Infections and Microorganisms

• HIV: human immunodeficiency virus.
• AIDS: acquired immune deficiency syndrome.
• There is no human reverse transcriptase, so is an ideal target for treating HIV. There are two major classes:

  • Nucleoside reverse transcriptase inhibitors (NRTIs): bind the active site and inhibit the enzyme and cause chain termination
  • Non-nucleoside reverse transcriptase inhibitors (NNRTIs): bind to an allosteric site and are non-competitive

  
• The allosteric site that NNRTIs bind to is close to the active site, inducing a conformational change that reduces enzyme activity. NNRTIs are vulnerable to resistance when used alone.

  
• Human protease active sites are not symmetrical whereas HIV protease is a symmetrical homodimer with two identical protein subunits (is an aspartyl protease).
• HIV protease inhibitors must be transition state inhibitors that mimic the tetrahedral transition state of the mechanism. This must be stable to hydrolysis e.g., hydroxyethylamine isosteres.

  
• HIV protease inhibitor design:

  • Mimic tetrahedral transition state
  • C2 symmetry
  • Benzyl group retained for strong binding to S1
  • Symmetrical reaction intermediate
  • Remove alcohols to stabilise the molecule
  • Addition of valines increases activity
  • Axis of symmetry through a bond increasing separation of NH groups

  
• Ritonavir is an improved HIV drug with features such as:

  • Pyridine bioisosteres to improve polarity and water solubility - thiazolyl ring to avoid metabolism
  • Use R-OH instead of R-SH to increase hydrogen bonding

  
• Avoiding HIV resistance:

  • Administration of a single agent only works for a short time before resistance occurs
  • High active anti-retroviral therapy (HAART) is an approach consisting of at least three drugs against two different targets
  • Generally two NRTIs plus a protease inhibitor
• HIV is hard to eradicate because:

  • HIV infects human host cells - integration of viral DNA into the human DNA means that the virus can reactivate at any time
  • Reverse transcriptase is an error prone polymerase, allowing HIV to mutate quickly
• Antibiotic: an agent that either kills or inhibits bacterial growth.
• Bactericidal: kills bacteria by inhibition of cell wall biosynthesis.
• Bacteriostatic: prevents bacteria from growing and replicating.
• Bactericidal agents:

  • Penicillins
  • Cephalosporins
  • Vancomycin
  • Rifampicin
• Bacteriostatic agents:

  • Chloramphenicol
  • Sulphonamides
  • Tetracyclines
• 5 mechanisms of antibiotics:

  • Inhibition of cell metabolism (sulphonamides)
  • Inhibition of cell wall synthesis (β-lactams)
  • Interactions with plasma membrane (polymyxins)
  • Disruption of protein synthesis (chloramphenicol)
  • Inhibition of nucleic acid transcription and replication (rifamycins)
• Bacterial wall biosynthesis:

  • Made from peptidoglycan (branched polymer)
  • Amide linkages catalysed by peptidoglycan transpeptidase
  • Structure varies according to bacterial strain

  
• D-ala-D-ala has a similar structure to beta-lactam rings.
• Ring strain of beta-lactams makes the carbonyl group more reactive, especially when fuse to a five-membered ring (introduces torsional effects).
• Beta-lactamases are mutated from transpeptidases. They contain a serine residue and are able to hydrolyse the ester link.

  
• Beta-lactams are not exceptionally reactive acylating agents, but they do not randomly acylate biomolecules in vivo.

  
• Beta-lactamase inhibitors are known as suicide inhibitors.

  
• Disadvantages of beta-lactams:

  • Activity over a wide spectrum of bacteria
  • Sensitive to beta-lactamases
  • Some are sensitive and destroyed in the stomach
• Penicillin G is sensitive to acid for three main reasons:

  1. Strain in the four-membered ring (increased by the presence of the fused five-membered ring)
  2. Reactive carbonyl group (little resonance with lactam nitrogen)
  3. Neighbouring group participation from the acyl side chain

  
• Cephalosporins: inhibition of cell wall by inhibition of peptidoglycan transpeptidase, but are resistant to acid hydrolysis and lactamases.
• Cephalosporin modifications:

  1. 7-acylamino side chain
  2. 3-acetoxymethylene side chain
  3. Substitution at C7

  
• 1st gen cephalosporins

  • Cephalothin: poorly absorbed from gut, administered by IV
  • Cephaloridine: pyridine ring stable to metabolism with a good leaving group, zwitterion so soluble in water but poorly absorbed through the gut wall (IV)
• Generation of 7-aminocephalosporic acid (7-ACA):

  • Need to hydrolyse the unreactive secondary amide in the presence of a labile β-lactam ring

  
• Subsequent generations of cephalosporins:

  • Oximino-cephalosporins, significantly increases stability to some β-lactamases
  • Cefuroxime: much greater stability, resistant to esterases due to urethane group
  • Aminothiazole ring enhances penetration across the outer membrane of bacteria
  • May increase affinity for transpeptidase enzyme
  • Reserved for resistant infections

  
• Nucleoside: base with sugar attached.

  Nucleotide: base, sugar and phosphate.

  
• Chain terminators: modified nucleoside triphosphates that block the extension of the new DNA strand.

  
• Azidothymidine (AZT) is used in the treatment of HIV.

  • Phosphorylated to a triphosphate in the body
  • Two mechanisms of action; inhibits a viral enzyme (reverse transcriptase) and is added to growing DNA to act as a chain terminator

  
• Acyclovir: chain terminator made of an incomplete sugar ring (deoxyguanosine molecule) used to treat herpes and shingles.

  
• Nucleotides cannot be administered directly due to the charged phosphate group reducing cellular uptake.

  • Cellular enzymes dephosphorylate the 5' phosphate.
  • A blocked nucleotide or 'protide' can bypass the dependence on nucleotide kinases
  • This allows passive entry

  
• Protides require blocking groups on the phosphate group.

  • These groups must be lipophilic, stable in plasma, hydrolysable and have non-toxic by-products
  • Hydrolysis can be done either by phosphoramidase or esterases, based on phosphoramidate diesters that include an aromatic moiety and an amino acid
  • R groups can be modified to provide thousands of combinations, allowing optimisation for each nucleoside drug
• R groups on protides:

  • < or = 6 carbons: little or no antiviral activity
  • C7 and C9 = moderate activity
  • C8 and C10 = optimal for potent antiviral activity

  
• Prodrug conclusions:

  • They often bypass the rate limiting first phosphorylation step required for nucleoside drug activation
  • They can enter cells readily (in some cases better than parent nucleosides)
  • Effective anticancer and antiviral drugs