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Transcript of Antibiotics. Step 1: How to Kill a Bacterium. What are the bacterial weak points? Specifically,...
Step 1: How to Kill a Bacterium.
• What are the bacterial weak points?
• Specifically, which commercial antibiotics target each of these points?
• The bacterial cell wall allows the microorganism to maintain its shape
• The cell wall also provides structural support to withstand changes in osmotic pressure.
Classification 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.
Diagrams of the cell envelope structure of Gram-negative (left) and Gram-positive bacteria. Key: peptidoglycan layer (yellow); protein (purple); teichoic acid (green); phospholipid ( brown); lipopolysaccharide (orange).
Figure 1. General overview of lipopolysaccharide (LPS) on the outer membrane of a Gram-negative bacterium. LPS consists of 3 major components: the highly variable outer O-antigen segment; a more conserved core, which is divided into outer and inner segments; and the bioactive lipid A portion. Variation within the length of the LPS, due to mutational absence of specific structures, not only changes the phenotypic appearance of the bacterium (i.e., smooth [S], semi-rough [SR], or rough [R]), but may also change some bioactive responses by the host to the bacterium itself. (A) Some bacterial species contain an outer capsule that protects the bacterium from host defenses such as complement, lysis, and phagocytosis. (B) Outer lipid bilayer with LPS which is approximately 8 nm in width. (C) Peptidoglycan layer. (D) Inner bilipid membrane. Note: Additional lipoproteins, porin complexes, and additional membrane proteins established within and surrounding the inner and outer membranes have been removed to simplify the diagram (Raetz, 1992; Caroff et al., 2002).
http://student.ccbcmd.edu/courses/bio141/labmanua/lab6/images/gram_stain_11.swf
Gram-Staining Animation Link
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:
• The -Lactam Antibiotics
• Vancomycin
• Daptomycin
Antibiotics that Block Bacterial Protein Production Include:
• Rifamycins• Aminoglycosides• Macrolides and Ketolides• Tetracyclines and Glycylcyclines• Chloramphenicol• Clindamycin• Streptogramins• Linezolid (member of Oxazolidinone Class)
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.
LINKLINK
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.
• Gram-positive aerobic bacteria
• Gram-negative aerobic bacteria
• Anaerobic bacteria (both Gram + and -)
• Atypical bacteria
• Spirochetes
• Mycobacteria
General Classes of Clinically Important Bacteria Include:
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
• 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
• Gram-positive anaerobic bacilli– Clostridium difficile– Clostridium tetani– Clostridium botulinum
• Gram-negative anaerobic bacilli– Bacteroides fragilis
Atypical Bacteria of Clinical Importance Include:
• Chlamydia
• Mycoplasma
• Legionella
• Brucella
• Francisella tularensis
• Rickettsia
Spirochetes of Clinical Importance Include:
• Treponema pallidum
• Borrelia burgdorferi
• Leptospira interrogans
Mycobacteria of Clinical Importance Include:
• Mycobacterium tuberculosis
• Mycobacterium avium
• Mycobacterium leprae
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.
Mechanism of Action of -Lactam Antibiotics
• 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.
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.
Six P's by which the action of β-lactams may be blocked:
(1) penetration,
(2) porins,
(3) pumps,
(4) penicillinases (β-lactamases),
(5) penicillin-binding proteins (PBPs), and
(6) peptidoglycan.
