Assay development for high throughput screening of selectin ligand antagonists

描述（由申请人提供）：选择素及其糖缀合配体在全身小静脉中白细胞的滚动和粘附中起关键作用。虽然全身小静脉中的白细胞粘附是抵御感染的重要防线，但白细胞募集失调或过度募集可导致组织损伤，并有助于几种炎症性疾病的病理，包括缺血-再灌注损伤、自身免疫性和过敏性疾病、动脉粥样硬化、血栓形成和镰状细胞病。选择素配体的合成需要几种糖基转移酶的表达，这些酶可以修饰特定多肽或脂质的碳水化合物组成，从而实现高亲和力的选择素结合。在缺乏白细胞聚焦转移酶（FucT）的小鼠中，（1,3）病灶的作用已被清楚地证明。特别是，缺乏FucTVII的小鼠显示所有三种选择素的配体表达显著减少，这表明FucTVII可能是治疗干预的有用靶点。a(1,3)FucTs催化焦点的碳1和n -乙酰氨基葡萄糖的碳3之间形成α -异头糖苷键。我们开发了一种基于elisa的检测方法来快速评估细胞裂解物的FucT活性。在此，我们建议将该试验用于高通量筛选（HTS），以筛选抑制白细胞FucT活性的小分子量化合物。在这个实验中，新的糖蛋白3'sialyl- n - acetyllactosarine oligosaccharide受体将被来自HL60细胞裂解物的FucT活性聚焦，新合成的sialyl Lewis X将被HECA-452抗体特异性检测，然后是过氧化物酶偶联抗体。在Specific Aim 1中，我们提出优化384孔板中HTS的FucT检测。Specific Aim 2将使用统计参数Z'和少量化合物来验证FucT分析。在特异性目标3中，我们建议启动HTS并确定能够抑制细胞裂解物和活髓细胞中白细胞FucT活性的“命中”。我们将进行体外和体内反筛选研究，以进一步评估选定的“命中”或先导化合物的功效和特异性。这些研究可能为开发治疗或预防镰状细胞病和其他炎症性疾病的血管闭塞性发作的新药铺平道路。

DESCRIPTION (provided by applicant): 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.