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Antibiotics

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  1. Antibiotics

  2. Step 1: How to Kill a Bacterium. • What are the bacterial weak points? • Specifically, which commercial antibiotics target each of these points?

  3. Target 1: The Bacterial Cell Envelope

  4. Two types of bacteria • Gram-positive: Stained dark blue by Gram-staining procedure • Gram-negative: Don’t take up the crystal violet stain, and take up counterstain (safranin) instead, staining pink in the Gram procedure.

  5. Structure of the bacterial cell envelope. Gram-positive. Gram-negative.

  6. Gram staining animation http://student.ccbcmd.edu/courses/bio141/labmanua/lab6/images/gram_stain_11.swf

  7. Structure of peptidoglycan. Peptidoglycan synthesis requires cross-linking of disaccharide polymers by penicillin-binding proteins (PBPs). NAMA, N-acetyl-muramic acid; NAGA, N-acetyl-glucosamine.

  8. Antibiotics that Target the Bacterial Cell Envelope Include: • The b-Lactam Antibiotics • Vancomycin • Daptomycin

  9. Target 2: The Bacterial Process of Protein Production

  10. An overview of the process by which proteins are produced within bacteria.

  11. Structure of the bacterial ribosome.

  12. Antibiotics that Block Bacterial Protein Production Include: • Rifamycins • Aminoglycosides • Macrolides and Ketolides • Tetracyclines and Glycylcyclines • Chloramphenicol • Clindamycin • Streptogramins • Linezolid (member of Oxazolidinone Class)

  13. Target 3: DNA and Bacterial Replication

  14. Bacterial synthesis of tetrahydrofolate.

  15. Supercoiling of the double helical structure of DNA. Twisting of DNA results in formation of supercoils. During transcription, the movement of RNA polymerase along the chromosome results in the accumulation of positive supercoils ahead of the enzyme and negative supercoils behind it. (Adapted with permission from Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. New York: Garland Science, 2002:314.)

  16. Replication of the bacterial chromosome. A consequence of the circular nature of the bacterial chromosome is that replicated chromosomes are interlinked, requiring topoisomerase for appropriate segregation.

  17. Antibiotics that Target DNA and Replication Include: • Sulfa Drugs • Quinolones • Metronidazole

  18. Which Bacteria are Clinically Important?

  19. General Classes of Clinically Important Bacteria Include: • Gram-positive aerobic bacteria • Gram-negative aerobic bacteria • Anaerobic bacteria (both Gram + and -) • Atypical bacteria • Spirochetes • Mycobacteria

  20. Gram-positive Bacteria of Clinical Importance • Staphylococci • Staphylococcus aureus • Staphylococcus epidermidis • Streptococci • Streptococcus pneumoniae • Streptococcus pyogenes • Streptococcus agalactiae • Streptococcus viridans • Enterococci • Enterococcus faecalis • Enterococcus faecium • Listeria monocytogenes • Bacillus anthracis • Staphylococcus aureus • Streptococcus viridans

  21. Gram-negative Bacteria of Clinical Importance • Enterobacteriaceae • Escherichia coli, Enterobacter, Klebsiella, Proteus, Salmonella, Shigella, Yersinia, etc. • Pseudomonas aeruginosa • Neisseria • Neisseria meningitidis and Neisseria gonorrhoeae • Curved Gram-negative Bacilli • Campylobacter jejuni, Helicobacter pylori, and Vibrio cholerae • Haemophilus Influenzae • Bordetella Pertussis • Moraxella Catarrhalis • Acinetobacter baumannii

  22. Anaerobic Bacteria of Clinical Importance • Gram-positive anaerobic bacilli • Clostridium difficile • Clostridium tetani • Clostridium botulinum • Gram-negative anaerobic bacilli • Bacteroides fragilis

  23. Atypical Bacteria of Clinical Importance Include: • Chlamydia • Mycoplasma • Legionella • Brucella • Francisella tularensis • Rickettsia

  24. Spirochetes of Clinical Importance Include: • Treponema pallidum • Borrelia burgdorferi • Leptospira interrogans

  25. Mycobacteria of Clinical Importance Include: • Mycobacterium tuberculosis • Mycobacterium avium • Mycobacterium leprae

  26. Antibiotics that Target the Bacterial Cell Envelope • The b-Lactam Antibiotics

  27. Mechanism of action of β-lactam antibiotics. Normally, a new subunit of N-acetylmuramic acid (NAMA) and N-acetylglucosamine (NAGA) disaccharide with an attached peptide side chain is linked to an existing peptidoglycan polymer. This may occur by covalent attachment of a glycine () bridge from one peptide side chain to another through the enzymatic action of a penicillin-binding protein (PBP). In the presence of a β-lactam antibiotic, this process is disrupted. The β-lactam antibiotic binds the PBP and prevents it from cross-linking the glycine bridge to the peptide side chain, thus blocking incorporation of the disaccharide subunit into the existing peptidoglycan polymer.

