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PubMed1991

Crystallization of yeast orotidine 5'-monophosphate decarboxylase complexed with 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid.

Using an incomplete factorial experimental design, we have identified conditions for crystallization of yeast orotidine 5'-monophosphate decarboxylase (ODCase) in an unliganded state and complexed separately to two inhibitors: 6-azauridine 5'-monophosphate (aza-UMP) and 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid (BMP). Crystals of X-ray diffraction quality have been obtained of yeast ODCase complexed with BMP, a putative transition state analog inhibitor (Ki = 8.8 x 10(-12) M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 microM dimer) plus 450 microM BMP by microdialysis at 4 degrees C against a mother liquor which consisted of 0.1 M Na-PIPES-acetate (pH 6.4), 37.5 microM BMP, 5 mM mercaptoethanol, 1% polyethylene glycol 400, and 2.3 M ammonium sulfate. Crystals were analyzed using precession photography and were assigned to trigonal space group R32 with unit cell dimensions a = b = 115 A, c = 385 A. The crystal density is 1.245 g/cm3 indicating the presence of two ODCase: BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 A3/Da and 41% solvent content. The morphological habit of crystals of the ODCase:BMP complex changed when the initial ammonium sulfate concentration was increased in 0.05 M steps from 2.3 to 2.45 M. All of these crystals diffracted to at least 3.0 A resolution over a period of several weeks at room temperature and are isomorphous.

Ammonium Sulfate↗
PubMed1980

Abnormal regulation of purine metabolism in a cultured mouse T-cell lymphoma mutant partially deficient in adenylosuccinate synthetase.

The isolation and characterization of a mutant mouse T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which accounts for its resistance to adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. Furthermore, the intact cells of this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in situ. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.

Adenylosuccinate Synthase↗
PubMed1980

The genes for and regulation of the enzyme activities of two multifunctional proteins required for the de novo pathway for UMP biosynthesis in mammals.

UMP biosynthesis requires six enzyme activities. Five of these enzyme centers are clustered into two multienzymatic proteins which are known to, or appear to, sequester the intermediates carbamyl approximately P, carbamyl aspartate and orotidylic acid. The advantages of sequestering these intermediates appear to be a conservation of energy, since two intermediates, carbamyl approximately P and orotidylate, might otherwise be rapidly degraded in mammalian cells. Carbamyl-aspartate appears not to be degraded rapidly in mammalian cells but it can pass into the blood and could possible disrupt brain metabolism by action as an acetylaspartate analog, if it passes the blood-brain barrier. For this, and possible for other reasons, there may be advantages to the fact that these intermediates are not other reasons, there may be advantages to the fact that these intermediates are not readily released from Complex A and U. In addition, these multienzymatic proteins may have other kinetic advantages, some of which have been discussed above. Studies with intact cells illustrate that azauridine, a chemical designed originally as an antineoplastic drug, produces a "ripple" effect when it inhibits the last enzyme of this pathway which leads to a sequential accumulation of pools of the various intermediates or their metabolites. This same agent increases the amount of some of the enzymes of this biosynthetic pathway in cells exposed to this drug. Both of these effects can negate the effectiveness of this potential antineoplastic drug. Sophisticated drug design may depend on whole-cell studies, such as those discussed here, in addition to the classic studies on the inhibition of a single enzyme center to select drugs that may be without significant side effects when they are finally tested in animals.

Animals↗
PubMed1987

Cloning and expression of the OMP decarboxylase gene URA4 from Schizosaccharomyces pombe.

URA4, the gene coding for orotidine monophosphate decarboxylase (OMPdecase), has been cloned from the fission yeast by homologous complementation and restricted in an Escherichia coli-Schizosaccharomyces pombe (E. coli-S. pombe) replicative plasmid to a 1.76 kb HindIII fragment. This plasmid is maintained at a high copy number in S. pombe and allows OMPdecase expression in Saccharomyces cerevisiae (S. cerevisiae) as well as in E. coli. After characterisation by restriction mapping and Southern hybridisation, the cloned gene was used as a probe to measure URA4 transcription and to examine its regulation. Messenger RNA levels were measured by DNA/RNA filter-hybridisation with pulse labelled RNAs during 6-azauridine (6-AUR) inhibited growth in wild type and 6-AUR sensitive strains. We found that in S. pombe the OMP analogue 6-AUR does not regulate the level of OMPdecase formation as it does in S. cerevisiae but rather modifies the ratio of total polyA+ to polyA- RNAs in the cell. Based on these results and on corresponding enzyme activities this study demonstrates divergent pyrimidine pathway regulation in the two yeasts S. cerevisiae and S. pombe. Finally, we propose the use of the URA4 gene as a convenient selective marker for genetic engineering in S. pombe.