Category Parenteral Agents Oral Agents
Natural Penicillins Penicillin G Penicillin V
Antistaphylococcal penicillins
Nafcillin, oxacillin Dicloxacillin
Aminopenicillins Ampicillin Amoxicillin and Ampicillin
Aminopenicillin + -lactamase inhibitor
Ampicillin-sulbactam Amoxicillin-clavulanate
Extended-spectrum penicillin
Piperacillin, ticaricillin Carbenicillin
Extended-spectrum penicillin + -lactamase inhibitor
Piperacillin-tazobactam, ticaricillin-clavulanate
The Penicillins
INTRODUCTION TO PENICILLININTRODUCTION TO PENICILLIN
• Antibacterial agents which inhibit bacterial cell wall synthesisAntibacterial agents which inhibit bacterial cell wall synthesis• Discovered by Fleming from a fungal colony (1928) Discovered by Fleming from a fungal colony (1928) • Shown to be non toxic and antibacterialShown to be non toxic and antibacterial
• Isolated and purified by Florey and Chain (1938)Isolated and purified by Florey and Chain (1938)• First successful clinical trial (1941) First successful clinical trial (1941) • Produced by large scale fermentation (1944)Produced by large scale fermentation (1944)• Structure established by X-Ray crystallography (1945) Structure established by X-Ray crystallography (1945) • Full synthesis developed by Sheehan (1957)Full synthesis developed by Sheehan (1957)• Isolation of 6-APA (1958-60) Isolation of 6-APA (1958-60) - -
development of semi-synthetic penicillinsdevelopment of semi-synthetic penicillins• Discovery of clavulanic acid and Discovery of clavulanic acid and -lactamase inhibitors-lactamase inhibitors
http://archive.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/Spencer/spencer_cellwall.html
Acyl side Acyl side chainchain
6-Aminopenicillanic acid6-Aminopenicillanic acid(6-APA)(6-APA)
STRUCTURESTRUCTURE
Side chain varies depending on carboxylic acid present in fermentation mediumSide chain varies depending on carboxylic acid present in fermentation medium
-Lactam-Lactamringring
ThiazolidineThiazolidineringring
present in corn steep liquor
Penicillin GCH2 CO2H
Benzyl penicillin (Pen G)Benzyl penicillin (Pen G)
R =R =
Phenoxymethyl penicillin (Pen V)Phenoxymethyl penicillin (Pen V)
R =R =
CH2
O CH2
N
S Me
Me
HN
H H
CO2HO
C
O
R
Penicillin V(first orally active penicillin)
OCH2 CO2H
Shape of Penicillin GShape of Penicillin G
Folded ‘envelope’ shape Folded ‘envelope’ shape
H NO
NHC
O
R
H
S
CO2H
H
Me
Me
..
Properties of Penicillin GProperties of Penicillin G
• Active vs. Gram +ve bacilli and some Gram -ve cocciActive vs. Gram +ve bacilli and some Gram -ve cocci• Non toxicNon toxic• Limited range of activityLimited range of activity• Not orally active - must be injectedNot orally active - must be injected• Sensitive to Sensitive to -lactamases -lactamases
(enzymes which hydrolyse the (enzymes which hydrolyse the -lactam ring)-lactam ring)• Some patients are allergic Some patients are allergic • Inactive vs. Inactive vs. StaphylococciStaphylococci
Drug DevelopmentDrug Development
AimsAims• To increase chemical stability for oral administrationTo increase chemical stability for oral administration• To increase resistance to To increase resistance to -lactamases-lactamases• To increase the range of activityTo increase the range of activity
SARSAR
ConclusionsConclusions• Amide and carboxylic acid are involved in bindingAmide and carboxylic acid are involved in binding• Carboxylic acid binds as the carboxylate ionCarboxylic acid binds as the carboxylate ion• Mechanism of action involves theMechanism of action involves the-lactam ring-lactam ring• Activity related to Activity related to -lactam ring strain -lactam ring strain
(subject to stability factors)(subject to stability factors)• Bicyclic system increases Bicyclic system increases -lactam ring strain-lactam ring strain• Not much variation in structure is possibleNot much variation in structure is possible• Variations are limited to the side chain (R)Variations are limited to the side chain (R)
Bicyclic system essential
N
S Me
Me
HN
CO2H
O
C H H
O
R
Carboxylic acid essential
Cis Stereochemistry essential
Lactam essential
Amide essential
• Penicillins inhibit a bacterial enzyme called the transpeptidase Penicillins inhibit a bacterial enzyme called the transpeptidase enzyme which is involved in the synthesis of the bacterial cell enzyme which is involved in the synthesis of the bacterial cell wallwall
