Methotrexate (MTX), one of the earliest
cancer chemotherapy agents, continues to be used extensively in the treatment of
leukemia and a variety of other
tumors. The efficacy of this
drug results from its facile uptake by cells, rapid polyglutamylation and virtually stoichiometric inhibition of
dihydrofolate reductase (DHFR), a key
enzyme in cell replication. From the work of a multitude of biochemists, molecular biologists, organic chemists and pharmacologists, much is known about the mode of action of MTX and the mechanisms by which
tumors exhibit inherent or acquired resistance to this
drug. MTX enters cells primarily by a carrier-mediated active transport system whose principal substrate is
5-methyltetrahydrofolate, and additional
glutamates are added to the gamma-position of the parent
glutamate moiety. The tight binding of MTX to DHFR is defined from NMR and X-ray crystallographic studies of the
enzyme and its
drug or substrate complexes, supplemented by site-directed mutagenesis to confirm specific interactions. Resistance to the
drug, encountered in cell culture model systems or in
cancer patients, can result from an increased level of DHFR (due to gene amplification), mutant DHFR with reduced affinity for MTX, or decreased uptake or polyglutamylation of the
drug. Although DHFR is an extremely well-studied
enzyme, there is still some uncertainty about its kinetics, mechanism for reduction of
folate, multiple forms, and activation by a diverse group of agents.
Prodrug forms of MTX, e.g., MTX alpha-
phenylalanine, which can be activated by
carboxypeptidase A-
monoclonal antibody conjugates, offer promise for improved efficacy of the
drug by selective targeting to
tumors. The large body of information summarized above has aided in the development of other
folate antagonists, provides a paradigm for assessing the status of other
cancer chemotherapeutic agents in current use, and offers a platform from which to speculate about the future of the field.