What is the mechanism of action of methotrexate?

• A. Inhibition of topoisomerase II.
• B. Inhibition of dihydrofolate reductase, depleting thymidine and purine synthesis.
• C. Intercalation into DNA to prevent transcription.
• D. Alkylation of DNA causing cross-links.

Answer: (B)

Methotrexate works by blocking folate metabolism. It acts as a folate analog that inhibits dihydrofolate reductase (DHFR), preventing the regeneration of tetrahydrofolate. Without tetrahydrofolate, cells can’t efficiently produce thymidylate and purines, which are the building blocks of DNA. The result is a depletion of nucleotides needed for DNA synthesis, especially in rapidly dividing cells. Clinically, this effect is enhanced by polyglutamation inside cells, which traps the drug and prolongs its action, and can be mitigated in normal tissues with leucovorin rescue to bypass the DHFR block. This mechanism is distinct from actions like intercalating into DNA, alkylating DNA, or inhibiting topoisomerases, which are not how methotrexate works.