Antibiotics acting on Protein Synthesis E. Westhof

Transcription

1 Antibiotics acting on Protein Synthesis E. Westhof 1. Considérations générales et introductives 1

2 Facts about Antibiotics Alexander Fleming (1928) discovered penicillin Ignored until early 1940s with extensive use in war 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-organisms that inhibits other microorganisms Antimicrobial agents: antibiotics & synthetic/semisynthetic agents used to inhibit microorganisms Bactericidal (kills bacteria) vs bacteriostatic (stops active growth without affecting viability) 2

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 the organism and its susceptibility to antibiotics 3

4 Glycopeptides Penicillins Polymixines Antibiotic targets > 50% Oxazolidinones Rifamycins Sulfamides Diaminopyridines Quinolones Lincosamides Macrolides Streptogramins Tetracyclines Aminoglycosides Spectrum of Activity Narrow Spectrum Broad spectrum 4

5 Antibiotic Resistance is Increasing 5

6 The bacterial ribosome : atoms (C,N, O, P) 55 proteins 3 RNA (4600 nucleotides) 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 6

7 Only RNA in the reaction site The ribosome, a molecular machine, is a ribozyme and, like all other known ribozymes, the ribosome uses RNA-based recognition motifs not only for catalysis but also for decoding processes. 7

8 Les trois étapes de la synthèse 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. 8

9 Accuracy Error rate 1/10 4 But only select trna by codon/anticodon! 1 mismatch = 1 wrong H-bond! So..? Must be a proofreading step analogous to aars. Kinetic Proofreading First step is reversible Second step controlled by k 4 /k 3 ratio k 3 is constant k 4 depends on strength codonanticodon binding 9

10 Ribosome: inhibiteurs Mécanisme Eu(E)/Pro(P)caryote Inhibition Initiation Acide aurine tricarboxylique P fixation IF sur 30S Kasugamycine P fixationarn init. Streptomycine P formation du complexe d init. Liaison aa-arnt Tetracycline P fixation Streptomycine P erreur de séquence protéique Paramomycine P erruers dans la sélection des ARNt Formation de la liaison peptidique Sparsomycine P peptidyl transférase Chloramphénicol P id- fixe sur 50S Erythromycine P id-id Cycloheximide P translocation du pepptidyl-arnt Translocation Acide fusidique P dissociation EF-G-GDP Thiostreptone P GTPase EFTu et EFG sur ribosome Toxine diphtérique E eef2 par ADP ribosylation Terminaison Puromycine P/E accepteur du groupe peptidyle, fin prématurée Inactivation Ricine E inactive ARN28S 10

11 Inhibition of Translation Numerous antibiotics target translational machinery Simulates 3 end of trna Competes for A-site Ends nascent chain Binds to hydrophobic tunnel Blocks egress of peptide chain 11

12 Inhibition of Translation Numerous antibiotics target translational machinery Binds at peptidyl transferase site and inhbits reaction Binds to 16S rrna, stabilizes ribosome in conformation that increases affinity for aatrna Sterilization: Definitions Kill all microbes, viruses, and other life forms Disinfect, Decontaminate, Pasteurize: Reduce the levels of microbes, viruses, and other life forms 12

13 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 13

14 Kinetics of Killing Microbes die exponentially. Not simultaneously Decimal Reduction Time (D) Time required to reduce the population 1/ cells 10 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 14

15 a= typical mesophile b= typical thermophile 15

16 Selectivity Principles and Definitions 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 original inoculum 16

17 MINIMUM INHIBITORY CONCENTRATION (MIC) MIC lowest antibiotic concentration that inhibits growth MIC 90 concentration required to inhibit 90% 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 Disk Diffusion Test Determination of MIC Str Tet Ery Tetracycline (:g/ml) MIC = 2 :g/ml Chl Amp 17

18 Assessing the antimicrobial activity of a compound using the minimum inhibitory concentration method A compound to be tested is serially diluted into growth medium, inoculated with a culture and then incubated. The minimum inhibitory concentration is indicated in the lowest dilution of the compound which prevents growth as indicated by the arrow. 18

19 Disk-Diffusion Method Bacillus cereus inoculated soaked; alcohol no effect Gram Staining Gram positive Staphylococcus epidermidis Gram negative Escherichia coli 19

20 Culture Antimicrobial Drug Resistance Principles 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 20

21 Antimicrobial Drug Resistance Mechanisms Altered permeability Altered influx Gram negative bacteria Altered efflux tetracycline Inactivation Beta-lactamase Chloramphenicol acetyl transferase Antimicrobial Drug Resistance Mechanisms 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) 21

22 Survey of Antibiotics Protein Synthesis Inhibitors Target the bacterial ribosome. Bacterial 70S (50S/30S) Mammalian 80S (60S/40S) High levels may interact with mammalian ribosomes. 50S binders - Macrolides, Clindamycin, Chloramphenicol, Streptogramins. 30S binders - Aminoglycosides, Tetracyclines Mupirocin 22

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 GTP GTP Initiation Factors mrna f-met-trna Spectinomycin 3 P A GDP + Pi S 1 2 GTP 70S Initiation Complex Aminoglycosides 30S Initiation Complex 23

24 Review of Elongation of Protein Synthesis P A Tetracycline P A Tu GTP Tu GDP + Pi Ts GTP Tu Ts Ts GDP Chloramphenicol Fusidic Acid GDP + G GTP P A G GDP + Pi G GTP P A Erythromycin 24

25 Protein Synthesis Inhibitors Mostly bacteriostatic Selectivity due to differences in prokaryotic and eukaryotic ribosomes Some toxicity - eukaryotic 70S ribosomes 25