Folate antagonists, also known as antifolates, inhibit dihydrofolate reductase (DHFR), an enzyme involved in the formation of nucleotides. When this enzyme is blocked, nucleotides are not formed, which disrupts DNA replication and cell division. Methotrexate is the primary folate antagonist used as a chemotherapeutic agent. It may be used alone or in combination with other anticancer drugs.

Drug Discovery
In 1948, it was found that a diet with reduced levels of folic acid led to a decrease in leukemia cell counts. That discovery started the search for folate antagonists. The same year, a folate antagonist, aminopterin, was found to produce remissions in childhood leukemias. Methotrexate was discovered soon after, and it proved to be a more effective, less toxic folate analogue. Since then, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for breast cancer, osteogenic sarcoma, and leukemias. ⁽¹⁾

A Closer Look at Folate Antagonists

Folic acid is a growth( )factor that provides single carbons to the precursors used to form the nucleotides used in the synthesis of DNA and RNA. Folate antagonists, also known as antifolates, act by blocking the active site of dihydrofolate reductase (DHFR), an enzyme that reduces folic acid to its active form. Active folates are co-enzymes necessary for methylation in various metabolic processes, in which they deliver methyl groups (one-carbon units) to specific target molecules. The inhibition of the dihydrofolate reductase keeps the folic acid in an inactive state. A decrease in the amount of activated folates is thought to cause a decrease in methylation, inhibiting a necessary step in purine and thymidylate formation. When nucleic( )acid formation is compromised because of a lack of nucleotides, cell growth is disrupted. ⁽²⁾ Methotrexate is the most commonly used folate antagonist.

More Details:
Folic acid, the core structure of all folates, is not useful until it is chemically reduced. The enzyme that reduces folic acid is dihydrofolate reductase (DHFR). First, DHFR reduces the folic acid into dihydrofolate. Then, DHFR reduces dihydrofolate into tetrahydrofolate (active folate). It is this compound that is used as a donor of methyl groups. The methyl groups are attached to N-5 and/or N-10 of the tetrahydrofolate which carries the methyl groups to other compounds. The enzyme thymidylate synthase (TS) catalyzes transfer of the carbon from the tetrahydrofolate to the target molecules. In order to do so, TS must oxidize the folate ring of the tetrahydrofolate, which reverts it back into a dihydrofolate. For this process to repeat, cells must repeatedly use DHFR to reduce the dihydrofolate into the active tetrahydrofolate form. This requires continual DHFR activity.

Methotrexate inhibits the activity of DHFR by tightly, though reversibly, binding to it rendering it inactive. It enters the cell via specific folate receptors, the low pH folate transporter, or by reduced folate carriers. Once in the cell, methotrexate binds to DHFR. This binding reduces the amount of DHFR available to the cell, and stops the reduction of the tetrahydrofolate precursors, ie. folic acid and dihydrofolic acid. Without tetrahdryofolate, the active folate, the cell cannot create new purine and thymidine nucleotides for DNA synthesis. Without replication, cell growth is blocked.⁽²⁾