Inhibitors of Protein Synthesis

1. Inhibitors of Protein Synthesis • Bacterial cells are 50% protein by dry weight • Inhibition of protein synthesis leads to cessation of growth or cell death • Bacterial 70S ribosomes differ sufficiently from eukaryotic ribosomes to allow selective toxicity • Other properties of the antibiotics still produce side effects. • Ribosomes are cytoplasmic • Drug must successfully enter (and stay) in the cytoplasm and exert effect.

2. The antibiotics • Families • Aminoglycosides, macrolides, tetracyclines, etc. • Individual drugs • Chloramphenicol, clindamycin, mupirocin • Variety of structures • Mostly multi-ring nuclei with side chains • Vary in effectiveness against different bacteria • Variation not in site of attack but in successful entry into cells and variations in bacterial defense

3. Review of Protein synthesis • 30S subunit, Initiation factors, and mRNA come together. • fMet-tRNA binds to mRNA • 50S subunit binds to form initiation complex • 2nd aminoacyl tRNA arrives at A site • Amino acid transferred from 1st tRNA to aa of new tRNA (peptide bond formed) • Uncharged tRNA moves to E site, then leaves • Translocation of ribosome puts tRNA with growing chain in P site; elongation continues.

4. www.emunix.emich.edu/.../ genetics/transl4.htm • Simultaneous transcription/translation in bacteria • Even before transcription is completed, multiple ribosomes attach to mRNA creating polysomes.

5. Polysomes http://bass.bio.uci.edu/~hudel/bs99a/lecture21/polysome.gif

6. Representative sites of actions http://www.elmhurst.edu/~chm/vchembook/images2/652antibiotic.gif

7. Aminoglycosides As name implies, molecules comprised of amino sugars. Includes streptomycin, gentamycin, kanamycin, etc. Highly polar molecules, do not distribute well into body compartments. Administration iv and im only Narrow therapeutic index. Streptomycin http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/images/antibiotics/gentamicin.gif

8. Mechanism of action for aminoglycosides • Bactericidal, rare among protein synthesis inhibitors • Transport through the wall, through PG of G+, through porins or through OM directly in G-, disrupting OM. • Transport though cell membrane by carrier, using electrochemical gradient (uses energy). • Binds to ribosomes, keeping [free drug] low • Combination of membrane damage and inhibition of protein synthesis is bactericidal

9. Aminoglycoside enters cell

10. Aminoglycosides cont. • Aminoglycosides bind various sites on both ribosomal subunits • Freeze translation after initiation step, prevent polysome formation • Interfere with codon recognition, resulting in misreading • Sometimes “cured” by ribosomal protein mutation • Concentration dependent killing and post-antibiotic effect • Related to membrane damage and ribosome binding

11. Aminoglycoside resistance • Altered ribosomes • Mutations first observed with streptomycin • Inadequate transport of drug • Seen mostly in strict anaerobes • Enzymatic modification of drug • Acetylation, adenylation, or phosphorylation • Decrease transport and ribosome binding • Info on plasmids and transposons (e.g. Tn5)

12. Tetracyclines Note “tetra” Various family members created by modifying the left 3 rings: chlortetracycline, oxytetracycline, doxycycline… animal feed, aquaculture http://www.sp.uconn.edu/~terry/images/micro/tetracycline.gif Vary in lipid solubility, but enter cell by membrane carrier; through OM of G- via porins. Bind to ribosomes, block binding of next aa-tRNA, preventing further protein synthesis.

13. Resistance • Influx/efflux • Mutations in OM proteins retards entry • More significant, plasmid encoded protein responsible for efflux of drug: pumped out • Change in ribosome binding site • Plasmid encoded protein that binds to ribosome and blocks binding site (presumably without preventing tRNA binding itself)

15. Macrolides • Binds to 50S subunit • Binding is reversible • Either prevents transfer of peptide or access by next tRNA, preventing elongation. • Other family members: azithromycin, clarithromycin http://www.elmhurst.edu/~chm/vchembook/654antibiotic.html

16. Inhibition of 50S subunit, resistance • Macrolides, chloramphenicol, ketolides, clindamycin, and streptogramins are somewhat related, all bind to 50S subunit. • Two common forms of resistance • Alteration of ribosome binding site • Methylation of 23S rRNA at target • Plasmid encoded enzyme • Efflux of drug • Many Gram – also resistant due to OM

17. Streptogramins & Mupirocin • Streptogramins- from Streptomyces • Used as mixture of two related drugs • Low activity against most Gram – • Especially useful against MRSA and most VRE • Mupirocin • Topical • Binds to isoleucyl tRNA synthetase • Used to eliminate carriage of nasal MRSA

18. Pharmacodynamics • Aminoglycosides • Iv or im, poor absorption, renal excretion • Tetracyclines, macrolides, others • Oral administration generally possible, but food often interferes, e.g. mineral complexing • Generally pass into most body compartments • Renal, fecal excretion, many metabolized first • Some drugs concentrated in phagocytic cells • Should be useful for treating some intracellular bacterial infections.

19. Toxicity • Aminoglycosides • Renal toxicity most important and most common • Inhibition of phospholipases and other enzymes result in inhibition of prostaglandin synthesis • Ototoxicity • Damage to hair cells, tinnitus, loss of hearing • Vestibular damage, headache, nausea, dizzy • Chloramphenicol • Damage to mitochondrial membranes • Hematological effects, e.g anemia

20. Toxicity-2 • Tetracyclines • Photosensitization • Discoloration of early dentition • Macrolides • Good therapeutic index, but still various side effects • Major side effect is GI disturbance (yeah, I know) • Clindamycin • Broad spectrum problem: pseudomembranous colitis