Regulation of pyrimidine biosynthesis and its strong coupling to the purine system

The control of pyrimidine biosynthesis in a yeast mutant deficient for uracil, adenine, and histidine has been studied in vivo. The uracil mutation causes accumulation of ureidosuccinic acid and dihydroorotic acid in the cells. Accumulation is prevented when the pyrimidine nucleotide level in the cell is raised, apparently owing to feedback inhibition in the pyrimidine system. Investigation of the coupling of purine and pyrimidine systems shows that a high level of purine nucleotides can reverse inhibition in the pyrimidine internal feedback loop. Under certan conditions this reversal may affect only the first step of the pyrimidine system so that ureidosuccinic acid is synthesized and the next element of the pyrimidine pathway, dihydroorotic acid, is not synthesized. Other aspects of coupling between the pyrimidine system and other systems are presented.     

Synthesis and structure–activity relationship of 2-arylamino-4-aryl-pyrimidines as potent PAK1 inhibitors

2-Arylamino-4-aryl-pyrimidines were found to be potent inhibitors of PAK1 kinase. The synthesis and SAR are described. The incorporation of a bromide at the 5-position of the pyrimidine core and in combination with a 1,2-dimethylpiperazine pendant domain yielded a lead compound with potent PAK1 inhibition and anti-proliferative activity in various colon cancer cell lines.     

Protective Effect of Furocoumarins Against 254-nm Ultraviolet in Staphylococcus aureus

For Staphylococcus aureus, pretreatment with furocoumarins (FCs) protect cells against killing by far ultraviolet light (FUV; approximately 254 nm). This protective effect was evident in the repair-proficient, parental strain as well as in the repair-deficient variants in the following order of efficacy: 4,5′’,8-trimethylpsoralen << 8-methoxypsoralen ≅ angelicin < 3-carbethoxypsoralen. The extent of protection was greater in the parental strain, indicating that despite the protective effect, a certain number of lethal lesions are nevertheless produced, which would be repaired with greater efficiency in such a strain than in the repair-deficient ones. This protective effect could be attribute to the inhibition of the formation of cyclobutyl pyrimidine dimers. Although the energy-transfer concept could explain the inhibition of pyrimidine dimer formation, and thus the protective effect of FC against FUV, we cannot rule out the possibility that the differences in degree of protection afforded by the FC employed here are related to a subtle and complex combination of effects.     

Differential control of cell cycle,proliferation, and survival of primary T lymphocytes by purine and pyrimidine nucleotides

Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Activation of T lymphocytes is associated with an increase of purine and pyrimidine pools. However, the question of how purine vs pyrimidine nucleotides regulate proliferation, cell cycle, and survival of primary T lymphocytes following activation has not yet been specifically addressed. This was investigated in the present study by using well-known purine (mycophenolic acid, 6-mercaptopurine) and pyrimidine (methotrexate, 5-fluorouracil) inhibitors, which are used in neoplastic diseases or as immunosuppressive agents. The effect of these inhibitors was analyzed according to their time of addition with respect to the initiation of mitogenic activation. We showed that synthesis of both purine and pyrimidine nucleotides is required for T cell proliferation. However, purine and pyrimidine nucleotides differentially regulate the cell cycle since purines control both G(1) to S phase transition and progression through the S phase, whereas pyrimidines only control progression from early to intermediate S phase. Furthermore, inhibition of pyrimidine synthesis induces apoptosis whatever the time of inhibitor addition whereas inhibition of purine nucleotides induces apoptosis only when applied to already cycling T cells, suggesting that both purine and pyrimidine nucleotides are required for survival of cells committed into S phase. These findings reveal a hitherto unknown role of purine and pyrimidine de novo synthesis in regulating cell cycle progression and maintaining survival of activated T lymphocytes.     

