adenosine-kinase and tiazofurin

The growth inhibitory activity of tiazofurin toward adenosine kinase deficient Chinese hamster ovary (CHO) cells was partially reversed by the presence of nicotinamide riboside. Similarly, the formation of tiazofurin 5'-monophosphate and the active metabolite, tiazofurin 5'-adenine dinucleotide could be partially inhibited by 100 microM nicotinamide riboside in CHO cells and substantially inhibited (80-90%) in adenosine kinase deficient cells. Tiazofurin phosphorylating activity from CHO cell extracts was resolved into two peaks by DEAE-cellulose chromatography. The first peak of activity was identified as adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). The second peak of activity correlated with a previously described 3-deazaguanosine phosphorylating activity that was identified as a nicotinamide ribonucleoside kinase. Contaminating purine nucleoside phosphorylase was removed by sedimentation through a sucrose density gradient which also resolved the tiazofurin phosphorylating activity into two peaks, one requiring just ATP and the other requiring both ATP and IMP. Of the substrates tested with the lower density peak, nicotinamide riboside was most efficient and was the only natural substance that competed well with tiazofurin for phosphorylation, substantiating its suggested identity as a nicotinamide ribonucleoside kinase. The apparent Km value for nicotinamide riboside (2 microM) was significantly less than that for tiazofurin (13.6 microM). ATP was the best phosphate donor; CTP and UTP were utilized less efficiently and IMP did not support the reaction. The best substrate for the higher density peak of tiazofurin phosphorylation was inosine and both ATP and IMP were required for the reaction, suggesting its identity as a 5'-nucleotidase. In summary, it appears that adenosine kinase, nicotinamide ribonucleoside kinase, and 5'-nucleotidase may all contribute to the phosphorylation of tiazofurin in CHO cells.

Topics: Adenosine Kinase; Adenosine Triphosphate; Animals; Antimetabolites, Antineoplastic; Biotransformation; Cell Line; Cricetinae; Cricetulus; Female; Inosine Monophosphate; Kinetics; Niacinamide; Ovary; Phosphorylation; Pyridinium Compounds; Ribavirin; Ribonucleosides

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