Effect of direct suppression of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site on the interconversion of tetrahydrofolate cofactors to dihydrofolate by antifolates. Influence of degree of dihydrofolate reductase inhibition.
An important unresolved issue in antifolate pharmacology is the basis for the observation that the major portion of cellular tetrahydrofolate cofactors is preserved after dihydrofolate reductase activity is abolished by antifolates despite the fact that tetrahydrofolate cofactor-dependent purine and pyrimidine biosynthesis ceases. This has been attributed to feedback inhibition of thymidylate synthase by dihydrofolate polyglutamates that accumulate in the presence of antifolates. This report combines network thermodynamic modeling and experimental observations to evaluate the effects of direct inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site with a potent lipophilic quinazoline antifolate PD130883 on folate oxidation in cells. Computer simulations predict and the data indicate that marked PD130883 suppression of thymidylate synthase only slows the rate but not the extent of tetrahydrofolate cofactor interconversion to dihydrofolate upon complete suppression of dihydrofolate reductase with trimetrexate. These observations are consistent with earlier studies from this laboratory with fluorodeoxyuridine inhibition at the deoxyuridylate binding site. Hence, the much weaker inhibition by dihydrofolate polyglutamates at the level of thymidylate synthase cannot account for the apparent preservation of tetrahydrofolate cofactor pools in cells and has virtually no pharmacologic significance under conditions in which antifolates completely suppress dihydrofolate reductase. The extent of interconversion of tetrahydrofolate cofactors to dihydrofolate is strongly influenced by residual dihydrofolate reductase catalytic activity. Exposure of cells to 0.1 microM trimetrexate results in only approximately 60% of maximum dihydrofolate levels achieved when dihydrofolate reductase activity is abolished. Network thermodynamic simulations predict, and experiments verify, that inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate site by PD130883, when dihydrofolate reductase is only partially suppressed (approximately 85%) with 0.1 microM trimetrexate, substantially decreases (31-47%) the net level of interconversion of tetrahydrofolate cofactors to dihydrofolate. Further computer simulations predict that under conditions in which residual dihydrofolate reductase activity persists within the cells (more than about 5%), feedback inhibitory effects of dihydrofolate polyglutamates as well as other weak inhibitors of thymidylate synthase can significantly limit the extent of net interconversion of tetrahydrofolate cofactors to dihydrofolate and produce an apparent "compartmentation phenomenon" in which tetrahydrofolate cofactor pools are preserved within the cell in the presence of antifolates. Residual dihydrofolate reductase activity cannot, however, account for the partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure to high trimetrexate or methotrexate levels.(ABSTRACT TRUNCATED AT 400 WORDS)
Seither
R L
RL
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298.
Trent
D F
DF
Mikulecky
D C
DC
Rape
T J
TJ
Goldman
I D
ID
D000818:Animals; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D066298:In Vitro Techniques; D007939:Leukemia L1210; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D011799:Quinazolines / Q000494:pharmacology; D052999:Quinazolinones; D013763:Tetrahydrofolates / Q000378:metabolism*; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D016597:Trimetrexate; D014407:Tumor Cells, Cultured
D005493:Folic Acid Antagonists; D011799:Quinazolines; D052999:Quinazolinones; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C067627:PD 130883; D013940:Thymidylate Synthase; D016597:Trimetrexate; D008727:Methotrexate