Inhibitory action of the adenine analog, 4-aminopyrazolo(3,4-d)pyrimidine, in Crithidia fasciculata

The adenine analog 4-aminopyrazolo(3,4-d)pyrimidine inhibits the growth of the kinetoplastid (trypanosomatid) flagellate Crithidia fasciculata. This inhibition is partially overcome only by adenine (of a number of purines tested), with an inhibition index of 0.025. More effective reversal of inhibition is obtained with any of a number of naturally occurring pyrimidine compounds, up to a concentration of 0.18 mM. Higher concentrations of pyrimidines or addition of guanine, as well as adenine and uracil, to the medium increases inhibition. The analog (presumably as the ribonucleotide) was found not to be inhibitory to any enzyme of the pyrimidine biosynthetic pathway that could be tested. It is suggested that the analog competes with adenine for adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7), is converted to a ribonucleotide, and is incorporated into nucleic acid.     

Aminoimidazole carboxamide ribonucleoside toxicity: a model for study of pyrimidine starvation

Aminoimidazole carboxamide ribonucleoside (AIC-R), a purine precursor, has biphasic effects on the growth of Chinese hamster fibroblasts. At 200 microM AIC-R cell growth is almost completely arrested, while at 50 and 700 microM AIC-R cell growth is comparable to that observed in the absence of nucleoside. The growth inhibition produced by AIC-R is the consequence of inhibition of the orotate phosphoribosyltransferase-orotidylic decarboxylase (OPRT-ODC) reactions, as evidenced by a 87% reduction in the intracellular concentrations of UTP and CTP, accumulation of orotate in the medium, and restoration of normal growth by inclusion of 100 microM uridine in the medium. Inhibition of pyrimidine nucleotide synthesis at 200 microM AIC-R is associated with an 82% reduction in the intracellular concentration of PP-ribose-P and a 150% increase in the concentration of purine nucleotides. Restoration of cell growth to a normal rate at 700 microM AIC-R--a condition under which PP-ribose-P remains depressed and purine nucleotide concentrations are also depressed (40% of control)--and absence of toxicity at 50 microM AIC-R--a condition under which purine nucleotide concentrations are increased by 150% and PP-ribose-P concentration is normal--suggest that the inhibition of OPRT-ODC observed at 200 microM AIC-R is caused by the combination of the reduction in PP-ribose-P and increase in purine nucleotides. These studies provide a better understanding of the control of the OPRT-ODC reactions in the cell and provide additional insight into the basis of pyrimidine starvation induced by purine nucleosides.     

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences (<1 J/m², producing less than one pyrimidine dimer per replicon) rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates without noticeably affecting synthesis in multi-repliconsize intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher (>1 J/m², producing more than one dimer per replicon) cytotoxic fluences inhibited DNA synthesis in operating replicons presumably because the elongation of nascent strands was blocked where pyrimidine dimers were present in template strands. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences. indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation than the repair-deficient strains despite their ability to remove pyrimidine dimers. This analysis suggests that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery.     

Toxicity of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate in Salmonella typhimurium

Growth of Salmonella typhimurium pyrC or pyrD auxotrophs was severely inhibited in media that caused derepressed pyr gene expression. No such inhibition was observed with derepressed pyrA and pyrB auxotrophs. Growth inhibition was not due to the depletion of essential pyrimidine biosynthetic pathway intermediates or substrates. This result and the pattern of inhibition indicated that the accumulation of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate was toxic. This intermediate is synthesized by the sequential action of the first two enzymes of the pathway encoded by pyrA and pyrB and is a substrate for the pyrC gene product. It should accumulate to high levels in pyrC or pyrD mutants when expression of the pyrA and pyrB genes is elevated. The introduction of either a pyrA or pyrB mutation into a pyrC strain eliminated the observed growth inhibition. Additionally, a direct correlation was shown between the severity of growth inhibition of a pyrC auxotroph and the levels of the enzymes that synthesize carbamyl aspartate. The mechanism of carbamyl aspartate toxicity was not identified, but many potential sites of growth inhibition were excluded. Carbamyl aspartate toxicity was shown to be useful as a phenotypic trait for classifying pyrimidine auxotrophs and may also be useful for positive selection of pyrA or pyrB mutants. Finally, we discuss ways of overcoming growth inhibition of pyrC and pyrD mutants under derepressing conditions.     

