scholarly journals The derepression of enzymes of de novo pyrimidine biosynthesis pathway inBrevibacterium ammoniagenesproducing uridine-5-monophosphate and uracil

1991 ◽  
Vol 82 (3) ◽  
pp. 263-266 ◽  
Author(s):  
A.A. Nudler ◽  
A.G. Garibyan ◽  
G.I. Bourd
Keyword(s):  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Biosynthesis Pathway

scholarly journals Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells

PLoS Genetics ◽  
2020 ◽  
Vol 16 (11) ◽  
pp. e1009117
Author(s):  
M. Carmen Lafita-Navarro ◽  
Niranjan Venkateswaran ◽  
Jessica A. Kilgore ◽  
Suman Kanji ◽  
Jungsoo Han ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Poor Prognosis ◽  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Biosynthesis Pathway ◽  
Salvage Pathway ◽  
Glioblastoma Cells ◽  
Rdna Transcription ◽  
Nucleolar Stress

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH’s activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.

scholarly journals HGG-38. DE NOVO PYRIMIDINE SYNTHESIS INHIBITION INDUCES REPLICATION CATASTROPHE MEDIATED CELL DEATH IN DIFFUSE MIDLINE GLIOMA

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i25-i25
Author(s):  
Sharmistha Pal ◽  
Jakub Kaplan ◽  
Sylwia Stopka ◽  
Michael Regan ◽  
Benjamin Kann ◽  
...  
Keyword(s):  
Cell Death ◽  
Replication Fork ◽  
De Novo ◽  
Combined Treatment ◽  
Pyrimidine Biosynthesis ◽  
In Vivo Studies ◽  
Biosynthesis Pathway ◽  
Pyrimidine Nucleotides ◽  
A Genome ◽  
Fork Stalling

Abstract Diffuse midline gliomas (DMG) are aggressive and lethal pediatric brain tumors that cannot be cured by conventional therapeutic modalities. Using a genome wide CRISPR screen we identified the de novo pyrimidine biosynthesis pathway as a metaboilic vulnerability in DMGs. BAY2402234 is a small molecule inhibitor of DHODH -a rate liminting enzyme in the de novo pyrimidine biosynthesis pathway. BAY2402234 induces cell death in DMG cells at low nanomolar concentrations while sparing adult glioblastoma cells and normal astrocytes. Further investigations revealed drammatic reduction in cellular UMP pools, the precursor for all pyrimidine nucleotides, after DHODH inhibition, specifically in DMG cells. Cytotoxicity of DHODH inhibition in DMG cells is rescued by exogenous uridine, supporting UMP depletion as the mechanism underlying DMG cell death and also showing that cell death is an “on target” response to BAY2402234. Cell death induced by BAY2402234 is a consequence of replication fork stalling as evident by accumulation of chromatin-bound RPA foci and g-H2AX. Stalled replication forks eventually collapse, resulting in replication catastrophy and apoptosis. Cytotoxic effects of DHODH inhibition are further exacerbated by inhibition of the intra-S checkpoint protein, ATR. Combined treatment of DMG cells with DHODH and ATR inhibitors resulted in enhanced accumulation of chromatin-bound RPA, g-H2AX, replication fork collapse and apoptosis. Importantly, in vivo studies verify that both BAY2402234 (DHODHi), and BAY1895344 (ATRi), cross the blood-brain barrier, accumulate in the brain at therapeutically relevant concentrations, and induce DNA damage in intracranial DMG xenografts in mice. Taken together, our studies have identified DHODH inhibition as a DMG-specific vulnerability resulting in cell death; the mechanism of DHODHi-induced cell death led us to identify combined inhibition of DHODH and ATR as a synergistic therapy against DMG tumors.

scholarly journals A Carbamoyl Phosphate Synthetase II (CPSII) Deletion Mutant of Toxoplasma gondii Induces Partial Protective Immunity in Mice

2021 ◽  
Vol 11 ◽  
Author(s):  
Xunhui Zhuo ◽  
Kaige Du ◽  
Haojie Ding ◽  
Di Lou ◽  
Bin Zheng ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
De Novo ◽  
Parasite Growth ◽  
Pyrimidine Biosynthesis ◽  
Mrna Levels ◽  
Biosynthesis Pathway ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate

