College of Medicine, Swansea University

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

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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

148848@swan.ac.uk