Antibiotics that Inhibit Bacterial Protein Synthesis

I.                 Review of the principle reactions in protein synthesis- basic mechanism is the same for bacterial and mammalian cells.  The main difference is in ribosome structure.

A.     Initiation-  requires an initiation complex made up of ribosomes, mRNA, various initiation factors, and GTP for hydrolysis. The mRNA must have an initiation codon (AUG) that corresponds to the fMET-tRNA.

B.     Elongation-  requires an aminoacyl-tRNA, and the activity of elongation factors and a peptidyl transferase enzyme.  This step results in the elongation and transfer of the peptide chain to the acceptor “A” site of the ribosome.

C.    Termination- arrival of the terminator codon (UAA, UGA, UAG), the termination release factor, and the peptidyl transferase at the “A” site lead to hydrolysis of the completed protein from the “P” site.

D.    Bacterial (prokaryotic) vs. mammalian (eukaryotic) ribosome structure-

1.     Bacterial-         size  70S         sm. subunit  30S                     lg. subunit  50S

2.     mammalian-    size  80S         sm. subunit  40S         lg. subunit  60S

Space for the figure drawn on the board in class:

II.               Aminoglycosides (AG)- streptomycin, gentamicin, tobramycin, amikacin, neomycin

A.     Structure- all three ring structures

B.     Mechanism- interact with the 30S ribosome subunit to inhibit protein synthesis.  Bactericidal.

1.     Entry into bacteria- aminoglycosides are very polar, so they don’t cross membranes very easily.  Fortunately for us, there is an energy and oxygen dependent transport process that takes the aminoglycosides into the bacterial cells.  Since O2 is required, anaerobes don’t do this, and are insensitive to these drugs.  Some G(+) bugs are protected from these drugs by their thick peptidoglycan cell wall.  However, they may be effective if used with a drug that can damage the cell wall (PNCs).

2.     “Freezing” the 70S complex- occurs at higher drug levels.  By binding to the 30S subunit, the aminoglycosides cause the bacterial initiation complex to be frozen, & protein synthesis stops. The “monosomes” accumulate and can be degraded by ribonucleases.  The subunits (with drug still attached) are free to go freeze other mRNAs.

3.     Aminoglycoside binding site- aminoglycosides bind to a particular site (S12) on the 30S ribosome.  A substitution mutation of just one amino acid in that area prevents streptomycin from binding, but it is possible that not all the aminoglycosides would work this way.

4.     Misreading of the genetic code- occurs at lower drug levels.  Drug binding leads to misreading of the transcribed mRNA and yields a nonsense protein.  Happens often with some aminoglycosides (gentamicin).

C.    Microbial resistance mechanisms-

1.     Inactivating enzymes- there are at least 11 enzymes which are encoded on plasmids that can alter or stop the uptake of AGs.  These enzymes alter the AGs because they can acetylate -NH2 groups, or they can phosphorylate or adenylate -OH groups.

2.     Reduced uptake- altered cell wall may be used to keep drug out(try using it with PNC)or altered transport may cause drug to be pumped back out of the bacterial cell.

3.     Mutation of 30S subunit- mutation of the S12 site is only in labs, but there may be similar natural processes.

D.    Pharmacology-

1.     Absorption- very polar.  Don’t cross membranes well, so given IM or IV.  AGs are non-irritating to muscles and veins.  Can be given orally for intra-intestinal infection, or can be nebulized for direct action against microbes in alveoli (C.F. Pt with Pseudomonas).

2.     Excretion- eliminated unchanged by glomerular filtration.T1/2 correlates with creatnine clearance.

3.     Distribution- only in extracellular fluid (~25% of lean body wt.). AGs don’t get inside most cells or into the CSF unless the meninges are inflamed.  AGs can be given intrathecally for resistant G(-) meningitis.

E.     Adverse effects-

1.     Nephrotoxicity- with prolonged use because AGs bind to the renal cortex tissue, and the levels can build up.  Partially reversible.  Toxicity parallels potency.  Neomycin is too toxic for systemic use.

Neomycin>gentamicin, tobramycin>amikacin>streptomycin

2.     Ototoxicity- AGs accumulate in endolymph.  They interact with (-) charged phospho-inositides in the membranes of electrically excitable cells.  High frequency hearing goes first, then lower hearing loss and vertigo are possible with longer therapy.                    Not Reversible  unless caught very early.  Do regular audiograms with long therapy.  AGs do get across the placenta enough to damage the fetal ear, so don’t use in pregnancy.  The danger of ototoxicity is inversely related to potency.

