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Bacterial Protein Synthesis Bacterial Protein Synthesis InhibitorsInhibitors ( (Antimicrobials)Antimicrobials)

Reference: Reference: Gareth ThomasGareth Thomas

Week 14Week 14

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12. Bacterial Protein 12. Bacterial Protein Synthesis InhibitorsSynthesis Inhibitors

Antimicrobials)Antimicrobials)• Many protein inhibitors inhibit protein Many protein inhibitors inhibit protein

synthesis in both prokaryotic and synthesis in both prokaryotic and eukaryotic cells (Table 10.2). eukaryotic cells (Table 10.2).

• This inhibition can take place at any This inhibition can take place at any stage in protein synthesis. stage in protein synthesis.

• However, However, some inhibitors have a some inhibitors have a specific action in that they inhibit specific action in that they inhibit protein synthesis in prokaryotic cells protein synthesis in prokaryotic cells but not in eukaryotic cellsbut not in eukaryotic cells, or vice , or vice versa. versa.

• Consequently, a number of useful drugs Consequently, a number of useful drugs have been discovered that will inhibit have been discovered that will inhibit protein synthesis in bacteria protein synthesis in bacteria but but either either have no effect or a very much reduced have no effect or a very much reduced effect on protein synthesis in mammalseffect on protein synthesis in mammals..

• The structures and activities of the The structures and activities of the drugs that inhibit protein synthesis are drugs that inhibit protein synthesis are quite diverse. quite diverse.

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•Consequently, only a few of the Consequently, only a few of the more commonly used drugs and more commonly used drugs and structurally related compounds structurally related compounds will be discussed in greater detail will be discussed in greater detail in this section.in this section.

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prof. azaprof. aza

Table 10.2. Examples of drugs that inhibit protein synthesis.

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12.112.1.. Aminoglycosides Aminoglycosides

• Streptomycin (Figure 10.15) is a Streptomycin (Figure 10.15) is a member of a group of compounds member of a group of compounds known as aminoglycosides. known as aminoglycosides.

• These compounds have structures in These compounds have structures in which which aminoamino sugar residues sugar residues in the in the form of mono- or polysaccharides are form of mono- or polysaccharides are attached to a substituted attached to a substituted 1 ,3-1 ,3-diaminocyclohexane ring by modified diaminocyclohexane ring by modified glycosidic type linkages. glycosidic type linkages.

•The ring is either The ring is either streptidinestreptidine (streptomycin) or (streptomycin) or deoxy deoxy streptamine streptamine (kanamycin, (kanamycin, neomycin, gentamicin and neomycin, gentamicin and tobramycin).tobramycin).

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Figure 10.18. The structures of (a) streptomycin and (b) neomycin C.

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•Streptomycin Streptomycin iis as the first s as the first aminoglycoside discovered aminoglycoside discovered (Schatz and co-workers. 1(Schatz and co-workers. 19944) 44) from cultures of the soil from cultures of the soil ActinomyceteActinomycetess Streptomyces Streptomyces griseus. griseus.

• It acts by interfering with the It acts by interfering with the initiation of protein synthesis in initiation of protein synthesis in bacteria. bacteria.

• The binding of streptomycin to the The binding of streptomycin to the 30S ribosome inhibits initiation30S ribosome inhibits initiation and and also causes some amino acid-tRNA also causes some amino acid-tRNA complexes to complexes to misread the mRNA misread the mRNA codonscodons..

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•This results in This results in the insertion of the insertion of incorrect amino acid residues incorrect amino acid residues into the protein chain, which into the protein chain, which usually leads to the death of the usually leads to the death of the bacteriabacteria. .

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•The mode of action of the other The mode of action of the other aminoglycosides has been aminoglycosides has been assumed assumed to follow the same to follow the same pattern pattern even though most of the even though most of the investigations into the investigations into the mechanism of the antibacterial mechanism of the antibacterial action of the aminoglycocides action of the aminoglycocides have been carried out have been carried out

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prof. azaprof. aza

•The clinically used The clinically used aminoglycosides have structures aminoglycosides have structures closely related to that of closely related to that of streptomycin. streptomycin.

•They are They are essentially broad-essentially broad-spectrum antibiotics although spectrum antibiotics although the are normally used to treat the are normally used to treat serious Gram-negativeserious Gram-negative bacterial bacterial infections (see section 4.2.5.1). infections (see section 4.2.5.1).

