scholarly journals De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis

2012 ◽  
Vol 24 (4) ◽  
pp. 1549-1559 ◽  
Author(s):  
Sandra Witz ◽  
Benjamin Jung ◽  
Sarah Fürst ◽  
Torsten Möhlmann
Keyword(s):  
De Novo ◽  
Salvage Pathway ◽  
Nucleotide Synthesis ◽  
Pyrimidine Nucleotide

scholarly journals Inhibition of De Novo Pyrimidine Nucleotide Synthesis By the Novel DHODH Inhibitor PTC299 Induces Differentiation and/or Death of AML Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5152-5152
Author(s):  
Marla Weetall ◽  
Kensuke Kojima ◽  
Sujan Piya ◽  
Christopher Trotta ◽  
John Baird ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
De Novo ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Dna Breaks ◽  
Advisory Committees ◽  
Pyrimidine Nucleotides ◽  
Nucleotide Synthesis ◽  
Pyrimidine Nucleotide

Background: Pyrimidine nucleotides are generated either by de novo synthesis or the salvage pathway in which pyrimidine nucleotides are obtained from the diet. Resting cells typically acquire adequate pyrimidine nucleotides from the salvage pathway. Rapidly proliferating cells, however, are dependent on the de novo synthesis of pyrimidine nucleotides. PTC299 is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis that had previously been in clinical trials for treatment of solid tumors. Results: Using 15N-labelled glutamine, we show that PTC299 reduces de novo pyrimidine nucleotide synthesis in PTC299-sensitive AML cell lines resulting in a depletion of total pyrimidine nucleotides. In parallel to reduction in pyrimidine nucleotides, PTC 299 leads to accumulation of DHO, the substrate of DHODH and unexpectedly, an accumulation of N-carbamoyl aspartate the metabolite above DHO in the de novo pyrimidine nucleotide synthesis pathway. PTC299 was broadly active against leukemia and lymphoma lines, with 80% of the AML lines tested showing sensitivity. Treatment of AML cell lines with PTC299 induced differentiation as shown by increased CD14 and/or reduced proliferation. Using isogenic AML lines, we show that PTC299 reduces the proliferation of both p53 wildtype and p53 deficient leukemia calls with similar potency as measured by the concentration of PTC299 required to reduce cell number by 50% (CC50). In cells expressing wildtype p53, PTC299 increases p53 activation. However, p53- wildtype cells undergo increased apoptosis whereas p53-deficience cells undergo necrosis. PTC299 induced a G1/S cell cycle arrest, also independent of p53 status. PTC299 increased H2A.X (a marker of double stranded DNA breaks) in both p53 wildtype and p53 deficient cells. These data suggest that the depletion of nucleotides results in stalling at the replication fork, and subsequent DNA-breaks. Conclusion: De novo pyrimidine nucleotide synthesis is critical for AML survival and proliferation. Depletion of nucleotides results in reduced proliferation, triggering either differentiation and/or cell death. Disclosures Weetall: PTC Therapeutics: Employment. Trotta:PTC Therapeutics: Employment. Baird:PTC Therapeutics: Employment. O'Keefe:PTC Therapeutics: Employment. Furia:PTC Therapeutics: Employment. Borthakur:PTC Therapeutics: Consultancy; Janssen: Research Funding; AbbVie: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; NKarta: Consultancy; AstraZeneca: Research Funding; Xbiotech USA: Research Funding; Incyte: Research Funding; GSK: Research Funding; Oncoceutics, Inc.: Research Funding; Novartis: Research Funding; Agensys: Research Funding; BMS: Research Funding; Oncoceutics: Research Funding; Cantargia AB: Research Funding; Bayer Healthcare AG: Research Funding; Eisai: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Polaris: Research Funding; Merck: Research Funding; Cyclacel: Research Funding; Eli Lilly and Co.: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Arvinas: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Strategia Therapeutics: Research Funding. Spiegel:PTC Therapeutics: Consultancy.

scholarly journals Cellular pyrimidine imbalance triggers mitochondrial DNA–dependent innate immunity

2021 ◽  
Author(s):  
Hans-Georg Sprenger ◽  
Thomas MacVicar ◽  
Amir Bahat ◽  
Kai Uwe Fiedler ◽  
Steffen Hermans ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Cultured Cells ◽  
De Novo ◽  
Metabolic Response ◽  
Interferon Response ◽  
Type I ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
De Novo Pyrimidine Synthesis

