Antibiotics That Inhibit Protein Synthesis

Question 1

What are the 3 different sites on the ribosome?

Answer 1

Amino acyl, peptidyl, and exit sites are present at both subunits once
they come together. 30s has specific activity of initially binding to the mRNA and is
where the codon recognition occurs. The 50s subunit is involved in GTP hydrolysis which
is involved in an error correction mechanism.

Question 2

30s subunit

Answer 2

Functions of 30s SU: mRNA binding, codon recognition.

Question 3

50s subunit

Answer 3

Functions of 50s SU: GTP hydrolysis, peptide bond formation, channeling the protein through the peptide tunnel.

Question 4

Antibiotic Binding sites on Ribosomes:

Answer 4

30s: tetracyclines, aminoglycosides.
50s: Chloramphenicol, puromycin, macrolides.

Question 5

What are the two major categories of aspects affected by antibiotics?

Answer 5

Two major categories of aspects affected of antibiotics: cell wall and protein synthesis.

Question 6

What are the different ways to affect ribosomal protein synthesis?

Answer 6

Competition with tRNA for the A site (tetracyclines). Selectivity largely through selective uptake by active transport into prokaryotic cells.

Abnormal codon: anticodon leads to misreading of the message. (Aminoglycosides, gentamicin, amikacon).

Inhibition of transpeptidation (chloramphenicol).

Premature termination of peptide chain (purpmycin).

Inhibition of translocation (erythromycin).

Question 7

Aminoglycosides

Answer 7

.Aminoglycosides
Examples:Streptomycin,Gentamicin, Amikacin
Administration: Usually IV*
Never take with chloramphenicol
Mechanism: Inhibit translation initiation
Broadspectrum.Narrowtherapeutic index, used only when other drugs fail
Requires careful monitoring of plasma drug level

Question 8

Are aminoglycoside used as a first line of treatment?

Answer 8

Not used as a first line of treatment unless the organism is known to be
highly resistant to other antibiotics. Admin is usually IV, but if you have
a GI infection then you can take them orally and theyll stay there longer due to their low
absorption.

Question 9

What shouldn’t aminoglycoside a be given with?

Answer 9

Shouldnt take with chloramphenicol because they both inhibit
translation, and they do have some effects on eukaryotic protein
synthesis. You can take one or the other, but taking them both can make
you go over the therapeutic index.

Question 10

How can aminoglycosides combat GI infections?

Answer 10

Some aminoglycosides are given orally to combat GI infections

Question 11

Diseases for Which Tetracyclines are Drugs of First Choice

Answer 11

• Rickettsial diseases: Rocky Mountain Spotted Fever
• Lyme Disease (Borrelia): these first two are transferred
  by ticks. Bulls eye rash. If not treated
  it can lead to arthritis-like
  symptoms and CNS effects
• Chlamydia trachomatis
• Amoebic dysentary (Entamoeba histolytica):Not a bacteria, this is a protozoan parasite that
  can affect humans and other mammals. Can be
  acquired through infected water.
• Mycoplasma pneumonia :caused by mycoplasma pneumonae. Cannot survive extracellulary. Not affected by penicillin because it doesnt have a cell wall.

Question 12

Are tetracyclines toxic to us?

Answer 12

For some reason tetracycline can be taken up by amoeba cells but not by our cells. So its
fairly nontoxic to us.

Question 13

What stage of translation do tetracyclines target?

Answer 13

3 Different stages of protein synthesis: Initiation, Elongation, and termination. Tetracyclines targets elongation?, specifically
binding of tRNA to the A site.

Question 14

Chlamydia

Answer 14

Chlamydia: intracellular
obligate parasite that 
infects human cells. STD. 
Can lead to pelvic inflammatory
disease if untreated.
Can lead to sterility. 
Inflammation of the
fallopian tubes or
epidimitis. Can
lead to trachimitis or
conjuctivitis if given
birth to a baby, leading
to blindness in the
child.

Question 15

Tetracyclines

Answer 15

Tetracyclines
- Compete with tRNA for binding to A-site
- ProductofStreptomyces
- Administration:oral
- Bacteriostatic
-Selectivity:
- Broad spectrum antibiotic;used against both Gram positive and negative bacteria
- Sideeffects:incorporated into developing teeth, photosensitivity
- Mainrisk:superinfection
- resemble the structure of amino acids. Works against
both gram positive and negative. Selectivity is not because they specifically recognize the prokaryotic ribosomes, they
will actually bind to eukaryotic ribosomes, but they get transported very effectively into
prokaryotic organisms and not into eukaryotic cells.
Should not be given to children
who are still developing teeth,
otherwise it will incorporate
into the enamel and turn
black.