• The The -lactam ring is involved in the mechanism of inhibition-lactam ring is involved in the mechanism of inhibition• Penicillin becomes covalently linked to the enzyme’s active site Penicillin becomes covalently linked to the enzyme’s active site
leading to irreversible inhibitionleading to irreversible inhibition
Covalent bond formed Covalent bond formed to transpeptidase enzymeto transpeptidase enzymeIrreversible inhibitionIrreversible inhibition
N
S Me
Me
HN
H H
CO2HO
C
O
R
Nu
Enz
CHN
C
CO2H
HH
Me
MeS
HN
O
RO
Nu-Enz-H N
S Me
Me
HN
H H
CO2HO
C
H
Enz-Nu
O
R
Mechanism of actionMechanism of action
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-LysL-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis
NAM NAM NAMNAGNAG
NAM NAM NAMNAGNAG
NAM NAM NAMNAGNAG
Bond formationinhibited bypenicillin
D-AlanineTRANSPEPTIDASE
PENICILLIN
SUGARBACKBONE
NAM
L-Ala
NAG
D-Glu
L-Lys
D-Ala
D-Ala
Gly Gly Gly Gly Gly Gly GlyGlyGlyGlyL-Lys
NAG
D-Ala
D-Ala
D-Glu
L-Ala
NAM
SUGARBACKBONE
L-Lys Gly Gly
D-Ala
NAM
L-Ala
NAG
D-Glu
L-Lys Gly Gly Gly Gly GlyGlyGlyGly
NAG
D-Ala
D-Glu
L-Ala
NAM
Cross linkingCross linking
Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis
• Penicillin inhibits final crosslinking stage of cell wall Penicillin inhibits final crosslinking stage of cell wall synthesissynthesis
• It reacts with the transpeptidase enzyme to form an It reacts with the transpeptidase enzyme to form an irreversible covalent bondirreversible covalent bond
• Inhibition of transpeptidase leads to a weakened cell wall Inhibition of transpeptidase leads to a weakened cell wall
• Cells swell due to water entering the cell, then burst (lysis)Cells swell due to water entering the cell, then burst (lysis)
• Penicillin acts as an analogue of the D-Ala-D-Ala portion of Penicillin acts as an analogue of the D-Ala-D-Ala portion of the pentapeptide chain. the pentapeptide chain.
Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis
Alternative theoryAlternative theory- Pencillin mimics D-Ala-D-Ala.- Pencillin mimics D-Ala-D-Ala.
Normal MechanismNormal Mechanism
PeptideChain
D-Ala D-Ala CO2H
OH
PeptideChain
Gly
HOH
PeptideChain
PeptideChain
D-Ala GlyPeptideChain
O
D-Ala
Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis
Alternative theoryAlternative theory- Penicillin mimics D-Ala-D-Ala.- Penicillin mimics D-Ala-D-Ala.
Mechanism inhibited by penicillinMechanism inhibited by penicillin
OH
PeptideChain
Gly
H
Blocked H2OBlocked
Blocked Irreversibly blocked
HCR
O
CO2H
NH
OMe
Me
N
S
S
HN
O
O
Me
Me
NH
CO2H
C
O
R H S
HN
O
O
Me
Me
NH
CO2H
C
O
R H
Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis
Penicillin can be seen to mimic acyl-D-Ala-D-AlaPenicillin can be seen to mimic acyl-D-Ala-D-Ala
PenicillinPenicillin Acyl-D-Ala-D-AlaAcyl-D-Ala-D-Ala
HH
CO2H
HN
OMe
Me
N
SC
R
O
Me
CO2H
HN
O CH3
HN
HH
C
R
O
Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis
Penicillin Analogues - PreparationPenicillin Analogues - Preparation
1) By fermentation1) By fermentation • vary the carboxylic acid in the fermentation medium vary the carboxylic acid in the fermentation medium • limited to unbranched acids at the limited to unbranched acids at the -position i.e. RCH-position i.e. RCH22COCO22HH• tedious and slowtedious and slow
2) By total synthesis2) By total synthesis • only 1% overall yield (impractical)only 1% overall yield (impractical)
3) By semi-synthetic procedures3) By semi-synthetic procedures • Use a naturally occurring structure as the starting material for Use a naturally occurring structure as the starting material for
analogue synthesisanalogue synthesis
Penicillin Analogues - PreparationPenicillin Analogues - Preparation
Fermentation
Penicillin acylasePenicillin acylaseor chemical hydrolysisor chemical hydrolysis
O
CClR
Penicillin G
HC
HN
CO2HO Me
Me
N
S
O
CH2
6-APA
OCO2H
HHH2N
Me
MeS
N
Semi-synthetic penicillinsSemi-synthetic penicillinsN
S Me
Me
HN
H H
CO2HO
C
O
R
Penicillin Analogues - PreparationPenicillin Analogues - Preparation
ProblemProblem - How does one hydrolyse the side chain by chemical - How does one hydrolyse the side chain by chemical
means in presence of a labilemeans in presence of a labile -lactam ring?-lactam ring?
