12 th Lecture By Abdelkader Ashour, Ph.D. Phone: PHL 424 Antimicrobials

Inhibitors of bacterial protein synthesis, Overview  These agents are bacteriostatic, protein-synthesis inhibitors that target the ribosome  Examples: Chloramphenicol, tetracyclines, macrolides, clindamycin (A-site) (P-site) Chloramphenicol Θ Macrolides, clindamycin Θ Θ Tetracyclines

Inhibitors of bacterial protein synthesis, Chloramphenicol  It is an antibiotic produced by Streptomyces venezuelae  It is a potent inhibitor of microbial protein synthesis  MOA: It binds reversibly to the 50S subunit of the bacterial ribosome and thus inhibits the peptidyl transferase step of protein synthesis {i.e., it inhibits the transpeptidation reaction (1)} ……How?  The interaction between peptidyltransferase and its amino acid substrate cannot occur, and peptide bond formation is inhibited  It also can inhibit mammalian mitochondrial protein synthesis {mitochondrial ribosomes resemble bacterial ribosomes (both are 70S)}  The peptidyltransferase of mammalian mitochondrial ribosomes, but not of cytoplasmic ribosomes, is inhibited by chloramphenicol  inhibition of synthesis of proteins of the inner mitochondrial membrane  Mammalian erythropoietic cells are particularly sensitive to the drug  Much of the toxicity observed with this drug can be attributed to these effects

Inhibitors of bacterial protein synthesis, Chloramphenicol, contd.  Antimicrobial Actions:  It is a bacteriostatic broad-spectrum antibiotic that is active against both aerobic and anaerobic G+ve and G-ve organisms  It is active also against rickettsiae and mycoplasma  Haemophilus influenzae, Neisseria meningitidis and Bordetella pertussis are highly susceptible  Strains of S. aureus tend to be less susceptible  P. aeruginosa is resistant to even very high concentrations of the drug  Resistance:  It is usually caused by a plasmid-encoded acetyltransferase that inactivates the drug. Acetylated derivatives of chloramphenicol fail to bind to bacterial ribosomes  Resistance also can result from decreased permeability and from ribosomal mutation

Inhibitors of bacterial protein synthesis, Chloramphenicol, contd.  Therapeutic Uses:  Because of potential toxicity, bacterial resistance, and the availability of other effective drugs (e.g., cephalosporins), chloramphenicol is almost an obsolete as a systemic drug  It may be considered for treatment of serious rickettsial infections in children for whom tetracyclines are contraindicated, i.e., those under 8 years of age  It is an alternative to  -lactam antibiotics for treatment of meningococcal meningitis occurring in patients who have major hypersensitivity reactions to penicillin or bacterial meningitis caused by penicillin-resistant strains of pneumococci  It is used topically in the treatment of eye infections because of its wide antibacterial spectrum and its penetration of ocular tissues and the aqueous humor

Inhibitors of bacterial protein synthesis, Chloramphenicol, contd.  Side effects:  The most important adverse effect of chloramphenicol is on the bone marrow It affects the hematopoietic system in two ways: a dose-related toxicity that presents as anemia, leukopenia, or thrombocytopenia; and an idiosyncratic response manifested by aplastic anemia, leading in many cases to fatal pancytopenia  Dose-related, reversible erythroid suppression due to an inhibitory action of chloramphenicol on mitochondrial protein synthesis in erythroid precursors, which in turn impairs iron incorporation into heme  Nausea and vomiting, unpleasant taste, diarrhea, and perineal irritation may follow the oral administration of chloramphenicol  Oral or vaginal candidiasis may occur as a result of alteration of normal microbial flora

Inhibitors of bacterial protein synthesis, Chloramphenicol, contd.  Side effects, cont.d  Toxicity for Newborn Infants Newborn infants, especially if premature, lack an effective glucuronic acid conjugation mechanism for the degradation & detoxification of chloramphenicol, with subsequent inadequate renal excretion of unconjugated drug Consequently, when infants are given dosages above 50 mg/kg/d, the drug may accumulate, resulting in a serious illness termed gray baby syndrome, with vomiting, refusal to suck, passage of loose, green stools, cyanosis, ashen-gray color, flaccidity, hypothermia, shock and collapse Death occurs in about 40% of patients within 2 days of initial symptoms. Those who recover usually exhibit no sequelae  To avoid this toxic effect, chloramphenicol should be used with caution in infants, and the dosage limited to 50 mg/kg/d or less (during the first week of life) in full- term infants and 25 mg/kg/d in premature infants  Toxic effects have not been observed in the newborns when as much as 1 g of the antibiotic has been given every 2 hours to the mothers during labor