Question 16

How do the two ribosomal subunits work

Answer 16

30s binds to mRNA
which allows for 50s
to join and form the
70s. Then the tRNA
can bind. Specifically
the A site is the
target of 
tetracycline. There
are two different 
binding sites 
associated with
the A site that
tetracycline can 
bind to. Usually
with most
antibiotics its only
a single site, but
not the case here.

Question 17

Superinfection

Answer 17

Superinfection occurs when you treat with a broad spect antibiotic that ends up knocking out a lot of normal
flora, with the result of a rise in yeast injection from normally present fungi that are normally kept in check
by the bacteria in the normal flora.

Question 18

Ribosomal differences

Answer 18

different forms of ribosomes present in different groups of organisms. Mitochondria have their own organsms. Although they are simlar there is enough of a difference for antibiotics that target them to be selective towards what is desired. Bacteria have 50s and 30s to make 70s, while eukaryotes have 40s and 60s to make 80s.

Question 19

How can you identify which ribosomal subunit an antibiotic acts on?

Answer 19

Use ribosomal subunits from resistant and sensitive strains. Both sensitive should give high inhibition of amino acid incorporation. Sensitive strain of affected subunit + resistant subunit should give high inhibition. Resistant affected subunit + sensitive subunit should give low amount of inhibition.

Question 20

How can you identify a specific protein subunit being affected by an antibiotic?

Answer 20

Same strategy with the different ribosomal subunits but add back the protein sequence in question and should see a high degree of inhibition of amino acid incorp using protein seq from sensitive strains.

Question 21

How does streptomycin kill bacteria?

Answer 21

Two mechs:
1. Freeze protein synthesis at the stage of initiation.
2. Misreading of the mRNA resulting in misincorperation of amino
acids and dysfunctional and nonfunctional proteins as a result.

Question 22

Evidence for ribosomal freezing during translation initiation

Answer 22

In prokaryotic organisms, transcription and translation can occur simultaneously, you have
the formation of polysomes. Multiple ribosomes will be present on the mRNA. It was found that
as they decreased concentration of streptomycin in in vitro translation systems and compared the
percentage of monosomes to the bactercidal effect (decrease in viable cells), they saw that there was
a direct relationship between the number of monosomes and the decrease in viable cells. More monosomes
came with less viable cells. The idea was that a ribosome binding to the mRNA couldnt go forward, and so no
additional ribosomes can bind to the same mRNA because it is blocked by the previous ribosome. Unless you are
dealing with an operon that has multiple shine del garno sequences on it, youll only have one ribosome bindng
site.

Question 23

Evidence of misreading induced by aminoglycosides

Answer 23

Evidence of misreading came from in vitro translation experiments. They worked with polymers that had single nucleotides, so poly U or poly C. Then they looked at the proportion
of products in a controlled translation experiment versus one with streptomycin present. It takes less streptomycin to create a misread than it does with the translation initiation
freezing. When looking at the code for the poly U system, UUU is supposed to give only Phe, however even in the control experiment setup we see a rather large amount of
Tyr being incorperated as well. This just points to the fact that in an in vitro translation system some of the correction mechanisms are missing so you will get a higher error rate than in
vivo. Adding the streptomycin gives a significant greater error of incorporation of the amino acids.

Question 24

Flipping bases

Answer 24

30s ribosomal subunit has the 30s decoding site. Basically this is found within the 16s ribosomal RNA (depicted in yellow). There are a couple of bases that flip out upon contact with the mRNA that work to
assure that the tRNA makes correct contact with the mRNA so that you get a good anti-codon-codon match. So the bases flip out of the 16s ribosomal RNA and that causes a conformational change which is carried
over to the 50s subunit. So all of this is taking place in the 30s subunit, and causes a conformational change in the 50s which activates its ability to join the initial amino acid to subsequent amino acids. So this
affects peptide bond formation. If therre is not a good match between the tRNA and mRNA, the flipping does not occur and the tRNA exits. Streptomycin will cause flipping to occur even in the absence of a proper
match occuring.

