Biochemical and biological studies on 2-desamino-2-methylaminopterin, an antifolate the polyglutamates of which are more potent than the monoglutamate against three key enzymes of folate metabolism.
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)
Rosowsky
A
A
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Galivan
J
J
Beardsley
G P
GP
Bader
H
H
O'Connor
B M
BM
Russello
O
O
Moroson
B A
BA
DeYarman
M T
MT
Kerwar
S S
SS
Freisheim
J H
JH
D000217:Acyltransferases / Q000037:antagonists & inhibitors*; D000630:Aminopterin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects; D004334:Drug Administration Schedule; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005493:Folic Acid Antagonists; D019877:Hydroxymethyl and Formyl Transferases; D007939:Leukemia L1210 / Q000188:drug therapy; D008297:Male; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D051026:Phosphoribosylglycinamide Formyltransferase; D011799:Quinazolines / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; D011799:Quinazolines; D013876:Thiophenes; C058706:2-desamino-2-methylaminopterin; C031662:CB 3717; D005492:Folic Acid; D013940:Thymidylate Synthase; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000217:Acyltransferases; C068874:raltitrexed; D000630:Aminopterin; D008727:Methotrexate