Lec 4- Target Protein synthesis

Question 1

Antibiotics- Antibacterial agents

Answer 1

• Naturally occurring
• Semi-synthetic: Slight alterations to naturally occurring agents
• Synthetics: Synthesized in the laboratory
• Antibiotics should demonstrate Selective toxcitiy (the drug should be toxic to pathogen not the host)
• Differences in structure or metabolism between the pathogen and the host is used in the selective toxicity

Question 2

Think of any difference between a human host and a bacterial pathogen that would be a target for antibacterial agents

Answer 2

*

Question 3

Why are antibacterial drug much more abundant than antifungal, antiprotozoal and antihelmintic (small worms) drugs

Question 4

What was one of the first inhibitors of bacterial protein synthesis

Answer 4

• Streptomycin
• 1944 developed from fungi that killed bacteria
• This could treat penicillin resistant bacteria
• 1st that targeted bacterial protein sysnthesis

Question 5

Antibacterial agents- Ribosomes

Answer 5

• Aminoglycosides
• tetracyclines
• macrolides
• Oxazolidinones
• Fusidic acid
• we are expected to recognise parts of the molecules as opposed to the entire molecule

Question 6

Protein synthesis

Answer 6

• DNA is transcribed into mRNA within the nucleus
• mRNA diffuses out of the nucleus into the cytoplasm
• mRNA is then translated into protein by ribosomes (this is the step these antimicrobials work at)
• Prokaryotic protein
  
  • 2 main structures make up the ribosome
  • Small section (30S)- 16S rRNA + 21 protein
  • The large section (the 50S)- 5S/23S rRNA’s + 31 proteins
  • They come together to translate mRNA
  • NB 30S= moves more quickly through viscous solution so is smaller than 50S
  • NB- A large number of protein= a large number of targets for antimicrobials

Question 7

Eukaryotic and prokaryotic ribosomes differ in size and structure

Answer 7

• Prokaryotic 70S ribosome
  
  • 23S and 5S rRNA’s (34 proteins) Large section
  • 16S + 21 proteins- small section
• Eukarytoic 80S ribosomes
  
  • 28S,5.8S and 5S rRNA’s + 45 proteins Large section
  • 18S rRNA + 30 proteins Small section
• Eukaryotic and prokaryotic ribosomes differ relatively greatly, we can use target these differences to gain Selective toxicity and produce an effective antimicrobial
  *

Question 8

Bacterial protein synthesis

Answer 8

• 4 phases
  
  • Initiation
  • Elongation
  • Termination
  • Ribosome recycling
• mRNA is translated 5’=> 3’
• Proteins are synthesized from the N-terminus to the C-terminus
• Requires- Due to complexity good target for antimicrobial because as soon as 1 is inhibited the bacteria will die
  
  • 2 ribosome sub-units- 30S and 50S
  • 3 Initiation Factors- IF1, IF2 (GTP) and IF3
  • The initiator Met-tRNA-Met (Met= N-formylmethionine)
  • Elongation factors EF-Tu(GTP), EF-Ts, EF-G
  • mRNA
  • Very complicated hence why it is a good target, lots of oppertunities to disrupt

Question 9

Initiation

Answer 9

• Binding of IF-3 to the 30S ribosomal sub-unit promotes dissociation of the ribosome into its 2 subunits and allows 30S to form the initiation complex
• Initiation factor IF-1 binds specifically to the base of the A-site of the 30S ribosomal subunit
• This directs the initiator tRNA to the ribosomal P-site by blocking the A-site
• IF-2 is a small GTP-binding protein. IF-2-GTP binds the initiator ‘Met-tRNA-Met and helps it to dock with the small ribosome sub-unit
• IF-2-GTP is hydrolysed and IF-3 released and 50S sub-unit binds to complete the initiation complex
• Oxazolidinones: 5 membered rings, N, O, O (double bond)

Question 10

Elongation- the elongation phase of protein synthesis consists of a cycle process

Answer 10

• Met-tRNA enters the P-site
• This causes a conformational change which opens the A site for the new aminoacyl-tRNA to bind to codon
• EF-Tu brings aminoacyl-tRNA to ribosome A-site- GTP slowly hydrolysed and aminoacyl enters A site
• The amino acid at P-site is transferred to the amino acid at A site of the growing polypeptide chain- Transpeptidase
• The whole assembly moves on one codon along the ribosome, with the tRNA’s staying put- shift left one pocket

Question 11

Elongation

Aminoglycosides and tetracyclines

Answer 11

• Some aminoglycosides change the shape of 30S subunit- misreading mRNA
  
  • Leading to incorrect amino acids and so non-function
  • e.g. streptomycin and gentomycin
• Some tetracyclines (and some aminoglycosides) prevent amino acids from entering the ribosome at 30S subunit
  
