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1
Antibiotics acting on ProteinSynthesis
E. Westhof
1. Considérations générales etintroductives
2
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)
3
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
4
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
6
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
7
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.
8
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.
9
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
12
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
14
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
15
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
18
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.
19
Disk-Diffusion Method
Bacillus cereusinoculated soaked;alcohol no effect
Gram Staining
• Gram positive• Staphylococcus
epidermidis
• Gram negative• Escherichia coli
20
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
21
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)
22
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
23
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
24
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
25
Protein Synthesis Inhibitors
• Mostly bacteriostatic• Selectivity due to differences in prokaryotic
and eukaryotic ribosomes• Some toxicity - eukaryotic 70S ribosomes