Summary
We have designed, developed and
tested novel peptides, based on
synthetic derivatives of endogenous
Interleukin (IL)-13 signalling pathway
proteins; namely the IL-13 cytokine
and the decoy receptor IL-13Rα2, that
modulate IL-13 signalling; either
agonistically or antagonistically.
• Atopic asthma is a chronic disease of
high and increasing prevalence and a
cause of major morbidity in developed
countries.5
• IL-13 is an immunoregulatory cytokine
and a key mediator in the pathogenesis
of asthma.1-3
• IL-13 exerts its effects via a receptor
complex, shared with functionally similar
cytokine IL-4 (Figure 2); however IL-13
appears to be of greater significance in
the effector phase of allergic
inflammation.4
• Inhibition of IL-13 signalling has been
shown to reduce symptoms of chronic
airway inflammation in murine models.2,3
• This work aims to address the need for
novel and efficacious therapeutics;
specifically focussing on development of
peptide therapeutics to target IL-13 in
asthma.
Results
Flow cytometry analysis of STAT6
phosphorylation:-
1. Wynn TA. IL-13 effector functions. Annu Rev Immunol
2003;21:425-456.
2. Grünig G, Warnock M, Wakil AE, Venkayya R,
Brombacher F, Rennick DM, et al. Requirement for IL-13
independently of IL-4 in experimental asthma. Science
1998;282:2261-3
3. Wills-Karp M, Luyimbazi J, Xu X, Schofield B, Neben
TY, Karp CL, et al. Interleukin-13: central mediator of
allergic asthma. Science 1998;282:2258-60.
4. Corry DB. IL-13 in allergy: home at last. Curr Opin
Immunol 1999;11(6):610-4.
5. Woolcock AJ, Peat JK. The rising trends in asthma.
Chichester: Wiley 1997;206:122-4.
6. Rigoutsos I, Floratos A. Combinatorial pattern discovery
in biological sequences: the TEIRESIAS algorithm.
Bioinformatics 1998; 14(1):55-67.
7. Larkin MA et al. ClustalW and ClustalX version 2
(2007). Bioinformatics 2007 23(21): 2947-2948.
8. Pettersen EF et al. UCSF Chimera – a visualization
system for exploratory research and analysis. J Comput
Chem 2004;25(13):1605-12.
• Six peptides were engineered, based on
binding regions of IL-13 and IL-13Rα2.
• Preliminary flow cytometry analysis of
peptide effects on IL-13 signalling.
indicate that P3 and RP1 are promising
IL-13 cytokine-traps, worthy of further
investigation.
• Combining sequence pattern discovery
and structural modelling is an effective
tool for the design of therapeutic
peptides intended for the management
of asthma.
• The approach may be applicable to the
design of cytokine trap peptides for other
cytokine systems with alternative
therapeutic applications.
In silico design of cytokine-trap peptides for modulation of the Interleukin-13 signalling pathway in asthma
Nicola J. Purdy, William Walker, Julian M. Hopkin, Jonathan G.L. Mullins College of Medicine, Swansea University
Peptide 1:
LRELIEELVNITQ
Binding hot spots /
structural motifs
identified by:-
• Multiple sequence
alignment
• Combinatorial
sequence pattern
discovery
• 3D structural
modelling
Peptides
engineered -
based on
structural motifs
(binding hot
spots)
Laboratory Analysis
IL-13
IL-13 receptor
complex protein
sequences
IL-13 receptor
complex protein
structures
• Multiple sequence alignment program
ClustalW27 and combinatorial sequence
discovery algorithm TEIRESIAS6 were
used for detection of conserved protein
motifs.
• Molecular modelling platform UCSF
Chimera8 was employed in the
identification of ligand – receptor binding
regions.
• Amino acid sequences of binding
regions/motifs used in peptide design.
• A549 human lung epithelial cells were
treated with various doses of peptides
(µM range) and analysed by flow
cytometry (Figure 3).
• Candidate modulatory peptides further
tested at lower dose (nM range – Figure
4).
Bioinformatics
Cell line (A549) in
vitro assays:-
• ELISA
• qRT-PCR
• Flow cytometry Figure 2. The currently accepted model of IL-13 and IL-4 cytokine/receptor interactions.
IL-13 binds to IL-13Rα1 with low affinity, followed by recruitment of IL-4Rα to the complex,
resulting in the generation of a high affinity receptor. IL-13Rα2 is regarded as a
regulatory, non-signalling decoy receptor.
Introduction
Figure 3. Effect of peptides on IL-13 - induced STAT6 phosphorylation.
All values relative to STAT6 phosphorylation in untreated A549 lung
epithelial cell line (IL-13 control). Peptide concentrations a) = 100µM; b)
= 10µM; c) = 1µM. (n = 3 ± stdev)
Methods
Figure 1. Schematic overview of methodologies.
Figure 4. Inhibition of IL-13-induced STAT6 phosphorylation by peptide
RP1.
Multiple sequence alignment, combinatorial
sequence discovery and structural
modelling:-
• Four peptides were developed from IL-
13 motifs / potential binding ‘hotspots’
(peptides P1, P2, P3 and P4 – Figure 3 –
corresponding to IL-13 helices A, B, D
and C respectively).
• Two peptides were based on the decoy
receptor IL-13Rα2 (RP1 and RP2 –
Figure 3). Preliminary flow cytometry
results for RP1 at a lower concentration
range (nM) are shown in Figure 4.
Results Conclusions
0
20
40
60
80
100
1 10 100 1000 αIL-4Rα control
0
20
40
60
80
100
1 10 100 1000 αIL-4Rα control
0
20
40
60
80
100
1 10 100 1000 αIL-4Rα control
Peptide inhibition of IL-13-induced STAT6 phosphorylation
R1 Peptide [nM/mL]
% In
hibi
tion
Peptide R1 inhibition of IL-13-
induced STAT6 phosphorylation
RP1 peptide [nM/mL]
References
Swansea University
College of Medicine – Coleg Meddygaeth
This project is funded by the Welsh Government
National Institute for Social Care and Health
Research
Binding assays:-
• Surface plasmon
resonance
• FRET
• Florescence
polarisation
IL-13Rα1
Nicola J. Purdy
PhD student