Question 25

Conditions for which streptomycin is indicated as a primary line of treatment

Answer 25

• bacterial endocarditis (with penicillin in combo). Strep commonly causes this when colonizing the heart valves.
• Tularemia “rabbit fever”. Transmitted by fleas.
• plague. Also transmitted by fleas. Also by rats carrying fleas.

Question 26

Aminoglycoside dependence

Answer 26

Situation where bacterial strains can actually become dependent on
aminoglycosides for growth. So instead of preventing them from growing
this actually fascilitates their growth. This is associated with a single point
mutation in the bacteria that involves a codon misreading situation
where the incorrect amino acid is replaced by the correct amino acid. So you
have a bacteria that cannot grow because they have the point mutation
already in a key gene, and because of the misreading from aminoglycosides
it gets fixed. This is associated with very low concentrations of amino glycosides.
This is a warning to take the full drug regiment treatment to eliminate bacteria present,
because if you only take a low level you may end up selecting for bacteria that have
these mutations that would otherwise die off. Not the mechanisms seen
with anticancer drug dependence. Basically overriding the mutation. If you were giving a high enough level
of the aminoglycoside,you would basically be fucking up the rest of the proteins, but
if its at a low enough level you may end up just salvaging the defective protein and letting
them grow.

Question 27

Streptomycin adverse reactions

Answer 27

Streptomycin- Adverse Reactions: estimated that about half of individuals that take streptomycin have this
first listed side effect. It is usually reversible though if you go off of the drug.
- Damage to 8th cranial nerve “ototoxicity”
Loss of hearing/balance associated with direct damage to cochlea/vestibular organ
- Kidneydamage
- Peripheral neuropathy : loss of sensation or tingling stabbing
pain. Usually reversible.
- DrugAllergy: Dermatitis, fever, anaphylactic shock

Question 28

Chloramphenicol

Answer 28

Chloramphenicol
•Produced by Streptomyces
•MOA:binds to 50S SU and blocks peptide
bond formation
•Low therapeutic index;used as drug of last resort
- Serious adverse effects: worse than streptomycin and aminoglycosides
Bone marrow depression: generally reversible. Aplastic anemia (irreversible): Must get transfusions or bone
marrow transplant.Gray syndrome

Question 29

Gray baby syndrome

Answer 29

Seen in premature infants who are given chlloramphenicol. Primarily due to
drug metabolism. Chloramphenicol is usually eliminated via a conjugation
reaction that occurs in phase 2 drug metabolism. The transferase is not being
produced in adequate amounts in premature infants.

Body limpness, gray ashen color, low blood pressure, cyanosis, hypothermia,
cardiovascular collapse. Due to buildup of chloramphenicol resulting from poorly developed
glucuronide formation.

Question 30

Macrolide antibiotics

Answer 30

Macrolide Antibiotics

• Example:erythromycin
• Product of Actinomycetes
• Administration:oral or IV
• Inhibit translocation step of translation (translation elongation)
• Antibiotic spectrum similar to penicillin (primarily effective against Gram positives)
• Side effects minor:GI disturbance,allergies, rare jaundice and superinfections
• side effects are generally not that serious. Macrolides can be either bacteriostatic or bacteriocidal. They are acid labile, so give it with an enteric coating to prevent it from being degraded in the stomach by low pH.

Question 31

Erythromycin

Answer 31

Macrolide nucleus composed of lactone ring.

Question 32

Macrolide MOAs

Answer 32

• Block peptide exit channel.

- Inhibit 50s subunit assembly.

Question 33

Resistance mechs to protein synthesis inhibitors

Answer 33

Point mutations in ribosome proteins that alter drug binding affinity.
RNA methylation.

Question 34

Primary uses of Erythromycin:

Answer 34

• Mycoplasma pneumonia
• Legionella pneumonia
• Chlamydia conjuctivitis
• Camphylobacter gastroenteritis.

Question 35

Bacteriostatic

Answer 35

:
- Growth inhibition
(Sulfas)

Question 36

Bacteriocidal

Answer 36

:
- Killing 99.9% bacterial cell death within 24 hours.
(Betalactams-Penicillin, Streptomycin)

Question 37

Are betalactams bacteriostatic or bacteriocidal?

Answer 37

While betalactams can be bacteriostatic at lower concentrations, theyre generally considered bacteriocidal