Methotrexate Mechanism of Action: Enzyme Inhibition and Nucleotide Depletion
Methotrexate primarily inhibits dihydrofolate reductase (DHFR), which directly depletes tetrahydrofolate (THF) cofactors required for synthesis of thymidine nucleotides, specifically deoxythymidylate (dTMP). 1
Primary Enzymatic Target
- Methotrexate tightly binds to and inhibits dihydrofolate reductase (DHFR), blocking the reduction of dihydrofolate to tetrahydrofolate, the active form of folic acid 1, 2
- The drug-enzyme complex is slowly dissociable, meaning methotrexate remains bound to DHFR for extended periods 3
- This inhibition prevents the regeneration of tetrahydrofolate cofactors necessary for one-carbon transfer reactions in nucleotide synthesis 4
Specific Nucleotide Product Depleted
- The primary nucleotide product that becomes depleted is deoxythymidylate (dTMP), which is essential for DNA synthesis 4
- Methotrexate's polyglutamated form also directly inhibits thymidylate synthase (TYMS), the enzyme that converts deoxyuridylate (dUMP) to dTMP in the de novo pyrimidine biosynthetic pathway 4
- This dual mechanism—blocking both DHFR and TYMS—creates a synergistic depletion of dTMP, severely impairing DNA synthesis and repair 5
Secondary Enzymatic Effects
Beyond DHFR inhibition, methotrexate affects multiple folate-dependent enzymes:
- Aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC) is inhibited by polyglutamated methotrexate, leading to adenosine accumulation and anti-inflammatory effects 4
- 5,10-methylenetetrahydrofolate reductase (MTHFR) activity is influenced, affecting homocysteine remethylation 4
- These secondary effects contribute to methotrexate's immunosuppressive properties, particularly relevant in rheumatoid arthritis and psoriasis treatment 4
Clinical Implications of Mechanism
- The depletion of dTMP and purine nucleotides preferentially affects rapidly proliferating cells, including malignant cells, bone marrow, and mucosal tissues 1
- Folic acid supplementation (at least 5 mg/week) is recommended to mitigate toxicity without reducing efficacy, as it can partially restore folate cofactors while methotrexate continues to inhibit rapidly dividing cells 4
- Resistance to methotrexate develops through elevated DHFR activity, DHFR gene amplification, defective drug transport, or decreased polyglutamation 1, 2
Polyglutamation and Enhanced Activity
- Intracellularly, methotrexate undergoes polyglutamation (addition of glutamate residues) under enzymatic control of folylpolyglutamate synthetase (FPGS) 4
- Polyglutamated forms (MTX-Glu4 and MTX-Glu5) show prolonged intracellular retention and less dissociable binding to DHFR, with dissociation half-lives exceeding 100 minutes compared to 12 minutes for the parent compound 3
- These longer-chain polyglutamates are more efficient DHFR inhibitors and extend the duration of drug action in target cells 3