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.

The antibiotics Families Individual drugs Variety of structures 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

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.

www.emunix.emich.edu/.../ genetics/transl4.htm

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

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

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

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

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)

Tetracylcines 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.

Resistance Influx/efflux Change in ribosome binding site 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)

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

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

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

Pharmacodynamics Aminoglycosides Tetracyclines, macrolides, others 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.

Toxicity Aminoglycosides Chloramphenicol 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

Toxicity-2 Tetracyclines Macrolides Clindamycin 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