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

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

Control of pyrimidine nucleotide formation in Pseudomonas aurantiaca.

The control of pyrimidine nucleotide formation in the bacterium Pseudomonas aurantiaca ATCC 33663 by pyrimidines was studied. The activities of the pyrimidine biosynthetic pathway enzymes were investigated in P. aurantiaca ATCC 33663 cells and from cells of an auxotroph lacking orotate phosphoribosyltransferase activity under selected culture conditions. All activities of the pyrimidine biosynthetic pathway enzymes in ATCC 33663 cells were depressed by uracil addition to the minimal medium when succinate served as the carbon source. In contrast, all pyrimidine biosynthetic pathway enzyme activities in ATCC 33663 cells were depressed by orotic acid supplementation to the minimal medium when glucose served as the carbon source. The orotidine 5'-monophosphate decarboxylase activity in the phosphoribosyltransferase mutant strain increased by more than sixfold in succinate-grown cells and by more than 16-fold in glucose-grown cells after pyrimidine limitation showing possible repression of the decarboxylase by a pyrimidine-related compound. Inhibition by ATP, GTP, UTP and pyrophosphate of the in vitro activity of aspartate transcarbamoylase in ATCC 33663 was observed. The findings demonstrated control at the level of pyrimidine biosynthetic enzyme synthesis and activity for the P. aurantiaca transcarbamoylase. The control of pyrimidine synthesis in P. aurantiaca seemed to differ from what has been observed previously for the regulation of pyrimidine biosynthesis in related Pseudomonas species. This investigation could prove helpful to future work studying pseudomonad taxonomic analysis as well as to those exploring antifungal and antimicrobial agents produced by P. aurantiaca.

Biosynthesis of the uridine-derived nucleoside antibiotic A-94964: identification and characterization of the biosynthetic gene cluster provide insight into the biosynthetic pathway.

The natural product A-94964 is a uridine-derived nucleoside antibiotic isolated from Streptomyces sp. SANK 60404. In this study, we propose a biosynthetic pathway for A-94964 using gene deletion experiments coupled with in silico analysis of the biosynthetic gene cluster. This study provides insights into the unique biosynthetic pathway for A-94964.

Pyrimidine nucleotide synthesis in the emerging pathogen Pseudomonas monteilii.

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.

Dihydroorotate Dehydrogenase as a Target for the Development of Novel Helicobacter pylori-Specific Antimicrobials.

Helicobacter pylori (H. pylori) infection is the world's most common bacterial infection, affecting approximately 50% of the global population. H. pylori is the strongest known risk factor for stomach diseases, including cancer. Hence, treatment for H. pylori infection can help reduce the risk of these diseases. However, the emergence of drug-resistant strains of H. pylori and the occurrence of adverse effects resulting from current therapies have complicated the successful eradication of H. pylori infection. Although various antibiotics that target several bacterial enzymes have been discovered, dihydroorotate dehydrogenase (DHODH) may hold potential for the development of novel anti-H. pylori agents with reduced toxicity and side effects. Here we review the existing literature that has focused on strategies for developing novel therapeutic agents that target the DHODH of H. pylori.

Aspartate Rescues S-phase Arrest Caused by Suppression of Glutamine Utilization in KRas-driven Cancer Cells.

During G1-phase of the cell cycle, normal cells respond first to growth factors that indicate that it is appropriate to divide and then later in G1 to the presence of nutrients that indicate sufficient raw material to generate two daughter cells. Dividing cells rely on the "conditionally essential" amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates and as a nitrogen source for nucleotide biosynthesis. We previously reported that while non-transformed cells arrest in the latter portion of G1 upon Q deprivation, mutant KRas-driven cancer cells bypass the G1 checkpoint, and instead, arrest in S-phase. In this study, we report that the arrest of KRas-driven cancer cells in S-phase upon Q deprivation is due to the lack of deoxynucleotides needed for DNA synthesis. The lack of deoxynucleotides causes replicative stress leading to activation of the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which arrests cells in S-phase. The key metabolite generated from Q utilization was aspartate, which is generated from a transaminase reaction whereby Q-derived glutamate is converted to α-ketoglutarate with the concomitant conversion of oxaloacetate to aspartate. Aspartate is a critical metabolite for both purine and pyrimidine nucleotide biosynthesis. This study identifies the molecular basis for the S-phase arrest caused by Q deprivation in KRas-driven cancer cells that arrest in S-phase in response to Q deprivation. Given that arresting cells in S-phase sensitizes cells to apoptotic insult, this study suggests novel therapeutic approaches to KRas-driven cancers.

