Lecture 7 Antibacterial drugs targeting protein synthesis

Question 1

Name an example of a pre-ribosomal stage inhibitor

Answer 1

Mupirocin

Question 2

Name examples of a early stage protein synthesis inhibitor

Answer 2

linezolid

tertracyclines

Question 3

Name examples of mid stage protein synthesis inhibitors

Answer 3

aminoglycosides
chloramphenicol
lincosamides
macrolids
streptogramins

Question 4

Name an example of a late stage protein synthesis inhibitor

Answer 4

fusidic acid

Question 5

What are the 3 stages of translation?

Answer 5

1) Initiation
2) Elongation cycle
3) Termination

Question 6

Describe the elongation stage of translation.

Answer 6

methionine-carrying tRNA starts out in the middle slot of the ribosome, called the P site.

Next to it, a fresh codon is exposed in another slot, called the A site. The A site will be the “landing site” for the next tRNA, one whose anticodon is a perfect (complementary) match for the exposed codon.

Once the matching tRNA has landed in the A site, it’s time for the action: that is, the formation of the peptide bond that connects one amino acid to another. This step transfers the methionine from the first tRNA onto the amino acid of the second tRNA in the A site.
We now have two amino acids, a (very tiny) polypeptide! The methionine forms the N-terminus of the polypeptide, and the other amino acid is the C-terminus.

Once the peptide bond is formed, the mRNA is pulled onward through the ribosome by exactly one codon. This shift allows the first, empty tRNA to drift out via the E (“exit”) site. It also exposes a new codon in the A site, so the whole cycle can repeat.
[see slide for images]

Question 7

Why is targeting ribosomes selectively toxic to bacteria?

Answer 7

Differences in human and bacterial ribosomes

Bacterial ribosomes are smaller and made from different RNA

Question 8

What makes mupirocin selectively toxic?

Answer 8

Structural differences between eukaryotic and prokaryotic isoleucyl-tRNA synthatases

Question 9

What makes fusidic acid and tetracycline selectively toxic?

Answer 9

difference in uptake of antibiotic into eukaryotic and prokaryotic cells - preferential to bacteria

Question 10

What is meant by ‘mechanism-based toxicity’ and how does this occur with ribosome inhibitors?

Answer 10

Side effect due to the mechanism of action of the drug

Due to inhibition of mitochondrial protein synthesis which shares similarities with bacterial protein synthesis

Question 11

Describe the spectrum of mupirocin

Answer 11

Narrow spectrum

Gram +ve

Question 12

Describe the mechanism of action of mupirocin

Answer 12

Bacteriostatic
Acts as a substate analogue to inhibit isoleucyl tRNA synthetase
Does not interfere with the ribosome directly
Isoleucine cannot be incorporated into the chain = inhibition of protein synthesis
Works as a competitive inhibitor that is 8000x more potent

Question 13

In what form is mupirocin given?

Answer 13

Topically
Due to rapid de-esterification in kidney and liver = producing metabolites lacking antibiotic activity (hydrolysis of ester bond between monic acid and 9-hydroxynonanoic acid)
Due to topical treatment it is well tolerated

Question 14

What bacteria are sensitive to mupirocin?

Answer 14

staphylococcal and streptococcal skin infections

nasal carriage of MRSA

Question 15

Which antibiotics target specifically the 50s subunit?

Answer 15

Macrolids
Lincosamides
Streptogramins
Oxaxolidinones
Aminoglycosides (and also 30s)
Chloramphenicol

Question 16

Which antibiotics target specifically the 30s subunit?

Answer 16

Aminoglycosides (and also 50s)

Tetracyclines

Question 17

Name an example of an antibiotic in the oxazolidinone class and its spectrum of action

Answer 17

Linezolid and Tedizolid (2014)

gram +ve due to being removed by efflux in gram -ve’s

Question 18

Describe the mechanism of action of the oxazolidinone class

Answer 18

Only fully synthesis drug to target RNA
Binds to the 50s subunit at the site where it normally interacts with the 30s subunit = full subunit not formed
Other theory:
Probably interferes with binding or correct positioning of aminoacyl-tRNA in the ribosomal A site

Question 19

What is linezolid used for?

Answer 19

oral/iv treatment of pneumonia or skin/soft tissue infections

Question 20

What are the side effects of linezolid?

Answer 20

Skin reactions e.g. rashes, dermatitis
GI e.g. vomiting
Abnormalities in liver function
Reversible thrombocytopenia and anaemia (mechanism based toxicity)

Question 21

Why is Tedizolid and improvement to linezolid?

Answer 21

Reduced risk of myelosupression

Increased bacterial potency

Question 22

Describe the spectrum of tetracyclines

Answer 22

Broad

Lipophilic members e.g. doxycycline more active than hydrophobic members e.g. tetracycline

Question 23

Describe the mechanism of action of tetracyclines

Answer 23

Bind to 30s subunit

Prevents the association of aminoacyl-tRNA with the ribosomes (same as oxazolidinone but binds to a different subunit)

Question 24

What are the clinical uses for tetracyclines?

