Inhibitors of Tetrahydrofolate Synthesis

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—from precursors. Selective interference with bacterial biosynthesis of THF can be achieved with sulfonamides and trimethoprim.

Sulfonamides structurally resemble p- aminobenzoic acid (PABA), a precursor in bacterial DHF synthesis. As false substrates, sulfonamides competitively inhibit utilization of PABA, and hence DHF synthesis. Because most bacteria cannot take up exogenous folate, they are depleted of DHF. Sulfonamides thus possess bacteriostatic activity against a broad spectrum of pathogens. Sulfonamides are produced by chemical synthesis. The basic structure is shown in (A). Residue R determines the pharmacokinetic properties of a given sulfonamide. Most sulfonamides are well absorbed via the enteral route. They are metabolized to varying degrees and eliminated through the kidney. Rates of elimination, hence duration of effect, may vary widely. Some members are poorly absorbed from the gut and are thus suitable for the treatment of bacterial bowel infections. Adverse effects may include allergic reactions, sometimes with severe skin damage (p.74), and displacement of other plasma protein-bound drugs or bilirubin in neonates (danger of kernicterus, hence contraindication for the last weeks of gestation and in the neonate). Because of the frequent emergence of resistant bacteria, sulfon-

amides are now rarely used. Introduced in 1935, they were the first broad-spectrum chemotherapeutics.

Trimethoprim inhibits bacterial DHF reductase, the human enzyme being significantly less sensitive than the bacterial one (rarely, bone marrow depression). A 2,4- diaminopyrimidine, trimethoprim has bacteriostatic activity against a broad spectrum of pathogens. It is used mostly as a component of co-trimoxazole.

Co-trimoxazole is a combination of trimethoprim and the sulfonamide sulfomethoxazole. Since THF synthesis is inhibited at two successive steps, the antibacterial effect of co-trimoxazole is better than that of the individual components. Resistant pathogens are infrequent; a bactericidal effect may occur. Adverse effects correspond to those of the components.

Sulfasalazine. Although originally developed as an antirheumatic agent (p.332), sulfasalazine (salazosulfapyridine) is used mainly in the treatment of inflammatory bowel disease (ulcerative colitis and terminal ileitis or Crohn disease). Gut bacteria split this compound into the sulfonamide sulfapyridine and mesalazine (5-aminosali- cylic acid). The latter is probably the antiinflammatory agent (inhibition of prostaglandin and leukotriene synthesis, of chemotactic signals for granulocytes, and of H2O2 formation in mucosa), but must be present on the gut mucosa in high concentrations. Coupling to the sulfonamide prevents premature absorption in upper small-bowel segments. The cleaved-off sulfonamide can be absorbed and may produce typical adverse effects (see above). Delayed release (prodrug) formulations of mesalazine without the sulfonamide moiety are available.

Inhibitors of DNA Function

Deoxyribonucleic acid (DNA) serves as a template for the synthesis of nucleic acids. Ribonucleic acid (RNA) executes protein synthesis and thus permits cell growth. Synthesis of new DNA is a prerequisite for cell division. Substances that inhibit reading of genetic information at the DNA template damage the regulatory center of cell metabolism. The substances listed below are useful as antibacterial drugs because they do not affect human cells.

Gyrase inhibitors. The enzyme gyrase (topoisomerase II) permits the orderly accommodation of a ~ 1000 µm long bacterial chromosome in a bacterial cell of ~ 1 µm. Within the chromosomal strand, double-stranded DNA has a double helical configuration. The former, in turn, is arranged in loops that are shortened by supercoiling. The gyrase catalyzes this operation, as illustrated, by opening, underwinding, and closing of the DNA double strand such that the full loop need not be rotated.

Derivatives of 4-quinolone-3-carboxylic acid (green portion of ofloxacin formula) are inhibitors of bacterial gyrases. They appear to prevent specifically the resealing of opened strands and thereby act bactericidally. These agents are absorbed after oral ingestion. The older drug nalidixic acid affects exclusively Gram-negative bacteria and attains effective concentrations only in urine; it is used as a urinary tract antiseptic. Norfloxacin has a broader spectrum. Ofloxacin, ciprofloxacin, enoxacin, and others, also yield systemically effective concentrations and are used for infections of internal organs.

Besides gastrointestinal problems and allergy, adverse effects particularly involve the CNS (confusion, hallucinations, and seizures). Since they can damage epiphyseal chondrocytes and joint cartilages in laboratory animals, gyrase inhibitors should not be used during pregnancy, lactation, and peri-

ods of growth. Tendon damage including rupture may occur in elderly or glucocorti- coid-treated patients. In addition, hepatic damage, prolongation of the QT-interval with risk of arrhythmias, and phototoxicity have been observed.

Nitroimidazole derivatives, such as metronidazole, damage DNA by complex formation or strand breakage. This occurs in obligate anaerobic bacteria. Under these conditions, conversion to reactive metabolites that attack DNA takes place (e.g., the hydroxylamine shown). The effect is bactericidal. A similar mechanism is involved in the antiprotozoal action on Trichomonas vaginalis (causative agent of vaginitis and urethritis) and Entamoeba histolytica (causative agent of large-bowel inflammation, amebic dysentery, and hepatic abscesses). Metronidazole is well absorbed via the enteral route; it is also given i.v. or topically (vaginal insert). Because metronidazole is considered potentially mutagenic, carcinogenic, and teratogenic in humans, it should not be used for longer than 10 days, if possible, and should be avoided during pregnancy and lactation. Timidazole may be considered equivalent to metronidazole.

Rifampin (rifampicin) inhibits the bacterial enzyme that catalyses DNA template-di- rected RNA transcription, i.e., DNA-depen- dent RNA polymerase. Rifampin acts bactericidally against mycobacteria (Mycobacterium tuberculosis, M. leprae), as well as many Gram-positive and Gram-negative bacteria. It is well absorbed after oral ingestion. Because resistance may develop with frequent usage, it is restricted to the treatment of tuberculosis and leprosy (p.282). Rifampin is contraindicated in the first trimester of gestation and during lactation.

Rifabutin resembles rifampin but may be effective in infections resistant to rifampin.