Regio-selective chemical-enzymatic synthesis of pyrimidine nucleotides facilitates RNA structure and dynamics studies.

Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site-specific labeling tools to expedite NMR structural and dynamics studies are required. Using enzymes from the pentose phosphate pathway, we coupled chemically synthesized uracil nucleobase with specifically (13) C-labeled ribose to synthesize both UTP and CTP in nearly quantitative yields. This chemoenzymatic method affords a cost-effective preparation of labels that are unattainable by current methods. The methodology generates versatile (13) C and (15) N labeling patterns which, when employed with relaxation-optimized NMR spectroscopy, effectively mitigate problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and function: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro-tRNA (76 nt), and HIV-1 core encapsidation signal (155 nt).

Control of pyrimidine nucleotide formation in Pseudomonas fulva.

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 50-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.

Dysregulation of de novo nucleotide biosynthetic pathway enzymes in cancer and targeting opportunities.

Cancer is a disease of uncontrolled cell growth and a major cause of death worldwide. Many molecular events characterize tumor initiation and progression. Global gene expression analyses using next-generation sequencing, proteomics and metabolomics show genomic, epigenetic, and metabolite concentration changes in various tumors. Molecular alterations identified include multiple cancer-driving mutations, gene fusions, amplifications, deletions, and post-translational modifications. Data integration from many high-throughput platforms unraveled dysregulation in many metabolic pathways in cancer. Since cancer cells are fast-growing, their metabolic needs are enhanced, hence the requirement for de novo synthesis of essential metabolites. One critical requirement of fast-growing cells and a historically important pathway in cancer is the nucleotide biosynthetic pathway and its enzymes are valuable targets for small molecule inhibition. Purines and pyrimidines are building blocks of DNA synthesis and due to their excessive growth, cancer cells extensively utilize de novo pathways for nucleotide biosynthesis. Methotrexate, one of the early chemotherapeutic agents, targets dihydrofolate reductase of the folate metabolic pathway that is involved in nucleotide biosynthesis. In this review, we discuss the nucleotide biosynthetic pathways in cancer and targeting opportunities.

Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine deaminase.

In the presence of 10(-4) to 10(-5) molar adenosine, established cell lines of fibroblastic or lymphoid origin die of pyrimidine starvation. Less than lethal concentrations inhibit cell growth. Over a broad concentration range, the effects of adenosine are prevented by providing a suitable pyrimidine source. We suggest that the recently described immune deficiency disease associated with absence of adenosine deaminase may be the result of pyrimidine starvation induced by adenosine nucleotides in cells of the lymphoid system.

Antitrypanosomal effect of allopurinol: conversion in vivo to aminopyrazolopyrimidine nucleotides by Trypanosoma curzi.

The parasite Trypanosoma cruzi metabolizes allopurinol by a sequential conversion to allopurinol mononucleotide and aminopurinol mononucleotide. The latter is incorporated into RNA. This transformation of a widely used innocuous agent, allopurinol, into a toxic adenine analog appears to account for the antiprotozoan effect of allopurinol. These unique enzymatic activities appear to occur only in T. cruzi and the pathogenic lesihaminae. Allopurinol may serve as a model for the synthesis of similar antiprotozoan agents.

Dynamics of the cellular metabolome during human cytomegalovirus infection.

