Dihydrofolic acid, biosynthesis

En2ymatic reduction of folic acid leads to the 7,8-dihydrofolic acid (H2 folate) (2), a key substance in biosynthesis. Further reduction, cataly2ed by the en2yme dihydrofohc acid reductase, provides (65)-5,6,7,8-tetrahydrofohc acid (H folate) (3). The folate (3) is the key biological intermediate for the formation of other folates (4—8) (Table 2). 

Trimethoprim acts in the body by interfering with the action of hydrofolate reductase, an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid. This process is necessary for purine biosynthesis of live organisms and DNA, respectively. Reducing the dihydrofolic acid to tetrahydrofolic acid is also catalyzed in humans by dihydrofolate reductase. However, trimethoprim has thousands of more inhibitory effects with respect to bacterial enzymes than with respect of analogons enzymes of mammals, which is the main benefit of trimethoprim. 

A considerable number of enzymes occupy a central and crucial role in the activity of drugs. Dihydrofolate reductase, an enzyme involved in purine and amino acid biosynthesis, is the target of antibacterial sulfanilamides, which act both as bacteriostatics and antimalarials. These drugs act on the enzyme in different ways, some being so-called antimetabolites (i.e., reversible enzyme inhibitors). Some diuretics act on carbonic... 

Trimethoprim (Proloprim, Trimpex) interferes with the bacterial folic acid pathway by inhibiting the dihydrofolate reductase enzyme in susceptible bacteria (see Fig. 33-2). This enzyme converts dihydrofolic acid to tetrahydrofolic acid during the biosynthesis of folic acid cofactors. By inhibiting this enzyme, trimethoprim directly interferes with the production of folic acid cofactors, and subsequent production of vital bacterial nucleic acids is impaired. 

Methotrexate acts by inhibition of dihydrofolate reductase, the enzyme requisite for the reduction of dihydrofolic acid (3) to 5,6,7,8-tetrahydrofolic acid (4). In turn, (4) is a precursor to a series of enzyme cofactors (5-7) essential for the transfer of one carbon unit necessary for the biosynthesis of purines and pyrimidines and hence, ultimately, DNA. As an inhibitor of dihydrofolate reductase, methotrexate kills cells during the S phase of the cell cycle, when the cells are in the log phase of growth. Unfortunately, this cytotoxicity is non-selective, and rapidly proliferating normal cells, e.g., gastrointestinal epithelium cells and bone marrow, are dramatically affected as well. In addition, recent use of high dose methotrexate therapy with leucovorin rescue has led to additional clinical problems arising from a dose-related nephrotoxic metabolite, 7-hydroxy methotrexate (8). Finally, the very polar nature of methotrexate renders it virtually impenetrable to the blood-brain barrier, which can necessitate direct intrathecal injection in order to achieve therapeutic doses for the treatment of CNS tumours. 

Figure 2.4 Sulfamidochrysoidine 10 (Prontosil rubrum , Bayer) and related antibacterial sulfonamides act via the metabolite sulfanilamide 11, which is an antimetabolite of p-aminobenzoic acid in the bacterial biosynthesis of dihydrofolic acid.

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. 

The two steps involved are the biosynthesis of dihydropteroic acid catalyzed by dihy-dropteroate synthetase and inhibited by sulfonamides and sulfones (Chapter 2), and the reduction of dihydrofolic acid by DHFR, which can be inhibited by MTX, PM, TM, and other DHFR inhibitors. Hitchings proposed such combinations, which ideally should pro-... 

Trimethoprim/sulfamethoxazole is an antibiotic combination. Sulfamethoxazole (SMZ) inhibits bacterial synthesis of dihydrofolic acid by competing with PABA. Trimethoprim (TMP) blocks production of tetrahydrofolic acid by inhibiting the enzyme dihydrofolate rednctase. This combination blocks two consecutive steps in bacterial biosynthesis of essential nncleic acids and proteins and is nsnally bactericidal. 

A further step in the pathway leading from the pteroates to folic acid and on to DNA bases requires the enzyme dihydrofolate reductase. Exogenous folic acid must be reduced stepwise to dihydrofolic acid and then to tetrahydrofolic acid, an important cofactor essential for supplying a 1-carbon unit in thymidine biosynthesis and, ultimately, for DNA synthesis (Fig. 38.5). The same enzyme also must reduce endogenously produced dihydrofolate. Inhibition of this key... 

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... 

Differences between bacteria and Man in the absorption and the biosynthesis of dihydrofolic acid 2.14) and its derivatives are so great that the whole system of sulfonamide chemotherapy rests on it (Section 9.3.1). In brief, pathogenic bacteria can synthesize their requirements of folic acid, but cannot absorb preformed folic acid in their nutriment. Man, on the other hand, cannot synthesize this coenzyme, but has no difficulty in absorbing it from food. 

There are antagonists of the biosynthesis of dihydrofolic acid, and antagonists of its utilization. The history of the discovery of the antibacterial sulfonamides, typical antagonists of biosynthesis, was given in Sections 2.1 and 6.3.1. In 1940, Woods showed that the anti-bacterial action of sulfanilamide depended on its competition with j -aminobenzoic acid (P.7), which is a natural metabolite (Woods, 1940). Later this competition was shown to take place at the site on the enzyme dihydrofolate synthetase, which uses j -aminobenzoic acid to build up the molecule of dihydrofolic acid (2.74) (G.M. Brown, 1962). 

