Transcript Inhibitors of Cell Wall Synthesis

Drugs,
Microbes, Host
– The Elements
of
Chemotherapy
Chapter 12
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Learning Objectives
• Identify and explain desirable characteristics of
antibiotics
• Identify and describe the five cellular targets of
antibiotics
• List and describe antibiotics targeting bacterial
cell wall, plasma membrane, protein and nucleic
acid synthesis, and folic acid biosynthesis.
Learning Objectives
• Describe how selective toxicity is achieved in
targeting fungi, viruses and helminthes. Give
examples of antifungal, antiviral, and antihelmintic
drugs.
• Describe five mechanisms of antibiotic resistance,
and explain how it can be acquired by bacteria.
• Describe the Kirby-Bauer and dilution assays for
antibiotic sensitivity
History of Antibiotics
• Paul Erlich: “magic bullet”, Salvarsan.
• Gerhard Domadk: Prontosil
• Alexander Fleming: Penicillin
Properties of Antimicrobial Agents
• Synthetic antimicrobials, antibiotics,
semisynthetic drugs (sources)
• Desirable characteristics
• Selective toxicity (target the pathogen)
• Few side effects (low toxicity)
• Narrow spectrum (leaves normal biota)
• Localization and stability in host
• Shelf life and cost
Possible Adverse Reactions
• Toxicity to organs
• Allergies
• Disruption of normal
flora
• Other adverse effects
Therapeutic Index
• What is the best drug to use?
• Lowest risk of side effects versus
• Highest probability of killing the pathogen
• 50 µg is toxic and 5 µg is effective; T.I. = 10
• 50 µg is toxic and 1 µg is effective; T.I. = 50
• Higher T.I. are better
Targets of Antibiotics
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1. Cell wall inhibitors
Block synthesis and repair
Penicillins
Cephalosporins
Carbapenems
Vancomycin
Bacitracin
Fosfomycin
Isoniazid
2. Cell membrane
Cause loss of selective permeability
Polymyxins
Daptomycin
Ribosome
Substrate
Enzyme
Product
Site of action
50S subunit Erythromycin
Clindamycin
Synercid
Pleuromutilins
Site of action
30S subunit
Aminoglycosides
Gentamicin
Streptomycin
Tetracyclines
Glycylcyclines
Both 30S
and 50S
Blocks initiation of protein
synthesis
Linezolid
3. DNA/RNA
Inhibit replication and transcription
Inhibit gyrase(unwinding enzyme)
Quinolones
Inhibit RNA polymerase
Rifampin
4. Protein synthesis inhibitors
acting on ribosomes
mRNA
DNA
5. Folic acid synthesis
Block pathways and inhibit
metabolism
Sulfonamides (sulfa drugs)
Trimethoprim
Folate Synthesis Inhibitors
Cell Wall
Inhibitors
• Target
peptidoglycan
synthesis
• Bactericidal
• Active against
young growing
cells
Inhibitors of Cell Wall Synthesis :
Penicillins
Nucleus
• Block cross-linking of
peptidoglycan
R Group
Betalactam
Thiazolidine
S
CO
N
CH3
CH3
N
Nafcillin
O
COOH
•Beta-lactam ring
•Different spectra of action
•Often cause allergic
reactions
Inhibitors of Cell Wall Synthesis :
Penicillins
• Penicilinaseresistant penicillins
(methicillin)
• Extendedspectrum
penicillins
(ampicillin)
• Penicillins + lactamase
inhibitors
(clavulanic acid)
(augmentin)
Inhibitors of Cell Wall Synthesis:
Penicillins
Figure 20.8
Inhibitors of Cell Wall Synthesis:
Cephallosporins
• Isolated in 1940s from the
mold Cephallosporium
acremonium.
O
C
CH2
N7
S
O
R Group2
Cephalothin
(first generation*)
S or O
5
6
4
3
N1
2
COOH
• -lactam ring, resistant to
penicillnases.
• Broad spectrum (2nd, 3rd,
and 4th generations more
effective against gramnegatives, 5th generations
effective against MRSA)
Basic Nucleus
R Group1
O
CH2
CH3
CH2
N
OC
Cefotiam (second
Ngeneration)
N
S
NH2
N
CH2
CH2
N
CH3
O
N
OH
CH
C
COONa
CH2
S
Moxalactam
(third
N
generation)
N
N
CH3
NH2
Cefepime
(fourth
generation)
S
O
N
C
C
NH
CH2
N
• Less allergenic.
• Administered parenterally
CH3
N
S
N
CH3
OCH3
OH
N
N
NH2
S
N
N
O
O
*New improved versions of drugs are referred to as new “generations.”
Ceftobiprole
(fifth
generation)
NH
Inhibitors of Cell Wall Synthesis:
Other Beta-Lactam Antibiotics
• Carbapenems: powerful, but potentially very
toxic.
• Reserved as a last line of defense for
pneumonia, septicemia, urinary tract infections
• NDM-1 gene in G- bacteria causes resistance to
carbapenems.