Viral replication requires energy and macromolecular precursors derived from the metabolic network of the host cell. Despite this reliance, the effect of viral infection on host cell metabolic composition remains poorly understood. Here we applied liquid chromatography-tandem mass spectrometry to measure the levels of 63 different intracellular metabolites at multiple times after human cytomegalovirus (HCMV) infection of human fibroblasts. Parallel microarray analysis provided complementary data on transcriptional regulation of metabolic pathways. As the infection progressed, the levels of metabolites involved in glycolysis, the citric acid cycle, and pyrimidine nucleotide biosynthesis markedly increased. HCMV-induced transcriptional upregulation of specific glycolytic and citric acid cycle enzymes mirrored the increases in metabolite levels. The peak levels of numerous metabolites during infection far exceeded those observed during normal fibroblast growth or quiescence, demonstrating that HCMV markedly disrupts cellular metabolic homeostasis and institutes its own specific metabolic program.

Antipyrimidine effects of five different pyrimidine de novo synthesis inhibitors in three head and neck cancer cell lines.

The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for their potential anticancer activity: acivicin inhibits carbamoyl-phosphate-synthase-II, N-(phosphonacetyl)-L- aspartate (PALA) inhibits aspartate-transcarbamylase, Brequinar sodium and dichloroallyl-lawsone (DCL) inhibit dihydroorotate-dehydrogenase, and pyrazofurin (PF) inhibits orotate-phosphoribosyltransferase. We compared their growth inhibition against 3 cell lines from head-and-neck-cancer (HEP-2, UMSCC-14B and UMSCC-14C) and related the sensitivity to their effects on nucleotide pools. In all cell lines Brequinar and PF were the most active compounds with IC50 (50% growth inhibition) values between 0.06-0.37 µM, Acivicin was as potent (IC50s 0.26-1 µM), but DCL was 20-31-fold less active. PALA was most inactive (24-128 µM). At equitoxic concentrations, all pure antipyrimidine de novo inhibitors depleted UTP and CTP after 24 hr exposure, which was most pronounced for Brequinar (between 6-10% of UTP left, and 12-36% CTP), followed by DCL and PF, which were almost similar (6-16% UTP and 12-27% CTP), while PALA was the least active compound (10-70% UTP and 13-68% CTP). Acivicin is a multi-target inhibitor of more glutamine requiring enzymes (including GMP synthetase) and no decrease of UTP was found, but a pronounced decrease in GTP (31-72% left). In conclusion, these 5 inhibitors of the pyrimidine de novo nucleotide synthesis varied considerably in their efficacy and effect on pyrimidine nucleotide pools. Inhibitors of DHO-DH were most effective suggesting a primary role of this enzyme in controlling pyrimidine nucleotide pools.

Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide.

Among all the new immunosuppressive molecules being investigated either preclinically or clinically, four stand out: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide (and its malononitriloamide analogs). Each drug has distinct mechanisms of immunosuppressive action, and in the past year significant advances have been made in our understanding of the actions of these drugs at the molecular and even atomic levels. Data from recent clinical trials demonstrate that these drugs very effectively suppress graft rejection or autoimmune diseases, validating the pivotal role played by each of their distinct molecular targets in the normal functioning of immune cells.

Human pyrimidine nucleotide biosynthesis as a target for antiviral chemotherapy.

The development of broad-spectrum, host-acting antiviral therapies remains an important but elusive goal in anti-infective drug discovery. To replicate efficiently, viruses not only depend on their hosts for an adequate supply of pyrimidine nucleotides, but also up-regulate pyrimidine nucleotide biosynthesis in infected cells. In this review, we outline our understanding of mammalian de novo and salvage metabolic pathways for pyrimidine nucleotide biosynthesis. The available spectrum of experimental and FDA-approved drugs that modulate individual steps in these metabolic pathways is also summarized. The logic of a host-acting combination antiviral therapy comprised of inhibitors of dihydroorotate dehydrogenase and uridine/cytidine kinase is discussed.

The crystal structure of Pyrococcus furiosus UMP kinase provides insight into catalysis and regulation in microbial pyrimidine nucleotide biosynthesis.

