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ANTIBIOTIC TARGETS AND MECHANISMS OF ACTION

Approximately23 unique classes and 18subclasses of clinically useful antibiotics representing approximately 100 antibiotics are used in clinical medicine. Although the classification scheme and number of antibiotics is complex and continues to expand as new classes emerge or existing classes are modified, their mechanisms of action target bacterial cell wall biosynthesis, folate synthesis, DNA replication, RNA transcription, and mRNAtranslation. These are logical targets critical to the survival of the microorganism. In addition, understanding of the mechanisms of action of antibiotics allows insight into strategies used by microorganisms to survive their toxic effects. Figure 12-1 shows the primary sites of antibacterial action for major classes of antimicrobial agents.

 

Inhibition of Bacterial Cell Wall Biosynthesis

Gram-positive and gram-negative bacteria have a multilayered cell wall structure composed of an inner cytoplasmic membrane, which overlies the cytoplasm, the peptidoglycan layer, and an outer membrane present only in gram-negative bacteria (Figure 12-2).The cytoplasmic membrane, composed of phospholipids and proteins, surrounds the cytoplasm, acts as an osmotic barrier, and is the location of the electron transport chain responsible for energy production. The peptidoglycan (murein) layer is composed of alternating N-acetyl-o-glucosamine (NAG) and N-acetyl-Dmuramic acid (NAM) chains that make up the glycan polysaccharide (Figure 12-3). In gram-positive bacteria, the peptidoglycan is substantially thicker and more multilayered than in gram-negative bacteria. The outer membrane of gram-negative bacteria is composed of Iipopolysaccharides, phospholipids, and porin proteins and is separated from the cytoplasmic membrane by a peri plasmic space.

Under normal growing conditions, peptidoglycan synthesis proceeds by the ligase-mediated formation of D-ala D-ala, a precursor used to form UDP-N-acetylmuramyl- pentapeptide (see Figure 12-3). This precursor molecule elongates peptidoglycan by transglycosylation of the glycan strands and elongates the peptide strands by transpeptidation. So strengthened, the peptidoglycan layer allows the microorganism to resist osmotic changes in its environment. ~-Iactam and glycopeptide antibiotics are examples of bacterial cell wall synthesis inhibitors. I3-lactam antibiotics, such as pen ems, cephems, carbapenems, and monobactams act by binding to penicillinbinding proteins (PBP), which are bifunctional transpeptidases/transglycosylases that cross-link peptidoglycan (Table 12-1).

The active moiety of B-Iactam is the four-membered B-lactam ring, a ring structure found in penicillins, cephalosporins, monobactams, and carbapenems (Figure 12-4). This four-membered ring represented by the structure labeled B-Iactam, functions as a structural analogue of the normal substrate acyl-D-alanyl-D-alanine and inhibits the transpeptidation reaction, resulting in bacterial lysis and cell death. The narrow to broad-spectrum antimicrobial activity, safety, and efficacy properties of B-Iactam antibiotics are typically enhanced by modification of moieties attached to the penicillin and cephalosporin ring structures (see Figure 12-4).

Glycopeptides, such as vancomycin and the investigational drug teicoplanin, act by complexing the non-cross-Iinked peptide strands of the peptidoglycan units having the pentapeptidyl tails ending in D-ala-Dala

(see Figure 12-3). This prevents their incorporation into the peptidoglycan chain by blocking the transpeptidation step. Glycopeptides bind to the substrate of the transpeptidation enzyme while penicillins bind to the enzyme mediating the transpeptidation reaction.

Glycopeptides are narrow-spectrum antibiotics, and the spectrum and clinical use are limited to gram-positive

microorganisms because they cannot penetrate the outer membrane of gram-negative bacteria. Vancomycin and teicoplanin are glycopeptides used to treat aerobic clinical infections caused by staphylococci, streptococci, and enterococci in the United States.

 

Inhibition of Folate Synthesis

Antibiotics are also capable of interfering with intracellular anabolic processes. The folic acid pathway provides the essential precursor molecule, pyridime thymidylate, needed in DNA biosynthesis. The pathway is mediated by two key enzymes: dihydropteroate synthase, which mediates the formation of 7,8 dihydropteroate from para-aminobenzoic acid. and dihydrofolate reductase, which mediates the formation of tetrahydrofolate (fHF) from dihydrofolate (Figure 12-5).

