Journal articles: 'Synthesis of pyrimidine nucleotides'

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Weetall, Marla, Kensuke Kojima, Sujan Piya, Christopher Trotta, John Baird, Kylie O'Keefe, Bansri Furia, Gautam M. Borthakur, and Robert Spiegel. "Inhibition of De Novo Pyrimidine Nucleotide Synthesis By the Novel DHODH Inhibitor PTC299 Induces Differentiation and/or Death of AML Cells." Blood 134, Supplement_1 (November 13, 2019): 5152. http://dx.doi.org/10.1182/blood-2019-124569.

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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.

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Szondy, Z., and E. A. Newsholme. "The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin." Biochemical Journal 261, no. 3 (August 1, 1989): 979–83. http://dx.doi.org/10.1042/bj2610979.

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The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and ‘half-maximal concentration’ values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.

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West, Thomas P. "Pyrimidine nucleotide synthesis inPseudomonascitronellolis." Canadian Journal of Microbiology 50, no. 6 (June 1, 2004): 455–59. http://dx.doi.org/10.1139/w04-028.

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Pyrimidine biosynthesis was active in Pseudomonas citronellolis ATCC 13674 and appeared to be regulated by pyrimidines. When wild-type cells were grown on succinate in the presence of uracil, the de novo enzyme activities were depressed while only four enzyme activities were depressed in the glucose-grown cells. On either carbon source, orotic acid-grown cells had diminished aspartate transcarbamoylase, dihydroorotase or OMP decarboxylase activity. Pyrimidine limitation of glucose-grown pyrimidine auxotrophic cells resulted in de novo enzyme activities, except for transcarbamoyolase activity, that were elevated by more than 5-fold compared to their activities in uracil-grown cells. Since pyrimidine limitation of succinate-grown mutant cells produced less enzyme derepression, catabolite repression appeared to be a factor. At the level of enzyme activity, aspartate transcarbamoylase activity in P. citronellolis was strongly inhibited by all effectors tested. Compared to the regulation of pyrimidine biosynthesis in taxonomically-related species, pyrimidine biosynthesis in P. citronellolis appeared more highly regulated.Key words: pyrimidine biosynthesis, regulation, Pseudomonas citronellolis, auxotroph, aspartate transcarbamoylase, inhibition.

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Shanmugasundaram, Muthian, Annamalai Senthilvelan, and Anilkumar R. Kore. "C-5 Substituted Pyrimidine Nucleotides/Nucleosides: Recent Progress in Synthesis, Functionalization, and Applications." Current Organic Chemistry 23, no. 13 (October 9, 2019): 1439–68. http://dx.doi.org/10.2174/1385272823666190809124310.

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The chemistry of C5 substituted pyrimidine nucleotide serves as a versatile molecular biology probe for the incorporation of DNA/RNA that has been involved in various molecular biology applications such as gene expression, chromosome, and mRNA fluorescence in situ hybridization (FISH) experiment, mutation detection on arrays and microarrays, in situ RT-PCR, and PCR. In addition to C5 substituted pyrimidine nucleotide, C5 substituted pyrimidine nucleoside displays a broad spectrum of biological applications such as antibacterial, antiviral and anticancer activities. This review focusses on the recent development in the synthesis of aminoallyl pyrimidine nucleotide, aminopropargyl pyrimidine nucleotide, fluorescent probes containing C5 substituted pyrimidine nucleotide, 2′-deoxycytidine nucleoside containing vinylsulfonamide and acrylamide modification, C5 alkenyl, C5 alkynyl, and C5 aryl pyrimidine nucleosides through palladium-catalyzed reaction, pyrimidine nucleoside containing triazole moiety through Click reaction, 5-isoxazol-3-yl-pyrimidine nucleoside, C5 azide modified pyrimidine nucleoside, 2′-deoxycytidine nucleotide containing photocleavable moiety, and uridine nucleoside containing germane and their biological applications are outlined.

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Hassan, Mohamed E. "Photochemical synthesis of C(5) alkyl and heteroaryl substituted pyrimidine nucleotides." Collection of Czechoslovak Chemical Communications 50, no. 10 (1985): 2319–23. http://dx.doi.org/10.1135/cccc19852319.

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A simple and direct method for the synthesis of C-5 modified nucleosides is described. Photoirradiation of 2'-deoxyuridine 5'-phosphate (I), in the presence of haloheteroarenes afforded the C-5 heteroaryl substituted nucleotides. 5-(2-Hydroxyethyl)nucleotide also obtained from photocoupling of I with 2-iodoethanol. Photoirradiation of 5-iodo-2-deoxyuridine 5'-phosphate (II), in the presence of methyl acrylate or acrylonitrile, gave 5-(2-methoxycarbonylethenyl) and 5-(2-cyanoethenyl) nucleotide, respectively.

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Froschauer, Elisabeth M., Nicole Rietzschel, Melanie R. Hassler, Markus Binder, Rudolf J. Schweyen, Roland Lill, Ulrich Mühlenhoff, and Gerlinde Wiesenberger. "The mitochondrial carrier Rim2 co-imports pyrimidine nucleotides and iron." Biochemical Journal 455, no. 1 (September 13, 2013): 57–65. http://dx.doi.org/10.1042/bj20130144.

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Mitochondrial iron uptake is of key importance both for organelle function and cellular iron homoeostasis. The mitochondrial carrier family members Mrs3 and Mrs4 (homologues of vertebrate mitoferrin) function in organellar iron supply, yet other low efficiency transporters may exist. In Saccharomyces cerevisiae, overexpression of RIM2 (MRS12) encoding a mitochondrial pyrimidine nucleotide transporter can overcome the iron-related phenotypes of strains lacking both MRS3 and MRS4. In the present study we show by in vitro transport studies that Rim2 mediates the transport of iron and other divalent metal ions across the mitochondrial inner membrane in a pyrimidine nucleotide-dependent fashion. Mutations in the proposed substrate-binding site of Rim2 prevent both pyrimidine nucleotide and divalent ion transport. These results document that Rim2 catalyses the co-import of pyrimidine nucleotides and divalent metal ions including ferrous iron. The deletion of RIM2 alone has no significant effect on mitochondrial iron supply, Fe–S protein maturation and haem synthesis. However, RIM2 deletion in mrs3/4Δ cells aggravates their Fe–S protein maturation defect. We conclude that under normal physiological conditions Rim2 does not play a significant role in mitochondrial iron acquisition, yet, in the absence of the main iron transporters Mrs3 and Mrs4, this carrier can supply the mitochondrial matrix with iron in a pyrimidine-nucleotide-dependent fashion.

