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

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

• 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.

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

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

The Dyes Used in Gram Staining

Methyl Violet 10B Safranin

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.

The Carbohydrate Building Blocks

N-Acetylglucosamine(NAG)

N-Acetylmuraminic Acid(NAM)

Antibiotics that Target the Bacterial Cell Envelope Include:

• The -Lactam Antibiotics

• Vancomycin

• Daptomycin

Target 2: The Bacterial Process of Protein Production

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

Structure of the bacterial ribosome.

Antibiotics that Block Bacterial Protein Production Include:

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

Target 3: DNA and Bacterial Replication

Bacterial synthesis of tetrahydrofolate.

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.

Antibiotics that Target DNA and Replication Include:

• Sulfa Drugs

• Quinolones

• Metronidazole

Which Bacteria are Clinically Important?

• 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

Antibiotics that Target the Bacterial Cell Envelope

• The -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.

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


• 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.


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


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


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


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


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


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


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


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)


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

Deep Intramuscular (IM) Formulations of Penicillin G

Penicillin G benzathine Penicillin G Procaine

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


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


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


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

Nafcillin

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.


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


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


• 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

Link

LINK

• 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


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


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.

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

Documents

bacterial species

bacterial ribosome

bacterial protein production

bacterial weak points

gramnegative bacterium

outer capsule

outer membranes

shapethe cell wall