HTS assay development for inhibitors of sickle cell

描述(申请人提供)：选择素家族的黏附分子在系统小静脉中白细胞的滚动和黏附中起关键作用。以往的研究表明，镰状红细胞可以与活化的内皮细胞上表达的P-选择素相互作用。其他研究表明，在镰状细胞病(SCD)的血管闭塞中，小静脉内白细胞的黏附起主要作用，缺乏P-和E-选择素基因的镰状小鼠可免受镰状血管闭塞的影响。选择素与白细胞上含有唾液酸化和岩藻糖基化的四糖唾液酸路易斯X(SLeX)的糖共轭配体结合。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。α(1，3)岩藻糖的作用已经在缺乏白细胞岩藻糖基转移酶(Fucts)的小鼠身上得到了很好的证明。特别是缺乏FucTVII的小鼠，所有三种选择素的配体表达都显著减少，这表明FucT可能是治疗干预的有用靶点。α(1，3)Fucts催化岩藻糖的碳1和N-乙酰氨基葡萄糖的碳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高通量筛选(HTS)抑制α(1，3)Fuct活性的小分子化合物。在本实验中，新糖蛋白3‘唾液酸乙酰乳糖胺低聚糖受体将被HL60细胞裂解产物岩藻糖化，作为白细胞Fuct活性的来源，新合成的唾液酸路易斯X将被HECA-452抗体和过氧化物酶结合抗体特异性检测。在具体目标1中，我们建议在高温超导的384孔板上测试Fuct试验的重复性和稳健性。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，我们建议启动原代HTS，并鉴定能够抑制细胞裂解物和活髓系细胞中白细胞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。这些研究可能为治疗或预防镰状细胞病血管闭塞发作的重要进展铺平道路。

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.