Inhibition of deoxyribonucleotide synthesis by pyridine carboxaldehyde thiosemicarbazones.

inhibited mammalian ribonucleoside diphosphate reductase in vitro. The inhibition was partially reversed by increasing the concentration of the dithiol substrate, but not of ferrous ion, magnesium ion, nucleotide substrate, or allosteric activator. Increasing the concentration of ferrous ion resulted in greater enzymic inhibition, a finding that is consistent with a model in which either the active form of the inhibitor is an iron chelate or the inhibitor is bound to an iron-charged enzyme at the site normally occupied by the dithiol substrate. The present study demonstrates that the ribonucleoside diphosphate reductase of Sarcoma 180 and the Novikoff rat tumor is also inhibited by the pyridine carboxaldehyde thiosemicarbazones and describes differences among the pyridine carboxaldehyde thiosemicarbazones in the mechanism of enzyme inhibition. Evidence that such a block occurs in intact tumor cells is provided by the finding that the conversion of ribonucleotides to deoxyribonucleotide forms was decreased in drug-treated cells. A preliminary report of this research has been presented (9). MATERIALS AND METHODS Studies with Intact Cells. Six-day growths of Sarcoma 180 ascites cells propagated in 9-to 1 1-week-old female CD-l mice (Charles River Breeding Laboratories, North Wilmington, Mass.) were incubated at a concentration of approximately 1.5 X 108 cells for 30mm at 37°in a totalvolume oflO ml of Robinson's medium minus calcium chloride (8) with inhibitors and cytidine-5-3 H (160 sg; 1.29 X 10†• cpm/pg). Inhibitors were dissolved in dimethyl sulfoxide ; the final concentration of solvent, which did not inhibit the incorporation of cytidine into nucleotide forms, did not exceed 4%. After incubation, the cells were collected by centrifugation, the cold 4% perchloric acid.soluble fraction was quantitatively collected and neutralized with KOH, and the KC1O4 was removed by centrifugation. The resulting supematant was concentrated and incubated for 2 hr at 37° with 0.06 M Tris-HC1, pH 9; 0.06 M MgSO4 ; and lyophiized crude Crotalus adamanteus venom extract, 7.5 mg/mi. The reaction was terminated by the addition of cold acetone:giacial acetic acid (9: 1, v/v), and the mixture was centrifuged. Ribonucleosides were separated from deoxyribonucleosides by chromatography on Whatman No. v/v), as described by Reichard (6). The location of deoxyribonucleosides was ascertained with ultraviolet light and was facilitated by the addition of carrier material SUMMARY The tumor-inhib itory agent 2-formylpyridine thiosemicarbazone (PT) and its 3-and 5-hydroxy derivatives (3-HP and 5-HP) inhibited the incorporation of cytidine-5-3 H into acid-soluble deoxyribonucleotides of Sarcoma 180 cells in vitro. These compounds also …