Mechanism of Action

 

Although initially complex in appearance, the mechanism of action of and structure of methotrexate shows the real life and very useful application of many basic principles of chemistry which will be studied throughout any GCSE, A-Level and Degree level course in the subject. For example:

  • Reduction

  • Enzyme Catalysis

  • Amine Groups

  • Heterocyclic chemistry

  • Peptide bonds and amino acids

  • Optical isomerism and the effect of this on selectivity

  • Basicity of different heteroatoms

  • The use of locant numbers to show the position of different functional groups

Folic acid in DNA synthesis

 As with all human cell reproduction, the abnormal reproduction of cells that causes cancer requires folic acid, a derivative of vitamin B, as a starting point. The structure of folic acid is given below.

The structure of folic acid. (Source: http://southmed.usouthal.edu)

 After absorption into the cell, folic acid is first reduced to dihydrofolate, FH2, by the enzyme dihydrofolate reductase and then to tetrahydrofolate, FH4, by the same enzyme.

Tetrahydrofolate acts as a carrier for methylene groups which bind to it on the nitrogen atoms N5 and N10. By carrying a methylene, CH, unit bonded to both N5 and N10 tetrahydrofolate is used for the conversion of 2-deoxyuridylate monophosphate (dUMP) to 2-deoxythymidylate monophosphate (dTMP).

 Tetrahydrofolate carrying a CH2 unit bonded to N5 and N10 (Adapted from: www.biology.ucsd.edu)

In this process the methylene, CH unit is reduced to a CH3 unit, which bonds with the dUMP, and the tetrahydrofolate is oxidised back to dihydrofolate. This rate limiting process for the replication of DNA is catalysed by the enzyme thymidylate synthetase.

Tetrahydrofolate also acts as a carrier for a carbonyl, CHO, group, which binds to nitrogen N10. This is one of the building blocks of purine which is required to make the amino acids guanine and adenine.

 Mechanism of action

The action of methotrexate relies on the fact that it is a structural analogue of folic acid. Methotrexate differs from folic acid only by an amine group in place of a hydroxyl group on the carbon C4 and a methyl group in place of a hydrogen atom on N10.

The structures of methotrexate and folate (folic acid). (Source:http://www.wellesley.edu)

 Methotrexate enters the cell via active uptake. The enantiomer L-methotrexate is absorbed forty times more readily than D-methotrexate, by the reduced folate carrier and the folate receptor protein, the mechanisms used to absorb folic acid. Inside the cell methotrexate is polyglutamated by folylpolyglutamate synthase (FPGS) by forming a peptide link between the carboxylic acid group of methotrexate and the amine group of glutamic acid. This prevents methotrexate from being able to leave the cell, greatly improving its ability to act by maintaining its intracellular concentration.

Methotrexate is a competitive inhibitor of dihydrofolate reductase and has a higher affinity for dihydrofolate reductase by a factor of 104 than dihydrofolate does, due to the greater basic strength of the amine group on carbon C4 of methotrexate compared with the hydroxyl group on C4 of folic acid. This produces an extra ionic bond with the enzyme increasing its affinity. Methotrexate has no efficacy for dihydrofolate reductase and so greatly reduces the amount of tetrahydrofolate produced.

Reduced amounts of tetrahydrofolate means the conversion of dUMP to dTMP is inhibited. With greatly reduced amounts of dTMP available the rapid reproduction of cancer cells is therefore prevented, as the DNA required to produce the cell cannot form properly. Similarly, the lack of tetrahydrofolate inhibits purine synthesis, restricting the production of the amino acids guanine and adenine and thereby also preventing the replication of cancer cells.