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Sprenger, Hans-Georg, Thomas MacVicar, Amir Bahat, Kai Uwe Fiedler, Steffen Hermans, Denise Ehrentraut, Katharina Ried, et al. "Cellular pyrimidine imbalance triggers mitochondrial DNA–dependent innate immunity." Nature Metabolism 3, no. 5 (April 26, 2021): 636–50. http://dx.doi.org/10.1038/s42255-021-00385-9.

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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.

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Chunduru, Jayendra, and Thomas P. West. "Pyrimidine nucleotide synthesis in the emerging pathogen Pseudomonas monteilii." Canadian Journal of Microbiology 64, no. 6 (June 2018): 432–38. http://dx.doi.org/10.1139/cjm-2018-0015.

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Regulation of pyrimidine biosynthesis by pyrimidines in the emerging, opportunistic human pathogen Pseudomonas monteilii ATCC 700476 was evident. When wild-type cells were grown on succinate in the presence of uracil or orotic acid, the activities of all 5 pyrimidine biosynthetic enzymes were depressed while the activities of 3 of the enzymes decreased in glucose-grown cells supplemented with uracil or orotic acid compared with unsupplemented cells. Pyrimidine limitation of succinate- or glucose-grown pyrimidine auxotrophic cells lacking orotate phosphoribosyltransferase activity resulted in more than a doubling of the pyrimidine biosynthetic enzyme activities relative to their activities in uracil-grown cells. Independent of carbon source, pyrimidine-limited cells of the pyrimidine auxotrophic cells deficient for dihydroorotase activity generally resulted in a slight elevation or depression of the pyrimidine biosynthetic enzyme activities compared with their activities in cells grown under saturating uracil conditions. Aspartate transcarbamoylase activity in P. monteilii was regulated at the enzyme activity level, since the enzyme was strongly inhibited by CTP, UMP, GMP, GDP, ADP, and UTP. In summary, the regulation of pyrimidine biosynthesis in P. monteilii could be used to control its growth or to differentiate it biochemically from other related species of Pseudomonas.

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Deval, Jérôme, Megan H. Powdrill, Claudia M. D'Abramo, Luciano Cellai, and Matthias Götte. "Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase." Antimicrobial Agents and Chemotherapy 51, no. 8 (May 14, 2007): 2920–28. http://dx.doi.org/10.1128/aac.00186-07.

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ABSTRACT Nonobligate chain terminators, such as 2′-C-methylated nucleotides, block RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3′-hydroxyl group can be excised in the presence of pyrophosphate (PPi), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2′-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2′-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2′-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2′-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.

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Pels Rijcken, W. R., G. J. M. Hooghwinkel, and W. Ferwerda. "Pyrimidine metabolism and sugar nucleotide synthesis in rat liver." Biochemical Journal 266, no. 3 (March 15, 1990): 777–83. http://dx.doi.org/10.1042/bj2660777.

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With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 14C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.

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Mendz, G. L., B. M. Jimenez, S. L. Hazell, A. M. Gero, and W. J. O'Sullivan. "De novo synthesis of pyrimidine nucleotides by Helicobacter pylori." Journal of Applied Bacteriology 77, no. 1 (July 1994): 1–8. http://dx.doi.org/10.1111/j.1365-2672.1994.tb03036.x.

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Balasubramaniyam, Thananjeyan, Kwnag-Im Oh, Ho-Seong Jin, Hye-Bin Ahn, Byeong-Seon Kim, and Joon-Hwa Lee. "Non-Canonical Helical Structure of Nucleic Acids Containing Base-Modified Nucleotides." International Journal of Molecular Sciences 22, no. 17 (September 2, 2021): 9552. http://dx.doi.org/10.3390/ijms22179552.

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Chemically modified nucleobases are thought to be important for therapeutic purposes as well as diagnosing genetic diseases and have been widely involved in research fields such as molecular biology and biochemical studies. Many artificially modified nucleobases, such as methyl, halogen, and aryl modifications of purines at the C8 position and pyrimidines at the C5 position, are widely studied for their biological functions. DNA containing these modified nucleobases can form non-canonical helical structures such as Z-DNA, G-quadruplex, i-motif, and triplex. This review summarizes the synthesis of chemically modified nucleotides: (i) methylation, bromination, and arylation of purine at the C8 position and (ii) methylation, bromination, and arylation of pyrimidine at the C5 position. Additionally, we introduce the non-canonical structures of nucleic acids containing these modifications.

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Lafita-Navarro, M. Carmen, Niranjan Venkateswaran, Jessica A. Kilgore, Suman Kanji, Jungsoo Han, Spencer Barnes, Noelle S. Williams, Michael Buszczak, Sandeep Burma, and Maralice Conacci-Sorrell. "Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells." PLOS Genetics 16, no. 11 (November 17, 2020): e1009117. http://dx.doi.org/10.1371/journal.pgen.1009117.

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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.

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Frosina, G., P. Fortini, O. Rossi, F. Carrozzino, A. Abbondandolo, and E. Dogliotti. "Repair of abasic sites by mammalian cell extracts." Biochemical Journal 304, no. 3 (December 15, 1994): 699–705. http://dx.doi.org/10.1042/bj3040699.

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Hamster cell extracts that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers, were used to study the repair of apurinic/apyrimidinic (AP) sites and methoxyamine (MX)-modified AP sites. Plasmid molecules were heat-treated at pH 5 and incubated with MX when required. The amount of damage introduced ranged from 0.2 to 0.9 AP sites/kb. Extracts were prepared from the Chinese hamster ovary CHO-9 cell line and from its derivative, 43-3B clone which is mutated in the nucleotide excision repair (NER) ERCC1 gene. AP and MX-AP sites stimulated repair synthesis by CHO-9 cell extracts. The level of synthesis correlated with the number of lesions and was of similar magnitude to the repair stimulated by 4.3 u.v. photoproducts/kb. Repair of AP and MX-AP sites was faster than the repair of u.v. damage and was independent of ERCC1 gene product. The high level of repair replication was due to a very efficient and rapid incision of plasmids carrying AP or MX-AP sites, performed by abundant AP endonucleases present in the extract. The calculated average repair patch sizes were: 7 nucleotides per AP site; 10 nucleotides per MX-AP site; 28 nucleotides per (6-4) u.v. photoproduct or cyclobutane pyrimidine dimer. The data indicate that AP and MX-AP sites are very efficiently repaired by base-excision repair in mammalian cells and suggest that MX-AP sites may also be processed via alternative repair mechanisms.

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Nikolov, E. N., and M. D. Dabeva. "Re-utilization of pyrimidine nucleotides during rat liver regeneration." Biochemical Journal 228, no. 1 (May 15, 1985): 27–33. http://dx.doi.org/10.1042/bj2280027.