Streptomycin>amikacin>gentamicin, tobramycin>neomycin

3.     Neuromuscular blockade- very rare, but happens when large doses of AGs are used in  conjunction with anesthesia, use of neuromuscular blocking agents, or in myasthenia gravis.  May be overcome by giving the Pt calcium salts.

F.     Antimicrobial spectrum- lots of G(-) bacteria, but for safety, other agents are usually used, unless resistance has developed.  Neomycin is used topically alone or in combo for minor soft tissue infections.

G.    Clinical use-

1.     drug monitoring-essential that plasma levels be taken regularly by fluorescent immuno-assay or radioimmunoassay, especially in Pts with renal failure.

2.     in combo.- AGs are used in combos for 2 reasons:

a.     additivity of spectrum

b.     synergy- against one particular, difficult to treat organism.  This is important with PNCs and vancomycins in the fight against G(+) bugs.

III.              Tetracyclines- doxycycline, minocycline

A.     Structure- 4 rings- hence the name

B.     Mechanism-

1.     Uptake- inhibition of protein synthesis in pro- and eukaryotic cells.  Selectively toxic because bacterial cell have active transport to take drug in.  We don’t.  Bactericidal.

2.     Effects on protein synthesis- bind to 30S subunit to prevent binding of the aminoacyl-t-RNA at the A site. (elongation can’t occur.)

C.    Microbial resistance- proteins gained via plasmids make an active export system to pump the drug out.  There is cross-resistance between all the tetracyclines.

D.    Pharmacology- adequate absorption in GI tract, but it is impaired by food (especially milk products and antacids) because tetracyclines can form less soluble complexes with di-and trivalent metal ions (calcium, magnesium, iron, and aluminum).  Minocycline and doxycycline are highly lipophilic, better absorbed, concentrate in adipose tissue, and have longer half-lives, so they can be administered in lower doses further apart.  May be administered IV.

1.     distribution- tetracyclines bind to tissues and to plasma proteins.  They penetrate into pleural, synovial, and ocular spaces well.  Good levels in CSF (~25% of plasma level).

2.     excretion- doxycycline and minocycline are metabolized by the liver.  All the others are eliminated via glomerular filtration, but are also passed in the feces.  All tetracyclines are concentrated in liver and excreted in bile, so some enterohepatic circulation can occur.

E.     Adverse effects-

1.     GI- upset and diarrhea due to irritation by the drug and changed gut flora.  May result in mucosal candidiasis or even pseudomembranous enterocolitis.  This occurs less with doxycycline, and doxycycline is also the safest for those with impaired renal function.

2.     Hepatotoxicity- pregnant women are at special risk with high doses.  Oxytetracycline and tetracycline are the least hepatotoxic.

3.     Phototoxicity- tetracyclines (esp. demeclocyline) absorb UV light and can cause photo-sensitivity.  Important if someone is travelling to the tropics and uses these drug for prophylaxis against malaria or diarrhea.  Also teens using it for acne & sunbathing.

4.     Effects on calcified tissues- chelating properties allow drug to complex with calcium in growing calcified tissues (like growing bone, so growth may be retarded but is reversible if you catch it early), and perinatal exposure can result in discolored  tooth enamel.  Okay to use these drugs if Pt is > 8 y.o.a.

F.     Spectrum- used to be good against everything.  Now, due to over-use, only used for atypical organisms… Chlamydia, Mycoplasma, Rickettsiae (RMSF), Borrelia burgdorferi (Lyme), Leptospira, and some parasites.  Still used in dermatology against Propionibacterium acnes.

IV.             Chloramphenicol-

A.     Structure- nitrobenzene moiety, linked to a derivative of dichloroacetic acid.

B.     Mechanism- binds to 50S subunit to inhibit protein synthesis by distorting the aminoacyl t-RNA in the A site so that it inhibits peptidyl transferase & prevents peptide bond formation.  Peptidyl transferase of the mammalian 80S ribosome isn’t sensitive to chloramphenicol, but mitochondrial ribosomes (70S) are.  This may explain some of the adverse effects.  Usually bacteriostatic, but can be bactericidal at high doses.

C.    Microbial resistance- chloramphenicol is inactivated by enzymes which reduce the nitro group, hydrolyze the amide linkage, or acetylate the drug (CAT enzyme- chloramphenicol acetyl transferase).  CAT gene is on a plasmid, and is easily passed between G(-) bugs.  Resistance is increasing among Salmonella, Shigella, and other organisms due to OTC availability of this drug in other countries.

