Methotrexate mechanism of action and resistance

Methotrexate is an inhibitor of dihydrofolate reductase. It undergoes reduction to FH4 via a reaction catalyzed by intracellular nicotinamide-adenine dinucleotide phosphate–dependent DHFR. MTX enters the cell by active-transport processes that normally mediate the entry of N5-methyl-FH4. At high concentrations, the drug can also diffuse into the cell. MTX has an unusually strong affinity for DHFR and effectively inhibits the enzyme. Like tetra hydrofolate itself, MTX becomes polyglutamated within the cell, a process that favors intracellular retention of the compound due to increased negative charge. MTX polyglutamates also potently inhibit DHFR. his inhibition deprives the cell of folate coenzymes and leads to decreased production of compounds that depend on these coenzymes for their biosynthesis. Although these molecules include the nucleotides adenine, guanine, and thymidine and the amino acids methionine and serine, depletion of thymidine is the most prominent effect. This leads to depressed DNA, RNA, and protein synthesis and, ultimately, to cell death The inhibition of DHFR can only be reversed by a thousandfold excess of the natural substrate, dihydrofolate or by administration of leucovorin, which bypasses the blocked enzyme and replenishes the folate pool. [Note: Leucovorin, or folinic acid, is the N5-formyl group–carrying form of FH4.] MTX is specific for the S phase of the cell cycle.