• Aminoglycosidic drugs Aminoglycosidic drugs are very water are very water soluble. soluble. They are usually They are usually administered as their water-soluble administered as their water-soluble inorganic salts but their polar nature inorganic salts but their polar nature means that themeans that theyy are are poorly absorbed poorly absorbed when administered orally.when administered orally.

• Once in the body they are easily Once in the body they are easily distributed into most body fluids. distributed into most body fluids.

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• However, their polar nature means However, their polar nature means that that they do not easily penetrate the they do not easily penetrate the central nervous system (CNS), bone, central nervous system (CNS), bone, fatty and connective tissuefatty and connective tissue. .

• Moreover, aminoglycosides Moreover, aminoglycosides tend to tend to concentrate in the kidney concentrate in the kidney where thewhere theyy are excreted by glomerular filtration.are excreted by glomerular filtration.

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Figure 19. Kanamycin.

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• Aminoglycoside-drug-resistant Aminoglycoside-drug-resistant strains of bacteria are not recognised strains of bacteria are not recognised as a serious medical problem. as a serious medical problem.

• They arise because dominant They arise because dominant bacteria strains have emerged bacteria strains have emerged that that possess enzymes that effectively possess enzymes that effectively inactivate the druginactivate the drug. .

•These enzymes act by catalysing These enzymes act by catalysing the the acylationacylation,, phosphorylation phosphorylation and adenylation of the drug and adenylation of the drug (see (see section 6.13). This results in the section 6.13). This results in the formation of inactive drug formation of inactive drug derivatives.derivatives.

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• The activity of the aminoglycosides The activity of the aminoglycosides is is related to the nature of their ring related to the nature of their ring substisubstittuents. uents.

• Consequently, it is convenient to Consequently, it is convenient to discuss this activity in relation to the discuss this activity in relation to the changes in the substituents of individual changes in the substituents of individual rings but, in view of the diversity of the rings but, in view of the diversity of the structures of aminoglycosides, it is structures of aminoglycosides, it is difficult to identify common trends. difficult to identify common trends.

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• As a result, this discussion will As a result, this discussion will bbe e largely limited to kanamycin (Figure largely limited to kanamycin (Figure 10.19). 10.19).

• However, the same trends are often However, the same trends are often true for other aminoglycosides whose true for other aminoglycosides whose structures consist of three rings, structures consist of three rings, including a central deoxystreptamine including a central deoxystreptamine residue.residue.

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• Changing the nature of the amino Changing the nature of the amino substituents at positions 2’ and 6’ of ring I substituents at positions 2’ and 6’ of ring I has the greatest effect on activity. has the greatest effect on activity.

• For exampleFor example, , kanamycin A, which has a kanamycin A, which has a hydroxy group at position 2’, and hydroxy group at position 2’, and kanamycin C, which has a hydroxy kanamycin C, which has a hydroxy group at position 6’, are both less group at position 6’, are both less active than kanamycin B, active than kanamycin B, which has which has amino groups at the 2’ and 6’ amino groups at the 2’ and 6’ positionspositions. .

•However, the removal of one or However, the removal of one or both of the hydroxy groups at both of the hydroxy groups at positions 3’ and 4’ does not have positions 3’ and 4’ does not have any effect on the potency any effect on the potency of the of the kanamycins.kanamycins.

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• Modifications to ring II (the Modifications to ring II (the deoxystreptamine ring) deoxystreptamine ring) greatly greatly reduce the potency of the reduce the potency of the kanamycinskanamycins. .

• However, N-acylation and alkylation of However, N-acylation and alkylation of the amino group at position I can give the amino group at position I can give compounds with some activitcompounds with some activityy

•For example, acylation of For example, acylation of kanamycin A gives 1-N-(L (-)-4- kanamycin A gives 1-N-(L (-)-4- amino-2-hydroxybutyryl) amino-2-hydroxybutyryl) kanamycin A (amikacin), which kanamycin A (amikacin), which has a potency of about 50% of has a potency of about 50% of that of kanamycin A (Figure that of kanamycin A (Figure 10.20). 10.20).