Carboxy-Lyases↗
PubMed1977

Increased intracellular phosphoribosylpyrophosphate and accelerated orotic acid decarboxylation in a mouse cell line resistant to purine and pyrimidine ribonucleosides.

A line of mouse fibroblasts (A9AU-1), originally selected for growth in the presence of 6-azauridine, has been found to be resistant to cytotoxic concentrations of adenosine, guanosine, and thymidine. A9AU-1 cells convert orotic acid to uridine 5'-monophosphate at twice the rate of the A9P line from which the A9AU-1 clone was selected. The resistant cells also excrete purines, synthesized de novo, into the medium at an increased velocity. The average intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP) concentration of the resistant line is 45% higher than that of the parental line. The elevated PRPP concentration is likely to be responsible for both the apparent acceleration of pyrimidine synthesis and the increased excretion of purines into the growth medium; it might also account, by one of the several possible mechanisms, for the resistance of the cells to cytotoxic concentrations of the various nucleosides.

Adenine Phosphoribosyltransferase↗
PubMed1995

UMP synthase activity expressed in deficient hamster cells by separate transferase and decarboxylase proteins or by linker-deleted bifunctional protein.

Segments of the human UMP synthase cDNA coding for the orotate phosphoribosyl transferase (OPRT) and orotidylate decarboxylase (ODC) domains of the bifunctional protein UMP synthase were produced by polymerase chain reaction techniques and cloned into a eukaryotic expression vector. The separate OPRT and ODC vectors, along with a selectable marker, were cotransfected into UMP synthase-deficient hamster cells (Urd-C) that require exogeneous uridine for growth. Transfected Urd-C cells surviving selection in media without added uridine were isolated and designated transferase decarboxylase Urd-C (TDU). All of the selected colonies contained DNA corresponding to the OPRT and ODC expression vectors. Two cell lines (TDU3 and TDU5) integrated many more copies of the OPRT and ODC vectors into their genomes compared to the other TDU lines. A 28.6-kDa ODC protein band and a 24.4-kDa OPRT band were detected on western blots with UMP synthase-specific polyclonal antiserum. The OPRT activity of the TDU lines was up to 8.7 times the OPRT activity of control CHL cells, and the ODC activity was up to 12.5 times control levels. Both OPRT and ODC activities in the monofunctional proteins were less heat stable than in the bifunctional UMP synthase protein. The monofunctional OPRT protein was less stable than the ODC protein at 45 degrees C. Growth of transfected cells in 6-azauridine resulted in striking increases in activity and temperature stability for the monofunctional ODC protein. A UMP synthase bifunctional protein was constructed with a deletion of the suspected linker region joining the two catalytic domains. The linker-deleted UMP synthase showed no significant change in either OPRT or ODC activity or temperature stability. The increased stability of the bifunctional protein may be a factor in its evolutionary selection in mammalian cells.

Animals↗
PubMed1983

A 13C tracer method for quantitating de novo pyrimidine biosynthesis in vitro and in vivo.

Gas chromatographic/mass spectrometric methods for the measurement of the flux through the de novo pyrimidine biosynthetic pathway by quantitating the incorporation of [13C]bicarbonate and 13CO2 into the uracil nucleotide pool in L1210 tumors are reported. Simultaneous measurements of the incorporation of [13C]bicarbonate and the more commonly used [14C]bicarbonate into uridine of L1210 cells in vitro showed that the two methods were comparable. A modification of the method was applied to in vivo studies where the incorporation of 13CO2 into the uracil nucleotide pool of L1210 tumors in mice was quantitated. The measurements were used to determine changes in the flux through the de novo pyrimidine pathway in animals pretreated with known inhibitors of the pathway. A comparison of control animals with those pretreated with 6-azauridine, acivicin, and pyrazofurin resulted in mean percentage inhibitions of 87, 95, and 94%, respectively. This technique should allow investigation of the respective contributions of salvage and de novo synthesis in the formation of pyrimidines in vivo and the effects of agents designed as enzyme inhibitors of the de novo pathway.

Animals↗
PubMed1996

Use of the yellow fever virus vaccine strain 17D for the study of strategies for the treatment of yellow fever virus infections.

