guanosine-triphosphate and mizoribine

Mizoribine induces the differentiation of promyelocytes by an unknown mechanism that relies on compromised guanine nucleotide synthesis. I have found that mizoribine also perturbs adenosine nucleotide levels in HL-60 promyelocytes, particularly ATP. To reconcile these observations with the known actions of mizoribine I have adapted an existing model of human purine metabolism composed as an S-system familiar from Biochemical Systems Theory. Mizoribine's actions were then simulated and compared with experimental data.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Cell Differentiation; Guanine Nucleotides; Guanosine Triphosphate; HL-60 Cells; Humans; Ribonucleosides; Signal Transduction

Ribavirin and mycophenolic acid (MPA) are known inhibitors of the IMPDH enzyme (E.C. 1.1.1.205). This enzyme catalyzes the conversion of inosine monophosphate to xanthine monophosphate, leading eventually to a decrease in the intracellular level of GTP and dGTP. The antiviral effect against bovine viral diarrhoea virus (BVDV) of 15 analogues related to MPA was determined. MDBK cells were infected with the cytopathic strain of BVDV in presence or absence of test compounds. Viral RNA was extracted from the cell supernatant fluids and quantified by RT-PCR. Ribavirin showed a potent antiviral effect against BVDV with 90% effective concentration (EC90) of 4 microM. MPA along with several analogues, including both its corresponding aldehyde and alcohol, and modifications in the length of the side chain (C2- and C4-derivatives) were tested. We have identified previously unreported IMPDH inhibitors that have potent anti-BVDV activity, namely: C6-MPAlc (5), C6-MPA-Me (7), C4-MPAlc (8), C4-MPA (10) and C2-MAD (20). Most of these compounds inhibited the IMPDH enzyme in the nanomolar range (4-800 nM) in cell-free assays. Some compounds, such as mizoribine, which is a potent inhibitor of IMPDH in vitro (enzyme 50% inhibitory concentration IC50=4 nM), had no detectable anti-BVDV activity up to 100 microM. The compounds were essentially non-toxic to a confluent monolayer of MDBK cells. However, in exponentially growing cells, they showed minimal toxicity at 100 microM over a 24 h period, but the toxicity was more pronounced after 3 days [50% cytotoxic concentration (CC50) value ranged from 5 to 30 microM].

Topics: Animals; Cattle; Cell Line; Computer Systems; Culture Media, Conditioned; Diarrhea Viruses, Bovine Viral; Dose-Response Relationship, Drug; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; Guanosine Triphosphate; IMP Dehydrogenase; Kidney; Molecular Structure; Mycophenolic Acid; NAD; Nucleosides; Reverse Transcriptase Polymerase Chain Reaction; Ribavirin; Ribonucleosides; Viral Plaque Assay; Virus Replication

Topics: Adenosine Triphosphate; Cell Differentiation; Cell Division; Enzyme Inhibitors; Guanosine Triphosphate; HL-60 Cells; Humans; IMP Dehydrogenase; Kinetics; Mycophenolic Acid; Ribonucleosides; Tumor Cells, Cultured

It has been reported that the immunosuppressant mizoribine (MZR) inhibits T cell proliferation by depleting intracellular guanine nucleotides via competitive inhibition of inosine 5'-monophosphate (IMP) dehydrogenase in the purine metabolism pathway. This study was performed to determine if the mechanism by which MZR suppresses the proliferation of mouse B cells and antibody production by these cells is dependent on the depletion of intracellular guanine nucleotides. Stimulation of purified splenic B cells of mice with lipopolysaccharide (LPS), a mitogen to B cells, increased both proliferation and antibody production. MZR suppressed both of these functions in a dose-dependent fashion. MZR also caused a decrease in the amount of intracellular guanosine 5'-triphosphate (GTP). When the cultures were grown on plates containing guanosine plus 8-aminoguanosine, the amount of intracellular GTP, which had been reduced by MZR, was restored. Furthermore, the repletion of GTP pools restored both proliferation and antibody production almost to their previous levels. These results suggest that MZR suppresses antibody production and proliferation of B cells by acting directly on B cells. Furthermore, it is suggested that the inhibitory effect of MZR on antibody production, as well as on T cell proliferation, is dependent on the decrease in intracellular guanine nucleotide pools of mouse B cells.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibody Formation; Antibody Specificity; B-Lymphocytes; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Guanosine; Guanosine Triphosphate; Immunosuppressive Agents; Lipopolysaccharides; Male; Mice; Mice, Inbred ICR; Ribonucleosides; Spleen

