BIOLOGICAL-ACTIVITY OF A NOVEL RATIONALLY DESIGNED LIPOPHILIC THYMIDYLATE SYNTHASE INHIBITOR

AG-331 fonyl)benzyl]N-6-methyl-2,6-diamino-benz[cd]indole glucuronate} is a novel lipophilic thymidylate synthase (TS) inhibitor. The proper ties of this compound were investigated in H35 rat hepatoma cells and in three variant cell lines resistant to antifolates by differing mech anisms. There was no evidence for any intracellular effect of AG-331 o n dihydrofolate reductase (DHFR); however, the low degree of cross-res istance found for the H35FF line, which has elevated TS levels, sugges ted that TS may not be the sole locus of action of AG-331 in hepatoma cells. TS-directed effects of AG-331 were suggested by the pattern of its inhibition of deoxyuridine incorporation into DNA and the lesser e ffects on purine incorporation. In addition, H35 cells treated with 10 mu M AG-331 were shown to accumulate in the S phase of the cell cycle , and this effect could be reversed by coadministration of thymidine. However, when treatments were conducted at a 5-fold higher concentrati on of AG-331, no S-phase block was apparent, suggesting the loss of a TS-directed effect at high inhibitor concentrations. Thymidine and fol inic acid also failed to protect cells against AG-331 cytotoxicity, su ggesting an alternate mode of action. Similar results were also obtain ed in protection experiments with a human hepatoma cell line, HEPG2, a lthough previous results obtained in colon- and breast-cancer cell lin es have suggested TS specific effects for AG-331. The possibility that biotransformation of AG-331 to other toxic species may occur in liver -derived cell lines has yet to be investigated.