guanosine-triphosphate and 2-amino-1-3-4-thiadiazole

GTP cyclohydrolase I exhibits a positive homotropic cooperative binding to GTP, which raises the possibility of a role for GTP in regulating the enzyme reaction (Hatakeyama, K., Harada, T., Suzuki, S., Watanabe, Y., and Kagamiyama, H. (1989) J. Biol. Chem. 264, 21660-21664). We examined whether or not the intracellular GTP level is within the range of affecting GTP cyclohydrolase I activity, using PC-12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. Since GTP cyclohydrolase I was the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in these cell lines, the intracellular activities of this enzyme were reflected in the tetrahydrobiopterin contents. We found that the addition of guanine or guanosine increased GTP but not tetrahydrobiopterin in these cells. On the other hand, three IMP dehydrogenase inhibitors, tiazofurin, 2-amino-1,3,4-thiadiazole, and mycophenolic acid, decreased both GTP and tetrahydrobiopterin in a parallel and dose-dependent manner, and these effects were reversed by the simultaneous addition of guanine or guanosine. There was no evidence suggesting that these inhibitors inhibited other enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin. Comparing intracellular activities of GTP cyclohydrolase I in the inhibitor-treated cells with its substrate-velocity curve, we estimated that the intracellular concentration of free GTP is 150 microM at which point the activity of GTP cyclohydrolase I is elicited at its maximum velocity. Below this GTP concentration, GTP cyclohydrolase I activity is rapidly decreased. Therefore GTP can be a regulator for tetrahydrobiopterin biosynthesis.

Topics: Animals; Antineoplastic Agents; Biopterins; GTP Cyclohydrolase; Guanine; Guanosine; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Kinetics; Mycophenolic Acid; Neuroblastoma; PC12 Cells; Ribavirin; Thiadiazoles; Tumor Cells, Cultured

Previous work from this laboratory has demonstrated a correlation between the inhibition by ribavirin (Rbv), mycophenolic acid (MPA), or 2-amino thiadiazole (TDA) of Sindbis virus replication in Aedes albopictus mosquito cells and a reduction in cellular GTP levels. This reduction in GTP results from the inhibition by these drugs of inosine monophosphate dehydrogenase (IMPDH), the first enzyme specific for the de novo synthesis of GMP. By serial passage of SV in A. albopictus cells in the presence of 25 microM MPA, we have now isolated viral mutants which are highly resistant not only to MPA but also to Rbv and TDA. For example, whereas 500 microM Rbv reduced the plaquing efficiency of SVSTD by at least 10(6)-fold, the same concentration of Rbv reduced the plaquing efficiency of the MPA-resistant mutants less than 5-fold. This is the first example of a viral mutant resistant to the antiviral compound Rbv.

Topics: Aedes; Animals; Clone Cells; DNA-Directed RNA Polymerases; Drug Resistance, Microbial; Guanosine Triphosphate; Mutation; Mycophenolic Acid; Nucleotidyltransferases; Ribavirin; Ribonucleosides; Sindbis Virus; Thiadiazoles

Topics: Aedes; Amanitins; Animals; Cells, Cultured; Dactinomycin; Guanosine Triphosphate; IMP Dehydrogenase; Ketone Oxidoreductases; Mycophenolic Acid; Niacinamide; Phosphorylation; Ribavirin; Ribonucleosides; Sindbis Virus; Thiadiazoles; Vesicular stomatitis Indiana virus; Virus Replication

The effects of 2-amino-1,3,4-thiadiazole [aminothiadiazole (NSC 4728)] on purine and pyrimidine ribonucleotide pools of L1210 ascites cells in vivo are presented and discussed as they relate to the site of action. Within 1 hr after administration of the drug, the levels of guanosine triphosphate, guanosine diphosphate, adenosine triphosphate, and adenosine diphosphate were reduced, whereas those of inosine monophosphate (IMP) and uridine triphosphate were increased. The most pronounced effects were the lowering of guanine ribonucleotide pools and the elevation of IMP. Aminothiadiazole produced a marked inhibition (approximately 95%) of the incorporation of [8-14C]inosine into guanine nucleotides, whereas only a slight inhibition (approximately 20%) of incorporation into adenine nucleotides was observed. These results suggest that the thiadiazole (or a metabolite thereof) inhibits the conversion of IMP to guanosine monophosphate; this conclusion is reinforced by the observation that mycophenolic acid, a known inhibitor of this conversion, produced effects on ribonucleotide pools similar to those produced by aminothiadiazole. Aminothiadiazole did not inhibit IMP dehydrogenase isolated from L1210 cells. The effects of the thiadiazole on nucleotide pools were prevented by simultaneous administration of nicotinamide. Since nicotinamide is known to prevent or reverse the antileukemic activity of aminothiadiazole, it is probable that the inhibition of synthesis of guanosine monophosphate is related to the antileukemic action of this agent.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cells, Cultured; Guanine Nucleotides; Guanosine Triphosphate; IMP Dehydrogenase; Inosine; Inosine Nucleotides; Leukemia L1210; Niacinamide; Ribonucleotides; Thiadiazoles; Uracil Nucleotides