2-Beta-D-Ribofuranosylthiazole-4-carboxamide (tiazofurin) is a new agent which has demonstrated remarkable antitumor activity in experimental systems. It is converted to a dinucleotide (tiazofurin adenine-dinucleotide, TAD) and appears to act as an analog of the natural cofactor, nicotinamide adenine-dinucleotide (NAD). In this form it is a potent inhibitor of IMP dehydrogenase (IMP:NAD oxidoreductase, EC 1.2.1.14) and results in depression of guanine nucleotide biosynthesis. The objectives of this proposal are to identify the enzymes involved in TAD biosynthesis from tiazofurin and to determine the primary target sites of its actionin Chinese hamster ovary (CHO) cells. Enzymes relevant to the metabolism of tiazofurin will be identified and characterized using conventional biochemical techniques and a series of tiazofurin-resistant lines of CHO cells. The effects of tiazofurin on the biochemical transformations of [14C]nicotinamide by CHO cells will be determined using high pressure liquid chromatographic (HPLC) techniques to detect and quantitate the formation of nicotinamide riboside, nicotinamide mononucleotide, NAD and NADP in extracts of appropriately incubated cells. The possible conversion of [3H]tiazofurin to the triphosphate derivative will be determined with HPLC and an authentic standard. Other potential sites of inhibition by TAD, such as poly(ADP-ribose) synthesis and dehydrogenases other than IMP dehydrogenase will be investigated directly and their roles in the action of this unique agent determined. Comparative studies will be carried out with the somewhat similar agent, ribavirin.
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