Preclinical cellular pharmacology of LY231514 (MTA): a comparison with methotrexate, LY309887 and raltitrexed for their effects on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells

Abstract

Summary LY231514 (N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-benzoyl]-L-glutamic acid) is a new folatebased antimetabolite currently in broad phase II clinical evaluation. Previous in vitro studies (C. Shih et al, Cancer Res 57: 1116-1123 have suggested that LY231514 could be a multitargeted antifolate (MTA) capable of inhibiting thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study compared LY231514 with methotrexate, raltitrexed and a glycinamide ribonucleotide formyltransferase inhibitor, LY309887, at 300, 100, 30 and 100 nM, respectively, for their effects on intracellular folate and at 100, 66, 20 and 30 nM respectively, for their effects on nucleoside triphosphate pools in CCRF-CEM cells.Methotrexate induced an accumulation of dihydrofolate species, together with a rapid depletion of ATP, GTP and all of the deoxynucleoside triphosphates. LY309887 caused an accumulation of 1 0-formyltetrahydrofolate, a rapid loss of ATP, GTP and dATP, but a slower loss in dCTP, dTTP and dGTP. Both LY231514 and raltitrexed had minimal effects on folate pools. In contrast, they caused rapid depletion of dTTP, dCTP and dGTP, but induced an accumulation of dATP at different rates, with raltitrexed doing so about 2.5 times faster. Most of the observed metabolic changes could be understood on the basis of current knowledge of folate and nucleotide metabolism. We concluded that LY231514 was distinct from methotrexate, LY309887 and raltitrexed based on their metabolic effects in CCRF-CEM cells, and that in this cell line the inhibitory effects of LY231514 were exerted primarily against the thymidylate cycle and secondarily against de novo purine biosynthesis.

Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications

“…When pemetrexed inhibits its target enzymes, there is no redistribution of folates within cells; the levels of folate substrates are not changed (90). In addition, inhibition of thymidylate synthase or of GARFT directly decreases the growth rate of tumor cells, as both seem to be limiting to DNA synthesis; that is, inhibition of 50% of either of these enzymes would decrease growth rates of tumors by 50%.…”

“…Hence, the accumulation of dUMP substantially, and sometimes completely, terminates inhibition of thymidylate synthase by FdUMP (102 -104). The same accumulation of dUMP occurs after exposure of tumor cells to pemetrexed (90), but because the excess dUMP binds to a different site on the enzyme (105), it does not inhibit pemetrexed binding and, in fact, may enhance the binding of pemetrexed polyglutamates to thymidylate synthase.…”

“…(b) Pemetrexed increases expression of the hENT1 nucleoside transporter that mediates gemcitabine influx (130). (c) Pemetrexed inhibition of thymidylate synthase in CCRF-CEM cells produces a rapid decrease in intracellular dCTP as well as dTTP and dGTP (90). This should increase the incorporation of gemcitabine, a cytidine analogue, into DNA.…”

Schedule-dependent synergism and antagonism between methotrexate and cytarabine against human leukemia cell lines in vitro

“…MTX, an inhibitor of dihydrofolate reductase, simultaneously reduced deoxynucleotide pools. 40 In cells treated with MTX followed by cytarabine, depletion of dCTP by MTX may have increased deoxycytidine kinase activity. The sequential treatment with MTX then cytarabine has been observed to augment the generation of ara-CTP and the formation of DNA strand breaks.…”

The effect of a novel transition state inhibitor of methylthioadenosine phosphorylase on pemetrexed activity

“…It is possible that the greater effect of MTAP suppression on methotrexate activity is due to the fact that this enzyme salvages not only adenine but methionine as well from methylthioadenosine. Unlike pemetrexed, which acts by direct suppression of tetrahydrofolate-dependent enzymes and does not deplete cellular folates (20), methotrexate depletes cells of tetrahydrofolate cofactors, including 5-methyltetrahydrofolate which is required for methionine synthesis (20,21). Hence, when the MTAP inhibitor is coadministered with methotrexate, there are two factors that limit the availability of methionine.…”