Bacterial-­‐Toxin Inhibi2on using Mul2valent Scaffolds
Sarah-Jane Richards & Matthew I.
Gibson
Department of Chemistry, University of Warwick, UK
S-J.Richards@warwick.ac.uk
www.warwick.ac.uk/go/gibsongroup
Joint CDT conference 2013 – Imperial College London Protein-Carbohydrate Interactions
Cell signalling
Fertilisation
Cellular adhesion of
•  Viruses
•  Bacterium
•  Bacterial toxins
Inflammation
You Houng, Nat. Mater., 2010 9 485 Gamblin, Chem. Rev, 2009, 109, 131 Miura, JPOLA, 2007, 45, 5031 Imberty, Chem. Eur. J., 2008, 14, 7490 Spain, Polym. Chem., 2011, 2, 60 Turnbull, B., Rev. Mol. Biotech., 2002, 90, 231 Affinity
Why Materials?
Predicted linear
response
Number of Binding Epitopes
Cell Surface Glycans – Materials
Science/multivalency
Kiessling, Angew, Chem., 2006, 45, 22348 Alexander et al, JACS, 2007, 129, 11014 Mammam, J. Med. Chem., 1995, 38, 4179 Spain et al. Polym. Chem., 2011, 2, 60 Glycopolymers by Post-Polymerisation Modification
OH
HO
HO
HN
n
O
O
N3
HO
Cu(I)Ligand
HN
n
O
N
N
N
Gibson, M. I. et al., J. Pol. Sci.
A., 2009, 47, 4332
O
OH
HO
OH
OH
Haddleton, D. M. et al., JACS, 2006,
128, 4823
Jones, M. W.; Polym. Chem., 2013,
Spain et al., J. Pol. Sci. A., 2007, 45, 2059
Gauthier et al., Angew. Chem. 2009, 48, 48
Practicalities
Scaffold synthesis can be inefficient
•  Monomer synthesis is not always
straightforward
•  Atom efficiency is poor
•  Copolymers require knowledge
of reactivity ratios
Variables:
Polymer Length þ
Carbohydrate þ
Linker Length X
Co-monomers X
Our Solution:
“Tandem post-polymerisation
modification”
HN
O
F
n
O
n
O
F
HN
HN
F
HN
n
O
x
O HN
y
O
n
O
N
N
N
R
F
F
OH
-  Easy to make 50 gram
scale
-  1 column/distillation
-  Compatible with RAFT/
ATRP
-  Quantitative
functionalisation with
non-hindered amines
-  Density control
Theato, P.; J. Pol. Sci. A., 2008, 46, 6677
-  Sequentially
modified
polymer libraries
Improved Synthesis with Poly(azlactones)
n
O
N
O
n
HN
HN
•  100 % Atom efficient
•  Quantitative conversion with unhindered amines
O
O
n
HN
O
O
HN
•  Scalable synthesis of monomer
•  One-pot, two step synthesis/postpolymerisation modification possible
Jones, M. W., Richards, S-J., Haddelton, D. M., Gibson, M. I.; Polym. Chem., 2013,4, 717
M.E Buck & D. M Lynn, Polym. Chem., 2012, 3, 66
Applications: Anti-adhesion Therapy
Interactions can be inhibited at
nM of glycopolymers
Influenza inhibition in mice
Becer et al. JACS,2010, 132, 15130
Hidari et al. Glycobiology 2008, 18, 779
Selective Binding of Cholera-Toxin
Enzymatic domain
Induces toxic effect
Carbohydrate
binding domain
Binds to epithelial cells
to promote cell uptake
Anti-adhesion therapy does not target
bacteria, so less evolutionary stress
Galectins – at least 13
β-D Galactose
Sigma-Aldrich – 8 Galactose-’specific’ lectins
How do we engineer a high-affinity binder for
cholera toxin, without total synthesis of
complex carbohydrates?
GM-1 ganglioside
Cholera Toxin
Peanut Agglutinin
w
w
Asn 14
w
Asn 90
Asp 80
Lys 91
Gly 213
Ser 211
Asp 83
w
Glu 51 w
Gln 56
w
w
w
Asn 127
Gly 104
w
w
Trp 88
Ile 58
His 13
Gly 33
w
Glu 11
Gln 61
w
Glu 129
w
Ile 101
Leu 212
Asn 41
Leu 31
6.3 Å
16 Å
Can glycan accessibility be used as a tool for lectin discrimination?
