Folate Antagonists

FOLATE ANTAGONISTS

Folate Metabolism In Microbial And Mammalian Cells

Folic acid is an important cofactor in all living cells. In the reduced form, tetrahydrofolate (THF), it functions as a carrier of single-carbon fragments, which are used in the synthesis of adenine, guanine, thymine and methionine (Figure 12.7). One important folate-dependent enzyme is thymidylate synthetase, which produces TMP by transfer of the methyl group from THF to UMP. In this and other folate-dependent reactions THF is converted to dihydrofolic acid (DHF), which must be reduced back to THF before it can participate again as a carbon fragment carrier. The enzyme responsible for the reduction of DHF to THF is dihydrofolate reductase (DHFR) which uses the nucleotide NADPH₂ as a cofactor. Bacteria, protozoa and mammalian cells all possess DHFR but there are sufficient differences in the enzyme structure for inhibitors such as trimethoprim and pyrimethamine to inhibit the bacterial and protozoal enzymes selectively without damaging the mammalian form. In the case of protozoa such as the Plasmodium species responsible for malaria, the DHFR is a double enzyme which also contains the thymidylate synthetase activity.

There is another fundamental difference between folate utilization in microbial and mammalian cells (Figure 12.7). Bacteria and protozoa are unable to take up exogenous folate and must synthesize it themselves. This is carried out in a series of reactions involving first the synthesis of dihydropteroic acid from one molecule each of pteridine and p-aminobenzoic acid (PABA). Glutamic acid is then added to form DHF, which is reduced by DHFR to THF. Mammalian cells do not make their own DHF, instead they take it up from dietary nutrients and convert it to THF using DHFR.

Sulphonamides

Sulphonamides (e.g. sulphamethoxazole and dapsone) are structural analogues of PABA (Figure 12.8). They competitively inhibit the incorporation of PABA into dihydropteroic acid and there is some evidence for their incorporation into false folate analogues, which inhibit subsequent metabolism. The presence of excess PABA will reverse the inhibitory action of sulphonamides, as will thymine, adenine, guanine and methionine. However these nutrients are not normally available at the site of infections for which the sulphonamides are used.

DHFR Inhibitors—Trimethoprim And Pyrimethamine

Trimethoprim is a selective inhibitor of bacterial DHFR. The bacterial enzyme is several thousand times more sensitive than the mammalian enzyme. Pyrimethamine, likewise, is a selective inhibitor of plasmodial DHFR. Both are structural analogues of the dihydropteroic acid portion of the DHF substrate (Figure 12.8). Crystal structures of the bacterial, plasmodial and mammalian DHFRs, each containing either bound substrate or the inhibitors, have been determined by X-ray diffraction studies. These show how inhibitors fit tightly into the active site normally occupied by the DHF substrate, forming a pattern of strong hydrogen bonds with amino acid residues and water molecules lining the site. Another DHFR is proguanil, a guanidine-containing prodrug which is metabolized in the liver to cycloguanil, an active selective inhibitor of plasmodial DHFR. Methotrexate is a potent DHFR inhibitor that has an analogous structure to the whole DHF molecule, including the glutamate residue. It has no selectivity towards microbial DHFR and therefore cannot be used to treat infections; however, it is widely used as an anticancer agent. A derivative of methotrexate that is used for treatment of Pneumocystis jirovecii infections in AIDS patients is trimetrexate. Although it is very toxic to mammalian cells, simultaneous administration of leucovorin (formyl-THF or folinic acid) as an alternative source of folate which cannot be taken up by the organism protects host tissues. DHFR inhibitors can be used in combination with a sulphonamide to achieve a double interference with folate metabolism. Suitable combinations with matching pharmacokinetic properties are sulphamethoxazole with trimethoprim (the antibacterial co-trimoxazole) and dapsone with pyrimethamine (the antimalarial Maloprim).