Lec 2- Target cell wall

Question 1

Sites of action of antibacterial agents

Answer 1

• Ribosome (protein synthesis)- aminoglycosides (gentamicin); tetracyclines; chloramphenicol; macrolides; azalides; lincosamides; oxazolidinones; mupirocin; fudisic acid; pleuromutilins
• Metabolism (DNA synthesis)- Anti-folates; sulphonamides; trimethoprim
• Cell wall- beta-lactams; glycopeptides
• Chromosome (DNA replication)- Quinolones; Rifampicin; Nitroimidazole; Nitrofurans
• Cell membrane disruption- Polymyxins; Daptomycin

Question 2

Main anti-bacterial agents- acting on the cell wall

Answer 2

• Beta-Lactams: Penicillin; cephalosporins; carbapenems; monobactams; clavulanic acid; sulbactam; tazobactam
• Glycopeptides: Vancomycin; Teicoplanin
• Others that act as anti-mycobacterial (TB) agents
  
  • Cycloserine (Petidoglyca)
  • Isoniazid (Mycolic acid)
  • Ethambutol (Arabinogalactan)

Question 3

Others that act as anti-mycobacterial (TB) agents and there targets

Answer 3

• Cycloserine
  
  • Target- Peptidoglycan
• Isoniazid
  
  • Target- Mycolic acid
• Ethambutol
  
  • Target- Arabinogalactan

Question 4

General bacterial cell wall structure

-Gram-negative

Answer 4

1. Inner membrane
2. Periplasmic space- this is where the peptidoglycan lies to improve wall strength
3. Outer membrane- extra cell membrane for drugs to cross (drugs can struggle to do this)
4. Phospholipid
5. Peptidoglycan- the presence of this in the membrane that makes a wall structure either gram-positive or negative
6. lipoprotein
7. protein
8. Lipopolysaccharide
9. Porins

Question 5

General bacterial cell wall structure

-Gram-Positive

Answer 5

1. Cytoplasmic membrane
2. Peptidoglycan- much larger than -ve bacteria
3. Phospholipid
4. Protein
5. Lipoteichoic acid
   NB- no extra membrane- one of the main difference

Question 6

General bacterial cell wall structure

Answer 6

• Beta-lactam and glycopeptide anti-biotics inhibit synthesis of the peptidoglycan component of the bacterial cell wall
• Peptidoglycan is essential to nearly all bacteria
• Peptidoglycan is unique to bacterial cell walls, no related polymer is found in mammalian cells

Question 7

Detail Gram-negative peptidoglycan

Answer 7

• M + G combine to make long strands- however, these individual strands are weak
• Cross-link with peptides between the sugar chains give the strength
• Transglycosylase- extends the glycan chains
• Transpeptidase- removes terminal D-alanine and cross-links the peptides
• D-amino acids do not occur in mammalian (another target that is different)

Question 8

Detail gram-positive cell wall structure

Answer 8

Question 9

Formation of cross-link (Gram-negative)

Answer 9

• Cross-linking shows how beta-lactamases work
• Nucleophilic attack of Transpeptidase (OH group) on D-alanine breaking the peptide bond
• This leaves a 4 amino acid chain attached to transpeptidase as well as a free D-alanine
• Diaminopimelic acid (3rd) on a different chain has a NH₂ group which then attacks the ester bond between D-alanine and transpeptidase
• This attack generates a new peptide (covalent) bond between the two chains
• This is UNIQUE to bacteria, hence no harm to the host

Question 10

Structures of penicillins G and V: Beta-lactam antibiotics

Answer 10

• Beta-lactam best-known agent to interfere with cross-link formation
• Beta-lactam is an amide bond in a cyclic formation, this is the main component
• Beta-lactam is very reactive because the normal bond angle for Carbon is 108’ but the angle of a square is 90’, this puts a lot of strain into the system which will be released when it reacts
• Penicillin G couldn’t be taken orally because the phenylacetic acid section of the molecule would be broken down in acid, this would then lose it effectivness-had to be given IV
• Penicillin V was far more acid stable

Question 11

Formation of cross-link (Gram-negative)

Answer 11

• Penicillins take the place of the D-alanie-D-alaine
• The transpeptidase then attacks the carbonyl of the penicillin
• This enzyme intermediate (Trans-penicillin) is stable, therefore

Question 12

Penicillin inhibition of transpeptidase

Answer 12

• Very similar structures
• 4 membered cyclic ring of the beta-lactam opens up into a 5 membered ring hence the increasing instability
• The 5 membered rings of the penicillin hinder the diaminopimelic acids ability to reach the ester bond to break and form an amide bond hence no cross-link

