Cytokines are small size proteins that, by diffusing in the extracellular space carry distinct signals (activation, inhibition, proliferation, differentiation), and provide cells a system to communicate. Current research increasingly shows that most cytokines, in addition to their specific receptors, bind to a class of molecules, collectively known as glycosaminoglycans, and in particular to one of them called heparan sulfate (HS). These long, unbranched polysaccharides are near ubiquitous constituents of cell surfaces, and are carried by a specialized family of glycoprotein: the proteoglycans. Cytokine-HS interactions importantly regulate cytokine-receptor binding and activation, signaling, storage and stabilization in the extracellular space, and/or induce structural changes that modify cytokines activity. Interactions with HS are thus crucial and impact the dynamic of the cytokines at the cell surface or in the extracellular matrix and thus, their proper tissue localization and many of their biological functions. Given the importance of HS-cytokine interactions both in normal and pathologic states, understanding the mechanism and the structural bases that drive these interactions and their functions represent an important issue. However, progresses in that field have been hampered by the extraordinary complexity of HS. In particular, the characterization of the protein-HS interface, the isolation of the corresponding binding domains, and the elucidation of the mechanism by which HS regulate cytokine activity and dynamic remain particularly challenging. Based on a preliminary determination of the structure that a cytokine: interferon-gamma (IFNg), recognizes along the HS chain, a set of glycoconjugate mimetics has been synthesized, and evaluated for their ability to interact with the protein. One of these molecules, composed of two N-sulfated octasaccharides (dp8), linked to each other through a 5 nm long spacer displays high affinity for the cytokine. This molecule, termed 2O10 inhibits the binding of IFNg to HS with an IC50 of 35-40 nM, and most interestingly also blocks the binding of the cytokine to its cell surface receptor. This achievement represented the first synthetic HS-like molecule that targets a cytokine, and provides a potentially powerful strategy to inhibit IFNg. Although this molecule enables us to define the structural organization of the binding site (i.e. two binding octasaccharides spaced apart by an internal domain of appropriate length), the sulfation profile of each of the dp8 (currently homogeneously sulfated) has not been address yet. Our working hypothesis, supported by preliminary results recently obtained, is that only a selected (and specific) number of sulfate groups are actually engaged in the complex with IFNg. By combining structural, chemical, analytical and biological approaches, the goal of our proposal is to characterize the molecular determinants involved in the interaction of IFNg with HS, and to investigate, at the cellular level, the biological relevance of this interaction. In particular, this project should significantly contribute to outline the importance of the sulfation profile within the octasaccharides that interact with IFNg. This important point has never been addressed before, due to the structural heterogeneity of natural HS molecules, but is now possible in the context of the 2O10 template that can be obtained in pure form and large amount by chemical synthesis. Based on these results, we want to enhance the affinity and specificity of such glycoconjugates towards IFNg. An ultimate goal of this work will be the development of an inhibitor of the cytokine, which could be then evaluated in a number of pathology in which the cytokine has been identified as a target. The new knowledge generated in this complex field of biology will be the basis of the development of new therapeutic strategies based on the use of 'small glycanic drugs' to control a fundamental immunologic process. This project results from a long-term and sustained collaboration carried out by partners 1 (glycobiologist) and 2 (organic chemist) in the investigation of the IFNg/HS interaction, bringing together structure/function analysis in the field of protein-HS interaction and HS carbohydrate chemistry. Coupled to partner 3 (mass spectrometry specialist), it will lead to a further step in the characterization of the IFNg/HS complex. Such knowledge and collaboration are shared and mastered by only a very small number of groups over the world. In this regard, the collaboration between the 3 partners, on which this proposal is based is rather unique and has already allowed addressing important issues in the field.