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The changes in the specific radioactivities of the pool of total acid-soluble uridine nucleotides and of uridine and cytidine components of total cellular and nuclear RNA were monitored in regenerating rat liver for 12 days after partial hepatectomy. Evidence is presented for the re-utilization of pyrimidine nucleotides derived from cytoplasmic RNA degradation for the synthesis of new RNA. The extent of recycling was assessed and the true rate of rRNA turnover determined more accurately. The reutilization of the uridine components of RNA was 7.0%/day during the proliferative and 3.2%/day during the post-proliferative phase, whereas that of the cytidine nucleotides was more pronounced (9.6%/day and 18.1%/day respectively). The results reveal the existence of partial compartmentalization of pyrimidine ribonucleoside triphosphate pools in the nucleus and cytoplasm of rat liver cells.

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Marobbio, Carlo Marya Thomas, Maria Antonietta Di Noia, and Ferdinando Palmieri. "Identification of a mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization." Biochemical Journal 393, no. 2 (December 23, 2005): 441–46. http://dx.doi.org/10.1042/bj20051284.

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Pyrimidine (deoxy)nucleoside triphosphates are required in mitochondria for the synthesis of DNA and the various types of RNA present in these organelles. In Saccharomyces cerevisiae, these nucleotides are synthesized outside the mitochondrial matrix and must therefore be transported across the permeability barrier of the mitochondrial inner membrane. However, no protein has ever been found to be associated with this transport activity. In the present study, Rim2p has been identified as a yeast mitochondrial pyrimidine nucleotide transporter. Rim2p (replication in mitochondria 2p) is a member of the mitochondrial carrier protein family having some special features. The RIM2 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes and its transport properties and kinetic parameters were characterized. It transported the pyrimidine (deoxy)nucleoside tri- and di-phosphates and, to a lesser extent, pyrimidine (deoxy)nucleoside monophosphates, by a counter-exchange mechanism. Transport was saturable, with an apparent Km of 207 μM for TTP, 404 μM for UTP and 435 μM for CTP. Rim2p was strongly inhibited by mercurials, bathophenanthroline, tannic acid and Bromocresol Purple, and partially inhibited by bongkrekic acid. Furthermore, the Rim2p-mediated heteroexchanges, TTP/TMP and TTP/TDP, are electroneutral and probably H+-compensated. The main physiological role of Rim2p is proposed to be to transport (deoxy)pyrimidine nucleoside triphosphates into mitochondria in exchange for intramitochondrially generated (deoxy)pyrimidine nucleoside monophosphates.

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Kartha, S., and F. G. Toback. "Purine nucleotides stimulate DNA synthesis in kidney epithelial cells in culture." American Journal of Physiology-Renal Physiology 249, no. 6 (December 1, 1985): F967—F972. http://dx.doi.org/10.1152/ajprenal.1985.249.6.f967.

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Adenine nucleotides infused into animals with acute renal failure appear to enhance recovery of kidney function and structure. To determine whether these compounds could act by a direct effect on renal cell metabolism, their capacity to stimulate DNA synthesis was evaluated in cultures of monkey kidney epithelial cells (BSC-1 line). AMP and ADP enhanced DNA synthesis by threefold more than was previously observed with other mitogens for these cells. Guanosine and inosine and their nucleotides and adenosine and ATP were also mitogenic but to a lesser extent, whereas pyrimidine derivatives were ineffective. In the presence of AMP, autoradiography of [3H]thymidine-labeled cells indicated that a greater number of cells entered the S phase of the cell cycle, and assessment of cell number revealed increased multiplication. The mitogenic effect of adenine nucleotides was not reproduced by agents that raise the cellular content of cAMP and was serum independent. Adenine nucleotides did not alter DNA synthesis when added to cultures of mouse fibroblasts. These results indicate that provision of exogenous purine nucleosides and nucleotides stimulate DNA synthesis in renal epithelial cells in culture.

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Fateev, Ilja V., Ekaterina V. Sinitsina, Aiguzel U. Bikanasova, Maria A. Kostromina, Elena S. Tuzova, Larisa V. Esipova, Tatiana I. Muravyova, Alexei L. Kayushin, Irina D. Konstantinova, and Roman S. Esipov. "Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis." Beilstein Journal of Organic Chemistry 14 (December 21, 2018): 3098–105. http://dx.doi.org/10.3762/bjoc.14.289.

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Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10–13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate specificity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two nucleotides were synthesized: 9-(β-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-Сl-AMP) using TthAPRT and 1-(β-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using TthНPRT.

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Pels Rijcken, W. R., B. Overdijk, D. H. van den Eijnden, and W. Ferwerda. "Pyrimidine nucleotide metabolism in rat hepatocytes: evidence for compartmentation of nucleotide pools." Biochemical Journal 293, no. 1 (July 1, 1993): 207–13. http://dx.doi.org/10.1042/bj2930207.

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Pyrimidine nucleotide metabolism in rat hepatocytes was studied by measurement of the labelling kinetics of the various intermediates after double labelling with [14C]orotic acid and [3H]cytidine, the precursors for the de novo and the salvage pathways respectively. For the uridine nucleotides, differences were found for the 14C/3H ratios in the UDP-sugars, in UMP (of RNA) and in their precursor UTP, suggesting the existence of separated flows of the radioactive precursors through the de novo and the salvage pathways. Higher ratios in the UDP-sugars, which are synthesized in the cytoplasm, and a lower ratio in UMP (of RNA) relative to the 14C/3H ratio in UTP indicated that UTP derived from orotic acid is preferentially used for the cytoplasmic biosynthesis of the UDP-sugars. Uridine, derived from cytidine, is preferentially used for the nuclear-localized synthesis of RNA. In contrast to these findings, the 14C/3H ratios in the cytidine derivatives CMP-NeuAc and CMP (of RNA), and in the liponucleotides CDP-choline and CDP-ethanolamine, were all lower than that in the precursor CTP. This indicates a preferential utilization of the salvage-derived CTP for the synthesis of the liponucleotides as well as for RNA and CMP-NeuAc. Similar conclusions could be drawn from experiments in which the intracellular amounts of several uridine- and cytidine-nucleotide-containing derivatives were increased by preincubating the hepatocytes with unlabelled pyrimidine nucleotides or ethanolamine. Based on these data, we propose a refined model for the intracellular compartmentation of pyrimidine nucleotide biosynthesis in which three pools of UTP are distinguished: a pool of de novo-derived molecules and a pool of salvage-derived molecules, both of which are channelled to the site of utilization; in addition an ‘overflow’ pool exists, consisting of molecules having escaped from channelling. An overflow pool could also be distinguished for CTP, but no discrimination between de novo and salvage-derived molecules could be made.

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Yao, Yixin, Yang Liu, Hui Guo, Makhdum Ahmed, Shabnam Bhuiyan, Krystle Nomie, Liang Zhang, and Michael Wang. "Metabolic Profiling Identifies De Novo Nucleotide Synthesis As a Potential Metabolic Vulnerability for Targeted Therapy Against Mantle Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 2945. http://dx.doi.org/10.1182/blood-2018-99-112192.

