tiazofurin and pyrazofurin

The exact route of metabolism of tiazofurin, a novel nucleoside with antitumor activity, is controversial. Using human cell lines severely deficient in salvage nucleotide enzymes, we were able to identify the route of activation in tiazofurin metabolism. With loss of adenosine kinase activity by mutation in two lymphoblastoid cell lines, CCRF-CEM and WI-L2, the growth sensitivity to tiazofurin decreased by 6- and 3-fold, respectively. In contrast, the mutant lines were about 3000- to 1500- and 16- to 4-fold more resistant to the structurally similar tiazofurin analogues pyrazofurin and ribavirin, respectively. Other mutants with defective deoxycytidine or uridine kinase activity showed normal sensitivity to all three analogues. Both cell lines with defective adenosine kinase activity accumulated about 50% wild-type levels of tiazofurin-5'-monophosphate and thiazole-4-carboxamide adenine dinucleotide analogue of tiazofurin at cytotoxic concentrations of the drug. Extracts of wild-type lymphoblasts catalyzed the phosphorylation of tiazofurin in the presence of adenosine 5'-triphosphate and Mg2+. Loss of adenosine kinase activity in the mutant extract eliminated this phosphorylating activity for tiazofurin consistent with the notion that adenosine kinase catalyzes phosphorylation of tiazofurin. However, an enzyme activity that catalyzed the phosphorylation of tiazofurin in the presence of inosine-5'-monophosphate as donor and Mg2+ was detected in the extracts of both wild-type cells and adenosine kinase-deficient mutants. The monophosphate donor specificity, divalent metal, high salt requirement, and nucleoside acceptor specificity of this enzyme activity paralleled that of a 5'-nucleotidase (EC 3.1.3.5) which catalyzes inosine phosphorylation. In addition, tiazofurin phosphorylation was competitively inhibited by inosine and the apparent Ki value was similar to the apparent Km value for inosine phosphorylation. These results indicate that two enzymes, adenosine kinase and a cytoplasmic 5'-nucleotidase, are functionally important anabolizing enzymes for tiazofurin in human cells.

Topics: 5'-Nucleotidase; Adenosine Kinase; Adenosine Triphosphate; Amides; Biotransformation; Cell Line; Drug Resistance; Humans; Inosine Monophosphate; Lymphocytes; Nucleotidases; Phosphorylation; Phosphotransferases; Pyrazoles; Ribavirin; Ribonucleosides; Ribose; Substrate Specificity

The postulation that the activity of key enzymes that reveal marked increases should be potential targets for anticancer chemotherapy (47) was supported by new evidence on the alterations of CDP reductase, CTP synthetase and OMP decarboxylase in hepatoma 3924A cell cultures. Inhibitors of these enzymes (VF-122, acivicin, pyrazofurin) and that of IMP dehydrogenase (tiazofurin) efficiently killed hepatoma 3924A cells in culture, as demonstrated by the clonogenic assay. Acivicin, pyrazofurin, tiazofurin and VF-122 were lethal against tumor cells in the exponential phase of growth with IC50 of 1.5, 5, 10 and 4.5 microM, respectively. All these antimetabolites exhibited cytotoxicity preponderantly against exponential-phase cultures, indicating that all the four drugs belong to Class II (phase-specific agents) in the Kinetic Classification of Anticancer Agents (38). Dibromodulcitol, a bifunctional alkylating agent, revealed cycle-specific cytotoxicity (Class III agent) against hepatoma 3924A, yielding IC50 values of 2.3 and 5.5 microM for exponentially and stationary growing cells, respectively. Using isobologram analysis on the survival data of 3924A cells, synergistic interaction was observed when DBD in combination with acivicin, pyrazofurin and tiazofurin was examined. DBD in combination with VF-122 exhibited additive lethality against hepatoma cells in culture. The synergistic and additive cytotoxicity in combinations of DBD with these antimetabolites was accompanied by the concurrent depletion of ribonucleotide and/or deoxyribonucleotide pools. The synergistic biological results of drug combinations of acivicin with DBD can be accounted for by the action of acivicin in inhibiting CTP synthetase, resulting in a synergistic decrease in CTP content, and by inhibition of DNA synthesis caused by DBD. The synergistic and additive depletion of UTP, CTP, dTTP and dCTP pools in the combinations of DBD with pyrazofurin may be responsible for the synergistic lethality of these combinations. Synergism, in terms of pool depletion, was observed for GTP and dCTP; summation was detected for dGTP when DBD and tiazofurin were given concurrently. The synergistic cytotoxicity of this drug combination may be a consequence of these alterations. The additive lethality of DBD-VF-122 drug combinations was reflected in the additive elevations of the ribonucleoside diphosphate concentrations. These observations indicate that treatments based on the Kinetic Classification and on the

Topics: Amides; Animals; Antimetabolites, Antineoplastic; Cell Division; Cell Line; Drug Synergism; Hydroxamic Acids; Isoxazoles; Liver Neoplasms, Experimental; Mitolactol; Pyrazoles; Rats; Ribavirin; Ribonucleosides; Ribose