Selectins and their glycoconjugated ligands play key role in the rolling and adhesion of leukocytes in systemic venules. While leukocyte adhesion in systemic venules represents an essential line of defense against infections, dysregulated or excessive leukocyte recruitment can cause tissue damage and contribute to the pathology of several inflammatory diseases including ischemia-reperfusion injuries, autoimmune and allergic diseases, atherosclerosis, thrombosis, and sickle cell disease. The synthesis of selectin ligands require the expression of several glycosyltransferases that modify the carbohydrate composition of specific polypeptide or lipid, allowing high-affinity selectin binding. The role of a (1,3)fucose has been clearly demonstrated using mice lacking leukocyte fucosyltransferases (FucT). In particular, mice deficient in FucTVII showed dramatic reductions in the expression of ligands for all three selectins, suggesting that FucTs may represent a useful target for therapeutic intervention. a(1,3)FucTs catalyze the formation of alpha anomeric glycosidic bond between carbon 1 of the fucose and carbon 3 of N-acetylglucosamine. We have developed an ELISA-based assay to evaluate rapidly the FucT activity from cell lysates. Herein, we propose to format this assay for high throughput screening (HTS) for small molecular weight compounds that inhibit leukocyte FucT activity. In this assay, the neoglycoprotein 3'sialyl-N-acetyllactosarhine oligosaccharide acceptor will be fucosylated by FucT activity derived from HL60 cell lysates and newly synthesized sialyl Lewis X will be detected specifically by the HECA-452 antibody followed by a peroxidase-conjugated antibody.
In Specific Aim 1, we propose to optimize the FucT assay for HTS in 384-well plates.
Specific Aim 2 will validate the FucT assay using the statistical parameter Z' and with a small collection of compounds.
In Specific Aim 3, we propose to initiate HTS and identify """"""""hits"""""""" that can inhibit leukocyte FucT activity in both cell lysates and live myeloid cells. We will perform in vitro and in vivo counter-screening studies to assess further the efficacy and specificity of selected """"""""hits"""""""" or lead compounds. These studies may pave the way to important progress in the development of new drugs for the treatment or prevention of vasoocclusive episodes in sickle cell disease, and in other inflammatory diseases. ? ? ?
| Méndez-Ferrer, Simón; Chow, Andrew; Merad, Miriam et al. (2009) Circadian rhythms influence hematopoietic stem cells. Curr Opin Hematol 16:235-42 |
| Wagner, Denisa D; Frenette, Paul S (2008) The vessel wall and its interactions. Blood 111:5271-81 |
| Mendez-Ferrer, Simon; Frenette, Paul S (2007) Hematopoietic stem cell trafficking: regulated adhesion and attraction to bone marrow microenvironment. Ann N Y Acad Sci 1116:392-413 |
