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

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1. Cell wall inhibitorsBlock synthesis and repair

PenicillinsCephalosporinsCarbapenemsVancomycinBacitracinFosfomycinIsoniazid

2. Cell membraneCause loss of selective permeability

PolymyxinsDaptomycin

3. DNA/RNA

Inhibit replication and transcriptionInhibit gyrase(unwinding enzyme) QuinolonesInhibit RNA polymerase Rifampin

Ribosome

mRNA DNA

4. Protein synthesis inhibitors acting on ribosomes

Site of action50S subunit Erythromycin

ClindamycinSynercidPleuromutilins

Site of action30S subunit

Aminoglycosides Gentamicin StreptomycinTetracyclinesGlycylcyclines

Both 30Sand 50SBlocks initiation of proteinsynthesis Linezolid

5. Folic acid synthesis

Block pathways and inhibitmetabolism Sulfonamides (sulfa drugs) Trimethoprim

Substrate

Enzyme

Product

Folate Synthesis Inhibitors

Cell Wall Inhibitors

• Target peptidoglycan synthesis

• Bactericidal

• Active against young growing cells

Inhibitors of Cell Wall Synthesis : Penicillins

• Block cross-linking of peptidoglycan

O

N

N

S

NucleusR Group

ThiazolidineBeta-lactam

Nafcillin

COCH3

CH3

COOH

•Beta-lactam ring•Different spectra of action•Often cause allergic reactions

Inhibitors of Cell Wall Synthesis : Penicillins

• Penicilinase-resistant penicillins (methicillin)

• Extended-spectrum penicillins (ampicillin)

• Penicillins + -lactamase inhibitors (clavulanic acid) (augmentin)

Inhibitors of Cell Wall Synthesis: Penicillins

Figure 20.8

Inhibitors of Cell Wall Synthesis: Cephallosporins

COOHCH2

65

43

2OC

O

Cephalothin(first generation*)

O

C

O

N

C

O

N

N N

NS N

SN

N N

N

N

N

S

S

C

O

OO

N

N

N

N

N

C

S

S

R Group1 R Group2Basic Nucleus

CH2 N7

S or O

CH2

NH2

CH3

Cefotiam (second generation)

CH2 CH2

CH3

CH3

OH CH

COONa

Moxalactam(third generation)

CH2

CH3

NH2Cefepime(fourthgeneration)NH

OCH3

CH2

CH3

NH2

OH Ceftobiprole(fifth generation)

NH

*New improved versions of drugs are referred to as new “generations.”

N1

• Isolated in 1940s from the mold Cephallosporium acremonium.

-lactam ring, resistant to penicillnases.

• Broad spectrum (2nd, 3rd, and 4th generations more effective against gram-negatives, 5th generations effective against MRSA)

• Less allergenic.

• Administered parenterally

• 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.

Inhibitors of Cell Wall Synthesis: Other Beta-Lactam Antibiotics

Other Inhibitors of Cell Wall Synthesis

• Polypeptide antibiotics

• Bacitracin: topical application against gram-positives

• 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

mRNAis misread,protein is incorrect

Formationof peptidebonds isblocked

30S

Ribosome is prevented from translocating

30S

tRNA isblocked,no protein issynthesized

30S

30S

Aminoglycosides

Chloramphenicol

Oxazolidinones

Tetracyclines

Erythromycin

50Saa aa

30S

mRNA

50Saaaa

PreventInitiation andBlock ribosomeassembly

50Saa

aa

mRNA

mRNA

50S

30S

30S

mRNA

mRNA

50Saa aa

30S

• Target 30S and 50S ribosomal subunits

• Side effect: damage to eucaryotic mitochondria.

Protein Synthesis Inhibitors

• Produced by Streptomyces

• Binds 30S, distorts the ribosome: causes translation errors

• Streptomycin: serious G- infections

• NeomycinTriple antibiotic

cream

• Side effects: oto- and nephrotoxicity

Inhibitors of Protein Synthesis:Aminoglycosides

Inhibitors of Protein SynthesisTetracyclines

• 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

• Binds 50S subunit: Prevents peptide bond formation

• Wide spectrum, cheap

• Toxicity: aplastic anemias (bone marrow supression)

Inhibitors of Protein Synthesis: Chloramphenicol

• New class of antibiotics, developed in 2000s

• Bind to 50S, prevent N-formyl-methionyl-tRNA binding to the ribosome: prevent initiation

• Linezolid: used to treat MRSA and VRE: drug of “last resort”.

Inhibitors of Protein Synthesis: Oxazolidinones

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 membrane, cause loss of selective permeability

• Target: ergosterol

• Nystatin

• Amphotericin B (used to treat systemic infections)

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O O

O

(a)

OH

OH OH

OH

OHOH OH

OH

Anti-Fungal Drugs

• Azoles

• Inhibit ergosterol synthesis

• Griseofulvin

• effective against ringworm

• inhibits microtubules

• prevents cell division

C

N

N

(b)

Cl

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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.

•Acyclovir• Disrupts Herpesviruses

replication• Purine analog

•Ribavirin• Blocks RNA synthesis

(RSV and hemorrhagic fever)

•Anti HIV agents:Reverse transcriptase

inhibitorsAZT

Antiviral Drugs: Inhibition of Nucleic Acid Synthesis

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

N

SR

O

R

O C

S

D1C1

BA C DX

1

1. Inactivation of a drug like penicillin by penicillinase, an enzyme that cleaves a portion of the molecule and renders it inactive.

2

4

3

5

2. The receptor that transports thedrug is altered, so that the drugcannot enter the cell.

3. Specialized membrane proteinsare activated and continuallypump the drug out of the cell.

4. Binding site on target (ribosome)is altered so drug has no effect.

5. The drug has blocked the usualmetabolic pathway (green), so themicrobe circumvents it by using analternate, unblocked pathway thatachieves the required outcome (pink).

1. Drug inactivation

Active penicillin

Penicillinase

Inactive penicillin

CH3

CH3

COOHOH

NH

COOH

2. Decreased permeabilityDrug

Normalreceptor

Differentreceptor3. Activation of drug pumps

Drug Inactivedrugpump

Activedrugpump

4. Change in drug binding site

5. Use of alternate metabolic pathwayDrug acts

Product

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

• Disk Diffusion Assay

• Kirby-Bauer

• Standardized conditions

• Zones of inhibition

• Larger zone indicates more susceptible

• Smaller zone indicates more resistant

S

R

R

I

I

S

1 2 3 40 5

Kirby-Bauer Disc Diffusion Test*Oxytetracycline 30g(R<17 mm;S 22mm)

Enrofloxacin 5 g(R < 17 mm;S 22 mm) Gentamicin 10 g

(R < 17 mm; S 21 mm)

OT30

GN10

CTX30

AMP10

C30

Cefotaxime 30 g(R < 14 mm; S 23 mm)

Ampicillin10g(R<14mm;S22mm)Chloramphenicol 30 g

(R < 21 mm; S 21 mm)

= Zone of Inhibition

= Antibiotic carrier (disc)

= Region of bacterial growth

Disc Diffusion Test (schematic).Example and evaluation of a sensitivity test, agar diffusion method.R = resistant, I = intermediate, S = sensitive

(b)

ENRmm

ENR5

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

• Drug diluted in series

• Inoculate and incubate

• Look for growth (MIC)

0

Same inoculum size of test bacteria added

Negativecontrol

0.2 0.4 0.8 1.6 12.86.43.2 g/ml

Increasing concentration of drug

No growth(a) Growth

(b)

Co

ntro

l

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