Assay development for high throughput screening of selectin ligand antagonists

描述(申请人提供)：选择素及其糖共轭配体在系统小静脉中白细胞的滚动和黏附中起关键作用。虽然白细胞在全身小静脉中的黏附是抵抗感染的一条基本防线，但失调或过度的白细胞募集可导致组织损伤，并导致几种炎症性疾病的病理，包括缺血再灌注损伤、自身免疫和过敏性疾病、动脉粥样硬化、血栓形成和镰状细胞疾病。选择素配体的合成需要几种糖基转移酶的表达，这些糖基转移酶可以改变特定多肽或脂类的碳水化合物组成，从而允许高亲和力的选择素结合。A(1，3)岩藻糖的作用已经在缺乏白细胞岩藻糖基转移酶(Fuct)的小鼠身上得到了清楚的证明。特别是，FucTVII基因缺陷的小鼠表现出所有三种选择素的配体表达显著减少，这表明FucT可能是治疗干预的有用靶点。α(1，3)Fucts催化岩藻糖碳1和N-乙酰氨基葡萄糖碳3之间形成α-异构糖苷键。我们已经建立了一种基于ELISA的方法来快速评估细胞裂解物中的Fuct活性。在此，我们建议将该方法用于抑制白细胞Fuct活性的小分子化合物的高通量筛选(HTS)。在本实验中，新糖蛋白3‘唾液酸氨基-N-乙酰乳糖胺低聚糖受体将被来自HL60细胞裂解产物的Fuct活性岩藻糖化，新合成的唾液酸路易斯X将被HECA-452抗体和过氧化物酶标记抗体特异性地检测到。在具体目标1中，我们建议在384孔板上优化HTS的Fuct测定。特殊目标2将使用统计参数Z‘和少量化合物集合来验证Fuct分析。在具体目标3中，我们建议启动HTS并鉴定能够抑制细胞裂解物和活髓系细胞中的白细胞Fuct活性的“HITS”。我们将进行体外和体内的反筛选研究，以进一步评估选定的“HITS”或先导化合物的有效性和特异性。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发作的新药方面取得重要进展铺平道路。

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.