Protein biosynthesis as a drug target

Question 1

Protein biosynthesis

Answer 1

DNA to RNA to Protein
Takes place in ribosomes
Prokaryote and Eukaryote ribosomes differ substantially in size and structure.
Eukaryote subunits are bigger and contain more rRNA in the large subunit, than prokaryote ribosomes.

Question 2

Tuberculosis

Answer 2

The storm front of what multi-antibiotic resistance may mean for infectious disease.
Multi-resistance Tb exists and is spreading, co-infection with HIV helps spread the disease.
Extensively Drug resistant (XDR) Tb, very difficult to treat.

Question 3

Directly observed treatment short course (DOTS)

Answer 3

Tb treatment is monitored directly - compliance is a key issue.

Question 4

Streptomycin

Answer 4

First agent active against against Tb

First aminoglycoside antibiotic

Question 5

Aminoglycosides

Answer 5

Contain amino sugars, bonded by glycoside linkage.
Streptomycin, Neomycin, Kanamycin, Gentamycin and Paromomycin.
Are irreversible inhibitors of bacterial protein synthesis.
Enter gram-negative bacteria via porin channels in their outer membrane.
They bind to the 30s small ribosomal subunit and disrupt protein synthesis by -
1. Interfering with peptide formation
2. Inducing mRNA misreading
3. Causing break-up of polysomes

Question 6

Paromomycin

Answer 6

A rare example of an antibiotic that is also used against protozoan infection (leishmaniasis).

Question 7

Aminoglycoside usage

Answer 7

Very poor oral availability.
Good intramuscular/intravenous availability.
Poor BBB penetration.
Fast renal clearance.
Less commonly used now.

Question 8

Chloramphenicol (Chlornitromycin)

Answer 8

Isolated from Streptomyces Venesuelae.
First synthetic antibiotic.
Inhibitor of bacterial protein synthesis.
Bacteriostatic gram-positive and gram-negative antibiotic due to common ribosomal structure.
Broad spectrum antibiotic.
Widespread resistance therefore no longer first line treatment for any infection in the developed world.
Drug of choice in many 3rd world countries as its cheap.
Binds the 50s subunit of bacterial ribosome, inhibits peptidyl transferase (a ribozyme) step of protein synthesis by binding to 23s rRNA.
Blocks the elongation of growing peptide chain by inhibiting the transfer of the chain to the next amino acid/tRNA unit.
Given orally, inactivated by glucuronidation in the liver.
Chloramphenicol palmitate is a prodrug with better intestinal absorption.
Distributes widely, can cross BBB.
Toxic due to bone marrow supression but used in treatment of bacterial meningitis and some rickettsial infections.
use today is superceded by other agents.

Question 9

Tetracyclines

Answer 9

Common basic structure comprises naphthacene ring system.
Inhibit bacterial protein synthesis, compete with tRNA for its binding site on 50s subunit of the ribosome, preventing elongation of the peptide chain.
Broad spectrum antibiotics.
second most widely used group after b-lactones (penicillins)
Uptake into microbes by passive diffusion - non specific transport, target all bacteria.

Question 10

Resistance to tetracyclins

Answer 10

Increased efflux through ABC transportersm plasmid-encoded.

Production of proteins that stop tetracyclines binding to the ribosome.

Question 11

Doxycycline (a tetracycline) against Malaria

Answer 11

Binds specifically to prokaryote RNA
Plasmodium is a protozoan eukaryote.
Parasites possess a prokaryote-derived unique organelle (the apicoplast)
Apicoplast rRNA is of bacterial origin, it contains its own ribosome to make proteins. the ribosome is therefore prokaryotic and susceptible to tetracyclines.

Question 12

The Apicoplast

Answer 12

A chloroplast like organelle found in apicomplexan parasites (plasmodium, toxoplasma).
Contains its own genome, although this mainly contains genes which are involved in its own transcription and translation.
It is essential to this group of organisms, therefore a drug target.
Bound by at least 3 membranes as it was aquired by secondary endosymbiosis.
it is not photosynthetic
Key biochemical pathways associated with the apicoplast -
1. Fatty acid synthesis (via prokaryotic type pathway)
2. Isoprenoid biosynthesis (non-mevalonate pathway)
3. Haem biosynthesis

Tetracyclines can diffuse across all the membranes needed to reach the apicoplast ribosome (which is prokaryotic)

Question 13

Macrolide antibiotics

Answer 13

Contain a macrocyclic lactone ring
Prototype - erythromycin
Poorly water soluble, rapidly inactivated at room temp.
Clarithromycin and azithromycin are semi-synthetic derivatives of erythromycin. Improved acid stability and oral availability. They bind the 50s rRNA, blocking aminoacyl translocation reactions.

Erythromycin is drug of choice against corynebacteria (diptheria etc.)

Used to treat a number of other gram-positive/negative infections.

Resistance -

1. Reduced uptake or increased efflux
2. Enzymes that hydrolyse the drug
3. Alternations to 50s rRNA (by a methylase)