5 R01 R01HG03950
HTS assay development for inhibitors of sickle cell
| Principal Investigator: |
Paul Frenette
MOUNT SINAI SCHOOL OF MEDICINE
DEPT OF MEDICINE
ONE GUSTAVE LEVY PL, BOX 1079
BOX 1079
|
Project Period: 09/23/2005 - 08/31/2008
Abstract (from grant application):
DESCRIPTION (provided by applicant): The selectin family of adhesion molecules plays key role in the rolling and adhesion of leukocytes in systemic venules. Previous studies have shown that sickle erythrocytes can interact with P-selectin expressed on activated endothelium. Other studies have revealed that the adhesion of leukocytes in venules of sickle cell mice played a major role in the vascular occlusion of sickle cell disease (SCD) and that sickle mice lacking both P-and E-selectin genes were protected from sickle vasoocclusion. Selectins bind to glycoconjugated ligands on leukocytes that contain sialyl Lewis X (sLex), a sialylated and fucosylated tetrasaccharide. The synthesis of selectin ligands requires the expression of several glycosyltransferases that modify the carbohydrate composition of specific polypeptide or lipid, allowing high-affinity selectin binding. The role of alpha (1,3)fucose has been well demonstrated using mice lacking leukocyte fucosyltransferases (FucTs). Mice lacking FucTVII, in particular, showed dramatic reductions in the expression of ligands for all three selectins, suggesting that FucTs may represent a useful target for therapeutic intervention. Alpha (1,3)FucTs catalyze the formation of an 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 in cell lysates. Herein, we propose to format this assay for high throughput screening (HTS) for small molecular weight compounds that inhibit alpha (1,3)FucT activity. In this assay, the neoglycoprotein 3'sialyl-Nacetyllactosamine oligosaccharide acceptor will be fucosylated by HL60 cell lysates as a source of leukocyte FucT activity 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 test the reproducibility and robustness of the FucT assay in 384-well plates for HTS. 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 the primary 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 treatment or prevention of vasoocclusive episodes in sickle cell disease.
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