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• In spite of this, amikacin is a useful In spite of this, amikacin is a useful drug for treating some strains of drug for treating some strains of Gram-negative bacteria because it is Gram-negative bacteria because it is resistant to deactivation by bacterial resistant to deactivation by bacterial enzymes. Similarly, 1-N-enzymes. Similarly, 1-N-ethylsisomicin (netilmicin) is as ethylsisomicin (netilmicin) is as potent as its parent aminoglvcoside potent as its parent aminoglvcoside sisomicin.sisomicin.

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• Changing the substituents of ring III Changing the substituents of ring III does not usually have such a great does not usually have such a great effect on the potency of the drug as effect on the potency of the drug as similar changes in ring I and II.similar changes in ring I and II.

• For example, removal of the For example, removal of the 2” 2” hydroxy group of gentamicin results hydroxy group of gentamicin results in a in a significant drop in activity. significant drop in activity.

• However, replacement of the 2” However, replacement of the 2” hydroxhydroxy y group of gentamicin (Figure group of gentamicin (Figure 21) by amino groups gives the highly 21) by amino groups gives the highly active seldomycins.active seldomycins.

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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.

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Figure 10.21. The structures of gentamicin.

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12.2. Chloramphenicol12.2. ChloramphenicolChloramphenicol was first isolated from the microorganism Streptomyces venezuela by Ehrlich and co-workers in 1947. It is a broad-spectrum antibiotic whose structure contains two asymmetric centres. However. only the D(-)-threo form is active.

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•Chloramphenicol can cause Chloramphenicol can cause serious side effects and so it is serious side effects and so it is recommended that it is only recommended that it is only used for specific infections. used for specific infections.

• It is often administered as its It is often administered as its palmitate in order to mask its palmitate in order to mask its bitter taste.bitter taste.

• The free drug is liberated from this The free drug is liberated from this ester by hydrolysis in the duodenum ester by hydrolysis in the duodenum chloramphenicol has a poor water chloramphenicol has a poor water solubility (2.5 gsolubility (2.5 g//dm-3 ) and So it is dm-3 ) and So it is sometimes administered in the form sometimes administered in the form of its of its sodium hemisuccinate salt (see sodium hemisuccinate salt (see section 3.7.4.2), which acts as a section 3.7.4.2), which acts as a prodrug.prodrug.

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prof. azaprof. aza

•ChChlloramphenicol acts by oramphenicol acts by inhibiting the elongation stage in inhibiting the elongation stage in protein synthesis in prokaryotic protein synthesis in prokaryotic cells. cells.

• It binds reversibly to the 50S It binds reversibly to the 50S ribosome subunit and is thought ribosome subunit and is thought to prevent the binding of the to prevent the binding of the aminoacyl-tRNA complex to aminoacyl-tRNA complex to the ribosome. the ribosome.

• However, its precise mode of action However, its precise mode of action is not understood.is not understood.

• Investigation of the activity of Investigation of the activity of analogues of chloramphenicol analogues of chloramphenicol showed showed that activity requires a para-that activity requires a para-electron-withdrawing group. electron-withdrawing group.

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• However, substituting the nitro group However, substituting the nitro group with other electron-withdrawing groups with other electron-withdrawing groups gave compounds with a reduced gave compounds with a reduced activity. Furthermore, modification of activity. Furthermore, modification of the side chain, with the exception of the side chain, with the exception of the difluoro derivative, gave the difluoro derivative, gave compounds that had a lower activity compounds that had a lower activity than chloramphenicol (Table 10.3). than chloramphenicol (Table 10.3).

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• These observations suggest that These observations suggest that D(-)-threo chloramphenicol has the D(-)-threo chloramphenicol has the optimum structure of those tested for optimum structure of those tested for activity.activity.

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• Investigation of the activity of Investigation of the activity of analogues of chloramphenicol analogues of chloramphenicol showed that activity requires a para-showed that activity requires a para-electron-withdrawing group. electron-withdrawing group. However, However, substituting the nitro group substituting the nitro group with other electron-withdrawing with other electron-withdrawing groups gave compounds with a groups gave compounds with a reduced activityreduced activity. .

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• Furthermore, modification of the side Furthermore, modification of the side chain, with the exception of the chain, with the exception of the difluoro derivativedifluoro derivative, gave compounds , gave compounds that had a lower activity than that had a lower activity than chloramphenicolchloramphenicol (Table 10.3). (Table 10.3).

• These observations suggest that These observations suggest that D(-)-threo chloramphenicol has the D(-)-threo chloramphenicol has the optimum structure of those tested for optimum structure of those tested for activity.activity.