Kiick et al.; Macromol. 2007, 40, 7103
Kiick et al; Biomac., 2006, 7 483
Glycopolymer Library
O
O
O
O
F
i)
O
F
F
F
O
+
H2N
HN
NH2
m
m = 0 or 2
m
Br
n
O
F
x
O
HN
dw/d(LogM)
HN
OH
O
HO
N3
OH
O
OH
HO
HO
x = 10, 25, 50, 100
y = 90, 75, 50, 0
N
-1
-1
Br
y
O
x
O
HN
HO
n = 25, 50, or 100
2.0
HO
O
2.5
O
ii)
Br
y
O
m
O
N
N
O
HO
-1
Mn - 7800 g.mol
Mn - 6100 g.mol
Mn - 7250 gmol
PDi - 1.19
PDi - 1.24
PDi - 1.32
1.5
1.0
0.5
0.0
1000
10000
-1
Mw (gmol )
1000
10000
1000
Mw (gmol-1)
Polymer
GP1
GP2
GP3
GP4
GP5
GP6
GP7
GP8
GP9
DP[a]
18
33
70
18
33
70
33
33
33
10000
Mw (gmol-1)
Linker[b]
Short
Short
Short
Long
Long
Long
Long
Long
Long
Density[c]
100
100
100
100
100
100
50
25
10
Mw/Mn[d]
1.29
1.27
1.26
1.32
1.28
1.27
1.23
1.21
1.20
OH
OH
Peanut Agglutinin
Cholera Toxin
25
25
MIC50 (µM Galactose)
MIC50 (µM Galactose)
30
Polymer length
20
15
10
5
0
GP1
GP2
GP3
Short Linker
GP4
GP5
GP6
Long Linker
20
15
10
5
0
GP1
GP2
GP3
Short Linker
GP4
GP5
GP6
Long Linker
O
O
O
F
i)
O
F
F
F
n = 25, 50, or 100
+
H2N
O
NH2
m
m = 0 or 2
HN
m
O
Br
n
O
F
x
O
HN
HO
HN
OH
HO
O
OH
O
ii)
Br
y
O
O
OH
N3
m
O
O
•  Degree of polymerisation
•  Linker length
•  Carbohydrate density
HO
HO
HO
x = 10, 25, 50, 100
y = 90, 75, 50, 0
Br
y
O
x
O
HN
N
N
N
O
HO
OH
OH
Peanut Agglutinin
Cholera Toxin
25
PNA [Galactose]
2.0
1.5
1.0
0.5
0.0
GP4
100 %
GP7
50 %
GP8
25 %
GP9
10 %
MIC50 (µM Galactose)
MIC50 (µM Galactose)
2.5
20
Ctx [Galactose]
15
10
5
0
GP4
100 %
GP7
50 %
GP8
25 %
GP9
10 %
Richards, S-­‐J., Jones, M. W., Hunabun, M. I., Haddelton, D. M.; Gibson, M. I.; Angew. Chem., 2012, 51, 7812 What is the ‘best’ polymer for lectin binding?
Absorption to protein functionalised
surface
•  Surface Plasmon Resonance (SPR)
•  Quartz Crystal Microbalance (QCM)
•  Enzyme-linked assays (ELISA)
Mass bound
How do you determine what is the best polymer? A
B
C
D
Inhibitor
E
F
100
P1
P2
P3
Δ f (Hz)
80
Molecular weight P3 > P2 > P1
•  Largest polymer shows smallest shifts
•  Does this imply weakest binding?
• What is effect of polymer chain length?
60
40
20
0
0.0
0.5
1.0
1.5
2.0
-1
[Polymer] (mg.mL )
QCM-d allows film properties to be probed
0.05 mg/mL
0.1 mg/mL
0.5 mg/mL
1.0 mg/mL
2.0 mg/mL
Rigid film
6
4
4
2
2
0
0
0
-10
-20
-30
-40
-50
Δ f (Hz)
Frequency
0.1 mg/mL
0.5 mg/mL
1.0 mg/mL
1.5 mg/mL
2.0 mg/mL
-6
-6
ΔD (x10 )
8
ΔD (x10 )
Flexible film
Dissipation
6
10
DP = 8
-60
-70
-80
0
-10
-20
-30
Δ f (Hz)
DP = 42
-40
-50
Solution phase inhibition
Surface Binding Affinity
0.10
6
5
0.04
0.02
0.00
DP =
-1
0.06
4
5
Ka x10 (M )
MIC50 (mM)
0.08
3
2
1
1
8
2
23
Polymer
A
0
3
42
DP =
8
8
23
23 DP
Polymer
42
42
< 6.5 nm
Increased
mass absorbed.
Lower KD
= ConA
= QCM Chip
B
> 6.5 nm
Single site binding
Flexible brush
Decreased
mass absorbed.
Higher KD
= Glycopolymer
Spanning binding sites
Rigid thin film
Gou.Y., Richards, S-J., Haddleton D. M., Gibson, M. I.; Polymer Chemistry, 2012, 3, 1634 Summary
•  Tandem Post-Polymerisation Modification
•  Multivalent inhibitors that have good affinity
AND specificity
A
< 6.5 nm
Increased
mass absorbed.
Lower KD
B
> 6.5 nm
Single site binding
Flexible brush
Decreased
mass absorbed.
Higher KD
Spanning binding sites
Rigid thin film
•  A number of techniques
are required to determine
the ‘best’ polymer.
Acknowledgements
Matthew
Gibson
Dave Haddleton
Mathew Jones
Yanzi Gou
Mark Hunabun
MIG Group Current Recent Collaborators •  Robert Deller •  Dan Phillips •  Caroline Moore •  Tom Congdon •  Alaina Emmanuella •  Lucienne OOen •  Daniel Mitchel •  Lewis Mann •  Rebecca Williams •  Dr Mat Jones •  MaOhew Summers •  Mark Hunaban •  Charline Wilmet •  Devian Patel •  Abdul Sahid •  Del Besra (B’ham) Bacterial-­‐Toxin Inhibi2on using Mul2valent Scaffolds
Sarah-Jane Richards & Matthew I.
Gibson
Department of Chemistry, University of Warwick, UK
S-J.Richards@warwick.ac.uk
www.warwick.ac.uk/go/gibsongroup
Joint CDT conference 2013 – Imperial College London