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Abstract Introduction: Cancer cells exhibit dramatic alterations in cellular metabolism, such as enhanced de novo nucleotide synthesis, to support cell growth, proliferation and survival. The abundance of the nucleotide pool as well as the level and activity of different rate-limiting enzymes belonging to the nucleotide synthetic pathway limit the maximal proliferative capacity of cells. Maintenance of an adequate pool of deoxyribonucleotide triphosphates is essential for DNA replication and DNA repair, and consequently, the genetic integrity of nuclear and mitochondrial genomes. We and others have demonstrated that mantle cell lymphoma (MCL) undergoes metabolic reprogramming to progress and develop resistance to targeted therapy; however, the contribution of de novo nucleotide synthesis to the development and progression of MCL remains poorly understood. In contrast, oncogenic Myc is demonstrated to be highly upregulated in a subset of MCL. In addition to its pro-glycolysis, pro-biogenesis and pro-tumor growth functions, oncogenic levels of Myc induce the expression of multiple genes involved in the nucleotide biosynthetic pathway (e.g., IMPDH2, CTPS1, and CAD). Myc-induced glutamine metabolism also increases the abundance and activity of different rate-limiting enzymes that produce the molecular precursors required for de novo nucleotide synthesis. The γ-nitrogen amide group of glutamine is an indispensable donor of nitrogen for de novo synthesis of both nucleobases purine and pyrimidine. Here, we hypothesize that a subset of MCL depends on de novo nucleotide synthesis for anabolic cell growth and cancer progression due to aberrant Myc expression and Myc-induced glutaminolysis. Methods: Primary MCL biopsy, apheresis, and blood specimens as well as MCL cell lines were utilized for metabolic and functional analyses. Liquid Chromatography Mass Spectrometry (LC-MS) metabolomics was employed to measure the steady-state level of metabolites. Western-blotting and real-time qPCR were utilized determine protein and gene expression levels. BrdU incorporation and the Cell-Trace Violet Cell Proliferation Assay were employed to assess DNA synthesis and cell proliferation. Cell viability was measured with the Cell Titer-Glo Cell Viability Assay. Pharmacological agents were employed to inhibit either de novo nucleotide synthesis or glutaminolysis. Results: Metabolomics profiling of steady-state levels of intracellular metabolites showed significant increases in N-carbamoyl aspartate/dihydroorotate and 5-phosphoribosyl-1-pyrophosphate (PRPP), which are critical intermediates in de novo pyrimidine and purine synthesis, respectively, as well as CTP, dUTP, dCTP, in a subset of MCL, indicating remarkably upregulated de novo nucleotide synthesis. The protein and mRNA levels of c-Myc and its target genes involved in the metabolism of nucleotides (IMPDH2, CTPS1, CAD) were significantly increased. Inhibition of pyrimidine synthesis with DON (6-diazo-5-oxo-L-norleucine), a CTPS1 inhibitor, dramatically reduced the pool of pyridine nucleotides, leading to remarkable apoptosis and halted cell proliferation of a subset of MCL cell lines. Consistent with c-Myc overexpression, increased glutamine uptake was also observed in a subset of MCL cell lines. Glutamine deprivation or pharmacological inhibition of glutamine metabolism showed a similar effect on the inhibition of pyrimidine synthesis as DON (6-diazo-5-oxo-L-norleucine), manifested by a significant reduction of pyrimidine nucleoside triphosphate levels, a dramatic increase in apoptosis, and retarded cell proliferation of a subset of MCL cell lines. Conclusions: Our preliminary results indicate that de novo nucleotide synthesis is upregulated in a subset of MCL with aberrant c-Myc expression. The expression of genes involved in nucleotide metabolism as well as glutaminolysis is also elevated in these cancer cells. Disruption of de novo nucleotide synthesis or glutaminolysis induces apoptosis and suppresses proliferation of a subset of MCL. Myc does not possess enzymatic activity and is considered "undruggable"; therefore, the inhibition of Myc target genes such as those involved in de novo nucleotide synthesis and glutaminolysis presents a promising alternative approach. Taken together, MCL dependency on de novo nucleotide synthesis may represent a metabolic vulnerability for targeted therapy for MCL. Disclosures Wang: AstraZeneca: Consultancy, Research Funding; Juno: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Research Funding; Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Dava Oncology: Honoraria; MoreHealth: Consultancy; Novartis: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Sharkin, Yuri. "New pyrimidine nucleotides as dATP mimics: Synthesis and some biochemical properties." Collection of Czechoslovak Chemical Communications 61, s1 (1996): 171–73. http://dx.doi.org/10.1135/cccc1996s171.

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22

Gołos, B., K. Misiura, M. Olesiak, A. Okruszek, W. J. Stec, and W. Rode. "Synthesis and interaction with thymidylate synthase of 5'-dithiophosphate and 5'-fluorothiophosphate of thymidine." Acta Biochimica Polonica 45, no. 1 (March 31, 1998): 83–86. http://dx.doi.org/10.18388/abp.1998_4289.

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Thymidine-5'-fluorothiophosphate, dTMP(S)-F, was synthesized by the oxathiaphospholane, and thymidine 5'-dithiophosphate, dTMPS2, by the dithiaphospholane, method. To estimate the role of 5'-phosphate group ionization in binding of pyrimidine nucleotides by thymidylate synthase, dTMP(S)-F was studied as an inhibitor of mouse tumour (L1210) enzyme, and its inhibitory properties were compared with those of dTMPS2, a close dTMP analogue. While dTMPS2 proved to be an inhibitor, competitive vs dUMP, with K(i)app = 94 microM, the 5'-fluorothiophosphate congener displayed no activity, indicating that the enzyme requires for binding the presence of a dianionic 5'-phosphate group in a nucleotide.

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23

Botha, Filip, Michaela Slavíčková, Radek Pohl, and Michal Hocek. "Copper-mediated arylsulfanylations and arylselanylations of pyrimidine or 7-deazapurine nucleosides and nucleotides." Organic & Biomolecular Chemistry 14, no. 42 (2016): 10018–22. http://dx.doi.org/10.1039/c6ob01917j.

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Evans, Mary E., Dean P. Jones, and Thomas R. Ziegler. "Glutamine inhibits cytokine-induced apoptosis in human colonic epithelial cells via the pyrimidine pathway." American Journal of Physiology-Gastrointestinal and Liver Physiology 289, no. 3 (September 2005): G388—G396. http://dx.doi.org/10.1152/ajpgi.00072.2005.

