Methotrexate: Mechanism of Action and Mechanisms of Resistance

What is Methotrexate?

Folic acid analogs comprise a class of antineoplastic agents of which methotrexate has gained the most widespread clinical use. These agents were the first to produce impressive remissions in acute leukemia and cures in choriocarcinoma in women.

Reduced folates (tetrahydrofolates) are the biologically active form of folates required as cosubstrates in one-carbon transfer reactions. Included in these reactions are several important enzymatic steps in the de novo synthesis of purines and pyrimidines.

Methotrexate (NSC-740), N-(4- {(2,4-diamino-6-pteridinyl)methyl]methylamino}benzoyl)-l-glutamic acid, C20H22N8O5, Mr 454.46, is a 2,4-diamino, N10-methyl analog of folic acid that is capable of inhibiting certain folate-requiring reactions.

Mechanism of Action and Mechanisms of Resistance

Methotrexate can inhibit dihydrofolate reductase (DHFR, Ki = 10−11 M), a key enzyme for the maintenance of biologically active intracellular reduced folate pools. The folate-requiring reactions utilize reduced folates, and all reactions except that catalyzed by thymidylate synthase maintain folates in a reduced state during carbon transfer. Thymidylate synthase, which catalyzes the methylation of deoxyuridylate to thymidylate (required for DNA synthesis), requires the transfer of a carbon group from the folate cofactor N5−10- methylene tetrahydrofolate with resultant oxidation of the folate to dihydrofolic acid. Oxidized folates must be reduced to the tetrahydro form by DHFR to be useful for intracellular metabolism. Inhibition of DHFR following methotrexate exposure ultimately leads to depletion of intracellular reduced folates. Cessation of first thymidylate and then purine nucleotide synthesis occurs as an indirect effect of methotrexate on reduced-folate levels. Methotrexate metabolites (polyglutamates) may also have direct inhibitory effects on folaterequiring enzymes, e.g., thymidylate synthase, and these effects may be important in inducing cytotoxicity.