prof. aza Bacterial Protein Synthesis Inhibitors (Antimicrobials) Reference: Gareth Thomas Week 14 prof. aza 12. Bacterial Protein Synthesis Inhibitors Antimicrobials) Many protein inhibitors inhibit protein synthesis in both prokaryotic and eukaryotic cells (Table 10.2). Many protein inhibitors inhibit protein synthesis in both prokaryotic and eukaryotic cells (Table 10.2). This inhibition can take place at any stage in protein synthesis. This inhibition can take place at any stage in protein synthesis. However, some inhibitors have a specific action in that they inhibit protein synthesis in prokaryotic cells but not in eukaryotic cells, or vice versa. However, some inhibitors have a specific action in that they inhibit protein synthesis in prokaryotic cells but not in eukaryotic cells, or vice versa. Consequently, a number of useful drugs have been discovered that will inhibit protein synthesis in bacteria but either have no effect or a very much reduced effect on protein synthesis in mammals. Consequently, a number of useful drugs have been discovered that will inhibit protein synthesis in bacteria but either have no effect or a very much reduced effect on protein synthesis in mammals. The structures and activities of the drugs that inhibit protein synthesis are quite diverse. The structures and activities of the drugs that inhibit protein synthesis are quite diverse. prof. aza Consequently, only a few of the more commonly used drugs and structurally related compounds will be discussed in greater detail in this section. Consequently, only a few of the more commonly used drugs and structurally related compounds will be discussed in greater detail in this section. prof. aza Table Examples of drugs that inhibit protein synthesis. prof. aza Aminoglycosides Streptomycin (Figure 10.15) is a member of a group of compounds known as aminoglycosides. Streptomycin (Figure 10.15) is a member of a group of compounds known as aminoglycosides. These compounds have structures in which amino sugar residues in the form of mono- or polysaccharides are attached to a substituted 1,3- diaminocyclohexane ring by modified glycosidic type linkages. These compounds have structures in which amino sugar residues in the form of mono- or polysaccharides are attached to a substituted 1,3- diaminocyclohexane ring by modified glycosidic type linkages. The ring is either streptidine (streptomycin) or deoxy streptamine (kanamycin, neomycin, gentamicin and tobramycin). The ring is either streptidine (streptomycin) or deoxy streptamine (kanamycin, neomycin, gentamicin and tobramycin). prof. aza Figure The structures of (a) streptomycin and (b) neomiycin C. prof. aza Streptomycin is as the first aminoglycoside discovered (Schatz and co-workers. 1944) from cultures of the soil Actinomycetes Streptomyces griseus. Streptomycin is as the first aminoglycoside discovered (Schatz and co-workers. 1944) from cultures of the soil Actinomycetes Streptomyces griseus. It acts by interfering with the initiation of protein synthesis in bacteria. It acts by interfering with the initiation of protein synthesis in bacteria. The binding of streptomycin to the 30S ribosome inhibits initiation and also causes some amino acid-tRNA complexes to misread the mRNA codons. The binding of streptomycin to the 30S ribosome inhibits initiation and also causes some amino acid-tRNA complexes to misread the mRNA codons. prof. aza This results in the insertion of incorrect amino acid residues into the protein chain, which usually leads to the death of the bacteria. This results in the insertion of incorrect amino acid residues into the protein chain, which usually leads to the death of the bacteria. prof. aza The mode of action of the other aminoglycosides has been assumed to follow the same pattern even though most of the investigations into the mechanism of the antibacterial action of the aminoglycocides have been carried out The mode of action of the other aminoglycosides has been assumed to follow the same pattern even though most of the investigations into the mechanism of the antibacterial action of the aminoglycocides have been carried out prof. aza The clinically used aminoglycosides have structures closely related to that of streptomycin. The clinically used aminoglycosides have structures closely related to that of streptomycin. They are essentially broad- spectrum antibiotics although the are normally used to treat serious Gram-negative bacterial infections (see section ). They are essentially broad- spectrum antibiotics although the are normally used to treat serious Gram-negative bacterial infections (see section ). Aminoglycosidic drugs are very water soluble. They are usually administered as their water-soluble inorganic salts but their polar nature means that they are poorly absorbed when administered orally. Aminoglycosidic drugs are very water soluble. They are