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

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.