UMP kinase (UMPK), the enzyme responsible for microbial UMP phosphorylation, plays a key role in pyrimidine nucleotide biosynthesis, regulating this process via feed-back control and via gene repression of carbamoyl phosphate synthetase (the first enzyme of the pyrimidine biosynthesis pathway). We present crystal structures of Pyrococcus furiosus UMPK, free or complexed with AMPPNP or AMPPNP and UMP, at 2.4 A, 3 A and 2.55 A resolution, respectively, providing a true snapshot of the catalytically competent bisubstrate complex. The structure proves that UMPK does not resemble other nucleoside monophosphate kinases, including the UMP/CMP kinase found in animals, and thus UMPK may be a potential antimicrobial target. This enzyme has a homohexameric architecture centred around a hollow nucleus, and is organized as a trimer of dimers. The UMPK polypeptide exhibits the amino acid kinase family (AAKF) fold that has been reported in carbamate kinase and acetylglutamate kinase. Comparison with acetylglutamate kinase reveals that the substrates bind within each subunit at equivalent, adequately adapted sites. The UMPK structure contains two bound Mg ions, of which one helps stabilize the transition state, thus having the same catalytic role as one lysine residue found in acetylglutamate kinase, which is missing from P.furiosus UMPK. Relative to carbamate kinase and acetylglutamate kinase, UMPK presents a radically different dimer architecture, lacking the characteristic 16-stranded beta-sheet backbone that was considered a signature of AAKF enzymes. Its hexameric architecture, also a novel trait, results from equatorial contacts between the A and B subunits of adjacent dimers combined with polar contacts between A or B subunits, and may be required for the UMPK regulatory functions, such as gene regulation, proposed here to be mediated by hexamer-hexamer interactions with the DNA-binding protein PepA.

Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2.

BACKGROUND: Francisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined. METHODS: We constructed a stable transposon mutagenesis library of virulent strain Schu S4 using a derivative of the EZ::TN transposon system. Approximately 2000 mutants were screened for the inability to invade, and replicate in the hepatic carcinoma cell line HepG2. These mutants were also tested for replication within the J774.1 macrophage-like cell line. RESULTS: Eighteen mutants defective in intracellular replication in HepG2 cells were identified. Eight of these mutants were auxotrophs; seven had mutations in nucleotide biosynthesis pathways. The remaining mutants had insertions in genes that were predicted to encode putative transporters, enzymes involved in protein modification and turnover, and hypothetical proteins. A time course of the intracellular growth of a pyrB mutant revealed that this mutant was only able to grow at low levels within HepG2 cells but grew like wild-type bacteria in J774.1 cells. This pyrB mutant was also attenuated in mice. CONCLUSION: This is the first reported large-scale mutagenesis of a type A strain of F. tularensis and the first identification of mutants specifically defective in intracellular growth in a hepatic cell line. We have identified several genes and pathways that are key for the survival and growth of F. tularensis in a hepatic cell line, and a number of novel intracellular growth-defective mutants that have not been previously characterized in other pathogens. Further characterization of these mutants will help provide a better understanding of the pathogenicity of F. tularensis, and may have practical applications as targets for drugs or attenuated vaccines.

Critical roles of glutamine as nitrogen donors in purine and pyrimidine nucleotide synthesis: asparaginase treatment in childhood acute lymphoblastic leukemia.

Asparaginase is a key component of the chemotherapy protocols used in the treatment of acute lymphoblastic leukemia (ALL). The current treatment protocols are remarkable in that childhood ALL cure rates are approaching 85%. As the name implies, asparaginase catalyzes the deamination of asparagine to aspartic acid. What is not generally realized is that asparaginase also catalyzes, essentially to the same extent, the removal of the amide nitrogen from glutamine to form glutamic acid. Glutamine is a required substrate for three enzymes involved in the de novo synthesis of purine nucleotides and two enzymes involved in the de novo synthesis of pyrimidine nucleotides. In this review, the specific roles of glutamine in the de novo synthesis of nucleotides are defined and an appropriate explanation for the cell cycle arrest and cytotoxicity induced in proliferating malignant lymphoblasts by asparaginase treatment is provided.

Mechanism of action of the novel anticancer agent 6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarbo xylic acid sodium salt (NSC 368390): inhibition of de novo pyrimidine nucleotide biosynthesis.