usually administered as their water-soluble inorganic salts but their polar nature means that they are poorly absorbed when administered orally. Once in the body they are easily distributed into most body fluids. Once in the body they are easily distributed into most body fluids. prof. aza However, their polar nature means that they do not easily penetrate the central nervous system (CNS), bone, fatty and connective tissue. However, their polar nature means that they do not easily penetrate the central nervous system (CNS), bone, fatty and connective tissue. Moreover, aminoglycosides tend to concentrate in the kidney where they are excreted by glomerular filtration. Moreover, aminoglycosides tend to concentrate in the kidney where they are excreted by glomerular filtration. prof. aza Figure 19. Kanamycin. prof. aza Aminoglycoside-drug-resistant strains of bacteria are not recognised as a serious medical problem. Aminoglycoside-drug-resistant strains of bacteria are not recognised as a serious medical problem. They arise because dominant bacteria strains have emerged that possess enzymes that effectively inactivate the drug. They arise because dominant bacteria strains have emerged that possess enzymes that effectively inactivate the drug. These enzymes act by catalysing the acylation, phosphorylation and adenylation of the drug (see section 6.13). This results in the formation of inactive drug derivatives. These enzymes act by catalysing the acylation, phosphorylation and adenylation of the drug (see section 6.13). This results in the formation of inactive drug derivatives. prof. aza The activity of the aminoglycosides is related to the nature of their ring substituents. The activity of the aminoglycosides is related to the nature of their ring substituents. Consequently, it is convenient to discuss this activity in relation to the changes in the substituents of individual rings but, in view of the diversity of the structures of aminoglycosides, it is difficult to identify common trends. Consequently, it is convenient to discuss this activity in relation to the changes in the substituents of individual rings but, in view of the diversity of the structures of aminoglycosides, it is difficult to identify common trends. prof. aza As a result, this discussion will be largely limited to kanamycin (Figure 10.19). As a result, this discussion will be largely limited to kanamycin (Figure 10.19). However, the same trends are often true for other aminoglycosides whose structures consist of three rings, including a central deoxystreptamine residue. However, the same trends are often true for other aminoglycosides whose structures consist of three rings, including a central deoxystreptamine residue. prof. aza Changing the nature of the amino substituents at positions 2 and 6 of ring I has the greatest effect on activity. Changing the nature of the amino substituents at positions 2 and 6 of ring I has the greatest effect on activity. For example, kanamycin A, which has a hydroxy group at position 2, and kanamycin C, which has a hydroxy group at position 6, are both less active than kanamycin B, which has amino groups at the 2 and 6 positions. For example, kanamycin A, which has a hydroxy group at position 2, and kanamycin C, which has a hydroxy group at position 6, are both less active than kanamycin B, which has amino groups at the 2 and 6 positions. However, the removal of one or both of the hydroxy groups at positions 3 and 4 does not have any effect on the potency of the kanamycins. However, the removal of one or both of the hydroxy groups at positions 3 and 4 does not have any effect on the potency of the kanamycins. prof. aza Modifications to ring II (the deoxystreptamine ring) greatly reduce the potency of the kanamycins. Modifications to ring II (the deoxystreptamine ring) greatly reduce the potency of the kanamycins. However, N-acylation and alkylation of the amino group at position I can give compounds with some activity However, N-acylation and alkylation of the amino group at position I can give compounds with some activity For example, acylation of kanamycin A gives 1-N-(L (-)-4- amino-2- hydroxybutyryl) kanamycin A (amikacin), which has a potency of about 50% of that of kanamycin A (Figure 10.20). For example, acylation of kanamycin A gives 1-N-(L (-)-4- amino-2- hydroxybutyryl) kanamycin A (amikacin), which has a potency of about 50% of that of kanamycin A (Figure 10.20). prof. aza In spite of this, amikacin is a useful drug for treating some strains of Gram- negative bacteria because it is resistant to deactivation by bacterial enzymes. Similarly, 1-N-ethylsisomicin (netilmicin) is as potent as its parent aminoglvcoside sisomicin. In spite of this, amikacin is a useful drug for treating some strains of Gram- negative bacteria because it is resistant to deactivation by bacterial enzymes. Similarly, 