E-selectin as anti-inflammatory drug target : expression, purification and charactrization for structural studies : assay development for antaginists evaluation

Abstract

E-, P- and L-selectin belong to the C-type lectin family of cell adhesion molecules that initiate inflammatory response. Infammation per se is a physiologocal defense mechanism, but excessive leukcyte extravasation leads to numerous pathoIogical and disease states, as well as metastatic cancer spread. Leukocyte tethering and rolling toward inflammatory site start with the interaction of selectins and the carbohydrate epitope of their glycoprotein ligands, sialyl Lewisx. Therefore inhibitors of selectin-ligand interaction are of high pharmaceutical interest as potent anti-inflammatory agents. Tetrasaccharide sialyl Lewisx serves as a lead strucure in chemical and computational search for selectin antagonists. Structural NMR and X-ray studies indicated binding mode of sialyl Lewisx with E-, and P-selectin, but improved structural studies with the second and third generation antagonists is missing. We expressed recombinant human E-, P- and L-selectin/IgG as secreted proteins in mammalian expression system and purified them to homogniety. Acitivity of the proteins was confirmed with blocking monoclonal antibodies and ligand binding confirmed by NMR. Bioassays were developed in cell-free and cell-based formats with E-selectin/IgG to evaluate inhibitory potencies of in-house synthesized selectin antagonists. Due to variation and instabilities on day-to-day and batch-to-batch basis, assays were used only for preliminary antagonists screen. To enhance further structural studies, we developed a new system for the expression of truncated form of human E-selectin (lectin and EGF-like domains). Initialy we tried to express these two domains in E.coli, but refolding of expressed inclusion bodies was inefficient. Therefore lectin and EGF-like domains of human E-selectin were expressed as secreted form in baculovirus-infeced insect cells with a flag-epitope on its C-terminus. Expressed protein (LecEGFFlag) was monomeric in solution, correctly folded and active, as confirmed in the reaction with monoclonal blocking antibodies, and NMR studies. Protein was expressed in two distinct glycosylation forms, with apparent molecular weigts of 19.96 kDa and 21.15 kDa. In addition, we developed for the first time a cell-free assay with truncated form of Eselectin (aforementioned LecEGFFlag) for the evaluation of of E-selectin inhibitors. In a proof-of-concept manner, three different E-selectin antagonists were tested and obtained IC50 values were in close agreement with published results. Reproducibility and stability of the assay on day-to-day and batch-to batch basis make it suitable not only for the preliminary screening, but also to quantify inhibitory potencies of E-selectin antagonists. Developed system is suitable for expression and similar characterization of P- and Lselectin as well.