PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 14. MECHANISM OF ACTION OF CHEMOTHERAPEUTIC AGENTS

The selectivity of antimicrobial agents is related to structural and metabolic differences between the Cells of MICROORGANISMS AND THE host Organism. A chemotherapeutic agent targets a specific site within the metabolic System of the microbial Cell. Such targets may include specific pathways of Cell wall synthesis, Protein Synthesis, nucleic acid synthesis, etc.

14.1 Inhibitors of Cell Wall Component Biosynthesis

14.1.1 Peptidoglycan Biosynthesis

Peptidoglycan (murein) is a vital structural component of the Introduction/37.html">Bacterial cell wall; consequently, the disruption of its biosynthesis by certain Antibiotics leads to microbial cell death or the cessation of cell Replication. Since structures similar to peptidoglycan are absent in mammalian cells, the selective action of these antibiotics is ensured. The peptidoglycan molecule consists of linear chains of alternating N-acetylmuramic acid and N-acetylglucosamine units cross-linked by peptide bridges. Peptidoglycan precursors are synthesized in the Cytoplasm, assembled on a carrier lipid molecule at the cytoplasmic membrane, and, in The First stage, incorporated into The Cell wall to form uncross-linked glycan chains. In the second stage, these chains are cross-linked to the pre-existing peptidoglycan by an enzyme localized on the outer surface of the cytoplasmic membrane. These Stages of peptidoglycan biosynthesis are common to all gram-positive and gram-negative Bacteria, although interspecies variations may involve certain details, such as the Amino Acid Composition of the peptide bridges.

This process has been studied in detail in Escherichia coli. Peptidoglycan precursors—N-acetylglucosamine and N-acetylmuramyl pentapeptide—are synthesized in the cytoplasm, each linked to a carrier molecule, uridine diphosphate (UDP), which provides the energy for the synthesis reactions. The pentapeptide linked to N-acetylmuramic acid contains L-Alanine, D-glutamic acid, meso-diaminopimelic acid, and two D-alanine residues at the C-terminus of the peptide. The lipid carrier is a highly lipophilic undecaprenyl phosphate, which resides in the cytoplasmic membrane and acts as an acceptor for peptidoglycan precursors on its inner surface. N-acetylmuramyl pentapeptide and N-acetylglucosamine are sequentially transferred from their UDP carrier to the lipid carrier, with the release of uridine monophosphate (UMP) and UDP, respectively, and The formation of a β-1,4-glycosidic bond. The disaccharide pentapeptide is attached to the lipid carrier by a pyrophosphate bond. In this form, it is translocated across the cytoplasmic membrane and attached to the existing cell wall peptidoglycan in the growth zone. Appropriate Genetic control ensures the preservation of the characteristic cell shape through the incorporation of newly synthesized elements at a specific locus.

It is hypothesized that a linear glycan attached to another lipid carrier molecule serves as the binding site in the cell wall for the newly translocated disaccharide pentapeptide. The linear glycan connects to it, forming a glycosidic bond between the N-acetylmuramic acid residue of the glycan and the N-acetylglucosamine of the newly translocated disaccharide pentapeptide. The transglycosylation reaction results in the extension of the linear glycan by one disaccharide unit and the release of the carrier lipid in the pyrophosphate form. The carrier loses one phosphate molecule through the action of pyrophosphorylase and returns to the cycle of disaccharide pentapeptide translocation from the cytoplasm to the cell wall.

Cross-linking takes place within the cell wall. The newly synthesized linear glycan is attached to the wall peptidoglycan via the formation of peptide bonds. This function is carried out by transpeptidases (TPases) located on the outer side of the cytoplasmic membrane. Initially, TPases bind to the D-alanyl-D-alanine residue of the pentapeptide in the linear glycan. The peptidase cleaves off the terminal D-alanine, and the TPase binds to the C-terminus of the remaining D-alanine, forming an active intermediate. The next stage of the transpeptidation reaction involves The transfer of the acyl group of the terminal D-alanine residue to the acceptor amino group of the diaminopimelic acid of the nearest peptidoglycan chain. A new peptide bond is formed between the carboxyl group of the D-alanine of the newly synthesized glycan chain and the amino group of the diaminopimelic acid of the existing peptidoglycan, while the TPase is released. It is assumed that The energy released during the Cleavage of the D-alanyl-D-alanine bond is utilized to drive the peptide cross-linking reaction.