AnswerAnswer - Activate the side chain first to make it more reactive - Activate the side chain first to make it more reactive
NoteNote - Reaction with PCl - Reaction with PCl55 requires involvement of nitrogen’s requires involvement of nitrogen’s
lone pair of electrons. Not possible for the lone pair of electrons. Not possible for the -lactam nitrogen.-lactam nitrogen.
N
SNH
OCO2H
CO
PhCH2PCl5
PENNC
Cl
PhCH2
ROH
PENNC
OR
PhCH2
H2O6-APA
Problems with Penicillin GProblems with Penicillin G
• It is sensitive to stomach acids It is sensitive to stomach acids
• It is sensitive to It is sensitive to -lactamases - -lactamases - enzymes which hydrolyse the enzymes which hydrolyse the --lactam ringlactam ring
• it has a limited range of activityit has a limited range of activity
Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity Reasons for sensitivityReasons for sensitivity
1) Ring Strain1) Ring Strain
H2O
H
N
S Me
Me
HN
H H
CO2HO
C
O
R
Relieves ring strainRelieves ring strain
CHN
HO2C
CO2H
HH
Me
MeS
HN
O
R
Acid or enzyme
N
S Me
Me
HN
H H
CO2HO
C
HO
O
R
Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity
2) Reactive 2) Reactive -lactam carbonyl group-lactam carbonyl group Does not behave like a tertiary amideDoes not behave like a tertiary amide
• Interaction of nitrogen’s lone pair with the carbonyl group is not possible Interaction of nitrogen’s lone pair with the carbonyl group is not possible • Results in a reactive carbonyl groupResults in a reactive carbonyl group
Tertiary amideTertiary amide
C
O
R
NR2 Unreactive
R
C
R
N
O
R
-Lactam-Lactam
Folded ringsystem
N
S
OH
Me
Me
CO2H
Impossiblystrained
N
SMe
Me
CO2H
O
Reasons for sensitivityReasons for sensitivity
X
Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity
3) Acyl Side Chain3) Acyl Side Chain - neighbouring group participation in the hydrolysis mechanism- neighbouring group participation in the hydrolysis mechanism
H
Furtherreactions
-HN
S
O
N
O
R R
O
N
O
S
HN
C N
O
S
N
H
R
O
H
Reasons for sensitivityReasons for sensitivity
C
E.W.G.
O
HN
O
S
N
H
Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity
ConclusionsConclusions
• The The -lactam ring is essential for activity and must be -lactam ring is essential for activity and must be retainedretained
• Therefore, cannot tackle factors 1 and 2Therefore, cannot tackle factors 1 and 2• Can only tackle factor 3Can only tackle factor 3
StrategyStrategyVary the acyl side group (R) to make it electron withdrawing to Vary the acyl side group (R) to make it electron withdrawing to decrease the nucleophilicity of the carbonyl oxygendecrease the nucleophilicity of the carbonyl oxygen
DecreasesDecreasesnucleophilicitynucleophilicity
N
S
HN
O
C
O
CH2PhOH
Penicillin VPenicillin V(orally active)(orally active)
Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity
ExamplesExamples
electronegativeelectronegativeoxygenoxygen
• Very successful semi-Very successful semi-synthetic penicillinssynthetic penicillinse.g. ampicillin, oxacilline.g. ampicillin, oxacillin
• Better acid stability and orally activeBetter acid stability and orally active• But sensitive to But sensitive to -lactamases-lactamases• Slightly less active than Penicillin GSlightly less active than Penicillin G• Allergy problems with some patientsAllergy problems with some patients
X = NH2, Cl, PhOCONH, Heterocycles, CO2H
C
HC
O
HN
O
S
N
X
R
H
Differences between Bicillin C-R and Bicillin L-A
Bicillin C-R 900/300 (penicillin G benzathine and penicillin G procaine injectable suspension) contains the equivalent of 900, 000 units of penicillin G as the benzathine and 300, 000 units of penicillin G as the procaine salts. It is available for deep intramuscular injection.
Bicillin L-A suspension in the disposable-syringe formulation is viscous and opaque. It is available in a 1 mL, 2 mL, and 4 mL sizes containing the equivalent of 600,000, 1,200,000 and 2,400,000 units respectively of penicillin G as the benzathine salt.
Bicillin L-A (2,400,000 units) is approved for treatment of syphillis, whereas Bicillin C-R (not sold in 2,400,000 unit size) is not.