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Glutamine (Gln) prevents apoptosis in intestinal epithelial cells, but the mechanism(s) remain unknown. Gln-derived metabolites include ammonia, glutamate (Glu), glutathione (GSH), and nucleotides. We previously showed that Gln potently inhibited apoptosis in cytokine-treated human colonic HT-29 cells; this effect was specific to Gln, unaffected by Glu, and unrelated to intracellular GSH. The current research examines mechanism(s) for Gln-induced antiapoptotic effects in HT-29 cells treated with TNF-α-related apoptosis-inducing ligand (TRAIL). Proliferating cells were treated with Gln or selected Gln metabolites for 24 h. Cells were then treated with TRAIL and Gln or its downstream metabolites, and apoptosis was assessed at 8 h after treatment. The purine and pyrimidine precursors inosine and orotate inhibited TRAIL-induced apoptosis. However, inhibition of purine synthesis with azaserine did not alter the potent antiapoptotic effect of Gln. In contrast, the pyrimidine synthesis inhibitor, acivicin, completely prevented this response. Supplementation with the pyrimidine uracil or the pyrimidine precursor orotate rescued the acivicin-induced blockade of Gln antiapoptotic action. Removal of bicarbonate, a substrate for pyrimidine synthesis, also inhibited the antiapoptotic effects of Gln. Uracil and thymine alone also significantly decreased TRAIL-induced apoptosis. The antiapoptotic effects of Gln were independent of DNA/RNA synthesis as measured by flow cytometry and bromodeoxyuridine incorporation. In conclusion, Gln prevents TRAIL-induced apoptosis in HT-29 cells through a mechanism involving the pyrimidine pathway. Our data also demonstrate the novel antiapoptotic effects of pyrimidine bases and their precursor orotate in these human intestinal cells.

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Suzuki, Ichirou, Sadao Kato, Tokuzou Kitada, Nobuhiro Yano, and Toshiki Morichi. "Growth of Lactobacillus bulgaricus in Milk. 2. Characteristics of Purine Nucleotides, Pyrimidine Nucleotides, and Nucleic Acid Synthesis." Journal of Dairy Science 69, no. 4 (April 1986): 971–78. http://dx.doi.org/10.3168/jds.s0022-0302(86)80490-8.

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26

Simakov, M. B., E. A. Kuzicheva, and I. L. Malko. "Abiogenic synthesis of pyrimidine nucleotides in solid state by vacuum ultraviolet radiation." Advances in Space Research 19, no. 7 (January 1997): 1059–62. http://dx.doi.org/10.1016/s0273-1177(97)00353-0.

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OLIVARES, J., and A. ROSSI. "Synthesis of pyrimidine nucleotides in rat myocardium: Potential role of blood cytidine." Journal of Molecular and Cellular Cardiology 20, no. 4 (April 1988): 313–22. http://dx.doi.org/10.1016/s0022-2828(88)80065-8.

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28

Switzer, Christopher. "A Missing Prebiotic Link: Discovery of a Plausible Synthesis of Pyrimidine Nucleotides." ChemBioChem 10, no. 16 (November 2, 2009): 2591–93. http://dx.doi.org/10.1002/cbic.200900516.

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29

Wunderlich, Christoph H., Romana Spitzer, Tobias Santner, Katja Fauster, Martin Tollinger, and Christoph Kreutz. "Synthesis of (6-13C)Pyrimidine Nucleotides as Spin-Labels for RNA Dynamics." Journal of the American Chemical Society 134, no. 17 (April 19, 2012): 7558–69. http://dx.doi.org/10.1021/ja302148g.

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30

Huang, M., and L. M. Graves. "De novo synthesis of pyrimidine nucleotides; emerging interfaces with signal transduction pathways." Cellular and Molecular Life Sciences (CMLS) 60, no. 2 (February 1, 2003): 321–36. http://dx.doi.org/10.1007/s000180300027.

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31

Gu, Xiaorong, Rita Tohme, Mendel Goldfinger, Benjamin K. Tomlinson, Nneha Sakre, Shannon Hanmer, Babal K. Jha, Jaroslaw P. Maciejewski, Amit Verma, and Yogenthiran Saunthararajah. "Venetoclax Inhibition of Pyrimidine Synthesis Guides Methods for Integration with Decitabine or 5-Azacytidine That Are Non-Myelosuppressive." Blood 136, Supplement 1 (November 5, 2020): 26–27. http://dx.doi.org/10.1182/blood-2020-143200.

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Venetoclax (Ven) administered daily with pulse-cycled parenteral decitabine (Dec) or 5-azacytidine (5Aza) is standard therapy for acute myeloid leukemia (AML) in the elderly. In practice, toxicity/myelosuppression is frequent, and prompts Ven dose reductions, but by guess-work, because the mechanism downstream of BCL2-inhibition by which Ven augments Dec/5Aza activity is unclear. For the first time, we show that Ven inhibits de novo pyrimidine synthesis, an effect that can guide its integration with Dec/5Aza in a way that enhances anti-AML activity without suppressing normal myelopoiesis. Dec and 5Aza are pro-drugs processed by pyrimidine metabolism into a deoxycytidine analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1), a pharmacodynamic effect that terminates malignant but not normal self-replication. We recently demonstrated that Dec- and 5Aza-resistance emerges automatically from adaptive responses of the pyrimidine metabolism network to Dec/5Aza-induced nucleotide perturbations, such that Dec/5Aza processing into DNMT1-depleting nucleotide is forestalled (Leukemia - https://rdcu.be/b58pS). A key element in this auto-resistance is upregulated de novo pyrimidine synthesis, that out-competes salvaged Dec/5Aza. De novo pyrimidine synthesis has an electron-transport dependent mitochondrial step executed by dihydroorotate dehydrogenase (DHODH): BCL2-inhibition by Ven depolarizes mitochondrial membranes - we examined for the first time Ven impact on DHODH/pyrimidine synthesis (others have focused on apoptosis and other metabolic consequences of mitochondrial depolarization). Consistent with Ven inhibiting DHODH/pyrimidine synthesis, both Ven and the direct DHODH inhibitor teriflunomide, at non-apoptotic concentrations, significantly decreased cytidine- and deoxycytidine triphosphate (CTP, dCTP) in AML cells (Fig1A). To see if this effect of Ven can counter Dec/5Aza resistance, we selected for THP1 AML cells double-resistant to Dec 0.5 μM and 5Aza 5 μM - these cells upregulated expression of de novo pyrimidine synthesis enzymes and contained uridine, cytidine and deoxycytidine at levels up to 7-fold higher than parental cells. The double-resistant AML cells were significantly cytoreduced by Ven at a clinically relevant concentration of 1 μM, even though parental THP1 AML cells were minimally sensitive (Fig1B). Consistent with inhibition of de novo pyrimidine synthesis as the mechanism, this action was significantly abrogated by cytidine supplementation (Fig1B). We previously showed that timed alternation of Dec with 5Aza, and incorporation of the cytidine deaminase inhibitor tetrahydrouridine (THU), counters metabolic Dec or 5Aza-resistance to extend non-cytotoxic DNMT1-depletion and survival in vivo (https://rdcu.be/b58pS). However, AML still eventually progresses, via upregulated de novo pyrimidine synthesis. In considering use of Ven to counter this mode of resistance, we reasoned that concurrent administration risks antagonism, because Ven can cause transient cytostasis, and DNMT1-depletion by Dec/5Aza is S-phase dependent. Prior Ven administration, however, creates effect-time sufficient to deplete endogenous nucleotides from AML cells and hence upregulate pyrimidine salvage (that uptakes Dec/5Aza) (Fig1A), as well as resumed cell cycle. Therefore, in vivo in a patient-derived xenotransplant model (PDX) of AML, we introduced Ven (at human equivalent dose), parsimoniously 2X/week, the day before each Dec or 5Aza administration, and at the time of overt AML progression on the base THU-Dec/5Aza regimen. This minimalist Ven application significantly extended survival (time-to-distress) even though it was initiated at progression (Fig1C). As expected, use of this regimen upfront in a PDX of Dec/5Aza-resistant AML produced even greater (several-fold) survival extension (treatment ongoing) vs the base regimen (Fig1D). A non-cytotoxic/non-myelosuppressive mechanism-of-action was confirmed by serial blood counts on-therapy. In practice, Ven dose in combination with Dec or 5Aza to treat AML is frequently empirically reduced because of toxicity/myelosuppression. Instead, Ven inhibition of de novo pyrimidine synthesis can guide mechanism-based, intermittent administration that avoids toxicity/myelosuppression yet enhances Dec/5Aza anti-AML activity. Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago.