Fio. 6. Biosynthesis of functional forms of folic acid FHi, dihydrofolic acid FH4, tetrahydrofolic acid FIGLU, formiminoglutamio acid FIG, formiminogly-oine. 

Sulphanilamide and its derivatives. The concept that a drug receptor could be an enzyme was extended from pharmacodynamics to chemotherapy when Woods (1940) demonstrated the reversal of the antibacterial action of sulphanilamide 2.12) by p-aminobenzoic acid 2,13) and pointed out that this reversal depended on the structural similarity of these two substances. Later, the receptor for sulphonamides was found to be the enzyme dihydrofolate synthetase, which incorporates p-aminobenzoic acid into the molecule of dihydrofolic acid (2.74), an essential coenzyme for the biosynthesis of purines and thymine, and hence of DNA. This enzyme was isolated and purified by G. Brown (1962), and these functions confirmed. 

Outstanding among drugs which inhibit the production of DNA from several stages back in the biosynthetic pathway are the sulphonamides and the 2,4-diaminopyrimidines used so much as antibacterials and anti-malarials. All of the chemotherapeutic sulphonamides, whether simple sulphanilamide (4.5a) or its more complex heterocyclic derivatives 4.5b) including sulphadiazine, competitively inhibit the enzymes dihydrofolate synthetase which produces dihydrofolic acid 2.14) (see p. 28). The basis of this inhibition, as outlined in Section 2.1 (p. 28), is the similarity in the steric and electronic properties of p-aminobenzoic acid 2,13) (which the enzyme is ready to build into new molecules of dihydrofolic acid) and the sulphonamides (4.5) which, when taken up by the enzyme, merely block it. The basis of the selectivity of these antibacterial sulphonamides depends on two factors, which reinforce one another (i) mammals lack the enzymes necessary for the synthesis of dihydrofolic acid, and hence they tolerate these sulphonamides very well (ii) pathogenic bacteria lack the permease with the aid of which mammals absorb dihydrofolic acid from the diet. Further relevant data will be foimd in Section 9.3. Dihydrofolic acid is only two steps away from the coenzyme required for biosynthesis of thymine and all the purine bases. Deprived of the substrates, especially thymine, bacteria soon die because they can make no new DNA. 

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

These are pyrimidine derivatives and are effective because of differences in susceptibility between the enzymes in humans and in the infective organism. Anticancer agents based on folic acid, e.g. methotrexate, inhibit dihydrofolate reductase, but they are less selective than the antimicrobial agents and rely on a stronger binding to the enzyme than the natural substrate has. They also block pyrimidine biosynthesis. Methotrexate treatment is potentially lethal to the patient, and is usually followed by rescue with folinic acid (A -formyl-tetrahydrofolic acid) to counteract the folate-antagonist action. The rationale is that folinic acid rescues normal cells more effectively than it does tumour cells. 

Formation of THF from dihydrofolate (DHF) is catalyzed by the enzyme dihydrofolate reductase. DHF is made from folic acid, a vitamin that cannot be synthesized in the body, but must be taken up from exogenous sources. Most bacteria do not have a requirement for folate, because they are capable of synthesizing folate, more precisely DHF, from precursors. Selective interference with bacterial biosynthesis of THF can be achieved with sulfonamides and trimethoprim. 

Folic Acid Antagonists. Folic acid antagonists block the biosynthesis of purine nucleotides. Methotrexate (7.76) is the prototypic fohc acid antagonist and functions by binding to the active catalytic site of dihydrofolate reductase, thereby interfering with the synthesis of the reduced form that accepts one-carbon units lack of this cofactor blocks the synthesis of purine nucleotides. As well as being used in the treatment of cancer, methotrexate has been used in the management of rheumatoid arthritis, psoriasis, and even asthma. 

Another group of inhibitors prevents nucleotide biosynthesis indirectly by depleting the level of intracellular tetrahydrofolate derivatives. Sulfonamides are structural analogs of p-aminobenzoic acid (fig. 23.19), and they competitively inhibit the bacterial biosynthesis of folic acid at a step in which p-aminobenzoic acid is incorporated into folic acid. Sulfonamides are widely used in medicine because they inhibit growth of many bacteria. When cultures of susceptible bacteria are treated with sulfonamides, they accumulate 4-carboxamide-5-aminoimidazole in the medium, because of a lack of 10-formyltetrahydrofolate for the penultimate step in the pathway to IMP (see fig. 23.10). Methotrexate, and a number of related compounds inhibit the reduction of dihydrofolate to tetrahydrofolate, a reaction catalyzed by dihydrofolate reductase. These inhibitors are structural analogs of folic acid (see fig. 23.19) and bind at the catalytic site of dihydrofolate reductase, an enzyme catalyzing one of the steps in the cycle of reactions involved in thymidylate synthesis (see fig. 23.16). These inhibitors therefore prevent synthesis of thymidylate in replicating... 

FIGURE 33-2 Folic acid metabolism in bacterial cells. Certain antibacterial drugs [e.g., sulfonamides and trimethoprim] inhibit the dihydrofolate synthetase and reductase enzymes, thus interfering with DNA biosynthesis. 

---