Question 13

Development of semi-synthetic penicillins

Answer 13

• While PenV and PenG were the first effective penicillins, they have limitations- better penicillins were needed but hard to synthesize
• When starved of phenylacetic acid, Penicillium chrysogenum produces the penicillin nucleus, 6-amino penicillanic acid (6-APA)- the hard bit
• 6-APA has little intrinsic activity
• 6-APA can be converted to active penicillin by reaction with an activated acid (e.g. acyl chloride)

Question 14

Structures of semi-synthetic penicillins

Answer 14

*

Question 15

Hydrolysis of penicillin by Beta-lactamase

-Penicillin Resistance

Answer 15

• One of the challenges is resistant to anti-microbial agents
• Bacterial mechanism against Beta-lactams is that the transpeptidase isn’t a transpeptidase but it would attack the beta-lactam with water, which isn’t stable in water hence hydrolysing the enzyme, inactivating it
• This is now known as the beta-lactamase
• Hydrolysis is not a new reaction (hence why we have penicillin for reconstitution), the beta-lactamase acts as a catalyst and speeds up the reaction by 10⁷ fold

Question 16

Beta-lactamase Inhibitors

Answer 16

• Still, contain a beta-lactam ring
• In the 5 membered ring instead of sulphur (penicillins) they have Oxygen or sulphone
• Sulbactam- a natural product
• Not anti-biotic just prevent hydrolysis of the beta-lactam

Question 17

Mechanism of Beta-lactamase inhibitors

Answer 17

• Beta-lactamase has an OH group which undergoes a nucleophilic attack of the carbonyl group of the 4 membered ring
• This opens the beta-lactam ring
• Because of the structure of clavulanic acid, this opening of the ring produces a reactive intermediate- causing stably cross-linked acyl-enzyme which is not rapidly hydrolysed by the enzyme
• Hence the beta-lactamase is not available to hydrolyse the penicillins

Question 18

Cephalosporins

Answer 18

• Cephalosporins (Cephems) are another class of beta-lactam antibiotic
• Cephalosporins C is a naturally produced antibiotic (by the fungus Acremonium)
• Ceph C can be chemically modified to a range of semi-synthetic cephalosporins- to give different properties, have 2 groups that can be modified
• Has beta-lactam ring, the difference between this and penicillins is that it contains 6 membered rings with sulphur and not 5 membered ring

Question 19

Semisynthetic cephalosporins

Answer 19

• Cephalosporin C limited clinical stability
• Semisynthetic cephalosporins(E.g. cephalothin, 1964) were produced to improve stability
  
  • 1st generation cephalosporins
• 2nd generation broader spectrum- more gram-negative activity
• Now on 5th generation (e.g. Ceftobiprole, Ceftaroline)
• 4th and 5th generation very broad spectrum

Question 20

Carbapenems

Answer 20

• Has Beta-lactam and COOH (mimic D-alanine)
• No sulphur in the 5 membered ring system (Its carbon) as well as a double bond
• Thienamycin is a naturally produced carbapenem antibiotic (Streptomyces)- most potent to date
• Broad-spectrum (G+ and G-) and resistance to Beta-lactamase
• Can be chemically modified to a range of semi-synthetic carbapenems:
  
  • Imipenem (sensitive to renal peptidase)
  • Meropenem (resistant to the renal peptidase

Question 21

Monobactams

Answer 21

• Nocardicin A is a naturally produced monobactam beta-lactam antibiotic
• Aztreonam is a synthetic monobactam, resistant to beta-lactamases and is the only clinically used monobactam
• Often tolerated by patients who have hypersensitivity to penicillins
• Doesn’t have the 5 or 6 membered rings hence good for hypersensitivity

Question 22

Adverse drug reaction

Answer 22

• “Mild” adverse drug reaction (ADR) (Hypersensitivity) to penicillins occur in about 1% of patients (diarrhoea, nausea, rash)
• 10% tend to report some of these effects but these are often not related to the penicillin
• This is a type I response (IgE)
• Believed to be due to “haptenization” of proteins
  
  • Due to the reactivity of beta-lactam ring, it reacts with nucleophiles on proteins, mainly albumin (HSA), Abs can then react with the modified albumin to create a response
• Cross-sensitivity is low between classes
• Severe anaphylaxis occurs in around 0.01%, mechanism unclear