5-Fluoropyrimidine-resistant mutants of pneumococcus

Three classes of 5-fluorpyrimidine-resistant mutants of Diplococcus pneumoniae have been characterized. The mutant strain upp is resistant to high concentrations of the fluoropyrimidine bases fluorouracil (FU) and fluorocytosine (FC); strain upp has a defective uridine monophosphate pyrophosphorylase. The mutant strain udk is resistant to inhibition by fluorouridine (FUR) and exhibits defective uridine kinase activity. The mutant strain fun is resistant to inhibition by the nucleosides fluorodeoxyuridine, fluorodeoxycytidine, and FUR, but shows normal activity for all pyrimidine pathway enzymes tested. This strain may be defective in the activity of a transport system that governs the cellular uptake of pyrimidine ribo- and deoxyribonucleosides. Biochemical studies on wild-type and fluoropyrimidine-resistant pneumococci are discussed with respect to the transport and early metabolism of preformed pyrimidine precursors by this organism.     

The control mechanism of thiamine biosynthesis. A model for the study of control of converging pathways

1. Thiamine or the pyrimidine moiety of thiamine added in excess to a growing culture of Salmonella typhimurium LT2 repressed subsequent thiamine synthesis in non-growing organisms. 2. A mutant unable to convert added pyrimidine moiety into thiamine was not repressible by the pyrimidine, showing that thiamine, not the pyrimidine, was the repressor. 3. Thiamine repression occurred at 40mmug. of thiamine/mg. dry wt. or above and de-repression occurred at 30mmug. of thiamine/mg. dry wt. or below. 4. Thiamine controlled the pyrimidine and thiazole pathways at the same concentration and to the same extent. 5. Biosynthesis of the thiazole moiety had, in contrast with biosynthesis of the pyrimidine moiety, an additional feedback inhibition control that allowed utilization of the exogenous thiazole. 6. The enzymes joining the pyrimidine and thiazole moieties were repressible by high concentrations of thiamine. 7. Thiamine was rapidly converted into thiamine pyrophosphate and this appeared to be the active repressor. 8. Theoretical aspects of control of converging pathways are discussed.     

The Concept of ‘Minimally Modified’ Antisense Oligonucleotides for Specific Inhibition of Gene Expression

Abstract

The design and use of minimally modified oligonucleotides for specific inhibition of gene expression is discussed. The “minimal” protection strategy is a combination of the end-capping technique and the protection of internal pyrimidine positions which are the major sites of endonuclease degradation. By reducing the number of phosphorothioate modifications needed to make the oligonucleotide resistant to nuclease degradation, non-sequence-specific effects, which are frequently observed with uniformly phosphorothioate-modified oligonucleotides, can be reduced.     

Indirect inhibition of mitochondrial dihydroorotate dehydrogenase activity by nitric oxide

Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate in the pyrimidine biosynthesis pathway. It is functionally connected to the respiratory chain, delivering electrons to ubiquinone. We report here that inhibition of cytochrome c oxidase by nitric oxide (NO) indirectly inhibits DHODH activity. In digitonin-permeabilized cells, DEA/NO, a chemical NO donor, induced a dramatic decrease in DHO-dependent O(2) consumption. The inhibition was reversible and more pronounced at low O(2) concentration; it was correlated with a decrease in orotate synthesis. Since orotate is the precursor of all pyrimidine nucleotides, indirect inhibition of DHODH by NO may significantly contribute to NO-dependent cytotoxicity.     

Pyrimidine biosynthesis and its regulation in the estrogen-stimulated chick oviduct.

