Inhibitors of folate biosynthesis

One of the most common infections in man is caused by the protozoan, Toxoplasma gondii, which is transmitted to humans when they consume raw or inadequately cooked, infected meat. Infected pregnant women can transmit the organism to the fetus. Cats are the only animals that shed oocysts that can infect other animals as well as man. The treatment of choice for this condition is the antifolate drug, pyrimethamine [peer i METH a meen] (see p. 353). A combination of sulfadiazine (see p. 289) and pyrimethamine is also efficacious. Leucovorin is often administered to protect against folate deficiency. Other inhibitors of folate biosynthesis, such as trimethoprim (see p. 293) and sulfamethoxazole (see p. 289) are without therapeutic efficacy in toxoplasmosis. [Note At the first appearance of a rash, pyrimethamine should be discontinued since hypersensitivity to this drug can be severe.]... 

Inhibitors of Folate Biosynthesis and Utilization — Evolutionary Changes As a Basis for Chemotherapy... 

A review on the potent inhibitors of de novo pyrimidine and purine biosynthesis summarizes the developments in this field <90MI 718-04) and another report is concerned mainly with the synthetic approaches to the various types of inhibitors of folate-dependent enzymes [Pg.729]

FIGURE 1. Biochemical pathways leading to the synthesis and utilization of thymidine monophosphate (TMP). TMP biosynthesis is effected both by the folate-dependent enzyme thymidylate synthetase (TS) and by thymidine kinase (TK). Using inhibitors of folate metabolism or lethal analogues of thymidine, the TS and TK pathways can be controlled to select for TK-competent or TK-deficient cells. For details, see text. 

Inhibit Enzymes Many drugs are competitive inhibitors of key enzymes in pathways. The statin drugs (lovastatin, simvastatin), used to control blood cholesterol levels, competitively inhibit 3-hvdroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in cholesterol biosynthesis. Methotrexate, an antineoplastic drug, competitively inhibits dihydrofolate reductase, depriving the cell of active folate needed for purine and deoxythymidine synthesis, thus interfering with DNA replication during S phase. 

Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme essential to the de novo purine biosynthetic pathway. It utilizes the cofactor 10-formyl tetrahydrofohc acid (10-formyl-THF) to transfer a formyl group to the primary amine of its substrate a-glycinamide ribonucleotide. Potent, and potentially selective, inhibitors of GARTfase and de novo purine biosynthesis have been shown to be promising as antitumor drugs. 

The mode of action of sulfanilamides became known around 1947, when the structure and biosynthesis of folic acid were elucidated. This compound is built by bacteria from the heterocyclic pteroyl moiety, p-aminobenzoate, and glutamate. p-Aminobenzene-sulfonamide (9.89, sulfanilamide) is a competitive inhibitor of the synthase enzyme, acting as an antimetabolite of p-aminobenzoate. Occasionally, the sulfanilamide can even be incorporated into the modified folate, resulting in an inactive compound and thus an inactive enzyme. This theory, proposed by Woods and Fildes in 1940, became the first molecular explanation of drug action. 

Tetrahydrofolate (Fig. le) and derivatives (Fig. If, g) are the biologically active forms of folate. This cofactor is involved in many, distinct enzymatic reactions, ranging from the amino acid metabolism, such as serine hydroxymethyltransferase (SHMT) (Fig. 5a), to nucleotide biosynthesis, such as thymidylate synthase (TS) (Fig. 5b) and dihydrofolate reductase DHFR (Fig. 5c). These enzymes are targets for anticancer drugs because they participate in the formation of thymidylate, the only nucleotide that cannot be obtained via the salvage reactions (30). Whereas the search for inhibitors of SHTM has only recently... 

The transfer of 1-carbon units at this oxidation level originally was thought to involve two derivatives of H4-folate, 10-formyl-H4-folate and 5,10-methenyl-H4-folate which acted as cofactors for the two transformylases in de novo purine biosynthesis [67-69]. However, recent work has shown that the glycinamide ribonucleotide transformylase (GAR TFase) from E. coli as well as from avian liver utilize 10-formyl-H4-folate as the actual cofactor [70,71]. The preference for 10-for-myl-H 4-folate was masked by the presence of the opposite, unreactive diastereomer (R at C-6 in H4-folate) which is an excellent competitive inhibitor of the enzyme. The apparent reactivity of the 5,10-methenyl-H4-folate in the same assay arose because of a contaminating cyclohydrolase activity capable of selectively hydrolyzing it to the correct diastereomer of 10-formyl-H4-folate. 

Two other validated targets for antimicrobial chemotherapy will be mentioned here, both of which are defined by pathway inhibition. The earliest antibiotics, the sulfonamides, are inhibitors of the folate biosynthetic pathway (24). Sulfamethoxazole and trimethoprim, targeting dihydropteroate synthase and dihydrofolate reductase, respectively, are each susceptible to rapid emergence of resistance but have been used successfully in combination. Recently, other enzymes in the pathway have begun to engender interest for target-based screening. Fatty acid biosynthesis is another pathway that has previously been... 

Soft Dihydrofolate Reductase (DHFR) Inhibitors. A series of esters was synthesized recently as possible dihydrofolate reductase (DHFR)inhibitors that are susceptible toward hydrolytic degradation (148,206-208). DHFR is involved in the reduction of dihydrofolate into tetrahydrofolate, and reduced folates are important cofactors in the biosynthesis of nucleic acids and amino acids. Hence, DHFR inhibitors can limit cellular growth. Consequently, classical DHFR inhibitors such as methotrexate (99) (Fig. 15.24) or nonclassical DHFR inhibitors such as trimetrexate (100) have shown antineoplastic or antiprotozoal... 

A complete understanding of sulfonamide action evolved over a 20-year period. The biosynthesis of the various folates in living cells had to be elucidated their functions in the scheme of things had to be worked out. The following discourse will consider the effects of sulfonamides, as well as that of another group of important enzyme inhibitors—the dihydrofolic acid reductase inhibitors. Figure 2-4 outlines the stratagem as it is presently understood. 

A key enzyme in pyrimidine biosynthesis, thymidylate synthetase, catalyzes the reductive methylation of 2 -deoxyuridylate (dUMP) to thymidylate (dTMP) with the concomitant conversion of 5,10-methylene-H4-folate to 7,8-dihydrofolate. The cofactor serves both as a 1-carbon carrier and a reductant. There is substantive evidence based on direct and indirect studies with the inhibitor 5 -fluoro-2 -deoxyuridylate, which will not be reviewed here, that the dUMP is covalently bound to the enzyme, possibly through a thiol group [59]. The intermediate ternary complex is hypothesized to have the following structure (10), where the CH2 unit is attached through the N-5 of H4-folate. The ring opening of the 5,10-methylene-H4-folate is... 

---