A unit of penicillin is defined as that amount which has the same activity as 0.6 g of pure penicillin G sodium salt.
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
(very limited spectrum against gram-negative bacteria)
Anaerobic bacteria
Clostridia spp. (except C. difficile), Antinomyces israelii
Spirochetes Treponema pallidum (syphilis)
Leptospira spp.
Problem 2 - Sensitivity to Problem 2 - Sensitivity to -Lactamases-Lactamases
Notes on Notes on -Lactamases-Lactamases• Enzymes that inactivate penicillins by opening Enzymes that inactivate penicillins by opening -lactam rings-lactam rings• Allow bacteria to be resistant to penicillinAllow bacteria to be resistant to penicillin• Transferable between bacterial strains (i.e. bacteria can Transferable between bacterial strains (i.e. bacteria can
acquire resistance)acquire resistance)• Important w.r.t. Important w.r.t. Staphylococcus aureusStaphylococcus aureus infections in hospitals infections in hospitals• 80% 80% StaphStaph. infections in hospitals were resistant to penicillin . infections in hospitals were resistant to penicillin
and other antibacterial agents by 1960and other antibacterial agents by 1960• Mechanism of action for lactamases is identical to the Mechanism of action for lactamases is identical to the
mechanism of inhibition for the target enzymemechanism of inhibition for the target enzyme • But product is removed efficiently from the lactamase active But product is removed efficiently from the lactamase active
sitesite
-LactamaseN
S Me
Me
HN
CO2HO
CH
O
R
CHN
CO2H
Me
MeS
HN
H
HO2C
O
R
BulkyBulkygroupgroup
Problem 2 - Sensitivity to Problem 2 - Sensitivity to -Lactamases-Lactamases
StrategyStrategy
EnzymeEnzyme
C
R
HN
O
CO2H
Me
MeS
N
H H
O
• Block access of penicillin to active site of enzyme by Block access of penicillin to active site of enzyme by introducing bulky groups to the side chain to act as steric introducing bulky groups to the side chain to act as steric shieldsshields
• Size of shield is crucial to inhibit reaction of penicillins withSize of shield is crucial to inhibit reaction of penicillins with--lactamases but not with the target enzyme (transpeptidase) lactamases but not with the target enzyme (transpeptidase)
orthoortho groups groups importantimportant
Problem 2 - Sensitivity to Problem 2 - Sensitivity to -Lactamases-Lactamases
ExamplesExamples - Methicillin (Beecham - 1960) - Methicillin (Beecham - 1960)
• Methoxy groups block access to Methoxy groups block access to -lactamases but not to transpeptidases-lactamases but not to transpeptidases• Active against some penicillin G resistant strains (e.g. Active against some penicillin G resistant strains (e.g. StaphylococcusStaphylococcus))• Acid sensitive (no e-withdrawing group) and must be injectedAcid sensitive (no e-withdrawing group) and must be injected• Lower activity w.r.t. Pen G vs. Pen G sensitive bacteria (reduced access Lower activity w.r.t. Pen G vs. Pen G sensitive bacteria (reduced access
to transpeptidase) to transpeptidase) • Poorer range of activityPoorer range of activity• Poor activity vs. some Poor activity vs. some streptococcistreptococci• Inactive vs. Gram - bacteriaInactive vs. Gram - bacteria
N
S Me
Me
HN
CO2H
O
MeO
OMe
H HC
O
Problem 2 - Sensitivity to Problem 2 - Sensitivity to -Lactamases-Lactamases
ExamplesExamples - Oxacillin - Oxacillin
• Orally active and acid resistantOrally active and acid resistant• Resistant to Resistant to -lactamases-lactamases• Active vs. Active vs. Staphylococcus aureusStaphylococcus aureus• Less active than other penicillinsLess active than other penicillins• Inactive vs. Gram - bacteriaInactive vs. Gram - bacteria• Nature of R & R’ influences absorption and plasma protein bindingNature of R & R’ influences absorption and plasma protein binding• Cloxacillin better absorbed than oxacillinCloxacillin better absorbed than oxacillin• Flucloxacillin less bound to plasma protein, leading to higher Flucloxacillin less bound to plasma protein, leading to higher
levels of free druglevels of free drug
Bulky and e- withdrawing
Oxacillin R = R' = HOxacillin R = R' = HCloxacillin R = Cl, R' = HCloxacillin R = Cl, R' = HFlucloxacillin R = Cl, R' = FFlucloxacillin R = Cl, R' = F
N
O
C
O
HN
OCO2H
Me