The peptidoglycan of E. coli and many bacilli possesses only 20–30% cross-linking. However, all free Peptides linked to N-acetylmuramic acid are tetrapeptides due to the action of D,D-carboxypeptidase (CPase), which, like TPase, cleaves the terminal D-alanine without forming a cross-link. In addition to these Enzymes, endopeptidase participates in peptidoglycan synthesis by cleaving peptide cross-links, potentially creating loci for the attachment of new peptidoglycan chains; transglycosylase attaches new glycan chains to the peptidoglycan, which are subsequently cross-linked by TPase.

14.1.2 Inhibitors of Peptidoglycan Biosynthesis

Inhibitors of peptidoglycan biosynthesis include Penicillins, Cephalosporins, and other β-lactams—such as cefoxitin (cephamycin), latamoxef (oxacephem), imipenem (carbapenem), and aztreonam (monobactam)—which suppress cross-link formation. Antibiotics such as vancomycin, teicoplanin, and cycloserine inhibit other stages of biosynthesis.

As structural analogues of β-alanyl-β-Alanine, β-lactams competitively inhibit TPases and CPases by forming covalent bonds with the active sites of these enzymes, accompanied by the cleavage of the β-lactam bond. The bond between the enzyme and the β-lactam is significantly stronger than that with its natural substrate. Furthermore, these antibiotics activate autolytic enzymes responsible for removing degrading cell wall components and separating daughter cells following division.

The action of β-lactams leads to morphological alterations in cells and ultimately results in cell death. The effect depends on the antibiotic concentration.

Vancomycin and teicoplanin exhibit an affinity for the β-alanyl-β-alanine portion of peptidoglycan precursors. They bind to this region and inhibit the transfer of the linear glycan in the cell wall to the disaccharide pentapeptide on its lipid carrier.

Thus, peptidoglycan assembly is halted prior to the transpeptidation stage. Due to their large molecular weight, these antibiotics do not penetrate gram-negative Bacterial cells and act exclusively on gram-positive microorganisms.

D-cycloserine acts at the stage of β-alanyl-β-alanine formation, inhibiting The activity of two enzymes: alanine racemase, which converts L-alanine to β-alanine, and synthetase, which forms the dipeptide. Cycloserine acts as a competitive inhibitor of these enzymes due to its structural similarity to one of the possible Conformations of β-alanine.

Echinocandins represent a new Class of antifungal antibiotics active against Candida spp.; their semisynthetic analogues are also effective against Aspergilli and Pneumocystis carinii, acting as non-Competitive Inhibitors of (1,3)-p-β-glucan synthase, an enzyme involved in the synthesis of glucan, the primary component of the fungal cell wall.

14.2 Inhibitors of Mycolic Acid and Arabinogalactan Synthesis in Mycobacteria

The cell wall of mycobacteria contains covalently linked peptidoglycan, arabinogalactan, and mycolic acids (hydroxylated Fatty acids). Non-covalently linked lipid components are represented by Glycolipids, Phospholipids, and Waxes. These lipid components provide mycobacteria with high resistance to antimicrobial agents.

Isoniazid is activated within mycobacterial cells by the catalase-peroxidase enzyme system. In its activated form, it inhibits the dehydrogenase that catalyzes the Formation of the double bond at C₂₄ of mycolic acid.

Ethambutol blocks arabinogalactan synthesis by inhibiting arabinosyltransferase, the enzyme responsible for the transfer of decaprenylarabinose during Polysaccharide synthesis.

14.3 Inhibitors of Protein Synthesis

Although protein synthesis processes in mammals and bacteria share many similarities, certain differences exist that ensure the selective action of numerous antibiotics whose antimicrobial activity is linked to the inhibition of protein synthesis. Many antibiotics selectively target prokaryotic ribosomal subunits (30 S or 50 S).

Aminoglycosides selectively interact with the 30S ribosomal subunit. Streptomycin binds to one of the 21 protein components of this subunit, which serves as the binding site for the initiation factor IF-3, thereby preventing the onset of protein synthesis. Streptomycin also disrupts the aminoacyl site of the 30S subunit, preventing aminoacyl-tRNA from positioning correctly. As a result, this leads to either the inhibition of protein synthesis or the misreading of METABOLISM/28.html">The Genetic Code.

Other aminoglycosides also bind to the 30S ribosomal subunit at a locus close to, but not identical with, the streptomycin-binding site, thereby inhibiting protein synthesis. The efficacy of aminoglycosides is enhanced by their active uptake by the bacterial cell. The process begins with the electrostatic binding of the antibiotic to the negatively charged cell surface. In the process, aminoglycosides damage the outer membrane of Gram-negative bacteria, which is accompanied by the release of certain lipopolysaccharides, Proteins, and phospholipids. This is followed by The Active Transport of the antibiotic across the cytoplasmic membrane and its binding to Ribosomes. Anaerobic microorganisms lack an active transport system for aminoglycosides and are therefore resistant to this class of antibiotics.

