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Methotrexate is an antimetabolite. A broad definition of antimetabolites would include compounds with structural similarity to precursors of purines or pyrimidines, or compounds that interfere with purine or pyrimidine synthesis.

Antimetabolites can cause DNA damage indirectly,

  • through misincorporation into DNA,
  • abnormal timing or progression through DNA synthesis,
  • or altered function of pyrimidine and purine biosynthetic enzymes.

 They tend to convey greatest toxicity to cells in S-phase (figure-1) and the degree of toxicity increases with duration of exposure.

 Action of methotrexate

Figure-1- Cell cycle summary and the action of methotrexate that acts in the S phase of the cell cycle

Common toxic manifestations include

  • stomatitis,
  • diarrhea, and
  • myelosuppression.

Mechanism of Action

Methotrexate inhibits Dihydro folate reductase, which regenerates reduced folates from the oxidized folates (figure-2 and 4)  produced when thymidine monophosphate is formed from deoxy uridine monophosphate (Figure-4) Without reduced folates, cells die a “thymine-less” death.

 Conversion of dihydro folate to tetra hydro folate

Figure-2- Methotrexate is a competitive inhibitor of Dihydrofolate reductase

Overview of role of folic acid in DNA synthesis

Folate is an essential vitamin, found in green leafy vegetables. It is essential for many biochemical processes in the body, including DNA synthesis and red blood cell synthesis

Tetrahydrofolate (THF) derived from the vitamin folic acid is the major source of 1-carbon units, used in the biosynthesis of many important biological molecules. This cofactor is a carrier of activated 1-carbon units at various oxidation levels (methyl, Methylene, Formyl, Formimino, and methenyl). These compounds can be interconverted as required by the cellular process. (See figure-3)

 Forms of tetrahydro folate

 Figure-3- forms of Tetrahydrofolate (THF).

Role of folic acid in the synthesis of pyrimidine nucleotides

All cells, especially rapidly growing cells, must synthesize Thymidylate (dTMP) for DNA synthesis. The difference between (T) and (U) is one methyl group at the carbon-5 position. Thymidylate is synthesized by the Methylation of uridylate (dUMP) in a reaction catalyzed by the enzyme Thymidylate synthase. This reaction requires a methyl donor and a source of reducing equivalents, which are both provided by N5, N10-methylene THF)-figure-3 and 4. For this reaction to continue, the regeneration of THF from Dihydro folate (DHF) must occur.

Many anti-cancer drugs act directly to inhibit thymidylate synthase, or indirectly, by inhibiting DHFR.

The enzyme Dihydro folate reductase (DHFR) is a target of the anticancer drugs aminopterin and Methotrexate.

These drugs are analogs of DHF and act as competitive inhibitors of DHFR. Inhibition of this enzyme prevents the regeneration of THF and blocks dTMP synthesis because of the lack of the methyl donor required for the reaction of thymidylate synthase.

The class of molecules used to inhibit thymidylate synthase are called the suicide substrates because they irreversibly inhibit the enzyme. Molecules of this class include 5-fluorouracil and 5-fluorodeoxyuridine. Both are converted within cells to 5-fluorodeoxyuridylate, FdUMP. It is this drug metabolite that inhibits thymidylate synthase.

 Thymidylate synthase


 Figure- 4-Thymidylate synthesized by the Methylation of uridylate (dUMP) in a reaction catalyzed by the enzyme thymidylate synthase. Tetrahydrofolate (THF) is regenerated from the dihydrofolate (DHF) product of the thymidylate synthase reaction by the action of dihydrofolate reductase (DHFR), an enzyme that requires NADPH. Methotrexate by  inhibiting DHFR impairs the regeneration of THF which is the metabolically active form of folic acid. Folic acid deficiency and impaired synthesis of dTMP and thereby decreased DNA synthesis is the outcome of methotrexate therapy.

Cells that are unable to regenerate THF suffer defective DNA synthesis and eventual death. For this reason, as well as the fact that dTTP is utilized only in DNA, it is therapeutically possible to target rapidly proliferating cells over non-proliferating cells through the inhibition of thymidylate synthase.

Role of folic acid in purine synthesis

THF is also required as a donor of two carbon atoms in the synthesis of the purine ring structure required for adenine and guanine. The carbon atoms donated by THF are indicated in Figure -5.Therefore, a lack of THF blocks the synthesis of the purine ring structure because of the lack of the ability of the cell to synthesize N10-formyl-THF.

 Purine ring

Figure-5- showing origin of the atoms of the purine base.

In summary, DNA synthesis requires synthesis of dTMP and the purines adenine and guanine. THF, derived from the vitamin folic acid, is required for the biosynthesis of these nucleotides.

Adverse effects of methotrexate

Methotrexate blocks the cell’s ability to regenerate THF, leading to inhibition of these biosynthetic pathways. The lack of nucleotides prevents DNA synthesis, and these cancer cells cannot divide without DNA synthesis.

Unfortunately, the effects of Methotrexate are nonspecific and other rapidly dividing cells such as epithelial cells in the oral cavity, intestine, skin, and blood cells are also inhibited. This leads to the side effects associated with methotrexate (and other cancer chemotherapy drugs) such as mouth sores, low white blood cell counts, macrocytic anemia, gastrointestinal discomfort, hair loss, skin rashes, and itching. Less frequent adverse effects include reversible increases in transaminases and hypersensitivity-like pulmonary syndrome. Chronic low-dose methotrexate can cause hepatic fibrosis.

Hematologic side effects  include myelosuppression which is one of the primary toxic effects of methotrexate. Methotrexate suppressed hematopoiesis has been reported to have caused anemia, plastic anemia, pancytopenia, leukopenia, neutropenia, thrombocytopenia, lymphadenopathy, and lymphoproliferative disorders including reversible hypogammaglobulinemia (which has been reported rarely).

Antidote of Methotrexate

Leukemia patients are often given the compound Leucovorin (N5-formyl THF) following treatment with the drug methotrexate. Leucovorin (N5-formyl THF, folinic acid) is used as an antidote for cells that have decreased levels of folic acid. Treatment of leukemia patients with methotrexate kills the tumor cells but also other normal rapidly dividing cells. N5-formyl THF is normally administered 24 hours following treatment with methotrexate; it can be converted to THF by these normal cells by bypassing the block caused by methotrexate. Therefore, these normal cells can synthesize deoxy thymidine and carry out DNA synthesis.

Alternative Drug

Pemetrexed is a novel folate-directed antimetabolite. It is “multitargeted” in that it inhibits the activity of several enzymes, including thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyl iransferase, thereby affecting the synthesis of both purine and pyrimidine nucleic acid precursors. To avoid significant toxicity to the normal tissues, patients receiving pemetrexed should also receive low-dose folate and vitamin B12 supplementation. Pemetrexed has notable activity against certain lung cancers and, in combination with Cisplatin, also against mesotheliomas.


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