Glycomimetic ligands of DC-SINGStructural insights into their molecular recognition by NMR and computational techniques

Resumen

DC-SIGN is a C-type lectin mainly expressed by immature dendritic cells (DCs), located in the skin or peripheral mucosal tissues. It presents a Carbohydrate Recognition Domain (CRD) at the C-terminus, responsible for the recognition of a broad spectrum of pathogens, including HIV-1. This virus exploit the internalization pathway of DC-SIGN to facilitate trans-infections of T cells and to invade the host immune system. Therefore, DC-SIGN is considered a very interesting target for developing new therapeutic agents able to inhibit this receptor. Glycomimetics, are a new class of small-molecule, designed as potential drugs, that mimic the bioactive function of carbohydrates and address the drawbacks of carbohydrate leads, namely their insufficient drug-like properties and weak binding affinities (that for monovalent carbohydrate are tipically in the mM-µM range). In this work appropriate structural techniques, such as high resolution Nuclear Magnetic Resonance (NMR) spectroscopy (mainly tr-NOESY and STD NMR) and computational methods (molecular modelling, CORCEMA-ST) have been combined to fully characterize the interactions of novel glycomimetics with DC SIGN ECD in solution. These compounds are based on two lead structures: the high-mannose oligosaccharides (Man9) and the L-fucose as part of the natural antigen LewisX. In particular, the objectives proposed and achieved in this thesis are: ¿ Identify at atomic level the ligand-receptor contacts that are key for the formation of the complex (ligand epitopes). ¿ Determine the affinities in solution. ¿ Investigate the existence of multiple modes of association. ¿ Characterize the bioactive conformations of the ligands bound to the receptor. The analysis of coupling constants and NOE signals (interprotonic distances), has allowed to restrict the range of possible conformations of the free ligands. This information, combined with conformational searches, molecular dynamics, and docking calculation, has led to three-dimensional model of the ligands in solution, as well as, allowed the characterization of their bioactive conformations and orientation of the ligands in the receptor binding site. STD NMR experiments were performed on each selected compound, in the presence of DC-SIGN (ECD) with an excess of ligand in the sample. The binding epitopes have been characterized using relative STD intensities, as introduced by Mayer and Meyer and the analysis of initial slope, proposed by Mayer and James. Furthermore, in order to obtain the dissociation constant (KD) a protocol based on STD NMR, developed in our group has been employed. The existence of multiple binding modes has been investigated using the protocol (Complete Relaxation and Conformational Exchange Matrix), actually, the multiplicity in molecular recognition of some ligands seems to be a peculiarity of the receptor DC-SIGN. CORCEMA-ST procedure enables the prediction of STD intensities from the Cartesian atomic coordinates of the ligand¿receptor complex, and represents an helpful tool for the quantitative structural interpretation of experimental STD NMR data.