Sulfamethoxazole blocks the step leading to the formation of 7,8 dihydropteroate by competitively inhibiting the binding of the structural analogue paraaminobenzoic acid with dihydropteroate synthase.

Trimethoprim blocks the step leading to formation of THF by preventing the dihydrofolate reductasemediated

recycling of folate coenzymes. Unlike other members of the current antibiotic classes. Sulfamethoxazole (SMZ) and trimethoprim (fMP) are completely synthetic molecules that do not exist in or ever have existed in nature. The spectrum of activity of folate

Interference of DNA Replication

The prokaryotic cell cycle consists of DNA replication followed immediately by cell division. In microorganisms such as Escherichia coli that divide in approximately 30 minutes under ideal growth conditions, DNA replication must be initiated and completed to ensure that each DNA duplex is delivered to each daughter cell. The enzymes necessary for DNA replication are topoisomerases I, II, III, and IV.

Quinolones are antibiotics that affect DNA replication by targeting topoisomerases II (DNA gyrase) and IV, enzymes considered important in controlling DNA topology, replication, and decatenation at the end of bacterial DNA replicative cycle. DNA gyrase and topoisomerase IV are tetrameric molecules composed of dimeric A and B subunits. The subunits of DNA gyrase are encoded by gyrA and gyrB, while the subunits of topoisomerase IV are encoded by par( and parE. The tetramers of DNA gyrase and topoisomerase IV are highly homologous, with gyrA homologous to par( and gyrB homologous to parE. Interestingly, the targets of quinolones appear to be selective, targeting DNA gyrase in gram-negative bacteria and topoisomerase IV in gram-positive bacteria, but newer quinolones appear to have high affinity for both targets.

Analyses of mechanism of action suggest quinolones interact with DNA gyrase-DNA complexes and topoisomerase IV-DNA complexes to trap the enzymes as stabilized

reaction intermediates forming barriers to further DNA replication. The qui no lanes and fIuoroquinolones are used to treat the Enterobacteriaceae, pseudomonads, and other non-Enterobacteriaceae, staphylococci, enterococci, neisseria, and streptococci species other than Streptococcus pneumoniae.

Interference of DNA Transcription

Transcription is the process by which a template DNA strand is copied into a functional RNA sequence, resulting in mature mRNA or structural RNA. The transcription of DNA into RNA is mediated by RNA polymerase; bacterial RNA polymerase is a core tetramer composed of an a subunit, two ~ subunits (~W), a y subunit, and a dissociable a subunit that controls transcription of particular gene classes.

Rifampin, a synthetic derivative of rifamycin B, is used in combination with other antibiotic classes to treat Mycobacterium tuberculosis by targeting transcription of DNA. The target of rifampin in M. tuberculosis is the RNA polymerase ~ subunit at an allosteric site, with the subsequent blocking of RNA chain elongation.

As a result, RNA transcription is aborted at the initiation step. Aerobic species treated with rifampin include staphylococci, enterococci, Haemophilus, and S. pneumoniae.

 

Interference of mRNA Translation

The cellular machinery of living organisms decodes mRNA into functional protein, a process defined as mRNA translation. Protein biosynthesis requires the sequential binding of the 305 and 505 ribosomal subunits to mRNA leading to translation of the genetic message. The initiation phase commences with initiation factors, proteins that bind to the 305 subunit, and the initiator transfer ribonucleic acid (tRNA), formylmethionyl tRNA, which binds to the P site of the 305 ribosomal subunit. This 305 subunit helps the bound initiator tRNAscan and find the start codon on mRNA.

Next, the 505 subunit binds to form the preinitiation complex. The codon immediately following the initiation codon dictates binding of the next tRNA to the ribosomal A site. Because protein syn-thesis is central to cellular function, it is an excellent target for antibiotic drug product development. Thus the bacteria ribosome is a primary target of numerous antibiotics with some targeting the 305 ribosomal subunit (Le., aminoglycosides,tetracyclines, glycylcycline) and others the 505 ribosomal subunit (Le., macrolides, oxazolidinones,streptogramins) (see Figure 12-1).

