[align=center]Antibiotics that inhibit the primary metabolites[/align]
Most microbiologists distinguish two groups of antimicrobial agents used in the treatment of infectious disease:
1- Antibiotics, which are natural substances produced by certain groups of microorganisms,
2- Chemotherapeutic agents, which are chemically synthesized.
A hybrid substance is a semi synthetic antibiotic, where in a molecular version produced by the microbe is subsequently modified by the chemist to achieve desired properties. Furthermore, some antimicrobial compounds, originally discovered as products of microorganisms, can be synthesized entirely by chemical means. They might be referred to as synthetic antibiotics to distinguish them from the chemotherapeutic agents .
The modern era of antimicrobial chemotherapy began in 1929 with Fleming's discovery of the powerful bactericidal substance penicillin, and Domagk's discovery in 1935 of synthetic chemicals (sulfonamides) with broad antimicrobial activity .
The most important property of an antimicrobial agent, from a host point of view, is its selective toxicity, i.e., that the agent acts in some way that inhibits or kills bacterial pathogens but has little or no toxic effect on the host. This implies that the biochemical processes in the bacteria are in some way different from those in the animal cells, and that the advantage of this difference can be taken in chemotherapy .
Characteristics of Antibiotics
Antibiotics are low-molecular weight substances that are produced as secondary metabolites by certain groups of microorganisms, especially Streptomyces, Bacillus, and a few molds (Penicillium and Cephalosporium). Antibiotics may have a cidal (killing) effect or a static (inhibitory) effect on a range of microbes. Antibiotics effective against procaryotes which kill or inhibit a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum. If effective mainly against Gram-positive or Gram-negative bacteria, they are narrow spectrum. If effective against a single organism or disease, they are referred to as limited spectrum .
A clinically-useful antibiotic should have as many of these characteristics as possible:
1- It should have a wide spectrum of activity with the ability to destroy or inhibit many different species of pathogenic organisms.
2- It should be nontoxic to the host and without undesirable side effects.
3- It should be nonallergenic to the host.
4- It should not eliminate the normal flora of the host.
5- It should be able to reach the part of the human body where the infection is occurring.
6- It should be inexpensive and easy to produce.
7- It should be chemically-stable (have a long shelf-life).
8- Microbial resistance is uncommon and unlikely to develop .
Many of the synthetic chemotherapeutic agents are competitive inhibitors of essential metabolites or growth factors that are needed in bacterial metabolism . Also called primary metabolites, such as phytosterols, acyl lipids, nucleotides, amino acids, and organic acids, are found in all plants and perform metabolic roles that are essential and usually evident .
Hence, these types of antimicrobial agents are sometimes referred to as anti-metabolites or growth factor analogs, since they are designed to specifically inhibit an essential metabolic pathway in the bacterial pathogen. At a chemical level, competitive inhibitors are structurally similar to a bacterial growth factor or metabolite, but they do not fulfill their metabolic function in the cell. Some are bacteriostatic and some are bactericidal .
Sulfonamides were introduced as chemotherapeutic agents by Domagk in 1935, who showed that one of these compounds (prontosil) had the effect of curing mice with infections caused by beta-hemolytic streptococci. Chemical modifications of the compound sulfanilamide gave compounds with even higher and broader antibacterial activity. The resulting sulfonamides have broadly similar antibacterial activity, but differ widely in their pharmacological actions .
Bacteria which are almost always sensitive to the sulfonamides include Streptococcus pneumoniae, beta-hemolytic streptococci and E. coli .
The sulfonamides (e.g. Gantrisin) and Trimethoprim are inhibitors of the bacterial enzymes required for the synthesis of tetrahydofolic acid (THF), the vitamin form of folic acid essential for 1-carbon transfer reactions. Sulfonamides are structurally similar to para aminobenzoic acid (PABA), the substrate for the first enzyme in the THF pathway, and they competitively inhibit that step .
Trimethoprim is structurally similar to dihydrofolate (DHF) and competitively inhibits the second step in THF synthesis mediated by the DHF reductase. Animal cells do not synthesize their own folic acid but obtain it in a preformed fashion as a vitamin. Since animals do not make folic acid, they are not affected by these drugs, which achieve their selective toxicity for bacteria on this basis .
Introduction to inhibition of cell metabolism
The drugs described in this chapter are effective against the enzyme- catalyzed reactions of intermediary metabolism in micro-organisms, but they ultimately affect the synthesis of macromolecules .
Drugs that show structural similarity to the normal substrate of an enzyme may bind to the active site of the enzyme in place of the substrate, but do not react to give a product. Such compounds are competitive inhibitors, and the degree of inhibition depends on the concentration on the substrate, the concentration of the inhibitors and the affinities of each for binding to the enzyme .
Folate is a coenzyme essential for cell growth, but most bacteria cannot transport folate, and most manufacture it denovo. By contrast, humans cannot make folate and must ingest it as a vitamin. Folate biosynthesis is thus an ideal target for antimicrobial drugs .
The discovery of sulfonamides:
The studies of Ehrlich on the selective binding of days to cell led to the discovery of the sulfonamide drugs. A reduction product of the day named streptozon introduced in 1935 namely sulfonamide, was effective as bacterial inhibitor both invitro and invivo. The reductive cleavage to sulfanilamide occurs in the liver as shown in (figure 1), and it was concluded that sulfanilamide was responsible for the antibacterial action of prontosil .
