What are the antibiotics that inhibit protein synthesis?
Macrolides. Lincosamides (clindamycin). Chloramphenicol. Tetracyclines. Aminoglycosides. Aminocyclitols. Oxazolidinones. Streptogramins
What is Erythromycin primarily effective against?
Gram (+): Streptomyces, Neisseria, Pasteurella
What class of drug is Erythromycin?
Macrolide (macrolactone glycoside): polyketide. 14, 15, of 16 atom lactone ring. 1 or more neutral or amino sugars attached
What is the Polyketide biosynthetic pathway?
Sequentially adds building blocks to extend chain. Pathway synthesizes many different antibiotics. Using different but similar enzymes in the pathway can produce variant antibiotics
What are the Macrolide Features?
1) 14-16 atom ring. 2) 1 oxygen. 3) No nitrogen. 4) Many short side chains. 5) 1 or more sugars
What is good about second generation macrolides?
Imrpove acid stability, BA, more predictable PK. Exampe: Clarithromycin
What are the characteristics of Clarithromycin?
Major drawback to erythromycin is low acid stability (low oral BA). Clarithromycin improves this aspect. Also fewer ADRs, longer half-life, easier dosing. Novel antibiotics with erythromycin-like structures can be developed biosynthetically
What are Azalides?
Like macrolides, but with N in ring. Example: Azithromycin
What are the characteristics of Azithromycin?
15 membered ring, with nitrogen; changes activity and properties significantly. Addition of nitrogen decreases potency (ability to bind to target) but more than counterbalanced by PK/PD effects. Enhanced activity against Gram (-) species
What is the PK/PD of Azithromycin?
Very rapid clearance from the blood; concentrated in the tissues where its needed (more than outweighing the decrease in potency). Low serum concentrations, high infection-site concentration. Prolonged period of action from single dose. Enhanced penetration of outer cell membrane. Generally ineffective against erythromycin-resistant strains
What are third generation macrolides?
Versions to counteract microbial resistance to macrolides. Ketolides: telithromycin
What is the primary use of Ketolides: Telithromycin?
Macrolide-resistant S. pneumoniae
What does the structure of Telithromycin look like?
Ketolides: like macrolides, but with =O replacing the C3 sugar. Telithromycin also has a synthetic hydrophobic side chain
What is some general information on Ketolides (Telithromycin)?
Synthetic hydrophobic side chains provide additional novel ribosome interactions. Result, 10 fold better binding to ribosomes resistant to erythromycin
What are the ADRs associated with Ketek (Telithromycin)?
Liver damage, deaths. Flawed Aventis trials, falsified data
How are the metabolic processes affected investigated by biochemists?
Identify the phase affected. Identify the ribosomal component affected. Identify the specific binding site, if there is one
What goes into identifying the phase affected?
Initiation vs. elongation/termination: Treat with the antibiotic. Prepare, analyze polysomes/ribosomes. All polysomes --> Elongation is blocked (only bound ribosomes). All monosomes --> Initiation is blocked (only free ribosomes)
What goes into identifying the ribosomal subunit affected?
Use resistant strains to figure out. THe subunit or protein assocaited with resistance is the drug target. 1) Purify ribosomes. 2) Separate into subunits. 3) Recombine, do in-vitro protein synthesis. 4) Purify ribosomes, dissociate and purify indivdual resistant proteins. 5) Recombine, do in-vitro protein synthesis
What goes into determining the genetic difference in the segment, relative to wild type?
Identify resistant segment. Sequence segment. Identify mutated nucleotide(s). Identify mutated gene. Mutated gene is drug target
What is the MOA of Macrolides?
Inhibit protein synthesis at early step; prevents the growth of nascent chain. Binds to the 50S subunit, 1:1. Binds to a pocket in the rRNA (binds near the opening to the peptide exit channel, at the base of a deep cleft in ribosome). Binding influence by L4, L22, L15, L16 (BUT BINDING IS TO THE rRNA)
Which domain on ribosomes are most affected by antibiotics?
What is the biochemistry of macrolide binding?
Initial binding complex undergoes conformational change to form a much more stable, LONG-LIVED COMPLEX (Ribosome-Inhibitor LLC). Ratio of Ribo-Inh LLC to Ribo-Inh provides a measure of potency of binding to the target (for erythromycin, the ratio is ~10; for tylosin (C16 macrolide), the ratio is ~600
What are the details of Macrolide inhibition?
1) Binds near the opening to the peptide exit channel, at the base of a deep cleft in ribosome. 2) Binding involves rRNA domains V (peptide transfer center) and II. 3) Binding prevents egress of nascent peptide, leading to premature release. Does not specifically inhibit peptidyl transferase. 4) C16s bind stronger, more contacts. 5) rProteins L22 and L4 are located near tunnel opening (but not close enough to bind); Mutations may alter rRNA folding, thus changing the shape of the binding site (the "lock")
Why are humans not sensitive to macrolides?
Because key nucleotide A2058 is "G" in human 28S rRNA. Furthermore mitochondrial protein synthesis is not affected because its 23S rRNA also has a G at A2058
How does resistance to Macrolides occur by resistance due to target modification?
Co-resistance to erythromycin, lincosamide, and streptogramins was identified - Termed MLSb. Due to the expression of an RNA methyltransferase enzyme. Specifically methylates A2058 in the 23S rRNA (a pivotal site in erythromycin binding, gene encoding the protein is called ErmC). Subsequently, several other Erm genes have been identified