WP4 - Binding and Structural Studies


WP4 will analyze at the molecular level the binding process of the synthetic tools prepared by this framework to the receptor DC-SIGN. These studies will provide the fundamental structural, kinetic and thermodynamic parameters of binding. The resulting analysis of this data will allow direct understanding of the mode-of-action of DC-SIGN and of the potential activity of each of these chemical systems.

a. The CNRS-IBS group will measure the interaction among synthesized multivalent systems (CRD-monomeric and ECD-tetrameric) and functonalized surface of a Biosensor based on SPR. The binding mode and the affinity between both the Biosensor surface and the multivalent systems will be evaluated. Simultaneously this group will provide DC-SIGN proteins to approach in the evaluation of the compounds properties by NMR or MS. Indeed, the binding mode in the CRD binding site will be studied and evaluated based on the crystals obtained from these ligands with the DC-SIGN-CRD.

b. The Madrid Team will apply tools such as flow cytometry (FACS) and ELISA assays to measure in vitro binding affinities of different multivalent systems with DC-SIGN expressed on cells. Also, competition experiments between the potential synthetic inhibitors and the pathogen glycoproteins will be performed. This group will produce these pathogen glycoproteins and cell lines expressing the receptor DC-SIGN needed for these assays.

c. The Amsterdam group will also analyse binding activity of glycoforms to immature, mature dendritic cells as well as macrophages and compare binding to transfectants of DC-SIGN and L-SIGN Furthermore, this group will determine the DC-SIGN lectin specificity of the glycoforms that do not interfere with other DC expressed C-type lectins such as Langerin present on Langerhans cells. It has recently been documented that specifically Langerin is an important natural barrier to viral infections and its function should therefore not be inhibited by the glycoforms.

d. The Seville team will contribute to this WP using Nuclear Magnetic Resonance (NMR). Using these tools, the 3D structure and dynamics of the carbohydrate and carbohydrate mimic ligands and of the multivalent systems prepared will be studied. NMR bioaffinity methods will be employed to analyze the interaction between these systems and the DC-SIGN receptor provided by the CNRS-IBS team, thus to determine the parts of the molecule necessary for the recognition.

e. The company Anterio will provide the modelling studies of the selected ligands with the CRD of the protein DC-SIGN. Docking studies and molecular mechanics simulations of the resulting complexes that include NMR-derived constraints will allow generating working models of the DC-SIGN–ligand interaction and will be instrumental for ligand refinement. This information together with the NMR data would provide a conformational picture of bound ligands into the CRD.

f. The Oxford Group will contribute using native-MS techniques. Mass spectrometry under ‘native’ (non-denaturing representative of those found in solution of biological relevance) conditions allows observation of ligand-protein interactions directly and allows an estimate of their affinity. The Oxford Group has already used this technique to observe protein-carbohydrate ligand interactions in glycodendriprotein systems (mass up to 300,000 Da).

All these data from NMR, Modelling, MS, etc. will provide very valuable information for the rational design of new ligands and to optimize the current design of the multivalent systems.