Antibiotics

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Antibiotics
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Step 1 How to Kill a Bacterium.

• What are the bacterial weak points?
• Specifically, which commercial antibiotics target
  each of these points?

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Target 1 The Bacterial Cell Envelope
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Two types of bacteria

• Gram-positive Stained dark blue by Gram-staining
  procedure
• Gram-negative Dont take up the crystal violet
  stain, and take up counterstain (safranin)
  instead, staining pink in the Gram procedure.

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Structure of the bacterial cell envelope.
Gram-positive. Gram-negative.
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Gram staining animation
http//student.ccbcmd.edu/courses/bio141/labmanua/
lab6/images/gram_stain_11.swf
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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.
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Antibiotics that Target the Bacterial Cell
Envelope Include

• The b-Lactam Antibiotics
• Vancomycin
• Daptomycin

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Target 2 The Bacterial Process of Protein
Production
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An overview of the process by which proteins are
produced within bacteria.
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Structure of the bacterial ribosome.
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Antibiotics that Block Bacterial Protein
Production Include

• Rifamycins
• Aminoglycosides
• Macrolides and Ketolides
• Tetracyclines and Glycylcyclines
• Chloramphenicol
• Clindamycin
• Streptogramins
• Linezolid (member of Oxazolidinone Class)

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Target 3 DNA and Bacterial Replication
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Bacterial synthesis of tetrahydrofolate.
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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, 2002314.)
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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.
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Antibiotics that Target DNA and Replication
Include

• Sulfa Drugs
• Quinolones
• Metronidazole

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Which Bacteria are Clinically Important?
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General Classes of Clinically Important Bacteria
Include

• Gram-positive aerobic bacteria
• Gram-negative aerobic bacteria
• Anaerobic bacteria (both Gram and -)
• Atypical bacteria
• Spirochetes
• Mycobacteria

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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
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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

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Anaerobic Bacteria of Clinical Importance

• Gram-positive anaerobic bacilli
• Clostridium difficile
• Clostridium tetani
• Clostridium botulinum
• Gram-negative anaerobic bacilli
• Bacteroides fragilis

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Atypical Bacteria of Clinical Importance Include

• Chlamydia
• Mycoplasma
• Legionella
• Brucella
• Francisella tularensis
• Rickettsia

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Spirochetes of Clinical Importance Include

• Treponema pallidum
• Borrelia burgdorferi
• Leptospira interrogans

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Mycobacteria of Clinical Importance Include

• Mycobacterium tuberculosis
• Mycobacterium avium
• Mycobacterium leprae

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Antibiotics that Target the Bacterial Cell
Envelope

• The b-Lactam Antibiotics

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Mechanism of action of ß-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 ß-lactam antibiotic, this process
is disrupted. The ß-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.
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Mechanism of penicillin-binding protein (PBP)
inhibition by ß-lactam antibiotics. PBPs
recognize and catalyze the peptide bond between
two alanine subunits of the peptidoglycan peptide
side chain. The ß-lactam ring mimics this
peptide bond. Thus, the PBPs attempt to catalyze
the ß-lactam ring, resulting in inactivation of
the PBPs.
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• Six P's by which the action of ß-lactams may be
  blocked
• penetration,
• porins,
• pumps,
• penicillinases (ß-lactamases),
• penicillin-binding proteins (PBPs), and
• peptidoglycan.

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The Penicillins
Category Parenteral Agents Oral Agents
Natural Penicillins Penicillin G Penicillin V
Antistaphylococcal penicillins Nafcillin, oxacillin Dicloxacillin
Aminopenicillins Ampicillin Amoxicillin and Ampicillin
Aminopenicillin b-lactamase inhibitor Ampicillin-sulbactam Amoxicillin-clavulanate
Extended-spectrum penicillin Piperacillin, ticaricillin Carbenicillin
Extended-spectrum penicillin b-lactamase inhibitor Piperacillin-tazobactam, ticaricillin-clavulanate
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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

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http//www.microbelibrary.org/microbelibrary/files
/ccImages/Articleimages/Spencer/spencer_cellwall.h
tml
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STRUCTURE
Side chain varies depending on carboxylic acid
present in fermentation medium
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Shape of Penicillin G
Folded envelope shape
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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