We have employed the attenuated vaccine strain 17D of yellow fever virus (YFV) to evaluate the inhibitory effect of a selected series of compounds on YFV in Vero cells. Use of the vaccine strain does not require high-level microbiological containment facilities and should allow extensive screening. In addition, YFV may serve as a model for other flaviviruses including hepatitis C virus (HCV), and thus strategies for the treatment of YFV infections may apply to flavivirus infections in general. In the present study, several compounds belonging to different classes of nucleoside analogues and polyanions were evaluated for their inhibitory effect on the replication of YFV. Compounds that are targeted at: (i) IMP dehydrogenase (ribavirin, EICAR, tiazofurin, selenazofurin and mycophenolic acid), (ii) OMP decarboxylase (pyrazofurin and 6-azauridine), (iii) CTP synthetase (carbodine and cyclopentenyl cytosine), (iv) dihydrofolate reductase (methotrexate) and the (v) sulfated polymers (dextran sulfate and PAVAS) proved inhibitory to the replication of YFV. Mycophenolic acid (EC50: 0.08 microgram/ml). EICAR (EC50: 0.8 microgram/ml) and methotrexate (EC50: 0.07 microgram/ml) were the most effective. The findings that EICAR and mycophenolic acid, despite their potent anti-YFV activity, had little or no effect on the replication of the bunyavirus Punta Toro or herpes simplex virus in Vero cells, indicates that their anti-YFV activity is rather specific and does not merely result from cytotoxicity. Inhibitors of S-adenosylhomocysteine hydrolase (SAH hydrolase) and thymidylate synthase were found to be devoid of anti-YFV activity.

Animals↗
PubMed1994

Automated analytical systems for drug development studies. I--A system for the determination of drug stability.

An automated system consisting of a pH-stat, microdialysis sampling and a liquid chromatograph was assembled to measure the rate of rapid chemical reactions. 2',3',5'-Triacetyl-6-azauridine was used as a model compound to validate the performance of the automated system. Buffer catalysis was minimized by using a non-catalytic concentration of borate buffer along with a pH-stat to maintain the pH during the kinetic run. The microdialysis sampling technique permitted sample quenching and buffering of the solutions to a pH compatible with the LC column materials. The combination of microdialysis sampling and rapid LC analysis allowed reactions with a half-life of approximately 1 min to be sampled every 30 s. The rates of hydrolysis of the drug, measured at different conditions of temperature (37-70 degrees C) and pH (9.0-10.5) using the automated system, compared well with the previously determined values.

Autoanalysis↗
PubMed2003

Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus.

The outbreak of SARS warrants the search for antiviral compounds to treat the disease. At present, no specific treatment has been identified for SARS-associated coronavirus infection. We assessed the antiviral potential of ribavirin, 6-azauridine, pyrazofurin, mycophenolic acid, and glycyrrhizin against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with SARS admitted to the clinical centre of Frankfurt University, Germany. Of all the compounds, glycyrrhizin was the most active in inhibiting replication of the SARS-associated virus. Our findings suggest that glycyrrhizin should be assessed for treatment of SARS.

Animals↗
PubMed2002

Comparison of colorimetric, fluorometric, and visual methods for determining anti-influenza (H1N1 and H3N2) virus activities and toxicities of compounds.

Methods have been developed previously for rapid evaluation of compounds for antiviral activity in 96-well microplates, which include visual quantitation of antiviral activity based upon inhibition of virus-induced cytopathic effect (CPE) or by less subjective colorimetric or fluorometric means. In the present studies we compared a number of colorimetric (crystal violet, MTT [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide], and neutral red) and fluorometric (Alamar Blue, bisbenzimide [Hoechst 33258], fluorescein diacetate, and rhodamine 6G) methods to visual scoring of antiviral activity in influenza A virus infections in Madin Darby canine kidney (MDCK) cells. Toxicity determinations using these same methods were also made for anti-influenza inhibitors and other compounds known to inhibit cell proliferation. Against influenza A/Texas/36/91 (H1N1) and A/Sydney/05/97 (H3N2) viruses, visual scoring and dye or stain methods produced results that were not significantly different from each other in deriving 50% virus-inhibitory concentrations (EC(50) values) for six anti-influenza compounds (amantadine, rimantadine, ribavirin, RWJ-270201 [BCX-1812], oseltamivir carboxylate, and zanamivir), with the exception of Alamar Blue which quantified lower EC(50) values than expected. In uninfected replicating cells, the visual and Alamar Blue methods underestimated the 50% cytotoxic concentrations (IC(50) values) of ribavirin, 1-beta-D-arabinofuranosylcytosine, and 6-azauridine, but more accurately assessed the toxicities of amantadine, rimantadine, and cycloheximide. Visual scoring, coupled with the use of one of these dyes or stains except Alamar Blue, can be used to accurately and rapidly quantify the anti-influenza virus activities and toxicities of potential new influenza virus inhibitors. These methods should also be applicable to evaluating antiviral effects against other lytic virus infections.

