Ribosomal Antibiotics

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Ribosomal Antibiotics

• Amber Von Ruden
• May 1, 2007

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Overview

• History of antibiotics
• Reasons for antibiotic resistance
• Function of ribosomes in protein-biosynthesis
• Ribosomal features that can be targeted
• Use of X-ray crystallography
• Discussion of 2 specific antibiotics that
  directly involve nucleic acids
• Antibiotic selectivity and toxicity

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Antibiotics

• Discovered by chance in 1928 by Alexander Fleming
• Development of penicillin and others rapidly
• Most antibiotics not used to treat human
  infectious diseases
• Antibiotics used in agriculture and other areas
• - Livestock weight gain
• - Increased yield of aqua cultures like
  shrimp
• Up to 80 of all antibiotics have non-therapeutic
  uses

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Antibiotic Resistance

• Side effect of non-therapeutic use is
  contamination of the environment with traces of
  antibiotics that remain active in soil
• Microorganisms exposed to low levels but not
  killed and develop resistance
• Hospitals have community acquired infections and
  are heavily affected with outbursts of multiply
  resistant bacteria Staphylococcus aureaus where
  70-90 of its strains are resistant against most
  common antibiotics
• Need to develop new classes of antimicrobial
  compounds not yet affected by resistance

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Antibiotics

• Compounds which are produced by microorganisms to
  defeat other organisms so a resistance mechanism
  must exist for each antibiotic
• Must target a crucial element of the life cycle
  of the organism being attacked
• Studying the mechanism of antibiotics and
  resistance can help facilitate the development of
  new drugs
• Most ribosomal antibiotics work by blocking a
  functional center of an enzyme during protein
  biosynthesis

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Protein-Biosynthesis

• Process responsible for translation of genetic
  code
• All active cell components are produced by the
  ribosome
• Inhibition of this process is destructive for any
  organism
• Recently developed antibiotics attack ribosomal
  protein synthesis

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Function of Ribosome

• 2 subunits assemble
• to produce protein
• Involves mRNA
• - Carries genetic code
• Involves tRNA
• - Brings amino acids
• Protein strand grows

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Inititation

• Small ribosome subunit attaches to the mRNA
• Initiation complex is complete after tRNA binds
  at that site
• This attracts the large ribosomal subunit which
  will bind to the small subunit with A and P site

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Elongation

• Inititator tRNA moves to the P site leaving the A
  site open for the next tRNA
• After binding to the A site the peptide bond
  forms between Met and Pro
• Empty tRNA carrying Met leaves and the tRNA
  carrying the Pro moves to the P site
• Ribosome moves to the next triplet code from the
  5 to 3 direction and the process carries on

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Elongation

• Another tRNA moves into the A site in order to
  add another amino acid to the peptide chain
• Continues to read the code from the 5' to the 3'
  and amino acids are added to the growing peptide
  chain
• This continues until the stop codon is reached

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End of Translation

• When the ribosome encounters a stop codon there
  is no tRNA attracted
• The ribosome separates and leaves the mRNA

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Features Targeted by Antibiotics

• Decoding site provided by small subunit during
  elongation and controls translation fidelity
• Peptidyl-transferase center (PTC) - catalytic
  site in large subunit
• Exit tunnel found in large subunit
• Mobile elements
• - mRNA threading and progression
• - Protein passage

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Experiments to Elucidate Ribosome Structure

• Electron microscopy using a combination of
  micrographs from one or more particles
• Sequencing of rRNA and secondary structure
  prediction
• Antibody-tagged ribosomal proteins localized by
  electron microscopy and neutron diffraction
• Crystallization and X-ray diffraction of ribosome
  and subunits

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X-ray Crystallography

• Several recent high and medium-resolution
  structures of ribosomal particles and their
  complexes with antibiotics
• Led to basic concepts in antibiotic-binding modes
  at the molecular level to help develop
  antibiotics and understand resistance
• May lead to suggestions concerning antibiotics
  inhibitory activity, selectivity, and toxicity

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Limitations of X-ray

• Availability of crystals
• Pathogenic bacteria ribosomes have not yet been
  crystallized so must use prokaryotes to use as
  models
• Reliable method but dissimilarities represent a
  variability in drug-binding modes

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Examples of Ribosomal Antibiotics
Helped reveal novel antibiotic properties!
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Function of Two Antibiotics
These involve nucleic acids!
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PTC Mobility

• Universally conserved nucleotides
• A2602 and U2585 are important for
• peptide bond formation
• Bulge toward center of PTC and
• facilitate and anchor rotatory motion
• Favorable ribosomal targets
• Conformational changes with
• binding of antibiotics

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Sparsomycin

• Targets A2602
• Universal antibiotic agent
• Correlation between the 2 binding sites and
  ribosomal functional state
• Stacks to flexible A2602 and causes
  conformational changes in entire PTC

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Phenol-alanine-sparsomycin

• Derivative that carries a p-hydroxyl benzyl
  function can be labeled with radioactive iodine
  to study the interaction with the ribosome
• Mode of action implies occurrence of chemical
  reaction involving sulfoxide moiety
• Initiated by imadazole-activated intermediate
  resulting in covalent bonding of the drug through
  the sulfur atom to either the peptide or
  ribosomal component

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Synercid

• Interacts partially with nonconserved nucleotides
  
• High level of selectivity against bacterial
  pathogens
• Injectable drug consisting of 2 components SA and
  SB

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SA Component - Dalfopristin

• Binds to the PTC to induces remarkable
  conformational alterations
• 180 flip of the U2585 base paralyzes its
  ability to anchor rotatory motion and direct the
  protein into the exit tunnel

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SB Component - Quinupristin

• Macrolide that binds to the common
  marcrolide-binding pocket
• Its bulkiness does not allow it to efficiently
  block the exit tunnel

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Components Working Together

• Likely that the dalfopristin will be expelled or
  relocated so together can stabilize this
  nonproductive flipping positioning of U2565
• Quinupristin can block the way out of
  dalfopristin
• Greatly enhances the antimicrobial activity

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Two Components of Synercid

• Quinupristin takes a passive role in blocking the
  tunnel
• Dalfopriston has a more dynamic role by hindering
  the motion of the vital nucleotide at the active
  site U2585
• Used in combination to treat infections by
  staphylococci and by vancomycin-resistant
  Enterococcus faecium

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Antibiotic Selectivity

• Drug selectivity is key for therapeutical
  effectiveness
• Universal drugs (Sparsomycin) are useless for
  combating infections but may be used for
  antitumor treatments
• Even lower degrees of selectivity can cause
  toxicity
• Structures of antibiotics complexed with
  ribosomes provide tools for investigating
  selectivity principles

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Adenine vs. Guanine

• Structural differences may come down to the
  identity of a single nucleotide
• Difference between A and G was found to dictate
  the preference of binding and level of activity
• The interactions that occur between these base
  pairs for an antibiotic paromomycin in the A site
  of bacterial rRNA are not homologous with the
  identities of these base pairs in humans and
  could not be used to create efficient drug-target
  interactions

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Conclusions

• Ribosomal antibiotic research has been enriched
  by recent three-dimensional structural
  information
• Ribosomal antibiotics bind to only a single or
  few binding sites
• Most cause conformational changes
• Minute structural differences are responsible for
  antibiotic selectivity and efficiency

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