Inhibitors of the IL-8/Glycosaminoglycan Interaction

Glycosaminoglycans (GAGs), such as heparan sulphate (HS), represent the natural co-receptors of chemokines in vivo by which chemotactic gradients are established. In a previous project we have identified a heparan sulfate octasaccharide to be the optimum binding length for the pro-inflammatory chemokine interleukin-8 (IL-8), which itself was found to bind to GAGs with high affinity only when it is present in its monomeric form. In addition, a reliable 3-D model of an IL-8/GAG complex was computed. Based on these results, we propose in this project a combined approach synergising molecular biology and synthetic chemistry in order to develop inhibitors of the IL- 8/GAG interaction as well as to establish robust in vitro and in situ assays for the biological activity of such anti- inflammatory compounds. Inhibitors to be generated in the course of this project include (i) recombinant, genetically engineered IL-8 mutants and (ii) synthetic GAG oligosaccharides. The rationale behind these IL-8 mutants is to obtain proteins which bind to GAGs with higher affinity than wtIL-8 but which exhibit impaired neutrophil receptor binding activity. Such recombinant drugs would displace wtIL-8 from its natural co-receptor but would not be able to recruit neutrophils from the blood to the inflammatory site. IL-8-specific GAG oligosaccharides, on the other hand, shall compete with the natural GAG ligands for the IL-8 binding site thus antagonising co-receptor binding and preventing IL-8 from being immobilised on the endothelial wall. The synthesis of GAG oligosaccharides represents, however, an ambitious chemical task, as shown by the few published synthetic GAG oligosaccharides with biological activity. Both the recombinant proteins and oligosaccharides will be biophysically evaluated in vitro with respect to their affinity for GAGs and wtIL-8, respectively. In addition, cell-based assays will be established to define the IC50 values of the IL-8 mutants as well as of the GAG oligosaccharides using a competition set-up with stably transfected cells carrying the IL-8 natural co-receptor and monitoring the displacement of fluorescently labelled wtIL-8 by the inhibitors. By the end of the project we hope to be able to come up with (a) lead compound(s), the biological activity of which will be further investigated in vivo using mouse models in a close industrial collaboration. Exploring new anti-inflammatory approaches by inhibiting the IL-8/GAG interaction will thus yield novel insights into the basic structure/function relationship which governs this biologically important interaction.