Inhibitors of bacterial protein synthesis, Chloramphenicol, contd.  Drug interactions:  It inhibits hepatic microsomal enzymes that metabolize several drugs such as warfarin  Conversely, other drugs may alter the drug elimination. Concurrent administration of phenobarbital or rifampin, which potently induce CYPs, shortens its t 1/2 and may result in subtherapeutic drug concentrations  Like other bacteriostatic inhibitors of microbial protein synthesis, chloramphenicol can antagonize bactericidal drugs such as penicillins or aminoglycosides

Inhibitors of bacterial protein synthesis, Tetracyclines  Tetracyclines are broad-spectrum bacteriostatic antibiotics that inhibit protein synthesis  Oxytetracycline is a natural product produced by Streptomyces rimosus. Tetracycline is a semisynthetic derivative of chlortetracycline, produced by Streptomyces aureofaciens  Demeclocycline is the product of a mutant strain of Strep. Aureofaciens  Methacycline, doxycycline and minocycline all are semisynthetic derivatives

Inhibitors of bacterial protein synthesis, Tetracyclines, contd.  MOA:  Tetracyclines enter microorganisms in part by passive diffusion and in part by an energy-dependent process of active transport  Tetracyclines then bind reversibly to the 30S subunit of the bacterial ribosome, blocking the binding of aminoacyl-tRNA to the acceptor site on the mRNA-ribosome complex  This prevents addition of amino acids to the growing peptide

Inhibitors of bacterial protein synthesis, Tetracyclines, contd.  Antimicrobial actions:  Tetracyclines are active against many G+ve & G-ve bacteria, including anaerobes, rickettsiae, chlamydiae, mycoplasmas  They are also active against some protozoa, e.g., amebas  The antibacterial activities of most tetracyclines are similar except that tetracycline- resistant strains may remain susceptible to doxycycline or minocycline, drugs that are less rapidly transported by the pump that is responsible for resistance  Resistance mechanisms : 1.Decreased intracellular accumulation due to either decreased influx or increased efflux by an active transport protein pump … (the most important) 2.Ribosome protection due to production of proteins that interfere with tetracycline binding to the ribosome 3.Enzymatic inactivation of tetracyclines  Resistance is primarily plasmid-mediated and often is inducible

Inhibitors of bacterial protein synthesis, Tetracyclines, contd.  Pharmacokinetics  Tetracyclines mainly differ in their absorption after oral administration and their elimination  Absorption after oral administration is approximately 30% for chlortetracycline; 60– 70% for tetracycline, oxytetracycline, demeclocycline, and methacycline; and 95– 100% for doxycycline and minocycline  A portion of an orally administered dose of tetracycline remains in the gut lumen, modifies intestinal flora, and is excreted in the feces  Absorption occurs mainly in the upper small intestine and is impaired by food (except doxycycline and minocycline); by divalent cations (Ca 2+, Mg 2+, Fe 2+ ) or Al 3+ ; by dairy products and antacids, which contain multivalent cations, …..so!  The decreased absorption results from chelation of divalent and trivalent cations  Tetracyclines are 40–80% bound by plasma proteins. They are distributed widely to tissues and body fluids except for CSF (levels are 10–25% of those in serum)  Tetracyclines cross the placenta to reach the fetus and are also excreted in milk. As a result of chelation with calcium, tetracyclines are bound to—and damage— growing bones and teeth