Tetracyclines bind to the 30S ribosomal subunit and prevent aminoacyl-tRNA from binding to the aminoacyl site. The 40S ribosomal subunit of mammals also binds tetracyclines; however, the selective action of these antibiotics relies on differential permeability. Bacteria actively take up tetracyclines, achieving cytoplasmic concentrations up to 50 times higher than those in the external environment, whereas mammals lack such an active transport system.

Chloramphenicol selectively inhibits protein synthesis by binding to the aminoacyl site of the 50S ribosomal subunit and inhibiting its peptidyl transferase. This enzyme catalyzes the formation of a new peptide bond between the growing peptide chain at the peptidyl site of the ribosome and The amino acid on the aminoacyl-tRNA located at the aminoacyl site. Chloramphenicol also penetrates mammalian cells, but it does not bind to 80S ribosomes. Its ability to penetrate cells ensures its effectiveness in treating diseases caused by intracellular parasites, such as Salmonella typhi.

Erythromycin selectively binds to the 50S ribosomal subunit at a site closely adjacent to the chloramphenicol-binding site and inhibits translocation. Erythromycin does not prevent peptidyl-tRNA from leaving the aminoacyl site, but it inhibits the release of tRNA from the peptidyl site and blocks the movement of peptidyl-tRNA into it.

Lincomycin and clindamycin bind to the 50S subunit in a region close to the binding sites of chloramphenicol and erythromycin, blocking Polypeptide chain elongation by inhibiting peptidyl transferase.

14.4 Agents Affecting Chromosomal Function

DNA replication and Transcription in bacteria are fundamentally similar to these processes in mammals; nevertheless, important differences exist, which form the basis for the selective activity of certain antibacterial agents.

Quinolones (synthetic derivatives of nalidixic acid), such as norfloxacin and ciprofloxacin, inhibit DNA gyrase—an enzyme unique to bacteria—thereby blocking Chromosome replication without affecting gyrase-independent DNA Replication Mechanisms.

Metronidazole and nitrofurantoin. It is hypothesized that the action of these compounds relies on unstable metabolites generated through reduction within the bacterial cell. These unidentified products induce DNA strand breaks. Metronidazole exhibits specific activity against anaerobic bacteria, in which the aforementioned metabolite is produced under low redox potential conditions. Resistance to metronidazole has not been reported.

Rifampicin binds to one of the proteins comprising the RNA polymerase complex and blocks the action of this enzyme at the Transcription initiation stage. The antibiotic does not halt transcription processes that have already been initiated. Resistant strains emerge rapidly due to structural alterations in the RNA polymerase molecule, which prevents it from binding rifampicin.

Flucytosine (5-flucytosine) is an antifungal agent most active against Candida, Cryptococcus, and Torulopsis species. Within the fungal cell, flucytosine is deaminated to 5-fluorouracil, which inhibits RNA Synthesis; 5-fluorouracil itself penetrates cellular barriers poorly. Candida albicans converts flucytosine into 5-fluorodeoxyuridine monophosphate, which suppresses DNA Synthesis by inhibiting thymidylate synthetase.

14.5 Folate Antagonists

There are fundamental differences in folate metabolism between bacteria and mammals. Bacteria are incapable of absorbing exogenous folate and must synthesize it de novo. This biosynthetic pathway involves the synthesis of dihydropteroic acid from pteridine molecules and p-aminobenzoic acid. Subsequently, dihydropteroic and glutamic acids combine to form dihydrofolate (DHF). DHF is reduced to tetrahydrofolate (THF) by Dihydrofolate Reductase in the presence of NADPH₂. THF serves as a carrier of single-carbon radicals (—CHO and —CH₃) in The biosynthesis of adenine, guanine, thymine, and Methionine. Consequently, impaired THF synthesis disrupts the cell's ability to synthesize Nucleic Acids and Proteins, thereby inhibiting its growth. Mammalian cells are able to uptake dietary DHF and convert it to THF via dihydrofolate reductase (DHFR). Sulfonamides and trimethoprim selectively inhibit folate metabolism in bacteria. The former inhibit the synthesis of dihydropteroic acid, a pathway absent in mammals, whereas the latter exhibits selective affinity for bacterial DHFR. Both agents are active only in the absence of adenine, guanine, thymine, and methionine in the growth medium.

Sulfonamides, acting as structural analogues of p-aminobenzoic acid, competitively inhibit its incorporation into the reaction producing DHF.