The mechanism of action of sulfonamides
Sulfonamides inhibit growing micro-organisms and extracts of bacteria or yeast compete with this antibacterial action. In 1940, woods suggested that a metabolite or growth factor was similar in structure to sulfanilamide, and synthetic p-aminobenzoic acid (pABA) was highly effective in this role. The essential cell component coenzyme folate was, inhibited by sulfanilamide .
Sulfonamides inhibit the incorporation of pABA into a precursor of dihydrofolic acid (DHF) that should then be reduced by the enzyme dihydrofolate reductase (DHFR) to tetrahydrofolic acid (THF) as in (figure 2) .
Because pABA and sulfonamide compete for the same enzyme, the inhibitory effects of sulfonamides can be reversed if exogenous pABA is available in sufficiently high concentrations. Other drugs (e.g. trimethoprim) that inhibit the conversation of dihydrofolate to tetrahydrofolate also limit the supply of these biosynthetic components .
As well as being competitive inhibitors of this enzyme, sulfonamides may also act as substrates of dihydropteroate synthetase, and [ 35S] sulfonamide causes the accumulation of labeled analogs of folic acid .
Sensitive bacteria and resistance to sulfonamide:
Bacteria that are sensitive to sulfonamides include some strains of streptococci. Because the sulfonamides inhibit the synthesis of nucleotides and amino acids, the drugs are not effective if these compounds are available from exogenous sources (Figure 3) .
The role of sulfonamides in the competitive inhibition of the folic acid synthesis (Figure 3) implies that resistance may be due to acquired impermeability of the bacterial membrane to the drugs, or to increased production of the substrate (pABA) to overcome the inhibition, or to a change in the properties of dihydropteroic acid synthetase so that the enzyme binds to the inhibitor less readily. The increased intracellular concentration of pABA in resistant bacteria may be due to a loss of the normal end-product inhibition of pABA synthesis .
Pharmacology of sulfonamides:
The rate of excretion of sulfonamides and their metabolites depends on their properties and on kidney function. In kidney failure they may accumulate and doses must be reduced, or sulfonamides avoided when kidney function is impaired. The risk of formation of crystals in the urine (crystaluria) is reduced with highly water-soluble sulfonamides .
Other folic acid antimetabolites:
The search for useful chemotherapeutic agents has involved the synthesis of other competitors of p-ABA. The growth of the tubercle bacillus Mycobacterium tuberculosis was inhibited by p-aminosalicylic acid (pAS) (Figure 4). The drug is thought to inhibit folic acid synthesis as an analog of pABA. The specificity implies an ability of the target enzyme to bind pAS that is not found in other micro-organisms. Analogs of folate are formed from the pAS used in the treatment of M. tuberculosis .
The sulfon drug dapsone (4:4 diaminodiphenylsulfone ) (Figure 4) was able to inhibit M. tuberculosis. The mode of action is presumed to be similar to that of the sulfonamides and pAS. The derivative 4:4-diacetyldapsone (which has also been used as a repository antimalarial drug) has a longer duration of action in the treatment of leprosy than dapsone .
Dihydrofolate reductase inhibitors:
The enzyme DHFR reduces dihydrofolate to tetrahydrofolate, the active form of folic acid (Figure 2), which is essential to all cells, whether they manufacture folic acid (sulfonamide-sensitive bacteria) or require pre-synthesized folic acid (vitamin-requiring higher organism). Analogs that closely resemble folic acid (e.g. methotrexate) (Figure 5) are taken up by mammalian cells and inhibit the mammalian DHFR. Such folate analogs are not absorbed by bacteria and are therefore of no use in the chemotherapy of bacterial infections .
The methylation that forms the thymine base of thymidine (Figure 6) takes place at the level of the nucleoside monophosphate (dTMP), and is significant because the reaction converts methylene tetrahydrofolate to the oxidized dihydrofolate .
Trimethoprim and other diaminopyrimidines:
The 2:4 diaminopyrimidines are a series of drugs identified by screening for compounds that selectively inhibit bacterial DHFR.Trimethoprim binds to the bacterial DHFR, preventing the conversion of DHF into the useful form. The inhibition achieved depends on the nutrients available, and thymidine specifically reverses the effects. The result of reductase inhibition is the cessation of synthesis of purines, pyrimidines, methionine, glycine, histidine, pantothenic acid and N-formylmethionyl tRNA .
Trimethoprim was originally designated a sulfonamide potentiator , although it is increasingly used alone. It was proposed that the trimethoprim-sulfamethoxazole (cotrimoxazole) combination was less susceptible to resistance. Because of the rapid onset of activity of trimethoprim, the combination is reported to be more immediately effective than sulfonamides alone .
Fansider, proguanil and trimetrexate in eukaryotes:
The Fansider combination includes the effective inhibitor of protozoal DHFR, pyrimethamine with sulfadoxidine, and is used for the prophylaxis and treatment of malaria. Proguanil which has also been used in the prophylaxis of malaria, is metabolized in the liver to a substance that resembles pyrimethamine and is also a DHFR inhibitor in plasmodia. Trimetrexate has been used as an antitumor agent, but is also used to treat the fungus P. carinii in AIDS patients .
Pharmacology of the DHFR inhibitors:
Trimethoprim is largely excreted unmetabolized in the urine, and because it is a weak base, excretion is particularly rapid if the urine is acidic. The side-effects of trimethoprim are similar to those of sulfonamides. Cotrimoxazole is well absorbed from the gastrointestinal tract, and the combination is reported to give the optimum synergistic plasma levels of the two compounds .