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Antibiotics Step 1: How to Kill a Bacterium. What are the bacterial weak points? Specifically, which commercial antibiotics target each of these points? Target 1: The Bacterial Cell Envelope Two types of bacteria Gram-positive: Stained dark blue by Gram-staining procedure

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step 1 how to kill a bacterium
Step 1: How to Kill a Bacterium.
  • What are the bacterial weak points?
  • Specifically, which commercial antibiotics target each of these points?
two types of bacteria
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.
slide6

Gram staining animation

http://student.ccbcmd.edu/courses/bio141/labmanua/lab6/images/gram_stain_11.swf

slide7

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.

antibiotics that target the bacterial cell envelope include
Antibiotics that Target the Bacterial Cell Envelope Include:
  • The b-Lactam Antibiotics
  • Vancomycin
  • Daptomycin
antibiotics that block bacterial protein production include
Antibiotics that Block Bacterial Protein Production Include:
  • Rifamycins
  • Aminoglycosides
  • Macrolides and Ketolides
  • Tetracyclines and Glycylcyclines
  • Chloramphenicol
  • Clindamycin
  • Streptogramins
  • Linezolid (member of Oxazolidinone Class)
slide15

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.)

slide16

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.

slide19

General Classes of Clinically Important Bacteria Include:

  • Gram-positive aerobic bacteria
  • Gram-negative aerobic bacteria
  • Anaerobic bacteria (both Gram + and -)
  • Atypical bacteria
  • Spirochetes
  • Mycobacteria
gram positive bacteria of clinical importance
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
gram negative bacteria of clinical importance
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
anaerobic bacteria of clinical importance
Anaerobic Bacteria of Clinical Importance
  • Gram-positive anaerobic bacilli
    • Clostridium difficile
    • Clostridium tetani
    • Clostridium botulinum
  • Gram-negative anaerobic bacilli
    • Bacteroides fragilis
atypical bacteria of clinical importance include
Atypical Bacteria of Clinical Importance Include:
  • Chlamydia
  • Mycoplasma
  • Legionella
  • Brucella
  • Francisella tularensis
  • Rickettsia
spirochetes of clinical importance include
Spirochetes of Clinical Importance Include:
  • Treponema pallidum
  • Borrelia burgdorferi
  • Leptospira interrogans
mycobacteria of clinical importance include
Mycobacteria of Clinical Importance Include:
  • Mycobacterium tuberculosis
  • Mycobacterium avium
  • Mycobacterium leprae
slide29

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.

slide30

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.

slide31

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.
slide34

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
slide35

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

slide36

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

slide37

Shape of Penicillin G

Folded ‘envelope’ shape

slide38

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
slide39

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)
slide40

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

slide41

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

slide43

Cross linking

Mechanism of action - bacterial cell wall synthesis

slide44

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
slide45

Normal Mechanism

Mechanism of action - bacterial cell wall synthesis

Alternative theory- Pencillin mimics D-Ala-D-Ala.

slide46

Mechanism inhibited by penicillin

Mechanism of action - bacterial cell wall synthesis

Alternative theory- Penicillin mimics D-Ala-D-Ala.

slide47

Penicillin

Acyl-D-Ala-D-Ala

Mechanism of action - bacterial cell wall synthesis

Penicillin can be seen to mimic acyl-D-Ala-D-Ala

slide48

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
slide49

Penicillin acylase

or chemical hydrolysis

Fermentation

Semi-synthetic penicillins

Penicillin Analogues - Preparation

slide50

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.

slide51

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
slide52

Acid or

enzyme

Relieves ring strain

Problem 1 - Acid Sensitivity

Reasons for sensitivity

1) Ring Strain

slide53

Tertiary amide

Unreactive

b-Lactam

Folded ring

system

Impossibly

strained

Problem 1 - Acid Sensitivity

Reasons for sensitivity

2) Reactive b-lactam carbonyl group

Does not behave like a tertiary amide

X

  • Interaction of nitrogen’s lone pair with the carbonyl group is not possible
  • Results in a reactive carbonyl group
slide54

Further

reactions

Problem 1 - Acid Sensitivity

Reasons for sensitivity

3) Acyl Side Chain

- neighbouring group participation in the hydrolysis mechanism

slide55

Decreases

nucleophilicity

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

slide56

electronegative

oxygen

Penicillin V

(orally active)

Problem 1 - Acid Sensitivity

Examples

  • Better acid stability and orally active
  • But sensitive to b-lactamases
  • Slightly less active than Penicillin G
  • Allergy problems with some patients
  • Very successful semi-synthetic penicillins
  • e.g. ampicillin, oxacillin
slide58

b-Lactamase

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
slide59

Bulky

group

Enzyme

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)
slide60

ortho groups

important

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
slide61

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
slide62

Antistaphylococcal Penicillins include Nafcillin and Oxacillin (parenteral) as well as Dicloxacillin (oral)

slide63

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.
slide64

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
slide65

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)

slide66

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

slide67

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.
slide68

Helicobacter pylori

Helicobacter pylori is linked to stomach inflammation, which may also result in gastric ulcers and stomach cancer

slide69

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)
slide70

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

slide72

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
slide73

PEN

PEN

H

C

OH

O

C

O

O

O

O

PEN

C

C

O

O

C

C

H

H

C

M

e

2

2

3

O

Formaldehyde

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
slide74

The aminopenicillins include Ampicillin (parenteral) as well as

Amoxicillin and Ampicillin (both oral)