Biochemical and
biological studies have been carried out with
2-desamino-2-methylaminopterin (dmAMT), which inhibits
tumor cell growth in culture but is only a weak inhibitor of
dihydrofolate reductase (DHFR). Since it was possible that the species responsible for growth inhibition are polyglutamylated metabolites, the di-, tri-, and tetraglutamates of dmAMT were synthesized and tested as inhibitors of purified recombinant human DHFR, murine
L1210 leukemia thymidylate synthase (TS), chicken liver
glycinamide ribonucleotide formyltransferase (GARFT), and murine
L1210 leukemia aminoimidazolecarboxamide
ribonucleotide formyltransferase (AICARFT). The compounds with three and four gamma-glutamyl residues were found to bind two orders of magnitude better than dmAMT itself to
DHFR, TS, and AICARFT, with 50% inhibitory concentration values in the 200 to 300 nM range against all three
enzymes. In contrast, at a concentration of 10 microM, dmAMT polyglutamates had no appreciable effect on GARFT activity. These findings support the hypothesis that dmAMT requires intracellular polyglutamylation for activity and indicate that replacement of the 2-amino group by 2-methyl is as acceptable a structural modification in
antifolates targeted against DHFR as it is in
antifolates targeted against TS. In growth assays against
methotrexate (MTX)-sensitive H35 rat
hepatoma cells and MTX-resistant H35 sublines with a transport defect, dmAMT was highly cross-resistant with MTX, but not with the TS inhibitors N10-propargyl-5,8-dideazafolic
acid and N-(5-[N-(3,4-dihydro-2-methyl-4-ox-oquinazolin-6-yl)-N- methylamino]thenoyl)-
L-glutamic acid, implicating DHFR rather than TS as the principal target for dmAMT polyglutamates in intact cells. On the other hand, an H35 subline resistant to 2'-deoxy-5-fluorouridine by virtue of increased TS activity was highly cross-resistant to N10-propargyl-5,8-dideazafolic
acid and not cross-resistant to MTX, but showed partial cross-resistance to dmAMT. Both
thymidine and
hypoxanthine were required to protect H35 cells treated with concentrations of dmAMT and MTX that inhibited growth by greater than 90% relative to unprotected controls. In contrast, N10-propargyl-5,8-dideazafolic
acid and N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)-N-methylamino] thenoyl)-
L-glutamic acid required only
thymidine for protection. Like MTX, therefore, dmAMT appears to inhibit
purine as well as
pyrimidine de novo synthesis, and its effect on cell growth probably reflects the ability of dmAMT polyglutamates to not only block
dihydrofolate reduction but also interfere with other steps of
folate metabolism, either directly or indirectly via alteration of reduced
folate pools.(ABSTRACT TRUNCATED AT 400 WORDS)