Dihydrofolate bacterial synthesis

Pharmacology SMZ inhibits bacterial synthesis of dihydrofolic acid by competing with para-aminobenzoic acid. TMP blocks the production of tetrahydrofolic acid by inhibiting the enzyme dihydrofolate reductase. 

Sulfonamides were the first group of chemotherapeutic agents used for the prevention or treatment of bacterial infections in humans. Sulfonamides (e.g., sulfisoxazole) act by inhibiting bacterial synthesis of folic acid, a chemical required for synthesis of nucleic acid and protein. These drugs competitively inhibit the first step in the synthesis of folic acid—the conversion of para-aminobenzoic acid into dihydrofolic acid. Because humans absorb preformed folic acid from food, sulfonamide inhibition has only a minimal effect on hiunan cells. 

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. 

Both of these drugs interfere with bacterial synthesis of folic acid and the production of nucleotides. Sulfamethoxazole decreases the enzymatic conversion of PABA, and trimethoprim decreases the activity of dihydrofolate reductase. Thus, the actions are synergistic (see Figure p 2621. 

Sulfamethoxazole inhibits bacterial synthesis of dihydrofohc acid, and trimethoprim blocks the production of tetrahydrofolic acid by inhibiting the enzyme dihydrofolate reductase. Thus two consecutive steps ate blocked in the biosynthesis of nucleic acids and proteins essential to many bacteria. In vitro serial dilution tests have shown that the combination of sulfamethoxazole and trimethoprim [738-70-5] inhibits the growth of common urinary tract pathogens with the exception of Pseudomonas aeruginosa. Table 3 illustrates the enhanced effect of the combination over that of either agent alone. 

Both the sulfonamides and trimethoprim interfere with bacterial folate metabolism. For purine synthesis tetrahydrofolate is required. It is also a cofactor for the methylation of various amino acids. The formation of dihydrofolate from para-aminobenzoic acid (PABA) is catalyzed by dihydropteroate synthetase. Dihydrofolate is further reduced to tetrahydrofolate by dihydrofolate reductase. Micro organisms require extracellular PABA to form folic acid. Sulfonamides are analogues of PABA. They can enter into the synthesis of folic acid and take the place of PABA. They then competitively inhibit dihydrofolate synthetase resulting in an accumulation of PABA and deficient tetrahydrofolate formation. On the other hand trimethoprim inhibits dihydrofolate... 

Trimethoprim, a trimethoxybenzylpyrimidine, selectively inhibits bacterial dihydrofolic acid reductase, which converts dihydrofolic acid to tetrahydrofolic acid, a step leading to the synthesis of purines and ultimately to DNA (Figure 46-2). Trimethoprim is about 50,000 times less efficient in inhibition of mammalian dihydrofolic acid reductase. Pyrimethamine, another benzylpyrimidine, selectively inhibits dihydrofolic acid reductase of protozoa compared with that of mammalian cells. As noted above, trimethoprim or pyrimethamine in combination with a sulfonamide blocks sequential steps in folate synthesis, resulting in marked enhancement (synergism) of the activity of both drugs. The combination often is bactericidal, compared with the bacteriostatic activity of a sulfonamide alone. 

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... 

Mechanism of Action. Pyrimethamine blocks the production of folic acid in susceptible protozoa by inhibiting the function of the dihydrofolate reductase enzyme. Folic acid helps catalyze the production of nucleic and amino acids in these parasites. Therefore, this drug ultimately impairs nucleic acid and protein synthesis by interfering with folic acid production. The action of sulfadoxine and other sulfonamide antibacterial agents was discussed in Chapter 33. These agents also inhibit folic acid synthesis in certain bacterial and protozoal cells. 

A metabolic pathway that has received considerable attention is the conversion of 2 -deoxyuridine 5 -monophosphate (dUMP, 6.60) to thymidine 5 -monophosphate (TMP, 6.61) (Scheme 6.13). Without an adequate supply of TMP, a cell or bacterium cannot create DNA for cell division. Therefore, blocking TMP synthesis is an attractive method for slowing the advancement of certain cancers and bacterial infections. Important molecules in the methylation of dUMP are the various folic acid derivatives folic acid (FA, 6.62), dihydrofolic acid (DHF, 6.63), tetrahydrofolic acid (THF, 6.64), and N5, A1 "-methylene tetrahydrofolic acid (MTHF, 6.65) (Figure 6.23). These structures... 

