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Pyrazofurin Metabolism, Enzyme Inhibition, and Resistance in L5178Y Cells¹

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510

The 5'-phosphate derivative of the C-nucleoside pyrazofurin (ß anomer) is a competitive inhibitor of orotidylate decarboxylase with a K_i of 5 x 10^-9 M. Inhibition is very rapid and appears to require ionization on the heterocyclic ring, since maximal effects are seen above the pK_a of 6.6. Pyrazofurin equilibrates with the cell water of L5178Y leukemia cells within 2 to 3 min; subsequent phosphorylation to a mixture of mono-, di-, and triphosphates occurs with the higher phosphorylated forms predominating. The di- and triphosphate derivatives are inactive as inhibitors of orotidylate decarboxylase. Similar conversion to phosphorylated nucleotides occurs in mouse liver in vivo. The initial phosphorylation of pyrazofurin appears to occur via adenosine kinase, since adenosine, but not other nucleosides, inhibited the formation of the 5'-phosphate. Adenosine kinase activity copurifies with pyrazofurin kinase, and pyrazofurin inhibits adenosine phosphorylation. A mutant of the L5178Y cell line lacking adenosine kinase does not phosphorylate pyrazofurin and is not sensitive to this drug. A 10,000-fold pyrazofurin-resistant line of L5178Y has been selected and has been shown to retain permeability to pyrazofurin but to lack detectable adenosine kinase activity. This line is also 10,000-fold cross-resistant to methylmercaptopurine riboside, an analog also activated by adenosine kinase. The pyrazofurin- and 6-azauridine-resistant lines both exhibit 4- to 6-fold elevated levels of orotidylate decarboxylase activity, which may account for the minor cross-resistance observed.

¹ Supported by USPHS Grant CA 10748 and American Cancer Society Grant CH 67Q.

² To whom requests for reprints should be addressed, at Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Conn. 06510.

Received 2/16/79. Accepted 8/ 2/79.

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