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Antibiotics acting on ProteinSynthesis

E. Westhof

1. Considérations générales etintroductives

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Facts about Antibiotics

• Alexander Fleming (1928) discovered penicillin– Ignored until early 1940s with extensive use in war

• 1954 1 million kgs antibiotics produced in US– Now > 25 million kgs

• Humans consume 235 million doses/year– 20%-50% of that use is unnecessary

DEFINITIONS

• Antibiotic: substance produced by micro-organismsthat inhibits other microorganisms

• Antimicrobial agents: antibiotics & synthetic/semi-synthetic agents used to inhibit microorganisms

• Bactericidal (kills bacteria) vs bacteriostatic (stopsactive growth without affecting viability)

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Characteristics of Antibiotics

• Selective toxicity against bacteria• Each antibiotic has specific activity against

certain bacteria – Spectrum of activity(broad vs narrow)

• Determined by– Antibiotic’s concentration at site of infection– Susceptibility of the bacteria

• Obtaining a culture can identify theorganism and its susceptibility to antibiotics

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Antibiotic targets

Diaminopyridines

Quino-lones

PenicillinsGlycopeptides

Sulfamides

LincosamidesStrepto-gramins

Oxazolidinones

Macrolides

Tetra-cyclinesRifamycins

Polymixines

> 50%

Aminoglycosides

Spectrum of Activity

Narrow Spectrum Broad spectrum

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Antibiotic Resistance is Increasing

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The bacterial The bacterial ribosome :ribosome :

270 000 270 000 atoms atoms (C,N, O, P)(C,N, O, P)55 55 proteinsproteins3 RNA (4600 3 RNA (4600 nucleotidesnucleotides))

Un ribosome bactérien à 5.5 Å résolution

Le ribosome d’E. coli synthétise un polypeptide de 100 acides aminés en 5 secondes à 37 °C

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OnlyRNAin the reactionsite

The ribosome, a molecular machine,is a ribozyme

and,like all other known ribozymes,the ribosome uses RNA-based

recognition motifsnot only for catalysis

but alsofor decoding processes.

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Les trois étapes de lasynthèse protéique

1. Initiation (IF2 + GTP > GDP)

2. Elongation (EF-Tu + GTP > GDP)

3. Terminaison (RF-3 + GTP > GDP)

Energetic control of translation

• Energy released from GTP Hydrolysis (IF-2, EF-Tu, EF-G, RF-3)

• Not required for translation• Increases rates• Increases irreversibility

– drives conf. changes.

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Accuracy

• Error rate 1/104

• But only select tRNA by codon/anticodon!• 1 mismatch = 1 wrong H-bond!• So…..?

• Must be a proofreading step analogous toaaRS.

First stepis reversible

Second stepcontrolled byk4/k3 ratiok3 is constantk4 depends onstrength codon-anticodon binding

KineticProofreading

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Ribosome: inhibiteursMécanisme Eu(E)/Pro(P)caryote InhibitionInitiationAcide aurine tricarboxylique P fixation IF sur 30SKasugamycine P fixationARN init.Streptomycine P formation du complexe d’init.Liaison aa-ARNtTetracycline P fixationStreptomycine P erreur de séquence protéiqueParamomycine P erruers dans la sélection des ARNtFormation de la liaison peptidiqueSparsomycine P peptidyl transféraseChloramphénicol P id- fixe sur 50SErythromycine P id-idCycloheximide P translocation du pepptidyl-ARNtTranslocationAcide fusidique P dissociation EF-G-GDPThiostreptone P GTPase EFTu et EFG sur ribosomeToxine diphtérique E eEF2 par ADP ribosylationTerminaisonPuromycine P/E accepteur du groupe peptidyle, fin prématuréeInactivationRicine E inactive ARN28S

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Inhibition of Translation• Numerous antibiotics target translational machinery

Simulates 3’ end of tRNACompetes for A-site Ends nascent chain

Binds to hydrophobic tunnelBlocks egress of peptide chain

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Inhibition of Translation• Numerous antibiotics target translational machinery

Binds at peptidyl transferasesite and inhbits reaction

Binds to 16S rRNA, stabilizesribosome in conformation thatincreases affinity for aatRNA

Definitions

• Sterilization:– Kill all microbes, viruses, and other life forms

• Disinfect, Decontaminate, Pasteurize:

– Reduce the levels of microbes, viruses, andother life forms

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Definitions

• Bacteriostatic– Inhibit the growth of bacteria– May be still viable or metabolically active

• Bacteriocidal– Kill bacteria

• Fungicidal; Fungistatic• Viricidal; Viristatic

Bacteriolytic processes are Bacteriocidal

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Kinetics of Killing

• Microbes die exponentially.– Not simultaneously

• Decimal Reduction Time (D)– Time required to reduce the population 1/10.– 100 cells10 cells

