Cytotoxicity of a new uridine analog, 4-hydroxy-1-(beta-D-ribofuranosyl)-pyridazine-6-one, and its interaction with uridine kinase.

A new uridine analog, 4-hydroxy-1-(beta-D-ribonfuranosyl)-pyridazin-6-one (3-deaza-6-azaUrd), inhibited the growth of L1210 cells in culture, with a concentration to reduce growth rate to 50% of control of 7 X 10(-5) M. After treatment for 24 or 48 h with 5 X 10(-4) M 3-deaza-6-azaUrd, 80% of the cells were unable to resume growth when the analog was removed from the cultures; also, 99% of the cells were killed, as determined by colony formation in soft agar. Studies on the prevention of the cytotoxic effects of 5 X 10(-4) M 3-deaza-6-azaUrd showed that uridine or cytidine gave complete protection. 2'-Deoxycytidine also gave partial protection, but orotic acid or thymidine had no effect on the growth inhibition by 3-deaza-6-azaUrd. These results suggested that growth inhibition by 3-deaza-6-azaUrd might be due to interference in pyrimidine biosynthesis. Activation of 3-deaza-6-azaUrd to its 5'-phosphate derivative appeared to be catalyzed by uridine kinase. 3-Deaza-6-azaUrd was shown to complete with uridine for phosphorylation (Ki = 4.7 mM) and, therefore, to be a possible alternative substrate for uridine kinase from mouse kidney (Km for uridine = 82 microM). The enzyme was partially purified by streptomycin sulfate precipitation, ammonium sulfate fractionation, and gel filtration. This preparation was found to be free of pyrimidine nucleoside phosphorylase and uridine monophosphate kinase.