BIOCHEMICAL EFFECTS OF FOLATE-BASED INHIBITORS OF THYMIDYLATE SYNTHASE IN MGH-U1 CELLS

The TS-inhibitory effects induced by a 24-h exposure to the folate-bas ed TS inhibitors CB3717, C2-desamino analogs of CB3717 including D1694 , and BW1843U89 were quantitated using the MGH-U1 human bladder carcin oma. The effects of D1694 on the time course of TS inhibition and on i ntracellular deoxyuridine monophosphate (dUMP) accumulation and deoxyu ridine (dUrd) production were evaluated. D1694 and BW1843U89 were the most active TS inhibitors with IC50 values of 2.4 and 0.5 nM, respecti vely. The C2-desamino C2-methyl dideazafolates were 27-292 times more potent than the parent CB3717 as TS inhibitors. A methyl group at the C2 position of CB3717 had the most dramatic effect, whereas a thiazole substitution for a benzyl added a small benefit and N10 substitution had a limited impact on TS-inhibitory potency and clonogenic survival. There was a significant correlation between the IC50 values for TS in hibition and those for cytotoxic potency obtained for these drugs. LV and thymidine protected cells from these folate-based TS inhibitors. I ntracellular dUMP levels following 24 h D1694 (IC50) exposure increase d 7-fold. Levels of dUrd effluxing into the media increased up to 4.5 mu M following a 24-h exposure to D1694 (IC90). We conclude that (a) C 2-desamino C2-methyl dideazafolates are potent TS inhibitors, (b) TS i nhibition requires prolonged exposure with these folate TS inhibitors, (c) survival is correlated with inhibition of TS for the folate-based TS inhibitors and (d) the biochemical consequences of TS inhibition i nclude increased dUMP and dUrd levels.