  28. Mechanism of penicillin-binding protein (PBP) inhibition by β-lactam antibiotics. PBPs recognize and catalyze the peptide bond between two alanine subunits of the peptidoglycan peptide side chain. The β-lactam ring mimics this peptide bond. Thus, the PBPs attempt to catalyze the β-lactam ring, resulting in inactivation of the PBPs.

  29. Six P's by which the action of β-lactams may be blocked: • penetration, • porins, • pumps, • penicillinases (β-lactamases), • penicillin-binding proteins (PBPs), and • peptidoglycan.

  30. The Penicillins

  31. INTRODUCTION • Antibacterial agents which inhibit bacterial cell wall synthesis • Discovered by Fleming from a fungal colony (1928) • Shown to be non toxic and antibacterial • Isolated and purified by Florey and Chain (1938) • First successful clinical trial (1941) • Produced by large scale fermentation (1944) • Structure established by X-Ray crystallography (1945) • Full synthesis developed by Sheehan (1957) • Isolation of 6-APA by Beecham (1958-60) - development of semi-synthetic penicillins • Discovery of clavulanic acid and b-lactamase inhibitors

  32. http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/Spencer/spencer_cellwall.htmlhttp://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/Spencer/spencer_cellwall.html

  33. R = 6-Aminopenicillanic acid (6-APA) Benzyl penicillin (Pen G) R = Acyl side chain Thiazolidine ring Phenoxymethyl penicillin (Pen V) b-Lactam ring Penicillin G present in corn steep liquor Penicillin V (first orally active penicillin) STRUCTURE Side chain varies depending on carboxylic acid present in fermentation medium

  34. Shape of Penicillin G Folded ‘envelope’ shape

  35. Properties of Penicillin G • Active vs. Gram +ve bacilli and some Gram -ve cocci • Non toxic • Limited range of activity • Not orally active - must be injected • Sensitive to b-lactamases (enzymes which hydrolyse the b-lactam ring) • Some patients are allergic • Inactive vs. Staphylococci Drug Development • Aims • To increase chemical stability for oral administration • To increase resistance to b-lactamases • To increase the range of activity

  36. SAR • Conclusions • Amide and carboxylic acid are involved in binding • Carboxylic acid binds as the carboxylate ion • Mechanism of action involves the b-lactam ring • Activity related to b-lactam ring strain • (subject to stability factors) • Bicyclic system increases b-lactam ring strain • Not much variation in structure is possible • Variations are limited to the side chain (R)

  37. Mechanism of action • Penicillins inhibit a bacterial enzyme called the transpeptidase enzyme which is involved in the synthesis of the bacterial cell wall • The b-lactam ring is involved in the mechanism of inhibition • Penicillin becomes covalently linked to the enzyme’s active site leading to irreversible inhibition Covalent bond formed to transpeptidase enzyme Irreversible inhibition

  38. NAM NAM NAM NAM NAM NAM NAM NAM NAM NAG NAG NAG NAG NAG NAG L-Ala L-Ala L-Ala L-Ala L-Ala L-Ala L-Ala L-Ala L-Ala D-Glu D-Glu D-Glu D-Glu D-Glu D-Glu D-Glu D-Glu D-Glu L-Lys L-Lys L-Lys L-Lys L-Lys L-Lys L-Lys L-Lys L-Lys Bond formation inhibited by penicillin Mechanism of action - bacterial cell wall synthesis

  39. Cross linking Mechanism of action - bacterial cell wall synthesis

  40. Mechanism of action - bacterial cell wall synthesis • Penicillin inhibits final crosslinking stage of cell wall synthesis • It reacts with the transpeptidase enzyme to form an irreversible covalent bond • Inhibition of transpeptidase leads to a weakened cell wall • Cells swell due to water entering the cell, then burst (lysis) • Penicillin possibly acts as an analogue of the L-Ala-g-D-Glu portion of the pentapeptide chain. However, the carboxylate group that is essential to penicillin activity is not present in this portion

  41. Normal Mechanism Mechanism of action - bacterial cell wall synthesis Alternative theory- Pencillin mimics D-Ala-D-Ala.

  42. Mechanism inhibited by penicillin Mechanism of action - bacterial cell wall synthesis Alternative theory- Penicillin mimics D-Ala-D-Ala.

  43. Penicillin Acyl-D-Ala-D-Ala Mechanism of action - bacterial cell wall synthesis Penicillin can be seen to mimic acyl-D-Ala-D-Ala

  44. Penicillin Analogues - Preparation • 1) By fermentation • vary the carboxylic acid in the fermentation medium • limited to unbranched acids at the a-position i.e. RCH2CO2H • tedious and slow • 2) By total synthesis • only 1% overall yield (impractical) • 3) By semi-synthetic procedures • Use a naturally occurring structure as the starting material for analogue synthesis

  45. Penicillin acylase or chemical hydrolysis Fermentation Semi-synthetic penicillins Penicillin Analogues - Preparation

  46. Penicillin Analogues - Preparation Problem - How does one hydrolyse the side chain by chemical means in presence of a labileb-lactam ring? Answer - Activate the side chain first to make it more reactive Note - Reaction with PCl5 requires involvement of nitrogen’s lone pair of electrons. Not possible for the b-lactam nitrogen.