5-fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyridylate binding to thymidylate synthase by dihydropteroylpolyglutamates.

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Abstract
Ternary complex formation of thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyl-transferase, EC 2.1.1.45), 5-fluorodeoxyuridylate (FdUMP) and poly(.gamma.-glutamyl) conjugates of pteroate and methotrexate (MTX) was examined as a basis for the sequence-dependent synergism of the 5-fluorouracil-MTX combination in inhibiting viability of L1210 murine leukemia cells. A 1.4-log (25-fold) increase in the inhibition of soft agar colony formation was observed when MTX preceded 5-fluorouracil, as compared to the reverse sequence. L1210 cells converted 39% of the total intracellular MTX into MTX poly(.gamma.-glutamate)s within 4 h of exposure to 1 .mu.M MTX. MTX and MTX(.gamma.-glutamate) formed reversible ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8-dihydropteroylpentaglutamate and l-5,10-methylenetetrahydropteroylpentaglutamate stoichiometric binding of FdUMP to 2 sites on thymidylate synthase was observed. The dissociation constants for FdUMP in the ternary complexes formed in the presence of MTX, MTX(.gamma.-glutamate), 7,8-dihydropteroylpentaglutamate and l-5,10-methylenetetrahydropteroylpentaglutamate were estimated to be 370, 27, < 10 and < 10 nM respectively, by equilibrium dialysis. The sequence-dependent effect of MTX plus 5-fluorouracil on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and consequently a trapping of intracellular folates as dihydropteroylpolyglutamates, which increase the extent of FdUMP binding to thymidylate synthase.

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