Other Inhibitors of Cell Wall Synthesis
• Polypeptide antibiotics
• Bacitracin: topical application against grampositives
• Vancomycin: glycopeptide
• Important "last line" against MRSA, methicillin
resistant S. aureus (VRSA reported in July
2012)
Other Inhibitors of Cell Wall Synthesis
• Isoniazid – Mycolic acid formation inhibited.
• This is one of the main anti-tuberculosis drugs since
1954.
• Due to resistance, has to be part of multi-drug
therapy.
• Fosphomycin – PEP analog, blocks linking of glycan
and peptide portions of peptidoglycan.
• Treatment of urinary infections.
• Resistance and side effects prevent wider application
Protein Synthesis
Aminoglycosides
Inhibitors
• Target 30S and
50S ribosomal
subunits
50S
aa
aa
Chloramphenicol
30S
50S
aa
aa
Oxazolidinones
• Side effect: damage
to eucaryotic
mitochondria.
30S
mRNA
mRNA
is misread,
protein is
incorrect
Formation
of peptide
bonds is
mRNA blocked
Prevent
Initiation and
Block ribosome
assembly
30S
Tetracyclines
50S
tRNA is
blocked,
no protein is
synthesized
30S
30S
Erythromycin
aa 50S aa
mRNA
Ribosome is
prevented from
translocating
30S
30S
mRNA
Inhibitors of Protein Synthesis:
Aminoglycosides
• Produced by
Streptomyces
• Binds 30S, distorts
the ribosome: causes
translation errors
• Streptomycin:
serious G- infections
• Neomycin
Triple antibiotic
cream
• Side effects: oto- and
nephrotoxicity
Inhibitors of Protein Synthesis
Tetracyclines
• Broad spectrum:
tetracyclin,
doxycyclin
• Blocks the A site:
prevents tRNA entry
• Reversible reaction:
bacteriostatic
• Widespread
resistance
• Side effects
Inhibitors of Protein Synthesis:
Macrolide Antibiotics
• Bind near the P site: Prevent translocation
• Lactone ring + sugars
• Bacteriostatic
• Active against G+
• Erythromycin
• Azithromycin and clarithromycin
• Hepatotoxicity
Inhibitors of Protein Synthesis:
Chloramphenicol
• Binds 50S subunit:
Prevents peptide
bond formation
• Wide spectrum, cheap
• Toxicity: aplastic
anemias (bone
marrow supression)
Inhibitors of Protein Synthesis:
Oxazolidinones
• New class of antibiotics,
developed in 2000s
• Bind to 50S, prevent Nformyl-methionyl-tRNA
binding to the ribosome:
prevent initiation
• Linezolid: used to treat
MRSA and VRE: drug of
“last resort”.
Nucleic Acid Synthesis Inhibitors
• Block:
 Nucleotide synthesis
 DNA replication
 RNA transcription
• Chloroquine: crosslinking of double helix
• Quinolones: block DNA unwinding by inhibiting
helicase
• Purine and pyrimidine analogs (AZT): insert
into viral nucleic acid, block replication.
DNA Replication: Quinolones and
Fluoroquinolones
• Broad spectrum, high potency, readily absorbed.
• Mechanisms are conserved: lead to toxicity
• Inhibit DNA gyrase: prevent DNA synthesis
• Treatment of serious hospital acquired infections: urinary
tract infections, pneumonia.
• High risk for MRSA resistance development: not
recommended for community acquired infections
• Nalidixic acid used in DNA replication studies
• Ciprofloxacin – used in the 2001 anthrax attack
• Levofloxacin – wide spectrum, including anaerobes and
anthrax. May cause damage to muscles and tendons
Transcription of DNA (RNA
synthesis)
• Difficult target because the process is well
conserved
• Rifamycin, Rifampicin
• Bind bacterial RNA polymerase: inhibit RNA
synthesis
• Tratment: tuberculosis, MRSA
Antibacterial Antibiotics:
Injury to the Plasma Membrane
• Bind to phospholipid and lipid A, disrupt
membranes
• Poor selective toxicity
• Must be used topically
• Polymyxin B and E
• Topical (kidney toxicity)
• Combined with bacitracin and neomycin in
over-the-counter preparation.
Antibiotics and Biofilms
• Highly resistant due to:
• Poor penetration.
• Altered gene expression pattern.
• Strategies:
• Treatment of plastic surfaces with antibiotic
before insertion.
• Daptomycin (lipopeptide), adding DNase.
• Disruption of quorum sensing.
Anti-Fungal Drugs
• Eukaryotes: more
similar to human cells
• Polyenes bind
OH
membrane, cause loss O
of selective
OH
permeability
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• Target: ergosterol
OH
OH
OH
OH
O
OH
O
OH
(a)
• Nystatin
• Amphotericin B
(used to treat
systemic infections)
Anti-Fungal Drugs
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• Azoles
N
• Inhibit ergosterol
synthesis
N
• Griseofulvin
• effective against
ringworm
• inhibits
microtubules
• prevents cell
division
C
Cl
(b)
Antiparasitic Chemotherapy
• Quinolones (anti-malaria): disrupt different life
stages of plasmodium.