The growth inhibitory mechanisms of mizoribine, an immunosuppressive imidazole nucleoside used clinically to inhibit rejection reactions after renal transplantation and in the treatment of systemic lupus erythematosus and rheumatoid arthritis, were studied in human and murine cells. We found that (a) human cells were 20- to 60-fold more resistant than murine cells to both mizoribine and its aglycone, (b) adenine phosphoribosyltransferase (APRT)-deficient human cells were resistant to aglycone but not to mizoribine, (c) hypoxanthine phosphoribosyltransferase (HPRT)-deficient human cells were at least 100-fold more sensitive to both mizoribine and aglycone, and (d) the decrease in intracellular GTP broadly paralleled the cytotoxicity in each case. Therefore, data obtained from studies using non-human tissues should be interpreted carefully before clinical application. Results indicate that the growth inhibitory effect of the aglycone but not of mizoribine is mediated by APRT, and depletion of guanine nucleotides is responsible for the effects of both drugs. Our data also suggest that the drugs may reduce mutant HPRT-deficient somatic cells in vivo, and may cause enhanced adverse reactions in HPRT-deficient individuals. The drug may have altered effects in patients receiving other purine or pyrimidine analogs.

Topics: Adenine Phosphoribosyltransferase; Adenosine Triphosphate; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Division; Cell Line; Cell Line, Transformed; Cells, Cultured; Guanosine Triphosphate; Humans; Hypoxanthine Phosphoribosyltransferase; Immunosuppressive Agents; Mice; Mutation; Ribonucleosides

Inhibitors of IMP dehydrogenase (EC 1.2.1.14), including mizoribine (Bredinin) and mycophenolic acid, have significant antitumor and immunosuppressive activities. Studies were aimed at determining the mechanism by which intracellular GTP depletion induced by these agents results in inhibition of DNA synthesis. Incubation of human CEM leukemia cells for 2 hr with IC50 concentrations of either mizoribine (4 microM) or mycophenolic acid (0.5 microM) reduced cellular GTP levels an average of 68% or 58%, respectively, compared with the levels in control cells. Under similar conditions, mizoribine and mycophenolic acid decreased the amount of [3H]adenosine incorporated into primer RNA by 75% and 70%, respectively, relative to the untreated controls, but had no significant effect on total RNA synthesis. Repletion of the guanine nucleotide pools by coincubation of CEM cells with guanosine plus 8-aminoguanosine prevented both the inhibition of primer RNA synthesis and the inhibition of tumor cell growth induced by these agents. Additional studies demonstrated that GTP depletion alone was capable of directly inducing inhibition of primer RNA synthesis. Primer RNA synthesis was inhibited an average of 84% in whole-cell lysates that lacked GTP but contained all remaining ribo- and deoxyribonucleoside triphosphates. On an M13 DNA template, RNA-primed DNA synthesis catalyzed by the purified complex of DNA primase (EC 2.7.7.6) and DNA polymerase alpha (EC 2.7.7.7) was decreased an average of 70% in the absence of GTP, compared with synthesis in the presence of 0.5 mM GTP. These results provide evidence that mizoribine and mycophenolic acid inhibit DNA replication by inducing GTP depletion, which suppresses the synthesis of RNA-primed DNA intermediates.