Toxoplasma gondii is an obligate intracellular protozoan parasite. T. gondii primarily infection in pregnant women may result in fetal abortion, and infection in immunosuppressed population may result in toxoplasmosis. Carbamoyl phosphate synthetase II (CPSII) is a key enzyme in the de novo pyrimidine-biosynthesis pathway, and has a crucial role in parasite replication. We generated a mutant with complete deletion of CPSII via clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 in type-1 RH strain of T. gondii. We tested the intracellular proliferation of this mutant and found that it showed significantly reduced replication in vitro, though CPSII deletion did not completely stop the parasite growth. The immune responses induced by the infection of RHΔCPSII tachyzoites in mice were evaluated. During infection in mice, the RHΔCPSII mutant displayed notable defects in replication and virulence, and significantly enhanced the survival of mice compared with survival of RH-infected mice. We tracked parasite propagation from ascitic fluid in mice infected with the RHΔCPSII mutant, and few tachyzoites were observed at early infection. We also observed that the RHΔCPSII mutant induced greater accumulation of neutrophils. The mutant induced a higher level of T-helper type-1 cytokines [interferon (IFN)-γ, interleukin (IL)-12]. The mRNA levels of signal transducer and activator of transcription cellular transcription factor 1 and IFN regulatory factor 8 were significantly higher in the RHΔCPSII mutant-infected group. Together, these data suggest that CPSII is crucial for parasite growth, and that strains lack the de novo pyrimidine biosynthesis pathway and salvage pathway may become a promising live attenuated vaccine to prevent infection with T. gondii.

scholarly journals Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction

2013 ◽  
Vol 33 (2) ◽  
Author(s):  
JingXian Fang ◽  
Takeshi Uchiumi ◽  
Mikako Yagi ◽  
Shinya Matsumoto ◽  
Rie Amamoto ◽  
...  
Keyword(s):  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Complex Iii ◽  
Biosynthesis Pathway ◽  
M Cell ◽  
Pyrimidine Synthesis ◽  
Cycle Arrest ◽  
Craniofacial Dysmorphism ◽  
Dehydrogenase Gene ◽  
Interfering Rna

Some mutations of the DHODH (dihydro-orotate dehydrogenase) gene lead to postaxial acrofacial dysostosis or Miller syndrome. Only DHODH is localized at mitochondria among enzymes of the de novo pyrimidine biosynthesis pathway. Since the pyrimidine biosynthesis pathway is coupled to the mitochondrial RC (respiratory chain) via DHODH, impairment of DHODH should affect the RC function. To investigate this, we used siRNA (small interfering RNA)-mediated knockdown and observed that DHODH knockdown induced cell growth retardation because of G2/M cell-cycle arrest, whereas pyrimidine deficiency usually causes G1/S arrest. Inconsistent with this, the cell retardation was not rescued by exogenous uridine, which should bypass the DHODH reaction for pyrimidine synthesis. DHODH depletion partially inhibited the RC complex III, decreased the mitochondrial membrane potential, and increased the generation of ROS (reactive oxygen species). We observed that DHODH physically interacts with respiratory complexes II and III by IP (immunoprecipitation) and BN (blue native)/SDS/PAGE analysis. Considering that pyrimidine deficiency alone does not induce craniofacial dysmorphism, the DHODH mutations may contribute to the Miller syndrome in part through somehow altered mitochondrial function.

scholarly journals DDRE-32. THERAPEUTIC TARGETING OF A NOVEL METABOLIC ADDICTION IN DIFFUSE MIDLINE GLIOMA

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i13-i13
Author(s):  
Sharmistha Pal ◽  
Jakub P Kaplan ◽  
Sylwia A Stopka ◽  
Michael S Regan ◽  
Bradley R Hunsel ◽  
...  
Keyword(s):  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Biosynthesis Pathway ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
A Genome ◽  
De Novo Pyrimidine Synthesis ◽  
Diffuse Midline Glioma

