THE THYMIDYLATE SYNTHASE INHIBITOR, ICI-D1694, OVERCOMES TRANSLATIONAL DETAINMENT OF THE ENZYME

We have investigated the mechanism of inactivation of thymidylate synt hase (TS) by ICI D1694 (a folate-based quinazoline) in normal versus t umor-derived human mammary epithelial cells. ICI D1694 is a very poten t cytotoxic agent against these cells with IC50 values of 1-2 nM. Its growth inhibitory activity was completely reversed by the addition of thymidine, confirming that TS is its sole target in these cells. Remar kably, TS protein levels rose by 10-40-fold following treatment with I CI D1694, depending on cell type, while TS mRNA levels remained consta nt. The mechanism appears to be a release of ''detainment'' of TS tran slation, since addition of cycloheximide, a translational inhibitor, b locked the TS protein levels from rising. But coadministration of 5,6- dichlorobenzimidazole, a transcriptional inhibitor, did not overcome p rotein accumulation, nor did thymidine which overcomes growth inhibiti on by ICI D1694. 5,10-Methylenetetrahydrofolate (via folinic acid), ho wever, did block the effects of ICI D1694, showing that the drug has i ts effect upon both detainment and enzyme inhibition by binding to the folate substrate site of TS. In addition, in the presence of ICI D169 4, TS protein was no longer cell cycle-regulated as evident by its con stitutive expression in synchronized cells. This accumulation and cons titutive expression of TS induced by D1694 should increase drug resist ance under a clinical setting. We suggest that an ideal inhibitor of T S would target the TS allosteric site that binds to TS mRNA, responsib le for specific translation of the protein, thereby complimenting inac tivation of the enzyme.