Shiga Toxin
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Toxin structure and design of therapeutics Pertussis toxin vaccine design Shiga-like toxin drug design
The A-B Class of Toxins • • •
A (active) subunit: toxic enzymatic activity B (binding) subunit: binding to cell surface Examples: • • • • •
pertussis toxin (A1; B is heteropentamer) Shiga toxin family (A1B5) cholera toxin family (A1B5) diphtheria toxin (A1B1) ricin (A1B1)
A:B toxin mechanism
bacterium
host cell
Pertussis toxin structure and vaccine design
Pertussis toxin • •
Produced by Bordetella pertussis Essential part of whooping cough vaccines •
•
Acellular vaccines cause fewer reactions • •
•
but blamed for bad reactions to killed whole-cell vaccines contain chemically-detoxified pertussis toxin but immunogenicity may be reduced
Goal: design genetically-detoxified PT for use in next generation acellular vaccines
Pertussis toxin: A and B components •
Single A-subunit (S1) • •
•
ADP-ribosyltransferase activity Transfers ADP-ribose from NAD to Giα subunit of heterotrimeric G-proteins
B-subunit is heteropentamer (S2/S4,S3/S4,S5) •
binds to sialylated glycoproteins
B-subunit of pertussis toxin
Sialyl-lactose binding
Sialyl-lactose binding
ATP binding
A:B toxin mechanism
bacterium
host cell
Active site
Structural studies on enzyme mechanism • • •
PhD project of Mykhaylo Demydchuk Engineer S1 (A-subunit) to be its own substrate New structures show substrate and product binding •
first glimpse at peptide bound to any ADP-ribosyltransferase
Pertussis toxin vaccine design •
Engineer to preserve epitopes, eliminate activity • • •
cell binding dissociation active site residues
Collaborators on pertussis toxin work •
Structural work: • • • •
•
Penny Stein Bart Hazes Amechand Boodhoo Mykhaylo Demydchuk
University of Alberta
University of Cambridge
Biochemistry: • •
Glen Armstrong Stephen Cockle
University of Alberta Connaught Laboratories (now Sanofi Pasteur)
Shiga-like toxin structure and drug design
E. coli food poisoning • •
Infection by enterohaemorrhagic E. coli Clinical course • •
Diarrhea, possibly with blood (haemorrhagic colitis) Haemolytic uraemic syndrome (HUS)
Pathogenesis of E. coli food poisoning •
Relevant strains all produce Shiga-like toxins •
•
particularly O157:H7
Toxin-mediated cell damage • •
leads to thrombosis in microvasculature site of damage determines pathology
Possible treatments for E. coli food poisoning • • • •
Supportive therapy Antibiotics? Anti-diarrheal agents? Specific drugs?
Shiga-like toxins •
Shiga toxin family • •
Shigella dysenteriae-1: Shiga toxin Escherichia coli: Shiga-like toxins (SLTs) • •
•
AB5 subunit structure •
A-subunit attacks ribosome enzymatically •
•
•
SLT-I - nearly identical to Shiga toxin SLT-II variants - ~60% identity to SLT-I
related to ricin
B-subunit binds to cell-surface glycolipid: Gb3 or Gb4
Drug target: A or B?
Gb3: SLT receptor •
Location of receptor determines pathology
host cell
Strategies to exploit Gb3 binding • •
Block cell-surface binding Sequester toxin in digestive tract •
Pk-Synsorb
Side chains conserved in Shiga-like toxin family Side chains that vary in Shiga-like toxin family
Gb3 and Pk-MCO
Site 1 of SLT-IB:Gb3 complex
Site 2 of SLT-IB:Gb3 complex
Site 3 of SLT-IB:Gb3 complex
Relative importance of binding sites Binding site mutated
Mutation
Relative cytotoxicity
None
–
1
1 1
Asp17Glu Phe30Ala
10-3 10-5
2 2 2
Ala56Tyr Gly62Thr Gly62Ala
10-2 10-6 10-5
3
Trp34Ala
10-1
Site 2 is best for soluble Pk • •
Site 2 binding is strongest in crystal Site 2 binding is strongest in solution •
•
NMR (Steve Homans)
One site has >10-fold higher affinity • •
isothermal calorimetry (Eric Toone) mass spec (Dave Bundle)
Design of new ligands •
Based on Gb3 trisaccharide? •
isolated Pk-trisaccharide binds weakly (10mM)
Improving on Pk trisaccharide •
Increase valency • •
•
toxin binds up to 15 glycolipids on cell surface sites 1 and 2 are close together
Find novel non-carbohydrate ligands
Designing a bridge
Bridge-starfish molecule
Influence of valency on binding 1 0.01 1E-04 1E-06 1E-08 1E-10
Pk
Bridge
Starfish
Bridge Starfish
Half of bridge-starfish complex
Bridge-starfish complex
In vivo tests of Starfish compound • •
Starfish is non-toxic Co-administered Starfish protected mice against action of SLT-I •
•
didn’t protect against SLT-II
Related “Daisy” compound protected against both SLT-I and SLT-II
Future possibilities • •
New bridged carbohydrates Docking and screening of non-carbohydrate compounds
Collaborators on Shiga-like toxin work •
Structural work: • • • • • •
•
University of Alberta
University of Cambridge
Carbohydrates: • • •
•
Penny Stein Hong Ling Allan Sharp Amechand Boodhoo Raj Pannu Roger Dodd Glen Armstrong Dave Bundle Pavel Kitov
University of Alberta