MeS
N
R'
R
Me
H H
Antistaphylococcal Penicillins include Nafcillin and Oxacillin (parenteral) as well as Dicloxacillin (oral)
Gram-positive bacteria Some Staphylococcus aureus, Some Staphylococcus epidermidis
Problem 3 - Range of ActivityProblem 3 - Range of Activity
FactorsFactors1.1. Cell wall may have a coat preventing access to the cellCell wall may have a coat preventing access to the cell2.2. Excess transpeptidase enzyme may be presentExcess transpeptidase enzyme may be present3.3. Resistant transpeptidase enzyme (modified structure)Resistant transpeptidase enzyme (modified structure)4.4. Presence of Presence of -lactamases-lactamases5.5. Transfer of Transfer of -lactamases between strains-lactamases between strains6.6. Efflux mechanismsEfflux mechanisms
StrategyStrategy• The number of factors involved make a single strategy The number of factors involved make a single strategy
impossibleimpossible• Use trial and error by varying R groups on the side chainUse trial and error by varying R groups on the side chain• Successful in producing broad spectrum antibioticsSuccessful in producing broad spectrum antibiotics• Results demonstrate general rules for broad spectrum Results demonstrate general rules for broad spectrum
activity.activity.
Problem 3 - Range of ActivityProblem 3 - Range of Activity
1.1. R= hydrophobic results in high activity vs. Gram + bacteria R= hydrophobic results in high activity vs. Gram + bacteria and poor activity vs. Gram - bacteriaand poor activity vs. Gram - bacteria
2.2. Increasing hydrophobicity has little effect on Gram + activity Increasing hydrophobicity has little effect on Gram + activity but lowers Gram - activitybut lowers Gram - activity
3.3. Increasing hydrophilic character has little effect on Gram Increasing hydrophilic character has little effect on Gram + activity but increases Gram - activity+ activity but increases Gram - activity
4.4. Hydrophilic groups at the Hydrophilic groups at the -position (e.g. NH-position (e.g. NH22, OH, CO, OH, CO22H) H)
increase activity vs Gram - bacteriaincrease activity vs Gram - bacteria
Results of varying R in Pen GResults of varying R in Pen G
Problem 3 - Range of ActivityProblem 3 - Range of Activity
Examples of Aminopenicillins include:Examples of Aminopenicillins include:
Class 1 - NHClass 1 - NH22 at the at the -position-position
Ampicillin and Amoxicillin (1964)Ampicillin and Amoxicillin (1964)
Ampicillin Ampicillin ((Omnipen, Polycillin, Principen)
2nd most used penicillin2nd most used penicillinAmoxicillin (Amoxil)Amoxicillin (Amoxil)
H
C
H
O
HNC
NH2
HO
O
C
NH2
CHN
O
H
H
O
Problem 3 - Range of ActivityProblem 3 - Range of Activity
• Active vs Gram + bacteria and Gram - bacteria which do Active vs Gram + bacteria and Gram - bacteria which do not produce not produce -lactamases-lactamases
• Acid resistant and orally activeAcid resistant and orally active• Non toxicNon toxic• Sensitive to Sensitive to -lactamases-lactamases• Increased polarity due to extra amino groupIncreased polarity due to extra amino group• Poor absorption through the gut wall Poor absorption through the gut wall • Disruption of gut flora leading to diarrheaDisruption of gut flora leading to diarrhea• Inactive vs. Inactive vs. Pseudomonas aeruginosaPseudomonas aeruginosa
Examples of Aminopenicillins Include:Examples of Aminopenicillins Include:
PropertiesProperties
•Amoxicillin is sometimes used together with clarithromycin (Biaxin) to treat stomach ulcers caused by Helicobacter pylori, a Gram negative bacteria•Also, a stomach acid reducer (lansoprazole, or Prevacid) is sometimes added.
Clarithromycin (Biaxin)
Lansoprazole(Prevacid)
Amoxicillin
Helicobacter pylori
Helicobacter pylori is linked to stomach inflammation, which may also result in gastric ulcers and stomach cancer
•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)
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
• Note that urease converts the nitrogen of urea into the nitrogen of ammonia• Since the urea nitrogen is a resonance-stabilized amide, its nitrogen is non-
basic, • Conversely, ammonia is basic, with the conjugate acid ammonium ion having
a pKa of 9.4.• Thus the formation of ammonia neutralizes stomach acid in the general
vicinity of the bacterium.