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Table 10.3. The activity against Escherichia coli of some analogues chloramphenicol relative to chloramphenicol.

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12.3 TetracycLines12.3 TetracycLines

• Tetracyclines are a family of natural Tetracyclines are a family of natural and semisynthetic antibiotics and semisynthetic antibiotics isolated from various Streptomyces isolated from various Streptomyces species, the first member of the species, the first member of the group chlortetracycline being group chlortetracycline being obtained in 1945 by Duggar from obtained in 1945 by Duggar from Streptomyces aureofaciens.Streptomyces aureofaciens.

• A number of highly active A number of highly active semisynthetic analogues have also semisynthetic analogues have also been prepared from the naturally been prepared from the naturally occurring compounds (Table 10.4). occurring compounds (Table 10.4).

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Table 10.4.The structures of the tetracyclines.

• The tetracvclines are The tetracvclines are a broad-spectrum a broad-spectrum group of antibiotics active against group of antibiotics active against many Gram-positive and Gram-many Gram-positive and Gram-negative negative bacteria, rickettsiae, bacteria, rickettsiae, mycoplasmas, chlamydiae and some mycoplasmas, chlamydiae and some protozoa that cause malaria. A number protozoa that cause malaria. A number of the natural and semisynthetic of the natural and semisynthetic compounds are in current medical use.compounds are in current medical use.

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•The structures of the The structures of the tetracyclines are based on four-tetracyclines are based on four-ring system. Their structures are ring system. Their structures are complicated by the presence of complicated by the presence of up to six chiral carbons in the up to six chiral carbons in the fused-ring systemfused-ring system. These . These normally occur at positions 4, 4a, normally occur at positions 4, 4a, 5, 5a, 6 and 12a, depending on 5, 5a, 6 and 12a, depending on the symmetry of the structure. the symmetry of the structure.

• The configurations of these centres in the The configurations of these centres in the active compounds have been determined active compounds have been determined by X-ray crystallography (Table 10.4). This by X-ray crystallography (Table 10.4). This technique has also confirmed that technique has also confirmed that C1 to C1 to C3 and C11 to C12 were conjugated C3 and C11 to C12 were conjugated structures.structures.

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Table 10.4.The structures of the tetracyclines.

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amphoteric,amphoteric,

•Tetracyclines are amphoteric, Tetracyclines are amphoteric, forming salts with acids and forming salts with acids and bases. bases. They normally exhibit They normally exhibit three pKa. ranges of 2.5 —3.4 three pKa. ranges of 2.5 —3.4 (pKa1 ), 7.2-7.5 (pKa2.) and 9.1(pKa1 ), 7.2-7.5 (pKa2.) and 9.1—9.7 (pKa3.), —9.7 (pKa3.), the last being the the last being the range for the corresponding range for the corresponding ammonium salts. These values ammonium salts. These values have been assigned by Leeson have been assigned by Leeson and co-workers to the structures and co-workers to the structures shown in Table 10.4. shown in Table 10.4.

forming stable chelatesforming stable chelates

• These assignments have been supported These assignments have been supported by the work of Rigler and collegues. by the work of Rigler and collegues. However, the assignments for pKa2, and However, the assignments for pKa2, and pKa3, are opposite to those suggested pKa3, are opposite to those suggested by Stephens and collegues. by Stephens and collegues.

• Tetracyclines also have a strong affinity Tetracyclines also have a strong affinity for metal ions, forming stable chelates for metal ions, forming stable chelates with calcium, magnesium and iron ions. with calcium, magnesium and iron ions.

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affinity for metalsaffinity for metals

• These chelates are usually soluble in These chelates are usually soluble in water, which accounts for water, which accounts for the poor the poor absorption of tetracyclines in the absorption of tetracyclines in the presence of drugs and foods that presence of drugs and foods that contain these metal ions. contain these metal ions.

• HoweverHowever, , this affinity for metals this affinity for metals appears to play an essential role in appears to play an essential role in the action of tetracthe action of tetracyyclines.clines.

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by preventing protein elongationby preventing protein elongation

•Tetracyclines are transported Tetracyclines are transported into the bacterial cell by passive into the bacterial cell by passive diffusion and active transport. diffusion and active transport. Active transport requires the Active transport requires the presence of Mgpresence of Mg22 ions and ATP ions and ATP possibly as an energy source. possibly as an energy source.