Topics: Adenosine Triphosphate; Cell Survival; DNA Primase; DNA, Neoplasm; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Leukemia; Mycophenolic Acid; Nucleosomes; Ribonucleosides; RNA Nucleotidyltransferases; RNA, Neoplasm; Tumor Cells, Cultured

IMP dehydrogenase is a key enzyme in the de novo pathway of purine biosynthesis and is responsible for catalyzing the first step in the formation of guanine ribonucleotides from inosine monophosphate. Mizoribine, an immunosuppressive agent in wide-spread clinical use in Japan, has been demonstrated to inhibit this enzyme. We have investigated the effects of mizoribine on human T cell activation. Stimulation of purified human peripheral blood T lymphocytes with phorbol ester and ionomycin leads to a five-fold increase in guanine ribonucleotide levels over 72 hours. The addition of mizoribine to these cultures at concentrations that are achieved in vivo leads to a dose-dependent inhibition of proliferation and concomitant 50% decrease in guanine ribonucleotide levels, an effect that is reversible with the addition of guanosine, which repletes the GTP pool. Similar effects are seen with direct stimulation via the CD3/T cell receptor complex. Inhibition of proliferation occurs at the G1/S interface of the cell cycle and is additive to that produced by cyclosporine. In order to determine whether inhibition of IMP dehydrogenase is a common mechanism of immunosuppression for drugs such as azathioprine and 6-mercaptopurine that interfere with purine biosynthesis, we compared the effects of these agents on the metabolism of purified T lymphocytes. The results of these studies demonstrate that mizoribine and mycophenolic acid, a highly specific inhibitor of IMP dehydrogenase, inhibit proliferation directly by the depletion of guanine ribonucleotides; 6-mercaptopurine, on the other hand, has a mixed effect on adenine and guanine ribonucleotide pools, whereas azathioprine inhibits proliferation by a mechanism completely independent of its effects on the purine metabolic pathway. We conclude from these studies that inhibitors of IMP dehydrogenase have potential as specific immunosuppressive agents.

Topics: Azathioprine; Cell Cycle; Cells, Cultured; Guanosine Triphosphate; Humans; Immunosuppressive Agents; IMP Dehydrogenase; Lymphocyte Activation; Mercaptopurine; Ribonucleosides; T-Lymphocytes

To investigate whether GTP concentrations can be a regulatory step in exocytotic hormone secretion, we treated isolated rat islets with mycophenolic acid (MPA) or mizoribine, two selective inhibitors of de novo GTP synthesis. When islets were cultured overnight in purine-free medium containing the drug, MPA reduced GTP levels by up to 81 +/- 1%; guanine circumvented this block via the nucleotide "salvage" pathway. MPA concomitantly inhibited glucose (16.7 mM)-induced insulin secretion in batch-type incubations (or perifusions), by up to 68% at 50 micrograms/ml. Although the inhibition of secretion occurred over a similar concentration range as the reduction in total GTP content, the two variables were not directly correlated. However, the secretory effects also were prevented by adding guanine, but not hypoxanthine or xanthine, to the culture medium. Similar results for GTP content and insulin release were seen using mizoribine. Insulin content was modestly (-18%) reduced by MPA but indices of fractional release (release/insulin content) were also markedly impaired. Although MPA also reduced ATP levels more modestly (-39%) and increased UTP (+87%), these were not the cause of the secretory defect since adenine restored ATP and UTP nearly to normal, but did not alter the reduction in GTP content or insulin secretion. MPA also inhibited secretion induced by amino acid or by a phorbol ester but had virtually no effect on release induced by a depolarizing concentration of K+, suggesting that GTP depletion does not merely impede Ca+ influx or directly block Ca(2+)-activated exocytosis. However, a severe reduction of GTP content did not prevent the pertussis toxin-sensitive inhibition of insulin release induced by epinephrine, suggesting that the function of heterotrimeric GTP-binding proteins is not limited by ambient GTP concentrations. Although these studies do not elucidate the exact site(s) in the exocytotic cascade which depend on intact GTP stores, they do provide the first direct evidence that GTP is required (and can be rate limiting) for insulin release.

Topics: Adenosine Triphosphate; Animals; Chromatography, High Pressure Liquid; Epinephrine; Exocytosis; Guanosine Triphosphate; IMP Dehydrogenase; Insulin; Islets of Langerhans; Kinetics; Male; Mycophenolic Acid; Pertussis Toxin; Rats; Rats, Inbred Strains; Ribonucleosides; Uridine Triphosphate; Virulence Factors, Bordetella

Topics: Cell Cycle; Gene Expression; Guanine Nucleotides; Guanosine; Guanosine Triphosphate; Humans; Immunosuppressive Agents; In Vitro Techniques; Lymphocyte Activation; Ribonucleosides; T-Lymphocytes