  • e.g. tetracycline, minicycline and doxycycline

Question 12

Tetracycline structures

Answer 12

• How to recognise structure
  
  • 4 Six-membered rings in a row is a tetracycline

Question 13

Tetracycline biosynthesis

Answer 13

• Natural or semi-synthetics

Question 14

3rd generation, semi-synthetic tetracyclines

Answer 14

• 3rd generation glycylcycline tetracycline= Tigecycline
• Active against
  
  • MRSA
  • VRS
  • VRE
  • many G-
  • partly resistant to efflux mechanisms of resistance

Question 15

Properties and uses of tetracyclines

Answer 15

• Tetracyclines are orally absorbed, polyketide antibiotics used to treat chest infection, acne, periodontal disease (and other non-infection based conditions)
• They have broad-spectrum activity: G+, G- bacteria, anthrax, Yersinia, rickettsia, mycoplasma, spirochaetes, Neisseria meningitidis
• Doxycycline used in treating malaria
• Deposit in calcifying tissue (bone and teeth) causing staining, should not be used in children U8 or in pregnancy
• Good at binding metal Ions- Ca, Zn, Fe, Al- inactivates it
• Other effects- anti-inflammatory

Question 16

Transpeptidation

Answer 16

• The peptide bond formation involves nucleophilic attack of the amino N of the amino acid that is linked to the 3’ Hydroxyl of the terminal adenosine of the tRNA in the A site on the carbonyl C of the amino acid (with attached nascent polypeptide) in ester linkage to the tRNA in the P-site
  *

Question 17

Inhibitors of Transpeptidase- Puromycin

Answer 17

• PUROMYCIN- causes premature chain termination
• Puromycin resembles the aminoacyl-tRNA
• It has NH (not as reactive) instead of O that joins an amino acid to t-RNA
• Stronger amide bond not reactive enough- new peptide bonds formed slowly
• Ribosome stalls or incomplete chain released
• Poor selectivity between eukaryotic and prokaryotic cells- mainly used in cell culture

Question 18

Inhibitors of Transpeptidase- chloramphenicol

Answer 18

• Chloramphenicol: isolated in 1947 as a natural product of Streptomyces venezuelae, now produced synthetically
• D-three form is active (how many optical isomers are there)
• Pro-drug forms: palmitate and stearate esters improve taste
• Succinate improves solubility for injections
• Penetrates mammalian cells and CSF, a drug of choice for typhoid and meningococcal meningitis
• Selective action on peptidyl transferase of 50S subunit

Question 19

Inhibitors of transpeptidation

Answer 19

• Chloramphenicol- blocks 50S ribosome, preventing peptide bond formation
• Kanamycin (aminoglycoside) causes misreading of the code by interfering with the (wobble) base pairing- cause wrong t-RNA to bind therefore wrong amino acid is put into the protein causing non-functional proteins
  
  • Series of sugars (glyco), with some OH groups replaced with NH (Amino)

Question 20

Inhibitors of Transpeptidation- Kirromycin

Answer 20

• Aminoacyl-tRNA transfer is facilitated by 2 elongation factors, EF-Tu and EF-Ts in prokaryotes
• EF-Tu binds GDP (inactive when bound) and GTP (active when bound)
• EF-Tu-GTP binds and delivers an aminoacyl-tRNA to the A site on the ribosome
• As GTP on EF-Tu is hydrolyzed to GDP + Pi
  
  • EF-Tu undergoes a large conformational change and dissociates from the complex
• Kirromycin blocks dissociation of GDP from EF-Tu after hydrolysis
  
  • This prevents dissociation of EF-Tu from the ribosome and effectively stalls protein synthesis

Question 21

Inhibitors of transpeptidase- Fusidic acid- NOT IN EXAM

Answer 21

• Rediscovered in 1962 as a product of the fungus, Fusidium Cocciuneum (originally discovered as Cephalosporin P)
• Active only vs a limited number of G+: Staphylococcus (in); Streptococcus and Corynebacterium
• Penetrates bone well, used in staphylococcal osteomyelitis also for some MRSA infections
• Prevents translocation and EFG dependent cleavage of GTP
  
  • Also inhibits 80S EF-2 but doesn’t enter mammalian cells
• Fusidic acid inhibits the multiple turnover EF-G-GTPase
  
  • This stops ribosome movement

Question 22

Inhibitors of Translocation- Macrolide antibiotics

Answer 22

• Macrolides bind to 50S ribosome and prevent movement from one codon to the next, halting translation
• Erthromycin
  *

Question 23

Inhibitors of translocation- Biosynthesis of macrolides

Answer 23

Question 24

Inhibitors of chain termination

Answer 24

• The end translation occurs when the ribosome reaches one or more STOP codons (UAA, UAG, UGA)
• STOP codons are recognized by release factors RF1, RF2, RF3
• There is one final step in the overall cycle of protein synthesis namely, disassembly of the ribosome
• In bacteria this requires the participation of the ribosome recycling factor (RRF)
• RRF together with elongation factor G (EF-G) disassembles the post-termination ribosomal complex
• Inhibitors include Puromycin, Streptogramins and some macrolides