• Metronidazole: anti-protozoan drug (Entamoeba
histolytica, Giardia lamblia, Trichomonas
vaginalis)
• Mebendazole and albendazole: broad spectrum
anti-helmintic drugs, block glucose utilization
• Pyrantel: paralyzes muscles of intestinal
roundworms.
Anti-Virals: Inhibitors of Virus Entry
• Few antivirals
• Toxicity problems
• Amantadine, Relenza
and Tamiflu
• Inhibits the entry of
Influenza A virus
• Fuzeon
• Blocks binding of HIV
to the GP-41 receptor
• Drug resistance.
Antiviral Drugs: Inhibition of Nucleic
Acid Synthesis
• Acyclovir
• Disrupts Herpesviruses
replication
• Purine analog
• Ribavirin
• Blocks RNA synthesis
(RSV and hemorrhagic
fever)
•Anti HIV agents:
Reverse transcriptase
inhibitors
AZT
Antiviral Drugs:
Nucleoside and Nucleotide Analogs
Antiviral Drugs:
Nucleoside and Nucleotide Analogs
Anti-Viral Drugs: Inhibition of Viral
Assembly/Release/Spread
• Protease inhibitors
• Indinavir, saquinavir
• Used in combination with
reverse transcriptase
inhibitors
• Interferons prevent spread of
viruses to new cells
• Glycoprotein produced by
immune cells
• Viral hepatitis
Mechanism of Antibiotic Resistance
1. Drug inactivation
S
R
1
O
N
S
R
Penicillinase
COOH
O
C
OH
Active penicillin
N
H
CH3
CH3
COOH
Inactive penicillin
1. Inactivation of a drug like penicillin
by penicillinase, an enzyme that
cleaves a portion of the molecule and
renders it inactive.
2. Decreased permeability
Drug
Normal
receptor
2
Different
receptor
3. Activation of drug pumps
Drug Inactive
drug
pump
3
Active
drug
pump
4. Change in drug binding site
2. The receptor that transports the
drug is altered, so that the drug
cannot enter the cell.
3. Specialized membrane proteins
are activated and continually
pump the drug out of the cell.
4. Binding site on target (ribosome)
is altered so drug has no effect.
4
5. Use of alternate metabolic pathway
Drug acts
5
A
B
C
X
C1
D
Product
D1
5. The drug has blocked the usual
metabolic pathway (green), so the
microbe circumvents it by using an
alternate, unblocked pathway that
achieves the required outcome (pink).
Mechanisms of Acquiring Antibiotic
Resistance Genes
Transfer of Resistance
• Resistance (R) factors (plasmids): transferred
by conjugation, transformation or transduction
• Transpozons: duplicated and inserted from one
plasmid to another or from a plasmid to a
chromosome
Preventing Drug Resistance
• Limit drug use - less selective pressure
• Proper drug use - viruses are not affected, use
full dose to ensure elimination of pathogens
• Narrow range antibiotics - kill only the targeted
microbes; less likely complications
• Multiple drug treatments - drugs can work
synergistically; much less likely to get drug
resistance.
Diffusion Assays
Kirby-Bauer Disc Diffusion Test*
• Disk Diffusion Assay
Oxytetracycli
ne 30g
(R<17 mm;S
22mm)
Enrofloxacin 5 g
(R < 17 mm;S 22 mm)
• Kirby-Bauer
0
mm
ENR
1 5
2
3
Gentamicin 10 g
(R < 17 mm; S 21 mm)
4
S
OT
R
30
• Standardized
conditions
CTX
30
GN
I 10
AMP
I 10
S
R
• Zones of inhibition
C
30
Cefotaxime 30 g
Ampicillin10g
Chloramphenicol 30 g
(R < 14 mm; S 23 mm)
(R<14mm;S22mm)
(R < 21 mm; S 21 mm)
• Larger zone indicates
more susceptible
= Zone of
Inhibition
• Smaller zone indicates
more resistant
Disc Diffusion Test (schematic).
Example and evaluation of a sensitivity test,
agar diffusion method.
R = resistant, I = intermediate, S = sensitive
= Region of
bacterial
growth
ENR
5
= Antibiotic
carrier (disc)
(b)
E-Test Strips
• Drug gradient used
• Can determine MIC
• Read where the zone
touches the strip
• MIC: Minimal inhibitory concentration.
• MBC: Minimal bactericidal concentration.
Tube Dilution Assay
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Same inoculum size of test bacteria added
Control
• Drug diluted in series
0
• Inoculate and incubate
Negative
control
(a)
• Look for growth (MIC)
(b)
0.2 0.4 0.8 1.6 3.2 6.4 12.8
m g/ml
Increasing concentration of drug
Growth
No growth