D.    Pharmacology-

1.     Absorption- rapidly absorbed from GI.  May be given IV as the succinic ester prodrug.

2.     Distribution- lipophilic, so readily crosses membranes, and is widely distributed.  Concentrates in brain tissue.

3.     Metabolism and excretion- 90% inactivated in the liver by glucuronyl transferase.  Then, inactive glucuronide is excreted in the kidney tubules and the unchanged portion of the drug is filtered in the glomerulus.  T1/2 closely correlates with plasma bilirubin and dosage should be modified  in patients with impaired renal and liver function.

E.     Adverse effects-

1.     Hematologic toxicity- dose related bone marrow suppression (pancytopenia) is reversible.  Due to inhibited mito protein synth  and reduced Fe put into heme (reticulocytopenia)?

2.     Aplastic anemia- rare (1 in 30,000) idiosyncratic- high % of fatalities.  Onset may be delayed after Tx is begun.

3.     Fatal toxicity in neonates- “gray baby syndrome” severe respiratory and C/V toxicity due to inadequate maturation of glucuronide conjugation system and underdeveloped glomerular and tubular secretion of the unconjugated drug in the newborn.

F.     Niches- wide spectrum.  Activity against G(-), G(+), and anaerobic bacteria.  Active against rickettsia, chlamydia, and mycoplasma.

1.     + ampicillin for H. influenzae in pediatric meningitis or other bacterial meningitis when Dx is in doubt.

2.     Typhoid fever, although resistance is limiting this.

3.     Rocky Mountain Spotted Fever & other rickettsial Dz.

4.     Brain abscess

5.     Intra-abdominal infection

6.     PNC resistant strains of meningococcus and pneumococcus.

V.               Macrolide Antibiotics- erythromycin, azithromycin, clarithromycin, clindamycin

A.     Structure- erythromycin is the parent drug. Many membered lactone ring attached to 1+ deoxysugars

B.     Mechanism- bind 50S subunit, inhibiting protein synthesis.  Binding site overlaps that of chloramphenicol and clindamycin.  Bacteriostatic alternative to PNC in G(+) infections.

C.    Pharmacology-

1.     Absorption and distribution- Given orally with enteric coating so as not to be inactivated by stomach acid.  ~50% of oral dose is absorbed, the remainder passes through feces.  Also available as IV, but IM is painful.  Good penetration, except to CNS.

2.     Excretion- concentrated in liver and excreted in bile.  Some of the drug is inactivated by demethylation of in liver but is largely excreted in its active form.

D.    Adverse reactions-

1.     GI upset- N &V, diarrhea.  Erythromycin binds to motilin receptors, but newer drugs aren’t so bad.

2.     Allergic reaction- occasional.

3.     Hepatitis- may occur with high doses of erythromycin.  The estolate ester (form with improved oral absorption) can cause cholestatic hepatitis in adults.

4.     Prolonged QT interval may lead to V tach and Dupontes torsade potante.

E.     Azithromycin and clarithromycin- new macrolide antibiotics.  Clarithromycin is 6-methoxy-erythromycin.  Azithro is and “azalide” and contains a nitrogen ring.  Both are better absorbed than erythromycin.

1.     Distribution- Azithro is tissue avid.  High tissue levels but low serum levels.

2.     Excretion- same as erythromycin.

3.     Adverse effects- decreased GI side effects, otherwise similar.

F.     Clindamycin-

1.     Structure- amino acid (trans-L-4-n-propylhygrinic acid) attached to a sulphur-containing sugar (octose).  It is a chlorinated derivative of lincomycin and has a similar spectrum, increased antibacterial activity and rate of absorption. Lincomycin is used w/ animals.

2.     Mechanism- binds 50S subunit and suppresses protein synthesis.

3.     Pharmacology-

a.     Absorption and distribution- clindamycin ( & clinda w/ palmitate) are well absorbed orally. (clinda phosphate is given IM or IV).  Well distributed in tissue and bone but not CNS.  >90% bound to plasma proteins.

b.     Metabolism and excretion- metabolized to inactive products in the liver. Drug and metabolites excreted in urine and bile.

4.     Adverse effects-

a.     GI-  upset and “pill esophagitis” if not taken w/ plenty of water.

b.     Pseudomembranous colitis- due to suprainfection with C. difficile in the gut.  You should stop the clindamycin and maybe give oral vancomycin, metronidazole, or bacitracin.  Opiods will make it worse.

5.     Therapeutic uses-

a.     Abscesses (abdominal, pelvic, or lung) and bacteremia or pneumonia caused by anaerobes, especially B. fragilis.

b.     Diabetic foot infections of Staph., and Strep. osteomyelitis.

c.      Intrauterine infections, septic abortion, endometritis, and pelvic abscesses.  May be combined with an aminoglycoside.

d.     + pyrimethamine for encephalitis from Toxoplasma gondii in AIDS Pts.  Or + primaquine for P. carinii.

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