• Once in the bacteria, Once in the bacteria, tetracyclines tetracyclines act by preventing protein elongation act by preventing protein elongation by inhibiting the binding of the by inhibiting the binding of the aminoacyl-tRNA to the 30S subunit of aminoacyl-tRNA to the 30S subunit of the prokaryotic ribosome. the prokaryotic ribosome.

• This binding has also been shown to This binding has also been shown to require magnesium ions.require magnesium ions.

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•Tetracyclines also Tetracyclines also penetrate penetrate mammalian cells and bind to mammalian cells and bind to eukaryotic ribosomes. eukaryotic ribosomes.

•However, However, their affinity for their affinity for eukaryotic ribosomes is lower than eukaryotic ribosomes is lower than that for prokaryotic ribosomes that for prokaryotic ribosomes and and so they so they do not achieve a high do not achieve a high enough concentration to disrupt enough concentration to disrupt eukaryotic protein synthesiseukaryotic protein synthesis. .

bacterial resistancebacterial resistance

• Unfortunately, bacterial resistance to Unfortunately, bacterial resistance to tetracyclines is common. It is believed to tetracyclines is common. It is believed to involve three distinct mechanisms, involve three distinct mechanisms, namely: namely:

• active transport of the drug out of the active transport of the drug out of the bacteria by membrane spanning proteins; bacteria by membrane spanning proteins;

• enzymic oxidation of the drug; enzymic oxidation of the drug;

• and ribosome protectionand ribosome protection by chromosomal by chromosomal protein determinants.protein determinants.

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structure-activity relationships of structure-activity relationships of tetracyclinestetracyclines

•The structure-activity The structure-activity relationships of tetracyclines relationships of tetracyclines have been extensively have been extensively investigated and reported. investigated and reported.

•Consequently, the following Consequently, the following paragraphs give only a synopsis paragraphs give only a synopsis of these relationships.of these relationships.

•This synopsis only considers This synopsis only considers general changes to both the general changes to both the general structure of the general structure of the tetracyclines (Figure 10.22) and tetracyclines (Figure 10.22) and the substitution patterns of their the substitution patterns of their individual rings.individual rings.

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•Activity in the tetracyclines Activity in the tetracyclines requires four rings with requires four rings with a cis A/B a cis A/B ring fusion. ring fusion.

• Changes to the 4-dimethylamino Changes to the 4-dimethylamino group also usually reduce activitygroup also usually reduce activity. . This group must have an a-This group must have an a-configuration and partial conversion configuration and partial conversion of this group to its of this group to its β-epimeβ-epimer under r under acidic conditions at room temperature acidic conditions at room temperature significantly reduces activity.significantly reduces activity.

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Figure 10.22. General structure activity relationships in the tetracyclines.

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• In addition, either removal of the a-In addition, either removal of the a-dimethdimethyylamino group at position 4 or lamino group at position 4 or replacement of one or more of its replacement of one or more of its methyl groups by larger alkyl groups methyl groups by larger alkyl groups also reduces activity. also reduces activity.

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• Ester formation at C-12a gives Ester formation at C-12a gives inactive esters, with the exception of inactive esters, with the exception of the formyl ester, which hydrolyses in the formyl ester, which hydrolyses in aqueous solution to the parent aqueous solution to the parent tetracycline. Alkylation of C-11a also tetracycline. Alkylation of C-11a also gives rise to a loss of activity.gives rise to a loss of activity.

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• Modification of the substituents at Modification of the substituents at positions 5, 5a. 6. 7. 8 and 9 may positions 5, 5a. 6. 7. 8 and 9 may lead to similar or increased activity. lead to similar or increased activity.

• Minor changes to the substituents at Minor changes to the substituents at these positions tend to change the these positions tend to change the pharmacokinetic properties rather pharmacokinetic properties rather than activity (Table 10.5). than activity (Table 10.5).

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Table 10.5. 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 A number of active derivatives have been synthesibeen synthesizzed by electrophilic ed by electrophilic substitution of C-7 and C-9 but the substitution of C-7 and C-9 but the effect of introducing sueffect of introducing subbssttituents at ituents at C-8 has not been studied because C-8 has not been studied because this position is difficult to substitute.this position is difficult to substitute.

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