The objective of this study is to investigate apparent metabolic relationships between a unique nucleoside analog, tiazofurin, and the utilization of nicotinamide riboside, a potential precursor of NAD that has received minimal attention in the literature. We have several lines of evidence indicating that a cellular nicotinamide ribonucleoside kinase can phosphorylate both tiazofurin and 3-deazaguanosine. Tiazofurin can be phosphorylated by two other cellular enzymes as well, adenosine kinase and 5'-nucleotidase. We have developed methods of physically separating these three enzymes and propose here to purify, from human placenta, the nicotinamide ribonucleoside kinase with the objective of comparing its catalytic properties with those of adenosine kinase and 5'-nucleotidase from the same source. In an attempt to explain the unusual selectivity of tiazofurin we will determine the specific activity of nicotinamide ribonucleoside kinase in a variety of cell types and look for correlations with the toxicity of tiazofurin. Using toxic analogs of nicotinamide riboside, which will be prepared from commercially available NAD analogs, we will carry out several procedures designed to induce and select for mutant CHO cell lines having a deficiency of nicotinamide ribonucleoside kinase, which may be a relatively expendable salvage enzyme. Mutant cell lines will be useful in determining the role of this enzyme both in drug activation and in natural cell metabolism. Various biochemical parameters that could influence the activities of the three enzymes that phosphorylate tiazofurin will be investigated to determine their relative importance in the activation of the drug and to ascertain whether they can be used to effectively modulate its activity. Clinical studies will also be carried out in concert with a tiazofurin study in patients with leukemia (CML blast crisis). Attempts will be made to determine whether correlations exist between the ability of leukemia cells to utilize 14C-nicotinamide ribonucleoside and clinical response to tiazofurin.
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