What is the mechanism of action of Methotrexate (dihydrofolate reductase inhibitor)?

Mechanism of Action of Methotrexate

Methotrexate primarily works as a competitive inhibitor of dihydrofolate reductase (DHFR), decreasing folate cofactors required for nucleic acid synthesis, while also inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC), which increases endogenous adenosine and provides anti-inflammatory effects. 1

Primary Mechanisms

DHFR Inhibition

• Acts as a competitive inhibitor of dihydrofolate reductase (DHFR)
• Prevents reduction of dihydrofolates to tetrahydrofolates
• Tetrahydrofolates are essential carriers of one-carbon groups in purine nucleotide and thymidylate synthesis
• This inhibition interferes with DNA synthesis, repair, and cellular replication 2

Polyglutamation and Intracellular Retention

• Within cells, methotrexate undergoes polyglutamation (activation) under the enzymatic control of folylpolyglutamate synthetase (FPGS)
• Polyglutamated forms have increased intracellular retention
• Glutamate residues can be removed by γ-glutamyl hydrolase (GGH), creating a dynamic process 1

ATIC Inhibition and Adenosine Release

• In its polyglutamated form, methotrexate inhibits aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC)
• This inhibition results in increased levels of endogenous adenosine
• Adenosine acts on adenosine receptors (ADORA A1 and 2a) producing anti-inflammatory effects
• This mechanism is particularly important in rheumatoid arthritis treatment 1, 3

Cell Transport Mechanisms

• Methotrexate enters cells via the solute carrier family 19, member 1 (SLC19A1) transporter
• It can be actively transported out of cells by ATP-binding cassette transporters (ABCC1-4, ABCG2)
• These transport mechanisms affect drug efficacy and resistance patterns 1

Disease-Specific Mechanisms

In Inflammatory Conditions (Psoriasis, Rheumatoid Arthritis)

• Low-dose methotrexate (<25 mg per week) primarily decreases proliferation of lymphoid cells
• This direct immunosuppressive effect is the main mechanism for improvement in psoriatic disease 1
• In rheumatoid arthritis, the adenosine-mediated anti-inflammatory effect appears most significant 3
• Effects on immune function include decreased proliferation of stimulated mononuclear cells 2

In Cancer Treatment

• Higher doses target rapidly dividing malignant cells
• Inhibition of DNA synthesis in actively proliferating tissues
• High-dose methotrexate may overcome resistance caused by:
  • Impaired active transport
  • Decreased affinity of DHFR for methotrexate
  • Increased levels of DHFR from gene amplification
  • Decreased polyglutamation 2

Secondary Mechanisms

• Inhibition of thymidylate synthase (TYMS), affecting pyrimidine biosynthesis
• Influence on 5,10-methylenetetrahydrofolate reductase (MTHFR), affecting homocysteine metabolism
• Potential inhibition of histone deacetylases, suggesting epigenetic effects 4
• Effects on glyoxalase and antioxidant systems 4

Clinical Implications

• Understanding these mechanisms explains why folic acid supplementation reduces side effects without significantly compromising efficacy
• Resistance mechanisms include decreased polyglutamation and DHFR upregulation 5
• The complex mechanism explains methotrexate's diverse applications in cancer, autoimmune disorders, and inflammatory conditions

Methotrexate's multifaceted mechanism of action, particularly its dual effects on DHFR and ATIC pathways, explains its versatility as both an antineoplastic and immunosuppressive agent, with different mechanisms predominating at different dosages and in different disease states.