O
N
HNC
C
NH2
S Me
Me
CO2R
H H
H
O
Problem 3 - Range of ActivityProblem 3 - Range of Activity
Prodrugs of AmpicillinProdrugs of Ampicillin (Leo Pharmaceuticals - 1969) (Leo Pharmaceuticals - 1969)
PropertiesProperties• Increased cell membrane permeabilityIncreased cell membrane permeability• Polar carboxylic acid group is masked by the esterPolar carboxylic acid group is masked by the ester• Ester is metabolised in the body by esterases to give the free Ester is metabolised in the body by esterases to give the free
drugdrug
PIVAMPICILLINR = CH2OC
O
CMe3
TALAMPICILLINR = O
O
BACAMPICILLIN
R = CH
Me
OC
O
O CH2Me
Problem 3 - Range of ActivityProblem 3 - Range of Activity
MechanismMechanism
• Ester is less shielded by penicillin nucleusEster is less shielded by penicillin nucleus• Hydrolysed product is chemically unstable and degradesHydrolysed product is chemically unstable and degrades• Methyl ester of ampicillin is not hydrolysed in the Methyl ester of ampicillin is not hydrolysed in the
body - bulky penicillin nucleus acts as a steric shieldbody - bulky penicillin nucleus acts as a steric shield
ENZYME
PEN
C O CH2O
OH
H HPEN
C O CH2O
OC
O
CMe3
FormaldehydeFormaldehyde
PEN
C OH
O
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 (note improved spectrum against Gram-negative organisms, relative to Pen G and Pen V)
Anaerobic bacteria Clostridia spp. (except C. difficile), Antinomyces israelii
Spirochetes Borrelia burgdorferi
Ampicillin Amoxicillin
• Assigned Reading:• Antibiotics Basics for Clinicians by Alan R. Hauser, pp. 1-31.
• Lange, Roland P.; Locher, Hans H.; Wyss, Pierre C.; Then, Rudolf L. The targets of currently used antibacterial agents: lessons for drug discovery. Current Pharmaceutical Design (2007), 13(30), 3140-3154. Link
• Levy, Stuart B.; Marshall, Bonnie. Antibacterial resistance worldwide: Causes, challenges and responses. Nature Medicine (New York, NY, United States) (2004), 10(12, Suppl.), S122-S129. Link
• Projan S J; Shlaes D M Antibacterial drug discovery: is it all downhill from here?. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases (2004), 10 Suppl 4 18-22. Link
• Lowy, Franklin D. Antimicrobial resistance: the example of Staphylococcus aureus. Journal of Clinical Investigation (2003), 111(9), 1265-1273. Link
• Goodman and Gillman’s Pharmaceutical Basis of Therapeutics, 12th edition, Chapter 53, pp. 1477-1503.
• Homework questions
1. List the major antibiotic families and their respective mechanisms of action.2. With respect to antimicrobial resistance, what is meant by the term ‘selection density’? 3. Why is the presence of antibacterial agents in wastewater a problem and what is a
potential solution to this problem?4. The actual number of antibacterial targets is probably much larger than that predicted by
a genomic analysis. Explain.5. What is meant by a ‘promiscuous drug’? In what respect might the -lactam antibiotics
satisify this definition?6. Provide several reasons for the current decline in commercial pharmaceutical interest in
developing new antimicrobial products.7. What is ‘Levy’s Law of Antibiotics’?8. Name the gene responsible for the production of b-lactamase in Staphylococcus aureus
and its regulators. Name the gene responsible for methicillin resistance. What does this latter gene code for?
9. What is the difference between bacteriostatic agents and bacteriocidal agents? When might it be sufficient to use a bacteriostatic agent?
10. Name the three different general mechanisms whereby bacteria acquire resistance.11. Describe the mechanisms of both vertical and horizontal transfer of resistance
determinants.12. What is a synergystic combination? Describe such a combination used to treat
enterococcal endocardiditis.13. What is a superinfection?14. Compare the structures of Penicillin G, Penicillin V, Ampicillin, and Methicillin, labeling
the key differences and providing the reasons these differences resulted in an improved drug.