Animals↗
PubMed1980

Isolation and initial characterization of the single polypeptide that synthesizes uridine 5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate synthase.

UMP synthase, or multienzyme pyr-5,6 (orotate phosphoribosyltransferase:orotidine monophosphate decarboxylase), has been purified from Ehrlich ascites carcinoma to apparent homogeneity. The purification was achieved by the use of 5-[2-[N-(2-aminoethyl)carbamyl]ethyl]-6-azauridine 5'-monophosphate-agarose and phosphocellulose affinity columns linked in tandem by a flow dialysis system. The purified protein has amolecular weight of approximately 51500 as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Both enzyme activities cosediment with an S20,w value of 3.7 S, which corresponds to a molecular weight of about 50000. Two-dimensional electrophoresis of UMP synthase shows that the protein exists as two isomeric forms with isoelectric points of 5.85 (major form) and 5.65 (minor form). Both forms have the same molecular weight of 51500 and contain both active centers. These results clearly show that the last two enzyme activities of de novo UMP biosynthesis occur on a single polypeptide chain of approximately 51500 daltons and that this polypeptide exists in at least two isomeric forms.

Animals↗
PubMed1986

Ribose as the preferential target for the oxidized form of elliptinium acetate in ribonucleos(t)ides. Biological activities of the resulting adducts.

The covalent binding of the oxidized form of elliptinium acetate, an antitumor drug, to various ribonucleos(t)ides is described. In the absence of a strong nucleophile on the bases, e.g., a sulfhydryl group, the main target of this quinone imine derivative is the sugar moiety. With unmodified regular bases, the first electrophilic addition always occurs on the 2'-oxygen of ribose (more slowly for pyrimidine than for purine); in a second step, cyclization of the reoxidized product leads to a spiro derivative: only one stereoisomer is detected with purine nucleoside; the other stereoisomer appears as a minor product (10-20%) with nucleotides and pyrimidine nucleosides. With modified bases, no change is observed except for bases exhibiting an additional strong nucleophilic center: oxidized elliptinium alkylates thioguanine and thioguanosine on the sulfur atom and in this last case not on the ribose moiety. All spiro derivatives are less cytotoxic than the parent compound even if the base is an antimetabolite (azauridine); however, thio-elliptinium adducts maintain high cytotoxicity.

Alkaloids↗
PubMed2003

Practical synthesis of D- and l-2-cyclopentenone and their utility for the synthesis of carbocyclic antiviral nucleosides against orthopox viruses (smallpox, monkeypox, and cowpox virus).

Highly efficient and practical methodology for the syntheses of D- and l-4,5-O-isopropylidene-2-cyclopentenone (9 and 22), versatile intermediates for the synthesis of carbocyclic nucleosides, have been developed via a ring-closing metathesis reaction from d-ribose in eight steps. The utility of D- and l-4,5-O-isopropylidene-2-cyclopentenone is demonstrated by their application for the preparation of D-cyclopentyl-6-azauridine 12 and D-cyclopentenyl-5-halocytosine nucleosides (33-35) using Mitsunobu reaction to introduce pyrimidine bases as potential antiviral agents. Preliminary antiviral activity against orthopox viruses (smallpox, monkeypox, and cowpox virus) of the synthesized nucleosides are described.

Antiviral Agents↗
PubMed1983

No increase in second tumors after cytotoxic chemotherapy for gestational trophoblastic tumors.

We investigated the incidence of second tumors after cytotoxic chemotherapy in 457 long-term survivors treated for choriocarcinoma or an invasive mole between 1958 and 1978. Treatment was given according to regular intermittent schedules and over a mean period of four months, with no maintenance. Methotrexate was given to all but two patients, and 261 (57 per cent) also received other cytotoxic drugs, most commonly dactinomycin, vincristine, cyclophosphamide, mercaptopurine, and 6-azauridine. After a mean period of 7.8 years since the beginning of treatment and a total of 3522 patient-years of risk, second neoplasms had developed in only two women (acute leukemia in one and carcinoma of the breast in the other). This figure is less than the number of cases of cancer that would have been expected (3.5) in this group and suggests that the use of methotrexate as chemotherapy for choriocarcinoma is not carcinogenic in the medium term.

