1. Considérations générales et introductives · –30S ribosome (streptomycin,...
Transcript of 1. Considérations générales et introductives · –30S ribosome (streptomycin,...
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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
12
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