AbstractCytosolic mitochondrial DNA (mtDNA) elicits a type I interferon response, but signals triggering the release of mtDNA from mitochondria remain enigmatic. Here, we show that mtDNA-dependent immune signalling via the cyclic GMP–AMP synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS–STING–TBK1) pathway is under metabolic control and is induced by cellular pyrimidine deficiency. The mitochondrial protease YME1L preserves pyrimidine pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33. Deficiency of YME1L causes inflammation in mouse retinas and in cultured cells. It drives the release of mtDNA and a cGAS–STING–TBK1-dependent inflammatory response, which requires SLC25A33 and is suppressed upon replenishment of cellular pyrimidine pools. Overexpression of SLC25A33 is sufficient to induce immune signalling by mtDNA. Similarly, depletion of cytosolic nucleotides upon inhibition of de novo pyrimidine synthesis triggers mtDNA-dependent immune responses in wild-type cells. Our results thus identify mtDNA release and innate immune signalling as a metabolic response to cellular pyrimidine deficiencies.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide

2021 ◽  
Author(s):  
Yao Li ◽  
Li Yi ◽  
Sipeng Cheng ◽  
Yongshan Wang ◽  
Jiongjiong Wang ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Active Metabolite ◽  
Canine Distemper Virus ◽  
De Novo ◽  
Specific Inhibitor ◽  
Vero Cells ◽  
Canine Distemper ◽  
Nucleotide Synthesis ◽  
Pyrimidine Nucleotide ◽  
Rate Limiting

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

scholarly journals Pyrimidine nucleotide availability is essential for efficient photosynthesis, ROS scavenging, and organelle development

2021 ◽  
Author(s):  
Leo Bellin ◽  
Michael Melzer ◽  
Alexander Hilo ◽  
Diana Laura Garza Amaya ◽  
Isabel Keller ◽  
...  
Keyword(s):  
De Novo ◽  
Energy State ◽  
Chloroplast Ultrastructure ◽  
Low Energy ◽  
Ros Scavenging ◽  
Nucleotide Synthesis ◽  
Knock Down ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
De Novo Pyrimidine Synthesis

ABSTRACTDe novo synthesis of pyrimidines is an essential and highly conserved pathway in all organisms. A peculiarity in plants is the localization of the first committed step, catalyzed by aspartate transcarbamoylase (ATC), in chloroplasts. By contrast, the third step in the pathway is catalyzed by dihydroorotate dehydrogenase (DHODH) localized in mitochondria in eukaryotes, including plants. To unravel pathway- and organelle specific functions, we analyzed knock-down mutants in ATC and DHODH in detail. ATC knock-downs were most severely affected, exhibiting low levels of pyrimidine metabolites, a low energy state, reduced photosynthetic capacity and accumulated reactive oxygen species (ROS). Furthermore, we observed altered leaf morphology and chloroplast ultrastructure in the mutants. Although less affected, DHODH knock-down mutants showed impaired seed germination and altered mitochondrial ultrastructure. Our results point to an integration of de novo pyrimidine synthesis and cellular energy states via photosynthesis and mitochondrial respiration. These findings highlight the likelihood of further regulatory roles for ATC and DHODH in pathways located in the corresponding organelles.ONE-SENTENCE SUMMARYImpaired pyrimidine nucleotide synthesis results in a low energy state, affecting photosynthesis and organellar ultrastructure, thus leading to reduced growth, reproduction, and seed yield

scholarly journals Renal hypertrophy in experimental diabetes. The activity of the ‘de novo’ and salvage pathways of purine [corrected] synthesis

1988 ◽  
Vol 249 (3) ◽  
pp. 911-914 ◽  
Author(s):  
S Kunjara ◽  
S J Beardsley ◽  
A L Greenbaum
Keyword(s):  
Nucleic Acids ◽  
De Novo ◽  
Experimental Diabetes ◽  
Salvage Pathway ◽  
Phosphoribosyl Pyrophosphate ◽  
Renal Hypertrophy ◽  
Early Diabetes ◽  
Nucleotide Synthesis ◽  
Main Route ◽  
Salvage Pathways

Measurements were made of the activity of phosphoribosyl pyrophosphate amidotransferase (PPRibP-At, EC 2.4.2.14) and of adenine (APRT, EC 2.4.2.7) and hypoxanthine (HPRT, EC 2.4.2.8) phosphoribosyltransferases, representing the ‘de novo’ and salvage pathways respectively. PPRibP-At activity increased within 3 days of diabetes, whereas APRT and HPRT increased later. Incorporation of [14C]formate and of [8-14C]adenine into the nucleic acids of kidney slices showed that formate was incorporated earlier, and to a greater extent, than was adenine. These results indicate that, although the ‘de novo’ pathway for nucleotide synthesis is the main route in early diabetes, the salvage pathway assumes greater importance at later stages.

