Predictions of a network thermodynamics computer model relating to the mechanism of methotrexate rescue by 5-formyltetrahydrofolate and to the importance of inhibition of thymidylate synthase by methotrexate-polyglutamates.

Abstract

Computer modeling has been a valuable tool for clarifying the mechanism of action of antifolates. Some consequences of folyl and antifolyl polyglutamate synthesis can be addressed by adaptation of a network thermodynamic computer model of methotrexate action. Reversal or prevention of methotrexate cytotoxicity by 5-formyltetrahydrofolate has widely been assumed to occur through the delivery of reduced folate in substrate amounts for thymidylate synthesis, by-passing the effects of methotrexate at dihydrofolate reductase. This mechanism is inconsistent with experimental data which shows that "rescue" is a competitive phenomenon and that the transport process is incapable of delivering reduced folate at an adequate rate. Computer modeling studies are presented which predict that expansion of the total folate pool as folylpolyglutamates with "rescue" would reduce the inhibitory effect of MTX on thymidylate synthesis. Dihydrofolate polyglutamates could then accumulate to the high level needed to displace methotrexate from the small fraction of sites on dihydrofolate reductase that are sufficient to sustain tetrahydrofolate synthesis. Experimental studies with Ehrlich ascites tumor cells support this prediction. It is likely that a critical step in the protection of normal host tissues in high dose-rescue treatment regimens is the conversion of exogenously supplied 5-formyltetrahydrofolate to polyglutamyl derivatives and accumulation of total intracellular folate to higher than normal levels. Other computer simulations are presented which examine the potential significance of direct inhibition of thymidylate synthase by polyglutamyl forms of methotrexate. The model predicts that in cells with biochemical properties similar to methotrexate sensitive L1210 cells, inhibition of dihydrofolate reductase would still be the predominant site of action unless the thymidylate synthase Ki for a methotrexate polyglutamate is below about 0.1 microM. However, in methotrexate-resistant cells with elevated dihydrofolate reductase but normal membrane transport and polyglutamylation, thymidylate synthase may be the more important target enzyme.