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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)

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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
  enzymes active site leading to irreversible
  inhibition

Covalent bond formed to transpeptidase
enzyme Irreversible inhibition
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Mechanism of action - bacterial cell wall
synthesis
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Mechanism of action - bacterial cell wall
synthesis
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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

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Mechanism of action - bacterial cell wall
synthesis
Alternative theory- Pencillin mimics D-Ala-D-Ala.
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Mechanism of action - bacterial cell wall
synthesis
Alternative theory- Penicillin mimics D-Ala-D-Ala.
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Mechanism of action - bacterial cell wall
synthesis
Penicillin can be seen to mimic acyl-D-Ala-D-Ala
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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

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

• It is sensitive to stomach acids
• It is sensitive to b-lactamases - enzymes which
  hydrolyse the b-lactam ring
• it has a limited range of activity

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Problem 1 - Acid Sensitivity
Reasons for sensitivity
1) Ring Strain
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Problem 1 - Acid Sensitivity
Reasons for sensitivity
2) Reactive b-lactam carbonyl group Does not
behave like a tertiary amide
X

• Interaction of nitrogens lone pair with the
  carbonyl group is not possible
• Results in a reactive carbonyl group

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Problem 1 - Acid Sensitivity
Reasons for sensitivity
3) Acyl Side Chain - neighbouring group
participation in the hydrolysis mechanism
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Problem 1 - Acid Sensitivity
Conclusions

• The b-lactam ring is essential for activity and
  must be retained
• Therefore, cannot tackle factors 1 and 2
• Can only tackle factor 3

Strategy Vary the acyl side group (R) to make it
electron withdrawing to decrease the
nucleophilicity of the carbonyl oxygen
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Problem 1 - Acid Sensitivity
Examples

• Very successful semi-synthetic penicillins
• e.g. ampicillin, oxacillin

• Better acid stability and orally active
• But sensitive to b-lactamases
• Slightly less active than Penicillin G
• Allergy problems with some patients

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Natural penicillins include Penicillin G
(parenteral) and Penicillin V (oral)
Gram-positive bacteria Streptococcus pyogenes, Viridans group streptococci, Some Streptococcus pneumoniae, Some Enterococci, Listeria monocytogenes
Gram-negative bacterai Neisseria meningitidis, Some Haemophilus influenzae
Anaerobic bacteria Clostridia spp. (except C. difficile), Antinomyces israelii
Spirochetes Treponema pallidum Leptospira spp.
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Problem 2 - Sensitivity to b-Lactamases
Notes on b-Lactamases

• Enzymes that inactivate penicillins by opening
  b-lactam rings
• Allow bacteria to be resistant to penicillin
• Transferable between bacterial strains (i.e.
  bacteria can acquire resistance)
• Important w.r.t. Staphylococcus aureus infections
  in hospitals
• 80 Staph. infections in hospitals were resistant
  to penicillin and other antibacterial agents by
  1960
• Mechanism of action for lactamases is identical
  to the mechanism of inhibition for the target
  enzyme
• But product is removed efficiently from the
  lactamase active site

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Problem 2 - Sensitivity to b-Lactamases
Strategy

• Block access of penicillin to active site of
  enzyme by introducing bulky groups to the side
  chain to act as steric shields
• Size of shield is crucial to inhibit reaction of
  penicillins with b-lactamases but not with the
  target enzyme (transpeptidase)

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Problem 2 - Sensitivity to b-Lactamases
Examples - Methicillin (Beecham - 1960)

• Methoxy groups block access to b-lactamases but
  not to transpeptidases
• Active against some penicillin G resistant
  strains (e.g. Staphylococcus)
• Acid sensitive (no e-withdrawing group) and must
  be injected
• Lower activity w.r.t. Pen G vs. Pen G sensitive
  bacteria (reduced access
• to transpeptidase)
• Poorer range of activity
• Poor activity vs. some streptococci
• Inactive vs. Gram - bacteria

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Problem 2 - Sensitivity to b-Lactamases
Examples - Oxacillin
Oxacillin R R' H Cloxacillin R
Cl, R' H Flucloxacillin R Cl, R' F