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Nicoloff, Hervé, Aram Elagöz, Florence Arsène-Ploetze, Benoît Kammerer, Jan Martinussen, and Françoise Bringel. "Repression of the pyr Operon in Lactobacillus plantarum Prevents Its Ability To Grow at Low Carbon Dioxide Levels." Journal of Bacteriology 187, no. 6 (March 15, 2005): 2093–104. http://dx.doi.org/10.1128/jb.187.6.2093-2104.2005.

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ABSTRACT Carbamoyl phosphate is a precursor for both arginine and pyrimidine biosynthesis. In Lactobacillus plantarum, carbamoyl phosphate is synthesized from glutamine, ATP, and carbon dioxide by two sets of identified genes encoding carbamoyl phosphate synthase (CPS). The expression of the carAB operon (encoding CPS-A) responds to arginine availability, whereas pyrAaAb (encoding CPS-P) is part of the pyrR1BCAaAbDFE operon coding for the de novo pyrimidine pathway repressed by exogenous uracil. The pyr operon is regulated by transcription attenuation mediated by a trans-acting repressor that binds to the pyr mRNA attenuation site in response to intracellular UMP/phosphoribosyl pyrophosphate pools. Intracellular pyrimidine triphosphate nucleoside pools were lower in mutant FB335 (carAB deletion) harboring only CPS-P than in the wild-type strain harboring both CPS-A and CPS-P. Thus, CPS-P activity is the limiting step in pyrimidine synthesis. FB335 is unable to grow in the presence of uracil due to a lack of sufficient carbamoyl phosphate required for arginine biosynthesis. Forty independent spontaneous FB335-derived mutants that have lost regulation of the pyr operon were readily obtained by their ability to grow in the presence of uracil and absence of arginine; 26 harbored mutations in the pyrR1-pyrB loci. One was a prototroph with a deletion of both pyrR1 and the transcription attenuation site that resulted in large amounts of excreted pyrimidine nucleotides and increased intracellular UTP and CTP pools compared to wild-type levels. Low pyrimidine-independent expression of the pyr operon was obtained by antiterminator site-directed mutagenesis. The resulting AE1023 strain had reduced UTP and CTP pools and had the phenotype of a high-CO2-requiring auxotroph, since it was able to synthesize sufficient arginine and pyrimidines only in CO2-enriched air. Therefore, growth inhibition without CO2 enrichment may be due to low carbamoyl phosphate pools from lack of CPS activity.

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Kim, Seohyun Chris, Derek K. O’Flaherty, Lijun Zhou, Victor S. Lelyveld, and Jack W. Szostak. "Inosine, but none of the 8-oxo-purines, is a plausible component of a primordial version of RNA." Proceedings of the National Academy of Sciences 115, no. 52 (December 3, 2018): 13318–23. http://dx.doi.org/10.1073/pnas.1814367115.

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The emergence of primordial RNA-based life would have required the abiotic synthesis of nucleotides, and their participation in nonenzymatic RNA replication. Although considerable progress has been made toward potentially prebiotic syntheses of the pyrimidine nucleotides (C and U) and their 2-thio variants, efficient routes to the canonical purine nucleotides (A and G) remain elusive. Reported syntheses are low yielding and generate a large number of undesired side products. Recently, a potentially prebiotic pathway to 8-oxo-adenosine and 8-oxo-inosine has been demonstrated, raising the question of the suitability of the 8-oxo-purines as substrates for prebiotic RNA replication. Here we show that the 8-oxo-purine nucleotides are poor substrates for nonenzymatic RNA primer extension, both as activated monomers and when present in the template strand; their presence at the end of a primer also strongly reduces the rate and fidelity of primer extension. To provide a proper comparison with 8-oxo-inosine, we also examined primer extension reactions with inosine, and found that inosine exhibits surprisingly rapid and accurate nonenzymatic RNA copying. We propose that inosine, which can be derived from adenosine by deamination, could have acted as a surrogate for G in the earliest stages of the emergence of life.

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34

Schultheisz, Heather L., Blair R. Szymczyna, Lincoln G. Scott, and James R. Williamson. "Enzymatic De Novo Pyrimidine Nucleotide Synthesis." Journal of the American Chemical Society 133, no. 2 (January 19, 2011): 297–304. http://dx.doi.org/10.1021/ja1059685.

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35

YOSHIMURA, Yuichi, Akira MATSUDA, and Tohru UEDA. "Alternative synthesis of 2'-deoxy-6,2'-methano-pyrimidine nucleosides (nucleosides and nucleotides. XC)." CHEMICAL & PHARMACEUTICAL BULLETIN 38, no. 2 (1990): 389–92. http://dx.doi.org/10.1248/cpb.38.389.

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36

MATSUDA, AKIRA, HIROKO ITOH, KENJI TAKENUKI, TAKUMA SASAKI, and TOHRU UEDA. "Alkyl addition reaction of pyrimidine 2'-ketonucleosides: Synthesis of 2'-branched-chain sugar pyrimidine nucleosides. Nucleodides and nucleotides. LXXXI." CHEMICAL & PHARMACEUTICAL BULLETIN 36, no. 3 (1988): 945–53. http://dx.doi.org/10.1248/cpb.36.945.