Abstract Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer that is in need of urgent “outside the box” therapeutic approaches. Recent studies show that tumor cells adapt to stresses created by oncogenic mutations and these oncogene-induced adaptations create vulnerabilities that can be exploited to therapeutic ends. To uncover these oncogene-induced vulnerabilities in DMGs we conducted a genome-wide CRIPSR knockout screen in three DMG lines. The top common DMG dependency pathway that we discovered is de novo pyrimidine biosynthesis. Under normal conditions pyrimidine nucleotide needs are met through the salvage pathway. However, in DMG tumorigenesis, pyrimidine nucleotide synthesis is rewired such that the cells become dependent on the de novo biosynthesis pathway. De novo pyrimidine synthesis is catalyzed by CAD, DHODH and UMPS; all three genes are identified as dependencies in our screen and have been validated using shRNA mediated gene knockdown. Interestingly, DMG cells did not exhibit a dependency on the de novo purine biosynthesis pathway. Using a small molecule inhibitor of DHODH, BAY2402234 [currently studied in phase I trial for myeloid malignancies (NCT03404726)], we have demonstrated and validated, (i) efficacy and specificity of de novo pyrimidine synthesis inhibition in vitro in DMG cells; (ii) de novo pyrimidine addiction is not attributable to cell proliferation; (iii) DHODH inhibition induces apoptosis by hindering replication and inciting DNA damage; (iv) DHODH and ATR inhibition act synergistically to induce DMG cell death; and (v) critical in vivo efficacy. The in vivo experiment documents that BAY2402234 crosses the blood-brain barrier, is present in the brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in intracranial DMG tumors in mice, and prolongs survival of orthotopic DMG tumor bearing mice. Taken together, our studies have identified a novel metabolic vulnerability that can be translated for the treatment of DMG patients.

scholarly journals Original Chemical Series of Pyrimidine Biosynthesis Inhibitors That Boost the Antiviral Interferon Response

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Marianne Lucas-Hourani ◽  
Daniel Dauzonne ◽  
Hélène Munier-Lehmann ◽  
Samira Khiar ◽  
Sébastien Nisole ◽  
...  
Keyword(s):  
Metabolic Pathway ◽  
Defense Mechanisms ◽  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Interferon Response ◽  
Type I ◽  
Innate Defense ◽  
New Class ◽  
Biosynthesis Inhibitors ◽  
Tightly Coupled

ABSTRACT De novo pyrimidine biosynthesis is a key metabolic pathway involved in multiple biosynthetic processes. Here, we identified an original series of 3-(1H-indol-3-yl)-2,3-dihydro-4H-furo[3,2-c]chromen-4-one derivatives as a new class of pyrimidine biosynthesis inhibitors formed by two edge-fused polycyclic moieties. We show that identified compounds exhibit broad-spectrum antiviral activity and immunostimulatory properties, in line with recent reports linking de novo pyrimidine biosynthesis with innate defense mechanisms against viruses. Most importantly, we establish that pyrimidine deprivation can amplify the production of both type I and type III interferons by cells stimulated with retinoic acid-inducible gene 1 (RIG-I) ligands. Altogether, our results further expand the current panel of pyrimidine biosynthesis inhibitors and illustrate how the production of antiviral interferons is tightly coupled to this metabolic pathway. Functional and structural similarities between this new chemical series and dicoumarol, which was reported before to inhibit pyrimidine biosynthesis at the dihydroorotate dehydrogenase (DHODH) step, are discussed.

scholarly journals De novo transcriptome and tissue specific expression analysis of genes associated with biosynthesis of secondary metabolites in Operculina turpethum (L.)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bhagyashree Biswal ◽  
Biswajit Jena ◽  
Alok Kumar Giri ◽  
Laxmikanta Acharya
Keyword(s):  
Secondary Metabolite ◽  
De Novo ◽  
Differentially Expressed Gene ◽  
Homology Search ◽  
Biosynthesis Pathway ◽  
Terpenoid Biosynthesis ◽  
De Novo Transcriptome ◽  
Illumina Hiseq ◽  
Secondary Metabolite Biosynthesis ◽  
Root Tissues

AbstractThis study reported the first-ever de novo transcriptome analysis of Operculina turpethum, a high valued endangered medicinal plant, using the Illumina HiSeq 2500 platform. The de novo assembly generated a total of 64,259 unigenes and 20,870 CDS (coding sequence) with a mean length of 449 bp and 571 bp respectively. Further, 20,218 and 16,458 unigenes showed significant similarity with identified proteins of NR (non-redundant) and UniProt database respectively. The homology search carried out against publicly available database found the best match with Ipomoea nil sequences (82.6%). The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis identified 6538 unigenes functionally assigned to 378 modules with phenylpropanoid biosynthesis pathway as the most enriched among the secondary metabolite biosynthesis pathway followed by terpenoid biosynthesis. A total of 17,444 DEGs were identified among which majority of the DEGs (Differentially Expressed Gene) involved in secondary metabolite biosynthesis were found to be significantly upregulated in stem as compared to root tissues. The qRT-PCR validation of 9 unigenes involved in phenylpropanoid and terpenoid biosynthesis also showed a similar expression pattern. This finding suggests that stem tissues, rather than root tissues, could be used to prevent uprooting of O. turpethum in the wild, paving the way for the plant's effective conservation. Moreover, the study formed a valuable repository of genetic information which will provide a baseline for further molecular research.

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