• Orally active and acid resistant
• Resistant to b-lactamases
• Active vs. Staphylococcus aureus
• Less active than other penicillins
• Inactive vs. Gram - bacteria
• Nature of R R influences absorption and plasma
  protein binding
• Cloxacillin better absorbed than oxacillin
• Flucloxacillin less bound to plasma protein,
  leading to higher
• levels of free drug

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Antistaphylococcal Penicillins include Nafcillin
and Oxacillin (parenteral) as well as
Dicloxacillin (oral)
Gram-positive bacteria Some Staphylococcus aureus, Some Staphylococcus epidermidis
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Problem 3 - Range of Activity

• Factors
• Cell wall may have a coat preventing access to
  the cell
• Excess transpeptidase enzyme may be present
• Resistant transpeptidase enzyme (modified
  structure)
• Presence of b-lactamases
• Transfer of b-lactamases between strains
• Efflux mechanisms

• Strategy
• The number of factors involved make a single
  strategy
• impossible
• Use trial and error by varying R groups on the
  side chain
• Successful in producing broad spectrum
  antibiotics
• Results demonstrate general rules for broad
  spectrum activity.

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Problem 3 - Range of Activity
Results of varying R in Pen G

• R hydrophobic results in high activity vs. Gram
  bacteria and poor activity vs. Gram - bacteria
• Increasing hydrophobicity has little effect on
  Gram activity but lowers Gram - activity
• Increasing hydrophilic character has little
  effect on Gram
• activity but increases Gram - activity
• Hydrophilic groups at the a-position (e.g. NH2,
  OH, CO2H) increase activity vs Gram - bacteria

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Problem 3 - Range of Activity
Examples of Aminopenicillins include
Class 1 - NH2 at the a-position Ampicillin and
Amoxicillin (Beecham, 1964)
Ampicillin (Penbritin) 2nd most used penicillin
Amoxicillin (Amoxil)
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Problem 3 - Range of Activity
Examples of Aminopenicillins Include

• Active vs Gram bacteria and Gram - bacteria
  which do not produce b-lactamases
• Acid resistant and orally active
• Non toxic
• Sensitive to b-lactamases
• Increased polarity due to extra amino group
• Poor absorption through the gut wall
• Disruption of gut flora leading to diarrhea
• Inactive vs. Pseudomonas aeruginosa

Properties
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• Amoxicillin is sometimes used together with
  clarithromycin (Biaxin) to treat stomach ulcers
  caused by Helicobacter pylori, a Gram - bacteria
• Also, a stomach acid reducer (lansoprazole, or
  Prevacid) is sometimes added.

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Helicobacter pylori
Helicobacter pylori is linked to stomach
inflammation, which may also result in gastric
ulcers and stomach cancer
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• In the early 20th century, ulcers were believed
  caused by stress
• In 1982, Robin Warren and Barry Marshall, two
  Australian physicians, suggested link between H.
  pylori and ulcers
• Medical community was slow to accept (first
  abstract describing such results was rejected for
  a poster)

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In 2005, the two researchers, Barry Marshall and
J. Robin Warren, received the Nobel Prize in
medicine for their discovery of the bacterium
Helicobacter pylori and its role in gastritis and
peptic ulcer disease
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Problem 3 - Range of Activity
Prodrugs of Ampicillin (Leo Pharmaceuticals -
1969)

• Properties
• Increased cell membrane permeability
• Polar carboxylic acid group is masked by the
  ester
• Ester is metabolised in the body by esterases to
  give the free drug

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Problem 3 - Range of Activity
Mechanism

• Ester is less shielded by penicillin nucleus
• Hydrolysed product is chemically unstable and
  degrades
• Methyl ester of ampicillin is not hydrolysed in
  the
• body - bulky penicillin nucleus acts as a steric
  shield

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The aminopenicillins include Ampicillin
(parenteral) as well as Amoxicillin and
Ampicillin (both oral)
Gram-positive bacteria Streptococcus pyogenes, Viridans streptococci, Some Streptococcus pneumoniae, Some enterococci Listeria monocytogenes
Gram-negative bacteria Neisseria meningitidis, Some Haemophilus influenzae, Some Enterobacteriaceae
Anaerobic bacteria Clostridia spp. (except C. difficile), Antinomyces israelii
Spirochetes Borrelia burgdorferi