• Increase heat; Decrease D

Sterilization

• D time for vegetative cells– 0.1 to 0.5 minutes at 65°C– Boiling for extended times kills nearly all

species commonly encountered

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a= ‘typical’ mesophileb= ‘typical’ thermophile

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Principles and Definitions

• Selectivity– Selectivty vs toxicity

• Therapeutic index– Toxic dose/ Effective dose

• Categories of antibiotics– Bactericidal

• Usually antibiotic of choice– Bacteriostatic

• Duration of treatment sufficient for host defenses

Principles and Definitions• Antibiotic susceptibility testing (in vitro)

– Minimum inhibitory concentration (MIC)• Lowest concentration that results in inhibition of

visible growth– Minimum bactericidal concentration (MBC)

• Lowest concentration that kills 99.9% of the originalinoculum

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MINIMUM INHIBITORYCONCENTRATION (MIC)

• MIC – lowest antibiotic concentration thatinhibits growth

• MIC90 – concentration required to inhibit90% of the strains (isolates) tested– “the “MIC Benchmark”

• MBC – minimum bactericidal concentration– Lowest concentration that results in 99.9%

killing of organism

Antibiotic Susceptibility Testing

8 4 02 1 Tetracycline (:g/ml)

MIC = 2 :g/ml

Determination of MIC

Chl Amp

Ery

Str

Tet

Disk Diffusion Test

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Assessing the antimicrobial activity of a compound using the minimum inhibitory concentrationmethod A compound to be tested is serially diluted into growth medium, inoculated with a culture andthen incubated. The minimum inhibitory concentration is indicated in the lowestdilution of the compound which prevents growth as indicated by the arrow.

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Disk-Diffusion Method

Bacillus cereusinoculated soaked;alcohol no effect

Gram Staining

• Gram positive• Staphylococcus

epidermidis

• Gram negative• Escherichia coli

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Culture

Antimicrobial Drug ResistancePrinciples and Definitions

• Clinical resistance• Resistance can arise by mutation or by gene transfer (e.g. acquisition

of a plasmid)• Resistance provides a selective advantage• Resistance can result from single or multiple steps• Cross resistance vs multiple resistance

– Cross resistance -- Single mechanism-- closely related antibiotics– Multiple resistance -- Multiple mechanisms -- unrelated antibiotics

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Antimicrobial Drug ResistanceMechanisms

• Altered permeability– Altered influx

• Gram negative bacteria– Altered efflux

• tetracycline

• Inactivation– Beta-lactamase– Chloramphenicol acetyl transferase

Antimicrobial Drug ResistanceMechanisms

• Altered target site– Penicillin binding proteins (penicillins)– RNA polymerase (rifampin)– 30S ribosome (streptomycin, aminoglycosides,…)

• Replacement of a sensitive pathway– Acquisition of a resistant enzyme

(sulfonamides, trimethoprim)

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Survey of Antibiotics

Protein Synthesis InhibitorsProtein Synthesis Inhibitors

▶ Target the bacterial ribosome.Target the bacterial ribosome.▶ Bacterial Bacterial –– 70S (50S/30S) 70S (50S/30S)▶ Mammalian Mammalian –– 80S (60S/40S) 80S (60S/40S)

High levels may interact with mammalianHigh levels may interact with mammalianribosomes.ribosomes.

▶ 50S binders - Macrolides, Clindamycin,50S binders - Macrolides, Clindamycin,Chloramphenicol, Streptogramins.Chloramphenicol, Streptogramins.

▶ 30S binders - Aminoglycosides, 30S binders - Aminoglycosides, TetracyclinesTetracyclines

▶ MupirocinMupirocin

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Protein Synthesis Inhibitors• Bactericidal

– Aminoglycosides• Streptomycin, Kanamycin, Neomycin• Gentamicin, Tobramycin, Amikacin, Netilmicin

• Oxazolidone (Linezolid)

• Bacteriostatic– Chloramphenicol– Tetracyclines

• Doxycycline, Minocycline– Streptogramins

• Quinupristin/Dalfopristin (Synercid)– Macrolides

• Erythromycin, Azithromycin, Clarithromycin• Clindamycin

Review of Initiation of Protein Synthesis

30S 1 32 GTP

1 2 3 GTPInitiation Factors

mRNA

3

12 GTP

30SInitiationComplex

f-met-tRNA

Spectinomycin

Aminoglycosides

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GDP + Pi 50S

70SInitiationComplex

AP

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Review of Elongation of Protein Synthesis

GTP

AP

Tu GTP Tu GDP

Ts

TsTu

+

GDPTs

Pi

P ATetracycline

AP

Erythromycin

Fusidic Acid

Chloramphenicol

G GTPG GDP + Pi

G

GDP

AP

+GTP

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Protein Synthesis Inhibitors

• Mostly bacteriostatic• Selectivity due to differences in prokaryotic

and eukaryotic ribosomes• Some toxicity - eukaryotic 70S ribosomes