Exposure of cultured clone A human colon tumor cells to 25 to 75 microM of NSC 368390 [6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarbox yli c acid sodium salt, DuP 785] for 48 to 72 h resulted in a 99.9% cell kill as determined by clonogenic assay. Cells exposed to NSC 368390 became depleted in intracellular pools of uridine 5'-triphosphate and cytidine 5'-triphosphate. Both uridine 5'-triphosphate and cytidine 5'-triphosphate were decreased to 50% of levels in control cells at 3 h and were undetectable at 15 h after addition of 25 microM of NSC 368390 to the cultures. Similar effects were observed in L1210 leukemia cells. Addition of 0.1 mM of uridine or cytidine restored intracellular pools of uridine 5'-triphosphate and cytidine 5'-triphosphate to control levels and rescued clone A cells from NSC 368390 cytotoxicity. Addition of uridine circumvented NSC 368390 cytotoxicity in L1210 cells, but addition of cytidine did not. This result is consistent with the fact that L1210 cells lack cytidine deaminase and thus cannot form uridine or its anabolites from cytidine. These results indicated that NSC 368390 inhibits a step in the de novo biosynthetic pathway leading to uridine 5'-monophosphate. Therefore, the effects of NSC 368390 on the six enzymes that comprise the de novo pathway leading to the formation of uridine 5'-monophosphate were examined. The results showed that NSC 368390 was a potent inhibitor of dihydroorotate dehydrogenase, the fourth enzyme in the pathway; thus, this study demonstrates that NSC 368390 exerts its tumoricidal effect by inhibiting a step in de novo pyrimidine biosynthesis resulting in the depletion of critical precursors for RNA and DNA synthesis.

Metabolism and metabolic effects of 8-azainosine and 8-azaadenosine.

8-Azainosine (8-aza-HR) is of interest because of its activity against experimental tumors. Metabolic studies in cell cultures were performed with 8-aza-HR and with the structurally related nucleoside, 8-azaadenosine (9-beta-D-ribofuranosyl-8-azaadenine) (8-aza-AR), which has a lower degree of antitumor activity than does 8-aza-HR. In H. Ep. 2 cells and in Ca755 cells, both 14C-labeled nucleosides were metabolized to nucleotides of 8-azaadenine (8-aza-A) and 8-azaguanine (8-aza-G) and incorporated into polynucleotides as 8-aza-A and 8-aza-G. 8-Aza HR was incorporated primarily as 8-aza-G, whereas 8-aza-AR was incorporated about equally as 8-aza-A and 8-aza-G. In H. Ep. 2 cells, the extent of incorporation of 8-aza-HR as 8-aza-G was about one-half that found when [14C]-8-aza-G was the precursor. In the H. Ep. 2/FA/FAR cell line, 8-aza-AR and 8-aza-HR were metabolized similarly, in that both were incorporated into polynucleotides principally as 8-aza-G; apparently, in this cell line which is deficient in adenosine kinase and adenine phosphoribosyltransferase, 8-aza-AR is metabolized by conversion to 8-aza-HR. A cell line (H. Ep 2/8-aza HR), which was resistant to 8-aza-HR but sensitive to 8-aza-AR and which retained hypoxanthine (guanine)-phosphoribosyltransferase activity, metabolized 8-aza-HR to only a small extent. However, in this cell-line, 8-aza-AR was more extensively metabolized and was incorporated primarily as 8-aza-A. The failure of these cells to convert 8-aza-AR or 8-aza-HR to 8-aza-G indicates that the basis for resistance may be a change in the substrate specificities of the enzymes of guanosine monophosphate synthesis such that these cells no longer effectively convert 8-azainosine monophosphate to 8-azaguanosine monophosphate. 8-Aza-AR was a potent inhibitor of purine synthesis de novo, but 8-aza-HR, at concentrations much higher than the inhibitory concentration of 8-aza-AR, did not inhibit this process. In H. Ep. 2 cells, 8-aza-HR blocked the conversion of orotic acid to uridine nucleotides and caused an accumulation of orotidine. This inhibition of pyrimidine biosynthesis apparently does not contribute significantly to the cytotoxicity of 8-aza-HR because uridine provided no degree of reversal of its inhibition of the growth of cell cultures.