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37

Yang, Kailin, Xiuxing Wang, Qiulian Wu, Leo Kim, Andrew Morton, Ryan Gimple, Briana Prager, et al. "STEM-22. TARGETING PYRIMIDINE SYNTHESIS ACCENTUATES MOLECULAR THERAPY RESPONSE IN GLIOBLASTOMA STEM CELLS." Neuro-Oncology 21, Supplement_6 (November 2019): vi238. http://dx.doi.org/10.1093/neuonc/noz175.995.

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Abstract Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamyolase, dihydroorotase (CAD) or the critical downstream enzyme, dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity. Higher expression of pyrimidine synthesis genes portend poor prognosis of glioblastoma patients. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.

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38

West, Thomas P. "Pyrimidine nucleotide synthesis in Pseudomonas nitroreducens and the regulatory role of pyrimidines." Microbiological Research 169, no. 12 (December 2014): 954–58. http://dx.doi.org/10.1016/j.micres.2014.04.003.

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39

Wang, Xiuxing, Kailin Yang, Qiulian Wu, Leo J. Y. Kim, Andrew R. Morton, Ryan C. Gimple, Briana C. Prager, et al. "Targeting pyrimidine synthesis accentuates molecular therapy response in glioblastoma stem cells." Science Translational Medicine 11, no. 504 (August 7, 2019): eaau4972. http://dx.doi.org/10.1126/scitranslmed.aau4972.

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Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD) or the critical downstream enzyme dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity in vitro. Higher expression of pyrimidine synthesis genes portends poor prognosis of patients with glioblastoma. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.

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40

Rees, DC, J. Duley, HA Simmonds, B. Wonke, SL Thein, JB Clegg, and DJ Weatherall. "Interaction of hemoglobin E and pyrimidine 5' nucleotidase deficiency." Blood 88, no. 7 (October 1, 1996): 2761–67. http://dx.doi.org/10.1182/blood.v88.7.2761.bloodjournal8872761.

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A Bangladeshi family is described in which the genes for both hemoglobin E (Hb E) and pyrimidine 5′ nucleotidase deficiency are segregating. An individual homozygous for both these conditions has a severe hemolytic anemia, whereas family members who are homozygous for Hb E are asymptomatic and those homozygous for pyrimidine 5′ nucleotidase deficiency have the mild hemolytic anemia that is characteristic of this disorder. Globin-chain synthesis experiments have shown that the mechanism underlying the interaction between these two genotypes is a marked decrease in the stability of Hb E in pyrimidine 5′ nucleotidase-deficient red blood cells (RBCs). It has also been found that in the enzyme-deficient RBCs in which Hb E is highly unstable, free alpha-chains, though not beta E-chains, acoumulate on the membrane. In view of the increasing evidence that the hemolysis associated with pyrimidine 5′ nucleotidase deficiency results not only from an increase in the level of erythrocyte pyrimidines, but also from inhibition of the hexose monophosphate shunt activity in young erythrocytes, it is likely that the marked instability of Hb E in the enzyme-deficient cells results from oxidant damage acting on a mildly unstable Hb variant. These observations may have important implications for the better understanding of the pathophysiology of Hb E/beta-thalassemia, globally the commonest important form of thalassemia.

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Yang, Kailin, Xiuxing Wang, Qiulian Wu, Leo Kim, Andrew Morton, Ryan Gimple, Briana Prager, et al. "FSMP-08. TARGETING PYRIMIDINE SYNTHESIS ACCENTUATES MOLECULAR THERAPY RESPONSE IN GLIOBLASTOMA STEM CELLS." Neuro-Oncology Advances 3, Supplement_1 (March 1, 2021): i17. http://dx.doi.org/10.1093/noajnl/vdab024.072.

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Abstract Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD) or the critical downstream enzyme dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity in vitro. Higher expression of pyrimidine synthesis genes portends poor prognosis of patients with glioblastoma. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.

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42

Aldritt, S. M., P. Tien, and C. C. Wang. "Pyrimidine salvage in Giardia lamblia." Journal of Experimental Medicine 161, no. 3 (March 1, 1985): 437–45. http://dx.doi.org/10.1084/jem.161.3.437.

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We have found that the anaerobic protozoan parasite Giardia lamblia is incapable of de novo pyrimidine metabolism, as shown by its inability to incorporate orotate, bicarbonate, and aspartate into the pyrimidine nucleotide pool. Results from high performance liquid chromatography of pyrimidine and pyrimidine nucleoside pulse-labeled nucleotide pools and enzyme assays suggest that the parasite satisfies its pyrimidine nucleotide needs predominantly through salvage of uracil by a cytoplasmic uracil phosphoribosyltransferase. Exogenous uridine and cytidine are primarily converted to uracil by the action of uridine hydrolase and cytidine deaminase before incorporation into nucleotide pools. Direct salvage of cytosine occurs to a relatively limited extent via cytosine phosphoribosyltransferase. G. lamblia relies on salvage of exogenous thymidine for ribosylthymine monophosphate (TMP) synthesis, accomplished primarily through the action of a 100,000 g-pelletable thymidine phosphotransferase.

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R. Kore, Anilkumar, and Irudaya Charles. "Recent Developments in the Synthesis and Applications of C5-Substituted Pyrimidine Nucleosides and Nucleotides." Current Organic Chemistry 16, no. 17 (September 1, 2012): 1996–2013. http://dx.doi.org/10.2174/138527212803251622.

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Alvarado, Luigi J., Regan M. LeBlanc, Andrew P. Longhini, Sarah C. Keane, Niyati Jain, Zehra F. Yildiz, Blanton S. Tolbert, et al. "Regio-Selective Chemical-Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies." ChemBioChem 15, no. 11 (June 20, 2014): 1573–77. http://dx.doi.org/10.1002/cbic.201402130.

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KANDEEL, M., T. ANDO, Y. KITAMURA, M. ABDEL-AZIZ, and Y. KITADE. "Mutational, inhibitory and microcalorimetric analyses of Plasmodium falciparum TMP kinase. Implications for drug discovery." Parasitology 136, no. 1 (January 2009): 11–25. http://dx.doi.org/10.1017/s0031182008005301.

