Synthèse d’alpha-C-galactosylcéramides fluorés et évaluation de leurs propriétés immunorégulatrices pour le traitement des maladies auto-immunes systémiques – FluoroC-GalCer

Alpha-galactosylceramides (GalCer) are glycolipids extracted from a marin sponge which exhibited in vivo (animal and human) immunostimulating and immunoregulating properties. Their interaction with iNKT cells, which leads to the expansion and the activation of these cells, is responsible for these properties. The ceramide portion of GalCer molecules binds to the CD1d domain of Antigen Presenting Cells (APC) and the resulting complex is recognised by iNKT cells via an interaction between the galactose unit of GalCer and the TCR (T cell receptor). The iNKT cell is activated by this ternary CPA/GalCer/iNKT complex and thus produces large amounts of cytokines which have two different functions. The Th1 response (IFN-gamma, IL-2, ' cytokines) is pro-inflammatory and was used for anti-cancer and anti-infectious therapy whereas the Th2 response is immunoregulating and can be used for the treatment of systemic auto-immune diseases. The latter are indeed characterized by a depletion of iNKT cells and by a decline of their immunoregulating functions. The therapeutic potential of natural GalCer compounds is unfortunately eroded by their low in vivo stability (like most glycoconjugate molecules) and by their poorly selective immune response (almost no bias in the Th1/Th2 response). Our proposal thus concerns the synthesis of non-hydrolyzable GalCer surrogates designed for inducing a Th2-biased immune response in order to apply their immunoregulation properties to the treatment of systemic auto-immune diseases. In that purpose, CF2- and CFH-glycosidic analogues of GalCer (FluoroC-GalCer compounds), featuring shortened lipidic chains, will be synthesized (organic synthesis team, UMR 6014 Rouen). Their immune response will be evaluated using animal models of autoimmunity (immunology team, UPR 9021 Strasbourg) and a structural study (NMR and molecular modeling), aiming at the assessment of their mimicking properties, will be run (NMR/MolMod team, UMR 6014 Rouen). Our team has a long standing interest in the synthesis of fluorinated C-Glycosides, relying on the hypothesis that these surrogates, stable to chemical and enzymatic hydrolysis, are better sugar mimics than CH2-glycosides thanks to the effects of fluorine atoms. Our FluoroC-GalCer compounds will feature various lipidic chains lengths but short ones will be favoured. It has indeed been demonstrated that a Th2-biased response (immunoregulation) was generally obtained with short ceramide chains. A methodology recently developed by our group, based on the addition of electrophilic fluorinated radicals to glycals, will allow us to prepare alpha-CF2-galactose and alpha-CFH-galactose synthons which will be further functionalized to set up the ceramide chains. The synthesized FluoroC-GalCer compounds will afterwards be subjected to different biological tests in order to assess their immunoregulation properties and their therapeutic potential for the treatment of auto-immune diseases such as systemic lupus erythematosus (SLE). A first ex vivo test with healthy mice will allow us to measure the iNKT cells proliferation and the cytokine production (Th2/Th1 balance), followed by an in vivo experiment (lupus mice) which will assess the iNKT cell expansion and the evolution of the disease. Finally, an ex vivo test, using human cells from SLE patients, will allow us to evaluate the recognition of our molecules by human iNKT cells. A structural study of two FluoroC-GalCer compounds (CF2 and CFH with short lipidic chains) will also be completed (NMR/MolMod team, UMR 6014 Rouen). A conformational analysis, using molecular modeling and 2D-NMR spectroscopy, will allow us to determine if the conformational behaviour of the FluoroC-GalCers is close enough to their natural counterparts to consider them as appropriate surrogates. Finally, a simulation study of the interactions between FluoroC-GalCers and CD1d (docking) using molecular modeling will provide data regarding the binding abilities of our compounds. Both studies (conformational analysis and docking) will provide information that should be helpful to rationalize the immune response induced by the FluoroC-GalCer compounds. Moreover, the combination of these structural data with the results obtained by the Immunology team could help us to design in the future a second generation of ligands, more performing than these first analogues.