Adult↗
PubMed2000

Electrostatic stress in catalysis: structure and mechanism of the enzyme orotidine monophosphate decarboxylase.

Orotidine 5'-monophosphate decarboxylase catalyzes the conversion of orotidine 5'-monophosphate to uridine 5'-monophosphate, the last step in biosynthesis of pyrimidine nucleotides. As part of a Structural Genomics Initiative, the crystal structures of the ligand-free and the6-azauridine 5'-monophosphate-complexed forms have been determined at 1.8 and 1.5 A, respectively. The protein assumes a TIM-barrel fold with one side of the barrel closed off and the other side binding the inhibitor. A unique array of alternating charges (Lys-Asp-Lys-Asp) in the active site prompted us to apply quantum mechanical and molecular dynamics calculations to analyze the relative contributions of ground state destabilization and transition state stabilization to catalysis. The remarkable catalytic power of orotidine 5'-monophosphate decarboxylase is almost exclusively achieved via destabilization of the reactive part of the substrate, which is compensated for by strong binding of the phosphate and ribose groups. The computational results are consistent with a catalytic mechanism that is characterized by Jencks's Circe effect.

Amino Acid Sequence↗
PubMed1991

Hypoxanthine-maintained two-cell block in mouse embryos: dependence on glucose and effect of hypoxanthine phosphoribosyltransferase inhibitors.

The culture conditions under which hypoxanthine maintains a two-cell block in preimplantation mouse embryos were assessed. Hypoxanthine prevented embryo development past the two-cell stage at concentrations as low as 30 nM, and this inhibitory activity required the presence of D-glucose. The action of hypoxanthine plus D-glucose was reversed by glutamine and higher lactate. D-mannose substituted for D-glucose in supporting the inhibitory action of hypoxanthine, but L-glucose, D-fructose, and 2-deoxyglucose were much less effective. Other purine derivatives such as inosine and adenosine, but not xanthosine or uric acid, also blocked development at the two-cell stage at a concentration of 30 microM, and guanosine was inhibitory at higher doses. Assays of hypoxanthine phosphoribosyltransferase (HPRT) activity in lysates of four-cell embryos determined that the drugs 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP), but not 6-azauridine (6-AzaU), prevented salvage of hypoxanthine. In addition, MPTF and 6-MP produced a significant two-cell block, which did not depend upon the presence of hypoxanthine or D-glucose; whereas 6-AzaU was without effect. When embryos were cultured 2 days in the presence or absence of D-glucose, hypoxanthine salvage was significantly reduced in lysates of four-cell embryos exposed to D-glucose. D-glucose had no effect when added directly to the assay mixture. These data demonstrate that the ability of hypoxanthine to block embryo development at the two-cell stage depends on the presence of D-glucose or other glycolyzable sugars and suggest that inhibition of the purine salvage pathway promotes the two-cell block.

Animals↗
PubMed1978

Prolongation of rat cardiac allograft survival by donor pretreatment. Screening of antineoplastic drugs.

We have made preliminary investigations into the effect of 33 different drugs on the survival of rat cardiac allografts, the drugs being administered to the allograft donor. All drugs were administered at LD50 i.v. to the graft donor 6 hr prior to removal of the organ. Especially effective were alkylating agents and antimetabolites. Pretreatment with cyclophosphamide, busulfan, methotrexate, azauridine, or bromodeoxyuridine prolonged the survival from 7 to more than 20 days. Pretreatment with chlorambusil, mannomustine, mannosulfan, 1-(2-chlorethyl)-3-cyclohexyl-1-nitrosourea, DTC, fluoruracil, or hudroxyurea prolonged the survival to more than 14 days. Several other alkylating agents and antimetabolites prolonged the survival moderately, i.e., to approximately 10 days or more. Purine antagonists, mercaptopurine and azathioprine, were totally ineffective as were also anticancer antibiotics and vinca alkaloids. Pretreatment with procarbazine or methylprednisolone alone increased the survival only moderately, whereas pretreatment with both of these drugs together increased the survival up to 27 days.

Alkylating Agents↗