The active form of folate is the tetrahydro-derivative that is formed through reduction by dihydrofolate reductase. This enzymatic reaction (Figure 29.5) is inhibited by trimethoprim, leading to a decrease in the folate coenzymes for purine, pyrimidine, and amino acid synthesis. Bacterial reductase has a much stronger affinity for trimethoprim than does the mammalian enzyme, which accounts for the drug s selective toxicity. [Note Examples of other folate reductase inhibitors include pyrimethamine, which is used with sulfonamides in parasitic infections (see p. 353), and methotrexate, which is used in cancer chemotherapy (see p. 378).]... 

Tetrahydrofolic acid (THF) is a coenzyme in the synthesis of purine bases and thymidine. These are constituents of DNA and RNA and are required for cell growth and replication. Lack of THF leads to inhibition of cell proliferation. 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 it-more precisely DHF-ffom precursors. Selective interference with bacterial biosynthesis of THF can be achieved with sulfonamides and trimethoprim. 

Pyrimethamine and trimethoprim reversibly inhibit the second step in the synthesis of folic acid by inhibiting the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid. The trimethoprim-binding affinity is much stronger for the bacterial enzyme than the corresponding mammalian enzyme, which produces selective toxicity. A powerful synergism exists between either pyrimethamine or trimethoprim and sulfonamides (e g., sulfemethoxazole and trimethoprim) because of sequential blockage of the same biosynthetic pathway. 

Fig. 12.7 Pathways of folate metabolism and use in microbial cells (upper) and mammalian cells (lower). Bacterial and protozoal cells must synthesize dihydrofolic acid (DHF) from p-aminobenzoic acid (PABA). DHF is converted to tetrahydrofolic acid (THF) by the enzyme dihydrofolate reductase (DHFR). THF supplies single carbon units for various pathways including DNA, RNA and methionine synthesis. Mammalian cells do not make DHF, it is supplied from the diet, conversion to THF occurs via a DHFR enzyme as in microbial cells.

Another enzyme for which X-ray diffraction studies have aided in an analysis of the mode of action is the enzyme dihydrofolate reductase. This catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential coenzyme used in the synthesis of thymidylate, inosinate, and methionine. The antitumor agent methotrexate is a powerful inhibitor of dihydrofolate reductase, causing, on binding, a cellular deficiency of thymidylate (the cause of its antitumor activity). The crystal structures of the enzyme from two bacterial sources—Escherichia coli and Lactobacillus casei—and from chicken liver have been studied (88-90). Both the E. coli and L casei enzymes have been studied as complexes with methotrexate bound at the active site, and, in the case of the . casei enzyme, the cofactor, NADPH, was also present. 

Trimethoprim inhibits another enzyme, dihydrofolate reductase, in the same folic acid metabolic pathway. Folic acid is converted into folate, which then has to be converted into an activated form by dihydrofolate reductase. In this way, trimethoprim interferes with the conversion of folate into its activated form, which is a cofactor in the synthesis of bacterial DNA. Dihydrofolate reductase also occurs in host cells, but it is less sensitive to trimethoprim. Trimethoprim is used to treat urinary tract infections. It is also formulated in combination with a sulphonamide, when it is known as co-trimoxazole, to treat pneumonia in patients with HIV (see page 170). Due to the synergistic effect of the two drugs, this combination is more effective than either drug alone. 

The activity and use of sulfonamides has been potentiated by addition of synergists that also inhibit bacterial enzymatic synthesis. The best known is trimethoprim 5-[(3,4,5-trimethoxyphenyl)methyl -2,4-pyrimidinediamine 47181 182 that binds dihydrofolic acid reductase. 43 and 47 are listed as antimalarial drugs183. 

Make sure you know the specific enzymes in bacterial folic acid synthesis that are inhibited by sulfonamides and trimethoprim Sulfonamides inhibit dihydropteroate synthase dihydrofolate reductase is inhibited by trimethoprim. The answer is (A). 

Resistance to bacterial diaminopyrimidines results from chromosomal mutations or plasmid-mediated mechanisms and develops very rapidly. Resistance conferred by chromosomal mutations allows bacteria to utilize exogenous sources of folinic acid or thymidine, thereby overcoming the drug-imposed blockade. Plasmid-mediated mechanisms result in the synthesis of dihydrofolate reductase characterized by a reduced affinity for antibacterial diaminopyrimidines. 

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