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
• but blamed for bad reactions to killed whole-cell
vaccines
• Acellular vaccines cause fewer reactions
• 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 University of Alberta
• Bart Hazes
• Amechand Boodhoo
• Mykhaylo Demydchuk University of Cambridge
• Biochemistry:
• Glen Armstrong University of Alberta
• Stephen Cockle 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)
• SLT-I - nearly identical to Shiga toxin
• SLT-II variants - ~60% identity to SLT-I
• AB5 subunit structure
• A-subunit attacks ribosome enzymatically
• 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 Mutation Relative
site cytotoxicity
mutated
None – 1
1 Asp17Glu 10-3
1 Phe30Ala 10-5
2 Ala56Tyr 10-2
2 Gly62Thr 10-6
2 Gly62Ala 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:
• Penny Stein University of Alberta
• Hong Ling
• Allan Sharp
• Amechand Boodhoo
• Raj Pannu University of Cambridge
• Roger Dodd
• Carbohydrates:
• Glen Armstrong University of Alberta
• Dave Bundle
• Pavel Kitov
• Biochemistry:
• Jim Brunton University of Toronto