Effects of Chinese Herbal Medicines on DNA-Synthesizing Enzyme Activities in Mammary Tumors of Mice

1994 ◽  
Vol 22 (01) ◽  
pp. 43-50 ◽  
Author(s):  
Shinobu Sakamoto ◽  
Ryuta Furuichi ◽  
Manabu Matsuda ◽  
Hideki Kudo ◽  
Satoe Suzuki ◽  
...  
Keyword(s):  
Prolactin Level ◽  
De Novo ◽  
Herbal Medicines ◽  
Mammary Tumors ◽  
Serum Prolactin ◽  
Serum Prolactin Level ◽  
Nucleotide Synthesis ◽  
Chinese Herbal ◽  
Pyrimidine Nucleotide ◽  
Salvage Pathways

Sho-saiko-to (SST) and Juzen-taiho-to (JTT), Japanese modified Chinese herbal prescriptions, suppressed the activities of thymidylate synthetase and thymidine kinase involved in de novo and salvage pathways for pyrimidine nucleotide synthesis, respectively, in mammary tumors of SHN mice with the reduction of serum prolactin level. These results indicate that SST and JTT may have the anti-tumor effects on mammary tumors.

scholarly journals DDRE-29. DE NOVO PYRIMIDINE SYNTHESIS IS A TARGETABLE VULNERABILITY IN IDH-MUTANT GLIOMA

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i12-i13
Author(s):  
Diana D Shi ◽  
Adam C Wang ◽  
Michael M Levitt ◽  
Jennifer E Endress ◽  
Min Xu ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Synthetic Lethality ◽  
Nucleotide Metabolism ◽  
Metabolic Inhibitor ◽  
Lower Grade ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
Idh1 Mutations

Abstract 70–90% of lower-grade gliomas and secondary glioblastomas harbor gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1), causing overproduction of the oncometabolite (R)-2-hydroxyglutarate [(R)-2HG]. Although inhibitors of mutant IDH enzymes are effective in other cancers, including leukemia, they have shown guarded efficacy in preclinical and clinical brain tumor studies, thus underscoring the need to identify additional therapeutic targets in IDH mutant glioma. We sought to identify tumor-specific metabolic vulnerabilities induced by IDH1 mutations that could be exploited therapeutically. To uncover such vulnerabilities, we conducted a chemical synthetic lethality screen using isogenic IDH1 mutant and IDH1 wild-type (WT) glioma cell lines and a novel metabolic inhibitor screening platform. We discovered that IDH1 mutant cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). This vulnerability is specific because inhibitors of purine nucleotide metabolism did not score in our screen. We validated that the cytotoxicity of pyrimidine synthesis inhibitors is on-target and showed that IDH1 mutant patient-derived glioma stem-like cell lines are also hyperdependent on de novo pyrimidine nucleotide synthesis compared to IDH1 WT lines. To test pyrimidine synthesis dependence of IDH1 mutant gliomas in vivo, we used a brain-penetrent DHODH inhibitor currently undergoing evaluation in leukemia patients, BAY 2402234. We found that BAY 2402234 displays monotherapy activity against gliomas in an orthotopic xenograft model of IDH1 mutant glioma, with an effect size that compared favorably with radiotherapy. We also developed novel genetically engineered and allograft mouse models of mutant IDH1-driven anaplastic astrocytoma and showed that BAY 2402234 blocked growth of orthotopic astrocytoma allografts. Our findings bolster rationale to target DHODH in glioma, highlight BAY 2402234 as a clinical-stage drug that can be used to inhibit DHODH in brain tumors, and establish IDH1 mutations as predictive biomarkers of DHODH inhibitor efficacy.

scholarly journals Crystallographic approach to fragment-based hit discovery against Schistosoma mansoni purine nucleoside phosphorylase

2021 ◽  
Author(s):  
Muhammad Faheem ◽  
Napoleão Fonseca Valadares ◽  
José Brandão-Neto ◽  
Domenico Bellini ◽  
Patrick Collins ◽  
...  
Keyword(s):  
De Novo ◽  
Screening Strategy ◽  
New Drugs ◽  
High Price ◽  
Salvage Pathway ◽  
Purine Salvage ◽  
Nucleotide Synthesis ◽  
Fragment Screening ◽  
Lack Of Information ◽  
Purine Pathway

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness – which is limited to the parasite’s adult form – and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. 14 of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

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