Studies on the incorporation of radio-labeled precursors into orotic acid and the pyrimidine nucleotides of RNA have established the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the chick oviduct. Measurements of the rate of incorporation of precursors into orotic acid in minces of oviduct revealed the activity of the orotate pathway to be accelerated in response to estrogen-stimulated nucleic acid synthesis and tissue growth. These data indicate that extrahepatic tissues of avian species meet their requirements for pyrimidine nucleotides through de novo synthesis rather than depend upon the liver or other exogenous sources for a supply of preformed pyrimidines. An examination of the influence of pyrimidine and purine nucleosides on the incorporation of radio-labeled precursors into orotic acid yielded evidence that pyrimidine biosynthesis in the chick is quite sensitive to inhibition by both purines and pyrimidines; the data indicate the reaction catalyzed by carbamoylphosphate synthetase to be the site of inhibition in both cases.     

Synthesis, salvage, and catabolism of uridine nucleotides in boron-deficient squash roots

Previous work has provided evidence that plants may require boron to maintain adequate levels of pyrimidine nucleotides, suggesting that the state of boron deficiency may actually be one of pyrimidine starvation. Since the availability of pyrimidine nucleotides is influenced by their rates of synthesis, salvage, and catabolism, we compared these activities in the terminal 3 centimeters of roots excised from boron-deficient and -sufficient squash plants (Cucurbita pepo L.). Transferring 5-day-old squash plants to a boron-deficient nutrient solution resulted in cessation of root elongation within 18 hours. However, withholding boron for up to 30 hours did not result in either impaired de novo pyrimidine biosynthesis or a change in the sensitivity of the de novo pathway to regulation by end product inhibition. Boron deprivation had no significant effect on pyrimidine salvage or catabolism. These results provide evidence that boron-deficient plants are not starved for uridine nucleotides collectively. Whether a particular pyrimidine nucleotide or derivative is limiting during boron deprivation remains to be examined.     

Inhibition of pyrimidine incorporation without inhibition of DNA synthesis.

A putative lymphocytic chalone was tested measuring the incorporation of purine and pyrimidine nucleosides and by cytophotometry. The pyrimidine precursors were inhibited but not the purines. Thymidine and deoxycytidine incorporation even performed simultaneously with cytophotometry can be misleading in the analysis of the inhibition of cell division.     

Antitumor activity of new pyrazolo[3,4-d]pyrimidine SRC kinase inhibitors in Burkitt lymphoma cell lines and its enhancement by WEE1 inhibition

Burkitt lymphoma (BL) is a highly aggressive B cell neoplasm. Although intensive polychemotherapy regimens have proven very effective, they are associated with significant toxicities. Therefore, more rational therapies that selectively target the molecular abnormalities of BL are needed. Recent data suggest that the tyrosine kinase SRC could represent a therapeutic target for BL. We found that new pyrazolo[3,4-d]pyrimidine SRC inhibitors exerted a significant cytotoxic effect and induced apoptosis on two BL cell lines, as determined by MTS assays, cytofluorimetric analyses and caspase 3 assay. Notably, our SRC inhibitors proved to be more effective than the well-known SRC inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine] in BL cells. Moreover, our small molecules induced a G₂/M arrest in BL cells through a possible new mechanism, whereby SRC inhibition hinders an AKT-WEE1-cyclin-dependent kinase 1 (CDK1) axis, leading to inhibition of CDK1, the main trigger of entry into mitosis. By using a small-molecule inhibitor of WEE1, a crucial CDK1 negative regulator, we were able to shift the balance toward apoptosis rather than growth arrest and enhance the efficacy of the SRC inhibitors, suggesting a possible use of these selective drugs in combination for a safe and efficient treatment of BL.     

