Biochemistry of Methotrexate: Teaching an Old Drug New Tricks

Abstract

Chemotherapy, despite its shortcomings, continues to be one of the major modalities in the treatment of cancer. Most of the drugs in current use were discovered empirically, i.e., through screening programs. In recent years, however, a more rational approach for drug development has emerged, based upon a four-stage strategy: a) the first stage involves the identification of opportune targets. These are usually biochemical parameters that are closely related to cell replication, e.g., DNA (or RNA) or enzymes responsible for the synthesis of the nucleic acids and their nucleotide precursors. Illustrative of this approach is the work of Weber (1), who has developed a comprehensive program for identifying enzymes that are rate-limiting in the synthesis of purine or pyrimidine nucleotides and are present in increased amounts in tumor cells; b) Sites on the target for drug interaction are defined. For DNA, these are nucleotide sequences with enhanced susceptibility to intercalating or covalent-binding agents; specificity of these sites, however, is usually not stringent. Substrate-binding sites on enzymes offer better possibilities for specificity. Some insight into the dimensions of these sites and the amino acids that interact with ligands can be obtained by comparison of binding constants for substrates and inhibitors, by chemical modification with group-specific reagents, and by NMR measurements. Accurate three-dimensional pictures of the sites, however, require X-ray diffraction analyses of the crystalline enzymes. This latter technique has been employed in the elegant studies of Kraut and Matthews [reviewed in (2)] for visualizing the Methotrexate (MTX) binding site on dihydrofolate reductase; c) Information about the drug-binding sites, along with computer graphic modeling, then provides guidance for the chemical synthesis of compounds tailored to fit with a high degree of specificity and affinity. Montgomery, Robins, Hitchings and Elion are among the leaders in this field; d) Finally, promising compounds that interact satisfactorily with their targets are examined for other criteria necessary for clinical acceptance, viz., facile uptake by cells (via active transport or diffusion),cytotoxicity (with selectivity toward tumor cells), distribution to tumorbearing sites in the body, and favorable pharmacologic characteristics such as resistance to metabolism and/or excretion.