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SUMMARYPlasmodium falciparum thymidylate kinase (PfTMK) can tolerate a range of substrates, which distinguishes it from other thymidylate kinases. The enzyme not only phosphorylates TMP and dUMP but can also tolerate bulkier purines, namely, dGMP, GMP, and dIMP. In order to probe the flexibility of PfTMK in accommodating ligands of various sizes, we developed 6 mutant enzymes and subjected these to thermodynamic, inhibitory and catalytic evaluation. Kinase activity was markedly affected by introducing a larger lysine residue instead of A111. The lack of the hydroxyl group after inducing mutation of Y107F affected enzyme activity, and had a more severe impact on dGMP kinase activity. PfTMK can be inhibited by both purine and pyrimidine nucleosides, raising the possibility of developing highly selective drugs. Thermodynamic analysis revealed that enthalpic forces govern both purine and pyrimidine nucleoside monophosphate binding, and the binding affinity of both substrates was highly comparable. The heat produced due to dGMP binding is lower than that attributable to TMP. This indicates that additional interactions occur with TMP, which may be lost with larger dGMP. Targeting PfTMK not only affects thymidine nucleotide synthesis but may also affect purine nucleotides, and thus the enzyme represents an attractive antimicrobial target.

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Moffatt, Barbara A., and Hiroshi Ashihara. "Purine and Pyrimidine Nucleotide Synthesis and Metabolism." Arabidopsis Book 1 (January 2002): e0018. http://dx.doi.org/10.1199/tab.0018.

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Pesini, Alba, Eldris Iglesias, M. Pilar Bayona-Bafaluy, Nuria Garrido-Pérez, Patricia Meade, Paula Gaudó, Irene Jiménez-Salvador, et al. "Brain pyrimidine nucleotide synthesis and Alzheimer disease." Aging 11, no. 19 (September 27, 2019): 8433–62. http://dx.doi.org/10.18632/aging.102328.

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Нагиев, Э., E. Nagiev, С. Нагиева, S. Nagieva, Ф. Исмаилова, and F. Ismailova. "Study of Uridylic Nucleotides Contents and the Investigation Aspartate Carbamoyltransferase in Liver and Small Intestine Mucosa Exposed when Administered to Rats Orotic Acid and Perftoran." Medical Radiology and radiation safety 62, no. 5 (October 27, 2017): 5–10. http://dx.doi.org/10.12737/article_59f2ef130f5421.00591025.

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Purpose: Study of uridylic nucleotides content and aspartate carbamoyltransferase which was a key enzyme on the pathway for the synthesis of pyrimidine nucleotides in tissues of irradiated animals upon administration of orotic acid and perftorane was conducted. Material and methods: Studies were performed on random-bred albino rats subjected to a single γ-ray exposure at a total dose of 6 Gy. Orotic acid was injected as potassium salt in a dose of 60 mg / kg, perftoran salt in a dose of 1 ml / 100 g water. Results: A decrease in the content of UTP and UDP, as well as an increase in UMP after irradiation, especially on the 7th day, was established. The most pronounced changes in the studied biochemical parameters take place in the mucosa of the small intestine. The administration of orotic acid and perftorane to irradiated animals contributes to a significant correction of both the nucleotide content and the activity of aspartate carbamoyltransferase. Conclusion: The radiation leads to some decrease in the content of UDP and UTP. Changes in the content of nucleotides and activity of aspartate carbamoyltransferase in the mucosa of the small intestine are more pronounced in comparison with liver tissue. The combined administration of orotic acid and perftorane promotes the normalization of the content of nucleotides, and the activity of aspartate carbamoyltransferase in the liver and mucous membrane of the small intestine of irradiated animals.

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Dragon, Stefanie, Rainer Hille, Robert Götz, and Rosemarie Baumann. "Adenosine 3′:5′-Cyclic Monophosphate (cAMP)-Inducible Pyrimidine 5′-Nucleotidase and Pyrimidine Nucleotide Metabolism of Chick Embryonic Erythrocytes." Blood 91, no. 8 (April 15, 1998): 3052–58. http://dx.doi.org/10.1182/blood.v91.8.3052.3052_3052_3058.

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Terminally differentiating erythrocytes degrade most of their RNA with subsequent release of mononucleotides. Pyrimidine mononucleotides are preferentially cleaved by an erythrocyte-specific pyrimidine 5′-nucleotidase; deficiency of this enzyme causes hemolytic anemia in humans. Details of the regulation of its activity during erythroid differentiation are unknown. The present study arose from the observation that the immature red blood cells (RBCs) of mid-term chick embryos contain high concentrations of uridine 5′-triphosphate (UTP) (5 to 6 mmol/L), which decline rapidly from days 13 to 14 onward. We analyzed two key enzymes of RBC pyrimidine nucleotide metabolism: pyrimidine nucleoside phosphorylase (PNP) and pyrimidine 5′-nucleotidase (P-5′-N), to evaluate if changes of enzyme activity during embryonic development are correlated with changes of RBC UTP. Secondly, we tested if these enzymes are under hormonal control. The results show that embryonic RBCs contain only minimal activity of PNP. In contrast, P-5′-N increases from day 13 on, suggesting that the enzyme is a limiting factor in UTP degradation. Activation of β-adrenergic and A2A-adenosine receptors causes transcription-dependent de novo synthesis of P-5′-N. Because β-adrenergic and adenosine receptors are also found on adult erythroid cells, P-5′-N might be an enzyme of differentiating RBCs whose expression is in part controlled by adenosine 3′:5′-cyclic monophosphate (cAMP).

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Howard, Michael J., Nisha A. Cavanaugh, Vinod K. Batra, David D. Shock, William A. Beard, and Samuel H. Wilson. "DNA polymerase β nucleotide-stabilized template misalignment fidelity depends on local sequence context." Journal of Biological Chemistry 295, no. 2 (December 4, 2019): 529–38. http://dx.doi.org/10.1074/jbc.ra119.010594.

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DNA polymerase β has two DNA-binding domains that interact with the opposite sides of short DNA gaps. These domains contribute two activities that modify the 5′ and 3′ margins of gapped DNA during base excision repair. DNA gaps greater than 1 nucleotide (nt) pose an architectural and logistical problem for the two domains to interact with their respective DNA termini. Here, crystallographic and kinetic analyses of 2-nt gap-filling DNA synthesis revealed that the fidelity of DNA synthesis depends on local sequence context. This was due to template dynamics that altered which of the two template nucleotides in the gap served as the coding nucleotide. We observed that, when a purine nucleotide was in the first coding position, DNA synthesis fidelity was similar to that observed with a 1-nt gap. However, when the initial templating nucleotide was a pyrimidine, fidelity was decreased. If the first templating nucleotide was a cytidine, there was a significantly higher probability that the downstream template nucleotide coded for the incoming nucleotide. This dNTP-stabilized misalignment reduced base substitution and frameshift deletion fidelities. A crystal structure of a binary DNA product complex revealed that the cytidine in the first templating site was in an extrahelical position, permitting the downstream template nucleotide to occupy the coding position. These results indicate that DNA polymerase β can induce a strain in the DNA that modulates the position of the coding nucleotide and thereby impacts the identity of the incoming nucleotide. Our findings demonstrate that “correct” DNA synthesis can result in errors when template dynamics induce coding ambiguity.

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Journal articles on the topic 'Synthesis of pyrimidine nucleotides'

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