Aminoglycosides are cationic carbohydrate-containing molecules and their positive charge provides the basis for their interaction with a specific region of the 165 ribosomal ribonucleic acid (rRNA) in the 305 ribosomal subunit. The 305 subunit provides a highaffinity docking site mediated by hydrogen bonding to the various substituents in the aminoglycoside cyclitol ring. Binding of the aminoglycosides to the A-binding site on the 305 subunit prevents the docking of aminoacyl-tRNA, resulting in mistranslation and subsequent production of aberrant proteins. The incorporation of aberrant proteins into the cell wall also results in cell leakage and enhanced cellular penetration of additional antibiotic.

The clinically useful tetracyclines are members of the polyketide class of antibiotics and are represented by tetracycline, doxycycline, and minocycline. As shown in Figure 12-1, the tetracyclines target the 305

ribosomal subunit. Tetracyclines reversibly inhibit protein synthesis by binding to 165 ribosomal RNA near the amino-acyl tRNA acceptor (A) site, thus inhibiting the rotation of bound tRNA into the A site during

translation. This physical blocking by tetracycline results in premature release of tRNA and termination of

peptide bond formation.

Macrolides such as erythromycin, clarithromycin, and azithromycin target the 505 subunit specifically by binding to the peptidyltransferase cavity in the proximity of the A and P loops, near adenine 2058 of 235 rRNA.By blocking the exit tunnel of the elongating peptides, premature release of peptidyl-tRNA intermediates occurs and polypeptide translation ceases. Macrolides also prevent assembly of the 505 ribosomal subunit by binding to 235 rRNA. As shown in Figure 12-1, lincosamide and chloramphenicol also target the 505 ribosomal subunit, thus inhibiting mRNA translation and subsequently protein synthesis. Macrolides and tetracyclines allow initiation and mRNA translation to begin but act by inhibiting peptide elongation.

The most recently approved class of antibiotics targeting protein synthesis are oxazolidinone represented by linezolid, a streptogramin represented by dalfopristin-quinupristin, and a glycylcycline represented by tigecycline. Linezolid is thought to bind to the 50S ribosomal subunit of prokaryotes, preventing formation of the preinitiation complex with the 30S ribosomal subunit containing bound initiation factors. The 50S ribosomal target appears to be the ribosomal P site in the peptidyltransferase center, and by blocking this site, the first peptide-forming step is prevented and protein synthesis is terminated.

Linezolid differs from other protein synthesis inhibitors by blocking the initiation step while macrolides and tetracyclines block peptide chain elongation.

Streptogramin antibiotics are composed of a mixture of two classes of distinct molecules designated streptogramin A and streptogramin B. DaIfopristinquinupristin is a combination of these two streptogramins in a ratio of 70:30. DaIfopristin is a polyunsaturated macro lactone classified as a type A streptogramin, and quinupristin is a peptide macrolactone classified as a type B streptogramin.

Streptogramins disrupt translation of mRNA into protein by binding to the peptidyltransferase domain of the bacterial ribosome. DaIfopristin interferes with the elongation of the polypeptide chain by preventing binding of aminoacyl-tRNA to the ribosome and the formation of peptide bonds. Quinupristin stimulates the dissociation of the peptidyl-tRNA and is thought to interfere with the release of the completed polypeptide by blocking its exit tunnel through which it normally leaves the ribosome. DaIfopristin and quinupristin act synergistically as a result of the enhanced affinity of quinupristin for the ribosome. DaIfopristin induces a conformational change such that quinupristin binds with greater affinity. The natural streptogramins are produced as mixtures of daIfopristin and quinupristin, the combination of which is a more potent antibacterial agent than either type of compound alone.

Glycylcycline is represented by tigecycIine, which carries a glycylamido moiety attached to the 9-position of minocycIine. TigecycIine inhibits protein translation in bacteria by binding to the 30S ribosomal subunit and blocking entry of amino-acyl tRNA molecules into the A site of the ribosome, thus preventing incorporation of

amino acid residues into elongating peptide chains

table 12-1 figure 12-1 figure 12-2 figure 12-3 figure 12-4 figure 12-5