1-N-ethylsisomicin (netilmicin) is as potent as its parent aminoglvcoside sisomicin. prof. aza Changing the substituents of ring III does not usually have such a great effect on the potency of the drug as similar changes in ring I and II. Changing the substituents of ring III does not usually have such a great effect on the potency of the drug as similar changes in ring I and II. For example, removal of the 2 hydroxy group of gentamicin results in a significant drop in activity. For example, removal of the 2 hydroxy group of gentamicin results in a significant drop in activity. However, replacement of the 2 hydroxy group of gentamicin (Figure 21) by amino groups gives the highly active seldomycins. However, replacement of the 2 hydroxy group of gentamicin (Figure 21) by amino groups gives the highly active seldomycins. prof. aza Figure 20. An outline of the chemistry involved in the synthesis of the antibiotics amikacin and netilmicin. Cbz is frequently used as a protecting group for amines because it is easily removed by hydrogenation. prof. aza Figure The structures of gentamicin. prof. aza Chloramphenicol Chloramphenicol was first isolated from the microorganism Streptomyces venezuela by Ehrlich and co-workers in It is a broad-spectrum antibiotic whose structure contains two asymmetric centres. However. only the D(-)-threo form is active. prof. aza Chloramphenicol can cause serious side effects and so it is recommended that it is only used for specific infections. It is often administered as its palmitate in order to mask its bitter taste. Chloramphenicol can cause serious side effects and so it is recommended that it is only used for specific infections. It is often administered as its palmitate in order to mask its bitter taste. The free drug is liberated from this ester by hydrolysis in the duodenum chloramphenicol has a poor water solubility (2.5 g/dm-3 ) and So it is sometimes administered in the form of its sodium hemisuccinate salt (see section ), which acts as a prodrug. The free drug is liberated from this ester by hydrolysis in the duodenum chloramphenicol has a poor water solubility (2.5 g/dm-3 ) and So it is sometimes administered in the form of its sodium hemisuccinate salt (see section ), which acts as a prodrug. prof. aza Chloramphenicol acts by inhibiting the elongation stage in protein synthesis in prokaryotic cells. Chloramphenicol acts by inhibiting the elongation stage in protein synthesis in prokaryotic cells. It binds reversibly to the 50S ribosome subunit and is thought to prevent the binding of the aminoacyl-tRNA complex to the ribosome. It binds reversibly to the 50S ribosome subunit and is thought to prevent the binding of the aminoacyl-tRNA complex to the ribosome. However, its precise mode of action is not understood. However, its precise mode of action is not understood. Investigation of the activity of analogues of chloramphenicol showed that activity requires a para-electron- withdrawing group. Investigation of the activity of analogues of chloramphenicol showed that activity requires a para-electron- withdrawing group. prof. aza However, substituting the nitro group with other electron-withdrawing groups gave compounds with a reduced activity. Furthermore, modification of the side chain, with the exception of the difluoro derivative, gave compounds that had a lower activity than chloramphenicol (Table 10.3). However, substituting the nitro group with other electron-withdrawing groups gave compounds with a reduced activity. Furthermore, modification of the side chain, with the exception of the difluoro derivative, gave compounds that had a lower activity than chloramphenicol (Table 10.3). prof. aza These observations suggest that D(-)- threo chloramphenicol has the optimum structure of those tested for activity. These observations suggest that D(-)- threo chloramphenicol has the optimum structure of those tested for activity. prof. aza Investigation of the activity of analogues of chloramphenicol showed that activity requires a para-electron- withdrawing group. However, substituting the nitro group with other electron-withdrawing groups gave compounds with a reduced activity. Investigation of the activity of analogues of chloramphenicol showed that activity requires a para-electron- withdrawing group. However, substituting the nitro group with other electron-withdrawing groups gave compounds with a reduced activity. prof. aza Furthermore, modification of the side chain, with the exception of the difluoro derivative, gave compounds that had a lower activity than chloramphenicol (Table 10.3). Furthermore, modification of the side chain, with the exception of the difluoro derivative, gave compounds that had a lower activity than chloramphenicol (Table 10.3). These observations suggest that D(-)- threo chloramphenicol has the optimum structure of those tested for activity. These observations