Inhibitors of bacterial protein synthesis, Tetracyclines, contd.  Pharmacokinetics, contd.  Tetracyclines are excreted mainly in urine and bile. Some of the drug excreted in bile is reabsorbed from the intestine (enterohepatic circulation) and contributes to maintenance of serum levels  Doxycycline, in contrast to other tetracyclines, is eliminated by nonrenal mechanisms (excreted in feces), does not accumulate significantly in renal failure, and requires no dosage adjustment, making it the tetracycline of choice for use in the setting of renal insufficiency  Minocycline is recovered from urine and feces in significantly lower amounts than are the other tetracyclines, and it appears to be metabolized to a considerable extent. Its renal clearance is low. The drug persists in the body long after its administration is stopped, possibly due to retention in fatty tissues. Nonetheless, its half-life is not prolonged in patients with hepatic failure

Inhibitors of bacterial protein synthesis, Tetracyclines, contd.  Therapeutic Uses:  A tetracycline is the drug of choice in infections with Mycoplasma pneumoniae, chlamydiae (e.g., C. trachomatis ) and rickettsiae (e.g., Rocky Mountain spotted fever)  They may be employed in various G+ve and G-ve bacterial infections, including vibrio cholera infections, provided the organism is not resistant  Tetracyclines are used in combination regimens to treat gastric and duodenal ulcer disease caused by Helicobacter pylori  Tetracyclines are sometimes employed in the treatment of protozoal infections, e.g., those due to Entamoeba histolytica or Plasmodium falciparum  Tetracyclines have been used to treat acne. They may act by inhibiting propionibacteria, which reside in sebaceous follicles and metabolize lipids into irritating free fatty acids. The relatively low doses of tetracycline used for acne are associated with few side effects

Inhibitors of bacterial protein synthesis, Tetracyclines, contd.  Side effects :  Hypersensitivity reactions (drug fever, skin rashes) to tetracyclines are uncommon. Most adverse effects are due to direct toxicity of the drug or to alteration of microbial flora  Nausea, vomiting, and diarrhea are the most common reasons for discontinuing tetracycline medication. These effects are due to direct local irritation of the GIT  Tetracyclines are readily bound to calcium deposited in newly formed bone or teeth in young children  When the drug is given during pregnancy, it can be deposited in the fetal teeth, leading to discoloration and enamel dysplasia; it can also be deposited in bone, where it may cause deformity or growth inhibition. If the drug is given for long periods to children under 8 years of age, similar changes can result  Tetracyclines can probably impair hepatic function, especially during pregnancy, in patients with preexisting hepatic insufficiency and when high doses are given intravenously  Tetracyclines other than doxycycline may accumulate to toxic levels in patients with impaired kidney function

Inhibitors of bacterial protein synthesis, Aminoglycosides, MOA  Aminoglycosides diffuse through aqueous channels formed by porin proteins in the outer membrane of G-ve bacteria to enter the periplasmic space. Their transport across the cytoplasmic membrane depends on electron transport (membrane electrical potential is required to drive permeation of these antibiotics)  This transport is rate-limiting and can be inhibited by divalent cations (e.g., Ca 2+ & Mg 2+ ), a reduction in pH and anaerobic conditions. The last two conditions impair the ability of the bacteria to maintain the membrane potential, which is the driving force necessary for transport. Thus the antimicrobial activity of aminoglycosides is reduced markedly in the anaerobic environment of an abscess for example

Inhibitors of bacterial protein synthesis, Aminoglycosides, MOA  Once inside the cell, aminoglycoside binds to the 30S ribosomal subunit and interferes with initiation of protein synthesis by fixing the 30S-50S ribosomal complex at the start codon (AUG) of mRNA, leading to accumulation of abnormal initiation complexes, so- called streptomycin monosomes, blocking further translation of the message

Inhibitors of bacterial protein synthesis, Aminoglycosides, MOA  Aminoglycoside binding to the 30S subunit also causes misreading of mRNA, leading to: premature termination of translation with detachment of the ribosomal complex and incompletely synthesized protein (B) incorporation of incorrect amino acids (indicated by the X), resulting in the production of abnormal or nonfunctional proteins (C)  The resulting aberrant proteins may be inserted into the cell membrane, leading to altered permeability and further stimulation of aminoglycoside transport. This leads to leakage of small ions, followed by larger molecules and, eventually, by proteins from the bacterial cell. This progressive disruption of the cell envelope, as well as other vital cell processes, may help to explain the lethal action of aminoglycosides