Answer 24

Mostly given orally

Skin, soft tissue and intra-abdominal infections

Question 25

What are the adverse effect of tetracyclines?

Answer 25

GI disturbances - for oral products *Photosensitivity* CNS effects - mainly for minocycline *Deposition in developing teeth and bones* - due to collation of metal ions - therefore contraindicated for young children/late in pregnancy

Question 26

What is the mechanism of action of aminoglycosides?

Answer 26

Most bactericidal Either binds to 30s (streptomycin, spectinomycin) or both 30s and 50s subunits (kanamycin, tobramycin) Effects the proof reading by causing a mis-read of mRNA = production of abnormally folded proteins Insertion of abnormal proteins into the cytoplasmic membrane = membrane destabilisation and bacterial cell death

Question 27

What are the clinical uses of aminoglycosides?

Answer 27

Mostly given IV/IM Serious gram -ve infections mostly Given alone for: Severe sepsis caused my enterobactericeae and Ps.aeruginosa Plague and tulaemia Enterococcal endocarditis Used in combo for: Gram +ve and anaerobic infections TB

Question 28

What are the adverse side effects of aminoglycosides?

Answer 28

Toxicity - dependent on the specific aminoglycoside *Ototoxicity* - effects on vestibular and cochlear branches of the 8th nerve and destruction of inner hair cells, effects up to 10% of patients Nephrotoxicity Neuromuscular blockade

Question 29

What is the mechanism of action of chloramphenicol?

Answer 29

Broad spectrum Binds to 50s subunit Inhibits peptidyl transferase activity = stops peptide bond formation

Question 30

What are the clinical uses of chloramphenicol?

Answer 30

Clinical use restricted due to toxicity | Still has a role in the treatment of typhoid and paratyphoid fever caused by salmonella

Question 31

What are the adverse effects from chloramphenicol?

Answer 31

``` Bone marrow depression - can lead to aplastic anaemia , probably from inhibition of protein synthesis in human marrow cell mitochondria Grey syndrome (infants and babies) - due to build up of the drug, life threatening and presents as cyanosis ('grey skin'), abdominal distention, vomiting and circulatory collapse ```

Question 32

Name one of the worlds best selling macrolid

Answer 32

Azithromycin

Question 33

Describe the clinical use of azithromycin

Answer 33

Chlamydia | Respiratory infections - e.g. haemophilus influenzae, moraxella catarrhalis

Question 34

What is the mode of action of macrolids?

Answer 34

Binds to the 23s rRNA at the peptide exit site in the 50s subunit exit tunnel of the nascent chain = chain cannot leave the ribosome and protein cannot be made (premature dissociation of the peptidyl-tRNA from the P-site)

Question 35

What adverse effects are associated with macrolids?

Answer 35

Generally well tolerated | Can cause GI issues, jaundice and ototoxicity (rare)

Question 36

What is the mode of action of streptogramins?

Answer 36

2 structurally unreleated molecules acting synergistically Quinupristin (30%) and dalfopristin (70%) = Synercid Both bid to the 50s subunit but at different sites = form a stable complex Quinupristin inactivates peptidyltransferase Dalfopristin prevents positioning of peptidyl-tRNA at P site, inhibiting peptide bond formation

Question 37

What is the clinical use of streptogramins?

Answer 37

IV drug for vancomycin resistant E. faecium bacteremias | Skin and soft tissue infections caused by S. aureus

Question 38

What are the adverse effects associated with streptogramins?

Answer 38

Generally well tolerated

Question 39

Name examples of lincosamides

Answer 39

Clindamycin | Lincomycin

Question 40

What is the mechanism of action of lincosamides?

Answer 40

Binds to 50s subunit | Inhibits peptidyl transferase - but not the same binding site as chloramphenicol

Question 41

What is the spectrum of activity for lincosamides?

Answer 41

Unusual spectrum active against gram +ve aerobes but not gram -ve aerobes Very potent against anaerobes

Question 42

What are the clinical uses for lincosamides?

Answer 42

gram +ve infections, particularly staphylococcal infections | Combined to to treat anaerobic infections

Question 43

What adverse effects are associated with lincosamides?

Answer 43

Rashes Diarrhoea Pseudomembranous colitis - caused by C.Diff

Question 44

What is the mechanism of action of fusidic acid?

Answer 44

Normally EF-G is an enzyme that catalyses translocation of tRNA and mRNA down the ribosome at the end of each elongation Uses energy from the hydrolysis of GTP to GDP Fusidic acid binds to EF-G and locks in the protein at the translocation stage [see slide for image] [still unsure how this works properly - see textbook]

Question 45

What is the spectrum of activity of fusidic acid?

Answer 45

Limited to gram +ve organisms

Question 46

What are the clinical uses of fusidic acid?

Answer 46

topical, oral and IV therapy for staphylococcal infections | Usually given in combination with other agents to reduce risk of resistance

Question 47

What are the adverse effects caused by fusidic acid?

Answer 47

Side effects rare Rashes Jaundice