suggest that D(-)- threo chloramphenicol has the optimum structure of those tested for activity. prof. aza Table The activity against Escherichia coli of some analogues chloramphenicol relative to chloramphenicol. prof. aza 12.3 TetracycLines Tetracyclines are a family of natural and semisynthetic antibiotics isolated from various Streptomyces species, the first member of the group chlortetracycline being obtained in 1945 by Duggar from Streptomyces aureofaciens. A number of highly active semisynthetic analogues have also been prepared from the naturally occurring compounds (Table 10.4). Tetracyclines are a family of natural and semisynthetic antibiotics isolated from various Streptomyces species, the first member of the group chlortetracycline being obtained in 1945 by Duggar from Streptomyces aureofaciens. A number of highly active semisynthetic analogues have also been prepared from the naturally occurring compounds (Table 10.4). prof. aza Table 10.4.The structures of the tetracyclines. The tetracvclines are a broad-spectrum group of antibiotics active against many Gram-positive and Gram-negative bacteria, rickettsiae, mycoplasmas, chlamydiae and some protozoa that cause malaria. A number of the natural and semisynthetic compounds are in current medical use. The tetracvclines are a broad-spectrum group of antibiotics active against many Gram-positive and Gram-negative bacteria, rickettsiae, mycoplasmas, chlamydiae and some protozoa that cause malaria. A number of the natural and semisynthetic compounds are in current medical use. prof. aza The structures of the tetracyclines are based on four-ring system. Their structures are complicated by the presence of up to six chiral carbons in the fused-ring system. These normally occur at positions 4, 4a, 5, 5a, 6 and 12a, depending on the symmetry of the structure. The structures of the tetracyclines are based on four-ring system. Their structures are complicated by the presence of up to six chiral carbons in the fused-ring system. These normally occur at positions 4, 4a, 5, 5a, 6 and 12a, depending on the symmetry of the structure. The configurations of these centres in the active compounds have been determined by X- ray crystallography (Table 10.4). This technique has also confirmed that C1 to C3 and C11 to C12 were conjugated structures. The configurations of these centres in the active compounds have been determined by X- ray crystallography (Table 10.4). This technique has also confirmed that C1 to C3 and C11 to C12 were conjugated structures. prof. aza Table 10.4.The structures of the tetracyclines. prof. aza amphoteric, Tetracyclines are amphoteric, forming salts with acids and bases. They normally exhibit three pKa. ranges of 2.5 3.4 (pKa1 ), (pKa2.) and 9.19.7 (pKa3.), the last being the range for the corresponding ammonium salts. These values have been assigned by Leeson and co-workers to the structures shown in Table Tetracyclines are amphoteric, forming salts with acids and bases. They normally exhibit three pKa. ranges of 2.5 3.4 (pKa1 ), (pKa2.) and 9.19.7 (pKa3.), the last being the range for the corresponding ammonium salts. These values have been assigned by Leeson and co-workers to the structures shown in Table 10.4. forming stable chelates These assignments have been supported by the work of Rigler and collegues. However, the assignments for pKa2, and pKa3, are opposite to those suggested by Stephens and collegues. These assignments have been supported by the work of Rigler and collegues. However, the assignments for pKa2, and pKa3, are opposite to those suggested by Stephens and collegues. Tetracyclines also have a strong affinity for metal ions, forming stable chelates with calcium, magnesium and iron ions. Tetracyclines also have a strong affinity for metal ions, forming stable chelates with calcium, magnesium and iron ions. prof. aza affinity for metals These chelates are usually soluble in water, which accounts for the poor absorption of tetracyclines in the presence of drugs and foods that contain these metal ions. These chelates are usually soluble in water, which accounts for the poor absorption of tetracyclines in the presence of drugs and foods that contain these metal ions. However, this affinity for metals appears to play an essential role in the action of tetracyclines. However, this affinity for metals appears to play an essential role in the action of tetracyclines. prof. aza by preventing protein elongation Tetracyclines are transported into the bacterial cell by passive diffusion and active transport. Active transport requires the presence of Mg 2 ions and ATP possibly as an energy source. Tetracyclines are transported into the bacterial cell by passive diffusion and active transport. Active transport requires the presence of Mg 2 ions and ATP possibly as an energy source. Once in the bacteria, tetracyclines act by preventing