Abstract
5,6,7,8-Tetrahydro-N5,N10-carbonylfolic acid (LY354899) has been demonstrated to inhibit the dehydrogenase activity of C1-tetrahydrofolate synthase. This compound was only moderately antiproliferative toward CCRF-CEM lymphocytic leukemia cells in culture, but induced apoptosis after long incubation times. Slightly greater potency was observed in CEM cells adapted to grow in low folate media. Cell cycle alterations induced by LY354899 were unique relative to antifolates that inhibit either the purine or thymidine de novo biosynthetic pathways. Based on the observed changes in DNA content, we hypothesized that inhibition of the dehydrogenase resulted in two temporally distinct events: the first was a purineless-like effect and the second was a thymineless-like effect that resulted in apoptosis. To test this hypothesis, we combined LY354899 with the purine salvage metabolite, hypoxanthine. This combination resulted in an earlier and more dramatic apoptotic response, indicating that the thymineless effect had been potentiated. Biochemical analysis of ribo- and deoxyribonucleoside triphosphates confirmed that inhibition of the dehydrogenase activity initially resulted in decreased pools of deoxypurines and deoxypyrimidines, followed 16 hr later by an increase in deoxyadenosine triphosphate (dATP) and a further decrease in deoxythymidine triphosphate (dTTP). These studies demonstrate that the inhibition of the dehydrogenase activity of C1-tetrahydrofolate synthase may represent a viable target for the development of novel antifolates. The results are discussed in terms of deoxypurine and deoxypyrimidine biosynthesis.
Footnotes
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Send reprint requests to: Dr. John L. Tonkinson, Schleicher & Schuell, Inc., 10 Optical Avenue, Keene, NH 03431.
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↵1 This work was supported by Eli Lilly and Co., Indianapolis, IN 46285.
- Abbreviations:
- LY354899
- 5,6,7,8-Tetrahydro-N5,N10-carbonylfolic acid
- DHFR
- dihydrofolate reductase
- TS
- thymidylate synthase
- GARFT
- glycinamide ribonucleotide formyl transferase
- FPGS
- folylpolyglutamate synthetase
- THF
- tetrahydrofolate
- rh
- recombinant human
- ZD1694
- N-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ethyl)-N-methylamino]-2-thenoyl)-l-glutamic acid
- LY231514
- N-[4[2-(-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-l-glutamate sodium salt
- MTA
- multitargeted antifolate
- 10-formylTHF
- 10-formyltetrahydrofolate
- 5
- 10-methenylTHF, 5,10-methenyltetrahydrofolate
- 5
- 10-methyleneTHF, 5,10-methylenetetrahydrofolate
- LY309887
- 6R-2′, 5′-thienyldideazatetra hydrofolic acid
- HPLC
- high-performance liquid chromatography
- 7
- 8-DHF, 7,8-dihydrofolate
- PI
- propidium iodide
- Received November 5, 1997.
- Accepted May 14, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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