion; Purine-Nucleoside Phosphorylase; Genomics; Histone Deacetylases; Repressor Proteins; Protein-Arginine N-Methyltransferases
PubMed: 36913304
DOI: 10.18632/oncotarget.28376 -
Cancer Research Oct 2021Methylthioadenosine phosphorylase (MTAP) is a key enzyme associated with the salvage of methionine and adenine that is deficient in 20% to 30% of pancreatic cancer. Our...
Methylthioadenosine phosphorylase (MTAP) is a key enzyme associated with the salvage of methionine and adenine that is deficient in 20% to 30% of pancreatic cancer. Our previous study revealed that MTAP deficiency indicates a poor prognosis for patients with pancreatic ductal adenocarcinoma (PDAC). In this study, bioinformatics analysis of The Cancer Genome Atlas (TCGA) data indicated that PDACs with MTAP deficiency display a signature of elevated glycolysis. Metabolomics studies showed that that MTAP deletion-mediated metabolic reprogramming enhanced glycolysis and purine synthesis in pancreatic cancer cells. Western blot analysis revealed that MTAP knockout stabilized hypoxia-inducible factor 1α (HIF1α) protein via posttranslational phosphorylation. RIO kinase 1 (RIOK1), a downstream kinase upregulated in MTAP-deficient cells, interacted with and phosphorylated HIF1α to regulate its stability. experiments demonstrated that the glycolysis inhibitor 2-deoxy-d-glucose (2-DG) and the purine synthesis inhibitor l-alanosine synergized to kill MTAP-deficient pancreatic cancer cells. Collectively, these results reveal that MTAP deficiency drives pancreatic cancer progression by inducing metabolic reprogramming, providing a novel target and therapeutic strategy for treating MTAP-deficient disease. SIGNIFICANCE: This study demonstrates that MTAP status impacts glucose and purine metabolism, thus identifying multiple novel treatment options against MTAP-deficient pancreatic cancer.
Topics: Animals; Biomarkers, Tumor; Cell Line, Tumor; Cell Survival; Cellular Reprogramming; Computational Biology; Disease Models, Animal; Energy Metabolism; Gene Expression Profiling; Glycolysis; Heterografts; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Metabolic Networks and Pathways; Metabolomics; Mice; Models, Biological; Pancreatic Neoplasms; Positron Emission Tomography Computed Tomography; Prognosis; Purine-Nucleoside Phosphorylase; Purines
PubMed: 34385182
DOI: 10.1158/0008-5472.CAN-20-0414 -
Chembiochem : a European Journal of... Apr 2020N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate...
N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N-nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product l-alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid l-2,3-diaminopropionic acid (l-Dap) is synthesized and loaded onto a free-standing peptidyl carrier protein (PCP) domain in l-alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. These discoveries will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.
Topics: Alanine; Azo Compounds; Bacterial Proteins; Biosynthetic Pathways; Molecular Structure; Multigene Family; Streptomyces
PubMed: 31643127
DOI: 10.1002/cbic.201900565 -
Biomedicines Mar 2022Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that...
Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in purine salvage, we demonstrate that purine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo purine synthesis, attenuates stemness of -deficient GBM cells. This ALA-induced reduction in stemness is mediated in part by compromised mitochondrial function, highlighted by ALA-induced elimination of mitochondrial spare respiratory capacity. Notably, these effects of ALA are apparent even when the treatment was transient and with a low dose. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify purine supply as an essential component in maintaining mitochondrial function in GBM cells and highlight a critical role of mitochondrial function in sustaining GBM stemness. We propose that purine synthesis inhibition can be beneficial in combination with the standard of care for -deficient GBMs, and that it may be feasible to achieve this benefit without inflicting major toxicity.
PubMed: 35453502
DOI: 10.3390/biomedicines10040751