protein elongation by inhibiting the binding of the aminoacyl- tRNA to the 30S subunit of the prokaryotic ribosome. Once in the bacteria, tetracyclines act by preventing protein elongation by inhibiting the binding of the aminoacyl- tRNA to the 30S subunit of the prokaryotic ribosome. This binding has also been shown to require magnesium ions. This binding has also been shown to require magnesium ions. prof. aza Tetracyclines also penetrate mammalian cells and bind to eukaryotic ribosomes. Tetracyclines also penetrate mammalian cells and bind to eukaryotic ribosomes. However, their affinity for eukaryotic ribosomes is lower than that for prokaryotic ribosomes and so they do not achieve a high enough concentration to disrupt eukaryotic protein synthesis. However, their affinity for eukaryotic ribosomes is lower than that for prokaryotic ribosomes and so they do not achieve a high enough concentration to disrupt eukaryotic protein synthesis. bacterial resistance Unfortunately, bacterial resistance to tetracyclines is common. It is believed to involve three distinct mechanisms, namely: active transport of the drug out of the bacteria by membrane spanning proteins; enzymic oxidation of the drug; and ribosome protection by chromosomal protein determinants. Unfortunately, bacterial resistance to tetracyclines is common. It is believed to involve three distinct mechanisms, namely: active transport of the drug out of the bacteria by membrane spanning proteins; enzymic oxidation of the drug; and ribosome protection by chromosomal protein determinants. prof. aza The structure-activity relationships of tetracyclines have been extensively investigated and reported. The structure-activity relationships of tetracyclines have been extensively investigated and reported. Consequently, the following paragraphs give only a synopsis of these relationships. Consequently, the following paragraphs give only a synopsis of these relationships. This synopsis only considers general changes to both the general structure of the tetracyclines (Figure 10.22) and the substitution patterns of their individual rings. This synopsis only considers general changes to both the general structure of the tetracyclines (Figure 10.22) and the substitution patterns of their individual rings. prof. aza Activity in the tetracyclines requires four rings with a cis A/B ring fusion. Activity in the tetracyclines requires four rings with a cis A/B ring fusion. Changes to the 4-dimethylamino group also usually reduce activity. This group must have an a-configuration and partial conversion of this group to its -epimer under acidic conditions at room temperature significantly reduces activity. Changes to the 4-dimethylamino group also usually reduce activity. This group must have an a-configuration and partial conversion of this group to its -epimer under acidic conditions at room temperature significantly reduces activity. prof. aza Figure General structure activity relationships in the tetracyclines. prof. aza In addition, either removal of the a- dimethylamino group at position 4 or replacement of one or more of its methyl groups by larger alkyl groups also reduces activity. In addition, either removal of the a- dimethylamino group at position 4 or replacement of one or more of its methyl groups by larger alkyl groups also reduces activity. prof. aza Ester formation at C-12a gives inactive esters, with the exception of the formyl ester, which hydrolyses in aqueous solution to the parent tetracycline. Alkylation of C-11a also gives rise to a loss of activity. Ester formation at C-12a gives inactive esters, with the exception of the formyl ester, which hydrolyses in aqueous solution to the parent tetracycline. Alkylation of C-11a also gives rise to a loss of activity. prof. aza Modification of the substituents at positions 5, 5a and 9 may lead to similar or increased activity. Modification of the substituents at positions 5, 5a and 9 may lead to similar or increased activity. Minor changes to the substituents at these positions tend to change the pharmacokinetic properties rather than activity (Table 10.5). Minor changes to the substituents at these positions tend to change the pharmacokinetic properties rather than activity (Table 10.5). prof. aza Table The pharmacokinetic properties of tetracycline hyrochlorides. The values given are representative values only because variations between individuals can be quite large. A number of active derivatives have been synthesized by electrophilic substitution of C-7 and C-9 but the effect of introducing substituents at C- 8 has not been studied because this position is difficult to substitute. A number of active derivatives have been synthesized by electrophilic substitution of C-7 and C-9 but the effect of introducing substituents at C- 8 has not been studied because this position is difficult to substitute. prof. aza