Biochemistry: •
Jim Brunton
University of Toronto
The A-B Class of Toxins • • •
A (active) subunit: toxic enzymatic activity B (binding) subunit: binding to cell surface Examples: • • • • •
pertussis toxin (A1; B is heteropentamer) Shiga toxin family (A1B5) cholera toxin family (A1B5) diphtheria toxin (A1B1) ricin (A1B1)
A:B toxin mechanism
bacterium
host cell
Pertussis toxin structure and vaccine design
Pertussis toxin • •
Produced by Bordetella pertussis Essential part of whooping cough vaccines •
•
Acellular vaccines cause fewer reactions • •
•
but blamed for bad reactions to killed whole-cell vaccines contain chemically-detoxified pertussis toxin but immunogenicity may be reduced
Goal: design genetically-detoxified PT for use in next generation acellular vaccines
Pertussis toxin: A and B components •
Single A-subunit (S1) • •
•
ADP-ribosyltransferase activity Transfers ADP-ribose from NAD to Giα subunit of heterotrimeric G-proteins
B-subunit is heteropentamer (S2/S4,S3/S4,S5) •
binds to sialylated glycoproteins
B-subunit of pertussis toxin
Sialyl-lactose binding
Sialyl-lactose binding
ATP binding
A:B toxin mechanism
bacterium
host cell
Active site
Structural studies on enzyme mechanism • • •
PhD project of Mykhaylo Demydchuk Engineer S1 (A-subunit) to be its own substrate New structures show substrate and product binding •
first glimpse at peptide bound to any ADP-ribosyltransferase
Pertussis toxin vaccine design •
Engineer to preserve epitopes, eliminate activity • • •
cell binding dissociation active site residues
Collaborators on pertussis toxin work •
Structural work: • • • •
•
Penny Stein Bart Hazes Amechand Boodhoo Mykhaylo Demydchuk
University of Alberta
University of Cambridge
Biochemistry: • •
Glen Armstrong Stephen Cockle
University of Alberta Connaught Laboratories (now Sanofi Pasteur)
Shiga-like toxin structure and drug design
E. coli food poisoning • •
Infection by enterohaemorrhagic E. coli Clinical course • •
Diarrhea, possibly with blood (haemorrhagic colitis) Haemolytic uraemic syndrome (HUS)
Pathogenesis of E. coli food poisoning •
Relevant strains all produce Shiga-like toxins •
•
particularly O157:H7
Toxin-mediated cell damage • •
leads to thrombosis in microvasculature site of damage determines pathology
Possible treatments for E. coli food poisoning • • • •
Supportive therapy Antibiotics? Anti-diarrheal agents? Specific drugs?
Shiga-like toxins •
Shiga toxin family • •
Shigella dysenteriae-1: Shiga toxin Escherichia coli: Shiga-like toxins (SLTs) • •
•
AB5 subunit structure •
A-subunit attacks ribosome enzymatically •
•
•
SLT-I - nearly identical to Shiga toxin SLT-II variants - ~60% identity to SLT-I
related to ricin
B-subunit binds to cell-surface glycolipid: Gb3 or Gb4
Drug target: A or B?
Gb3: SLT receptor •
Location of receptor determines pathology
host cell
Strategies to exploit Gb3 binding • •
Block cell-surface binding Sequester toxin in digestive tract •
Pk-Synsorb
Side chains conserved in Shiga-like toxin family Side chains that vary in Shiga-like toxin family
Gb3 and Pk-MCO
Site 1 of SLT-IB:Gb3 complex
Site 2 of SLT-IB:Gb3 complex
Site 3 of SLT-IB:Gb3 complex
Relative importance of binding sites Binding site mutated
Mutation
Relative cytotoxicity
None
–
1
1 1
Asp17Glu Phe30Ala
10-3 10-5
2 2 2
Ala56Tyr Gly62Thr Gly62Ala
10-2 10-6 10-5
3
Trp34Ala
10-1
Site 2 is best for soluble Pk • •
Site 2 binding is strongest in crystal Site 2 binding is strongest in solution •
•
NMR (Steve Homans)
One site has >10-fold higher affinity • •
isothermal calorimetry (Eric Toone) mass spec (Dave Bundle)
Design of new ligands •
Based on Gb3 trisaccharide? •
isolated Pk-trisaccharide binds weakly (10mM)
Improving on Pk trisaccharide •
Increase valency • •
•
toxin binds up to 15 glycolipids on cell surface sites 1 and 2 are close together
Find novel non-carbohydrate ligands
Designing a bridge
Bridge-starfish molecule
Influence of valency on binding 1 0.01 1E-04 1E-06 1E-08 1E-10
Pk
Bridge
Starfish
Bridge Starfish
Half of bridge-starfish complex
Bridge-starfish complex
In vivo tests of Starfish compound • •
Starfish is non-toxic Co-administered Starfish protected mice against action of SLT-I •
•
didn’t protect against SLT-II
Related “Daisy” compound protected against both SLT-I and SLT-II
Future possibilities • •
New bridged carbohydrates Docking and screening of non-carbohydrate compounds
Collaborators on Shiga-like toxin work •
Structural work: • • • • • •
•
University of Alberta
University of Cambridge
Carbohydrates: • • •
•
Penny Stein Hong Ling Allan Sharp Amechand Boodhoo Raj Pannu Roger Dodd Glen Armstrong Dave Bundle Pavel Kitov
University of Alberta
Biochemistry: •
Jim Brunton
University of Toronto
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