Dihydrofolate reductase (DHFR) inhibitors

Dihydrofolate reductase, DHFR, whose role is to regenerate folic acid into its reduced form tetrahydrofolate, is necessary for bacteria, Plasmodia and normal and cancerous human cells. Inhibitors of dihydrofolate reductase have antibiotic, antimalarial and antineoplastic properties.

Inhibitors of DHFR must have a sufficient specificity for the DHFR of micro-organisms and that of cancerous cells for not inducing too many adverse effects in humans.


Trimethoprim is an inhibitor of DHFR of micro-organisms, but resistance to its action develops quickly when it is used alone, so it is usually combined with another antibiotic.

To reinforce its action and to prevent resistance development, trimethoprim is combined to a sulfonamide which inhibits the synthesis of folic acid. By this double effect, inhibition of the synthesis of folic acid and inhibition of DHFR, the combination used, generally trimethoprim-sulfamethoxaxole called co-trimoxazole is effective against many microorganisms: streptococci, staphylococci, Proteus, Enterobacter, Salmonellas, Shigella, Serratia, Brucella abortus, Pasteurella haemolytica, Nocardia etc, responsible of urinary, pulmonary, intestinal infections.

The combination trimethoprim-sulfamethoxaxole (Bactrim*), is generally well tolerated, but can induce adverse effects such as leukopenia, thrombocytopenia, anemia, agranulocytosis, evocative of a disorder of cellular maturation, probably secondary to an inhibition of the human DHFR. This combination can cause severe but rare dermatological reactions, Stevens-Johnson and Lyell syndromes. Trimethoprim can also cause hyperkalemia by decreasing urinary potassium elimination.


Pyrimethamine is a diaminopyridine derivative which inhibits in a rather selective way the DHFR of Plasmodium without inhibiting that of the patient. This inhibition blocks the cellular division of the parasite at the stage of schizont.

Pyrimethamine (Daraprim*) is different from trimethoprim by a greater specificity for the DHFR of Plasmodium malariae: the following table indicates the approximate concentration which is necessary for obtaining a 50% inhibition of the hepatic, bacterial and parasitic DHFR.


Hepatic enzyme of the rat

Bacterial enzyme (Escherichia coli)

Enzyme of plasmodium



2 500



250 000



Pyrimethamine is absorbed slowly, but completely, from the digestive tract. Its plasma half-life is long, about 100 hours.

The combination of pyrimethamine and an inhibitor of the synthesis of folic acid like sulfadoxine (Fansidar*) or sulfamethoxazole reinforces its antiparasitic activity. Pyrimethamine combined with sulfadiazine is also used for the treatment of toxoplasmosis.

The adverse effects of pyrimethamine are often anemia and those of the combination pyrimethamine-sulfonamide are sometimes severe skin reactions.

The resistance of Plasmodia to pyrimethamine comes from a mutation leading to the synthesis of a non susceptible DHFR by replacement of an amino acid by another.

Chloroguanide or Proguanil

Proguanil, (Paludrine*) itself, has no antimalarial activity, but one of its metabolites, cycloguanil, which has a triazine structure, inhibits rather specifically the DHFR of Plasmodia and prevents their division. Proguanil, used during pregnancy, does not seem to have induced fetal malformations.

Proguanil, combined with atovaquone, is marketed as antimalarial under the name of Malarone*.


Pentamidine (Pentacarinat*) is a diamine used in the treatment of African trypanosomiasis, leshmaniasis and pneumocystis pneumonia. It has several mechanisms of action among which the inhibition of the dihydrofolate reductase of parasites.


Methotrexate (MTX), analog of folic acid, was the first drug to obtain remissions of leukemias and to be effective in choriocarcinoma. It forms an inactive ternary complex with DHFR and NADPH. MTX is a nonspecific inhibitor of the dihydrofolate reductase (DHFR) of bacteria and cancerous cells as well as normal cells.

The inhibition of the DHFR, by preventing the reduction of folic acid, inactive precursor, into tetrahydrofolic acid and then in folinic acid, disturbs the synthesis of purine bases, adenine and guanine, and of a pyrimidine base, thymidine.

In addition to its inhibitory effect of DHFR, it can inhibit the amidophosphoryl-transferase which transforms phosphoribosyl pyrophosphate into 5-phosphoribosylamine, leading to the synthesis of IMP.

MTX penetrates into cells by active transport, but, when it is present at high concentration, a passive diffusion is possible. In cells, under the influence of folyl-polyglutamate synthetase, MTX bind one or more glutamate residues to form polyglutamate derivatives which persist a long time inside the cells.

MTX has not a great selectivity of action and its effects appear especially on the cells in fast division, cancerous cells, normal cells of the digestive epithelium tract and bone marrow.

The bioavailability of methotrexate taken by oral route is approximately 75%. It binds to plasma proteins at 35% and penetrates into the brain and the cerebral spinal fluid.

Administration of folinic acid, active form of folic acid, (LEDERFOLINE*), which is active without intervention of the DHFR, reduces the toxicity of methotrexate for normal tissues but reduces also its efficacy.

The treatments by very high doses of methotrexate, as those used in certain lymphoma and osteosarcoma, necessitate the maintenance of a high and alkaline diuresis and the administration of high doses of folinic acid, 24 or 36 hours after the discontinuation of the infusion of methotrexate, to thwart the toxicity of methotrexate.

The clinical uses of MTX are numerous:

•  High-dose, in cancerology: treatment of choriocarcinoma, leukemia, lymphoma, osteosarcoma, head and neck cancers and breast cancer, where it is used in combination with other antineoplastics.

•  Low-dose, - from 100 to 1000 times lower than those used in cancerology - it is used, perhaps because of its anti-inflammatory effect and its inhibitory effect of cell divisions, in the treatment of psoriasis, rheumatoid arthritis, inflammatory diseases of the digestive tract, of the liver, certain severe asthmas.

Adverse effects of methotrexate are frequent, especially in large doses.


Trimetrexate is an inhibitor of the bacterial, parasitic and human DHFR, more liposoluble than methotrexate. It was used for the treatment of Pneumocystis pneumonia in immunocompromised patients. To reduce its toxicity, it is used in combination with folinic acid. Adverse effects of trimetrexate are the same that those of methotrexate but probably more severe. It was marketed under the name of Neutrexin*.

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