Lec 13-Antibacterials: DNA synthesis and replication

Question 1

Metronidazole

Answer 1

• Enters the cell as a prodrug by passive diffusion and is activated by reduction of the nitro group in specific organelles in the protozoa and anaerobic bacteria
• The activated forms are cytotoxic and can interact with DNA molecule =
  
  • => Inhibition of DNA synthesis and DNA damage by oxidation
  • => single-strand and double-strand breaks
  • =>DNA degradation and cell death
• Metronidazole and vancomycin are the front line treatment for c.difficile

Question 2

Nitrofurantoin

Answer 2

• Similar mode of action to metronidazole
• Used in UTI’s

Question 3

Rifampicin

Answer 3

• A semi-synthetic antibiotic obtained by reacting 3-formylrifamycin with 1-amino-4-methylpiperazine
• Specifically inhibits bacterial RNA polymerase (responsible for DNA transcription) by forming a stable drug-enzyme complex
• The corresponding mammalian enzymes are not affected by rifampicin
• Bacterial resistance to rifampicin is caused by a mutation leading to a change in the structure of beta-subunit of RNA polymerase
• 9 chiral centres, imine bond (C=N) easily to hydrolyse

Question 4

Bacterial DNA replication

Answer 4

• Thymidylate synthase: Converts dUMP => dTMP
• DNA helicase: promotes strand separation at the replication fork
• DNA Polymerase III: a primary enzyme of replication nucleotides to the growing DNA chain and proofreads DNA (150 nucleotides/sec)
• Primase: Makes short RNA primers for lagging strand
• DNA Polymerase I: fills in gaps on lagging strand and removes RNA primers
• Topoisomerase I: relax supercoiled DNA
• Topoisomerase II: (DNA gyrase); promotes negatiuvesupercoiling, maintains the shape of the chromosome
• Topoisomerase III: removes supercoiling
• Topoisomerase IV: Similar function to II, removes knots and links behind the replication fork

Question 5

Quinolones inhibitors of DNA gyrase

Answer 5

• Quinolones block re-sealing of bacterial DNA strands on supercoiling causing the bacterial chromosome to break into multiple fragments
• The carboxyl COOH and carbonyl C=O are important for activity
• Quinolones 1000x more active against bacterial vs human gyrases
• Recognise N (6membered ring) with carbonyl and carboxyl groups attached to benzene
• Bending strands (need to coil not enough space)
• Both strands of one DNA is cut by DNA gyrase
• One strand is passed through the other and the DNA break is sealed

Question 6

Antigene oligonucleotides

Answer 6

• Tm is the melting temperature
• Antigene strategies require triplex-forming oligonucleotides (TFOs)
• Tm is the temperature value at 50% dissociation of triple helix into TFO and double helix or dissociation of double helix into the single-stranded coil
• We need Tm to be above 37 because of body temperature, otherwise it would just stay in dissociated form constantly
• If Tm is too high we target unwanted genes
• antisense oligonucleotides are complementary to the RNA sequence preventing translation of RNA and so subsequent protein production
• Antigene targets the gene instead of the RNA

Question 7

Watson-Crick paired duplex DNA

Answer 7

• Purine (adensosine and guanine) targeting achievable by two Hoogsteen hydrogen bonds
• Pyrimidine targeting restricted to One Hoogsteen hydrogen bond- instability due to less hydrogen bonding
• TFO design limited to polypurine targets unlike antisense design

Question 8

Hoogsteen-paired pyrimidine TFOs

Answer 8

• Pyrimidine TFOs from isomorphous triplex structures
• Need for protonation of cytosine N3 imparts pH instability
• Replacement of H by CH3 at C5 is stabilising but only marginally
  
  • This is because in Cytosine-Guanine hoogsteen paired pyrimidines one of the N groups is hydrogen bond acceptor, we need it to be a donor
  • We need to protonate the N by adding acidic pH BUT our body does work at acidic pH
  • We need to stabalise the R by putting an electron donor (CH3) to stabalise + charge

Question 9

pH-independent G, A-containing TFOs

Answer 9

• Form stable triplexes over a wider pH range, especially at pH 7.2
• G*G.C and A*A.T triplexes are NOT isomorphous; need improving
• Unmodified DNA backbone easily hydrolyzed in vivo; SHORT HALF-LIFE

Question 10

Targeting DNA gyrase in E.coli

Answer 10

• Stable pH 7.2 but Tm value is 30’C
• Bacterial growth inhibited in the presence of TFO vs absence (control)
• TFO is bacteriostatic whereas fluoroquinolones are bacterialcidal

Question 11

TFO Backbones

Answer 11

• LNA monomers are commercially available
• LNA resists enzymatic hydrolysis so longer half-life in vivo
• LNA is more A-DNA (and RNA) -like in conformation
• LNA oligomers form stable triplexes but
• ALL LNA backbone oligos suffer from self-pairing- stick to themselves
• The typical solution to self-pairing; form chimeric oligos-
• LNA = Locked Nucleic Acids