Assay development for high throughput screening of selectin ligand antagonists

描述（由申请方提供）：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。虽然白细胞粘附在系统性小静脉中代表了抵抗感染的基本防线，但调节异常或过度的白细胞募集可引起组织损伤并促成几种炎性疾病的病理学，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，这些糖基转移酶修饰特定多肽或脂质的碳水化合物组成，从而允许高亲和力的选择素结合。使用缺乏白细胞岩藻糖基转移酶（FucT）的小鼠已清楚地证明了α（1，3）岩藻糖的作用。特别地，FucTVII缺陷的小鼠显示出所有三种选择素的配体表达的显著降低，表明FucTs可能代表治疗干预的有用靶点。a（1，3）FucTs催化岩藻糖的碳1和N-乙酰葡糖胺的碳3之间的α异头糖苷键的形成。我们已经开发了一种基于ELISA的测定来快速评估来自细胞裂解物的FucT活性。在此，我们建议将该测定用于抑制白细胞FucT活性的小分子量化合物的高通量筛选（HTS）。在本试验中，新糖蛋白3 '唾液酸-N-乙酰乳肌氨酸寡糖受体将通过源自HL 60细胞裂解物的FucT活性岩藻糖基化，新合成的唾液酸刘易斯X将通过HECA-452抗体特异性检测，然后通过过氧化物酶偶联抗体检测。在具体目标1中，我们建议优化384孔板中HTS的FucT测定。具体目标2将使用统计参数Z'和少量化合物来验证FucT测定。在特定目标3中，我们提出启动HTS并鉴定可以抑制细胞裂解物和活髓样细胞中白细胞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”或先导化合物的疗效和特异性。这些研究可能为开发用于治疗或预防镰状细胞病和其他炎症性疾病中血管闭塞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.