Trimethoprim, a DHF analogue, inhibits dihydrofolate reductase; the bacterial enzyme is several thousand times more sensitive to it than the mammalian enzyme, rendering trimethoprim non-toxic at doses used for treating microbial infections. Trimethoprim is frequently administered in combination with sulfonamides, most commonly with sulfamethoxazole in the form of co-trimoxazole. Both steps disrupting folate metabolism are synergistic; thus, the combined use of these drugs is highly effective, although it does not prevent The Emergence of resistant microbial strains.

14.6 Antimicrobial Agents Acting on the Cytoplasmic Membrane

14.6.1 Bacterial Membranes

Bacterial membranes differ significantly from those of fungal and mammalian cells: bacterial membranes lack sterols, fungal membranes contain predominantly ergosterol, and mammalian membranes contain Cholesterol. However, the overall Structure of pro- and Eukaryotic Cell membranes is quite similar. In bacteria, a phospholipid bilayer (phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol) accounts for 20–30% of the membrane mass. Proteins (50–70% of the mass) are located on both surfaces of the membrane and span the lipid layer. Proteins and phospholipids are held together by ionic, hydrophobic, and Hydrogen Bonds.

Most membrane-active agents (alcohols, phenols, quaternary ammonium compounds, bisbiguanides) compromise membrane integrity, disrupting its metabolic Functions and causing cytoplasmic leakage. The action of these substances has low selectivity, meaning they exert a similar effect on mammalian membranes. Consequently, they are employed primarily not as therapeutics, but rather as disinfectants, antiseptics, and preservatives. Polymyxin is one of the membrane-active agents that can be used therapeutically against Infections caused by Pseudomonas spp.; however, its systemic toxicity limits its clinical utility. Polyenes and imidazoles are important antifungal agents. Their selective action is based on subtle differences in the composition and biosynthesis of fungal versus mammalian membranes.

14.6.2 Polyenes

Polyenes, among which amphotericin B and nystatin are The most significant, exhibit a strong affinity for ergosterol, ensuring their selective action against fungal cells. The hydrophobic region of the polyene binds to membrane sterols, causing the hydroxylated hydrophilic portion of the antibiotic molecule to be drawn into the interior of the membrane. This creates transmembrane channels through which cytoplasmic constituents (K⁺ ions, Amino Acids, NUCLEOTIDES) leak out of the cell. As the cytoplasmic pH drops precipitously, macromolecules degrade, leading to cell death. The affinity of polyenes for cholesterol is lower than for ergosterol; thus, mammalian cells suffer less damage, although intravenous administration of amphotericin B for systemic infections commonly affects the Kidneys.

14.6.3 Imidazoles

Imidazoles, of which miconazole and ketoconazole are the most widely used, possess a broad spectrum of activity, acting against dermatophytes, dimorphic Fungi, Yeasts, and Gram-positive bacteria, although they are not used clinically to treat bacterial infections. Their MECHANISM OF ACTION involves membrane damage resulting from the binding of imidazoles to Unsaturated fatty acids present in membrane phospholipids. In addition, imidazoles inhibit ergosterol biosynthesis—specifically the demethylation of the 14-α-methyl radical of lanosterol—which leads to the accumulation of lanosterol, exerting a cytotoxic effect on the cell. The synthesis of triglycerides and phospholipids is also suppressed. Finally, ketoconazole inhibits electron transport in the fungal Respiratory Chain under aerobic conditions by suppressing succinate and NADH oxidases. The Importance of this latter effect is underscored by the significantly lower inhibitory concentration of the drug required under aerobic compared to anaerobic conditions.

Allylamine derivatives (butenafine, terbinafine) inhibit squalene epoxidase in the fungal cell membrane, thereby suppressing ergosterol biosynthesis.

at an early stage, effective against dermatophytes, Candida spp.

14.6.4 Cationic Peptides

Cationic peptides are characterized by a high content of basic and hydrophobic amino acids, which makes their molecules amphipathic, i.e., possessing both positively charged hydrophilic and hydrophobic regions. They interact with the cytoplasmic membrane via electrostatic forces, penetrate its lipid layer, and induce structural rearrangement resulting in the formation of channels that disrupt membrane permeability. The selectivity of their action depends on the Lipid Composition of microbial and mammalian membranes. Cationic peptides can also target the outer membrane of Gram-negative bacteria by competitively displacing Ca²⁺ and Mg²⁺ ions, which leads to structural disruption, as well as by covalently binding to The sulfhydryl groups of cell envelope proteins.