The metabolism of pyrimidine compounds by Tetrahymena pyriformis

The pyrimidine requirements for growth of T. pyriformis and for reversal of the growth inhibition caused by folate deprivation have been studied. The effects of thymidine and 5-fluorodeoxyuridine have been shown to be quantitatively different from the effects of these compounds on growth and the rate of DNA synthesis in mammalian cells. Labelled nucleosides added to the medium have been found to be converted to the corresponding bases with the exception of deoxycytidine, which is first deaminated to deoxyuridine. As a result no deoxynucleosides other than thymidine specifically label DNA. The results allow deductions to be made concerning the enzymes involved in pyrimidine utilization by this organism. It is suggested that pyrimidine utilization is always channeled through uracil in the case of those compounds that can supply the pyrimidine requirement for growth.     

Production of a precursor to the pyrimidine moiety of thiamine.

The supernatant fluid from cultures of Escherichia coli W-11, a pur E mutant, prevented the inhibition of growth of E. coli B in a medium containing adenine or adenosine. Adenine inhibition was prevented more readily than adenosine inhibition. More than 90% of the biological activity of the supernatant fluid was recovered in the anionic fraction after treatment with Dowex-50 (NH4+). The cationic fraction, containing large amounts of 5-aminoimidazole ribonucleoside (AIRS), did not prevent adenine inhibition. The W-11 supernatant fluid was shown by bioautography to contain only one compound that prevented adenine inhibition. Proliferating and non-proliferating cultures produced only one compound that prevented adenine inhibition. The compound was shown to be an intermediate (int-1) in the biosynthesis of the pyrimidine moiety of thiamine, Int-1 was stable during sterilization at 121 C for 15 min, during concentration by either flask evaporation or lyophilization, and after storage for several days at 4 C or at -- 20 C. Int-1 was distinguishable from other known derivatives or intermediates of the pyrimidine moiety. A scheme is presented that illustrates the proposed relationship between int-1 and the synthesis of thiamine.     

Inhibition of de novo pyrimidine synthesis augments Gemcitabine induced growth inhibition in an immunocompetent model of pancreatic cancer

Leflunomide (Lef) is an agent used in autoimmune disorders that interferes with DNA synthesis. De Novo pyrimidine synthesis is a mechanism of Gemcitabine (Gem) resistance in pancreatic cancer. This study aims to assess the efficacy and changes in the tumor microenvironment of Lef monotherapy and in combination with Gem, in a syngeneic mouse model of pancreatic cancer.Methods: MTS proliferation assays were conducted to assess growth inhibition by Gem (0-20 nM), Lef (0-40 uM) and Gem+Lef in KPC (KrasLSL.G12D/+;p53R172H/+; PdxCretg/+) cells in vitro. An in vivo heterotopic KPC model was used and cohorts were treated with: PBS (control), Gem (75 mg/kg/q3d), Lef (40 mg/kg/d), or Gem+Lef. At d28 post-treatment, tumor burden, proliferation index (Ki67), and vascularity (CD31) were measured. Changes in the frequency of peripheral and intratumoral immune cell subsets were evaluated via FACS. Liquid chromatography-mass spectrometry was used for metabolomics profiling.Results: Lef inhibits KPC cell growth and synergizes with Gem in vitro (P<0.05; Combination Index 0.44 (<1 indicates synergy). In vivo, Lef alone and in combination with Gem delays KPC tumor progression (P<0.001). CTLA-4+T cells are also significantly decreased in tumors treated with Lef, Gem or in combination (Gem+Lef) compared to controls (P<0.05). Combination therapy also decreased the Ki67 and vascularity (P<0.01). Leflunomide inhibits de novo pyrimidine synthesis both in vitro (p<0.0001) and in vivo (p<0.05).Conclusions: In this study, we demonstrated that Gem+Lef inhibits pancreatic cancer growth, decrease T cell exhaustion, vascularity and as proof of principle inhibits de novo pyrimidine synthesis. Further characterization of changes in adaptive immunity are necessary to characterize the mechanism of tumor growth inhibition and facilitate translation to a clinical trial.