Identification of CCL17 neutraligands targeting atopic diseases

Introduction Chemokines constitute a family of small cytokines that attract and activate leukocytes during the inflammatory response. Among them, the β-chemokine CCL17 formerly known as thymus- and activation regulated chemokine (TARC), is involved in the development of atopic disorders such as asthma and atopic dermatitis. This chemokine exerts its biological effects by binding to and activating the CCR4 cell-surface receptor that belongs to the Gi-protein-coupled receptor family. Enhanced expression of CCL17 as well as elevated recruitment of CCR4 + Th2 cells have been observed in asthma and atopic dermatitis. To date, most therapeutic strategies have focused on disrupting the chemokine/receptor interaction through use of chemokine receptor antagonists. We have discovered a novel class of small chemical molecules called “neutraligands” that bind to the chemokine itself, not to the receptor, and neutralize its biological activity. The concept has already been fully validated with the discovery of “chalcone 4”, a small chemical that binds CXCL12, thereby preventing bronchial inflammation and hyperresponsiveness in animal models of asthma (Hachet-Haas et al., JBC 2008; Galzi et al., Pharmacol Ther 2010; Gasparik et al., ACS Med Chem Lett 2012; Daubeuf et al., JBC 2013). Our aim was to identify new CCL17 neutraligands, and evaluate their in vivo activity in mouse models of cutaneous and respiratory Th2-driven allergic inflammation. Methods We have set up a cell-based high throughput assay to identify new CCL17 neutraligands. HEK293 cells were transfected with the CCL17 receptor, CCR4, together with the G protein Gαqi5 that will increase the intracellular Ca 2+ response (FlexStation3) upon activation with CCL17. A library of pure natural substances, as well as a subset of the academic library of Strasbourg (1000 compounds) was screened. The hits were tested in two Th2-driven murine models: – an 8-day model of allergic hypereosinophilia to ovalbumin; – a model of allergic dermatitis to MC903 (calcipotriol). Results Two hit molecules were selected from the HTS assay, compounds A and B, with CCL17-neutralizing activity: when preincubated with the chemokine, they blocked CCL17-induced Ca 2+ responses (2 μg/mL; IC50s = 5 and 8 μM, respectively); by contrast, when preincubated with the cells, i.e. with the CCR4 receptor, the Ca 2+ responses were not affected, indicating the compounds were CCL17 neutraligands and not CCR4 receptor antagonists. We show that compounds A and B (350 μmol/kg, I.P.) significantly reduced the number of cells collected in broncho-alveolar lavage fluid, in particular eosinophils (40 and 60% inhibition, respectively) in the model of allergic hypereosinophilia. In addition, compounds A and B (350 μmol/kg) administered topically prevented MC903-induced ear redness and thickness, and decreased plasma IgE levels (by 90%). Conclusion Our results show successful identification of CCL17 neutraligands that efficiently control Th2 inflammation, and will help understand the role of CCL17 in asthma and atopic dermatitis.