A high-throughput sphingomyelinase assay using natural substrate.
A high-throughput sphingomyelinase assay using natural substrate.
复制标题
使用天然底物的高通量鞘磷脂酶测定。
DOI：
10.1007/s00216-012-6174-5
复制
发表时间：
2012
影响因子：
4.3
通讯作者：
Zheng,Wei
中科院分区：
文献类型：
--
作者：
Xu,Miao;Liu,Ke;Southall,Noel;Marugan,JuanJ;Remaley,AlanT;Zheng,Wei
鞘磷脂酶是一组水解酶，能裂解神经鞘磷脂，形成神经酰胺和磷胆碱。鞘磷脂是质膜的常见成分。神经酰胺是一种第二信使，几乎存在于所有类型的细胞中，并调节多种细胞功能，如增殖、分化、凋亡和炎症反应。最近，抑制鞘磷脂酶活性以降低神经酰胺浓度已成为一种潜在的治疗方法，用于治疗包括动脉粥样硬化、病原体感染、炎症、糖尿病和肥胖在内的多种疾病。为了有效地筛选用于鉴定新的鞘磷脂酶抑制剂的化合物集合，我们开发了一种利用1536孔板格式的天然底物鞘磷脂的高通量分析方法。该方法在pH=5.0的缓冲液中的信号/碱基比为6.1倍，在pH=6.5的缓冲液中为4.3倍，表明该方法是一种可靠的化合物文库筛选方法。用该方法筛选约300,000个化合物，鉴定出8个化合物为鞘磷脂酶抑制剂(IC50s = 1.7~38.2.μM)，它们在天然底物法和前荧光底物法中表现出不同的活性。结果表明，天然底物鞘磷脂酶试验用于筛选鞘磷脂酶抑制剂具有良好的稳定性和有效性。
Sphingomyelinases are a group of hydrolases that cleave sphingomyelin, a common component of plasma membranes, to form ceramide and phosphocholine. Ceramide is a second messenger that is present in virtually all cell types and regulates a variety of cellular functions such as proliferation, differentiation, apoptosis, and inflammation response. Inhibition of sphingomyelinase activity to reduce ceramide concentrations has recently emerged as a potential therapeutic approach for several diseases including atherosclerosis, pathogen infections, inflammation, diabetes, and obesity. To effectively screen compound collections for the identification of new sphingomyelinase inhibitors, we have developed a high-throughput assay utilizing the natural substrate sphingomyelin in 1,536-well plate format. The assay has a signal-to-basal ratio of 6.1-fold in pH 5.0 buffer and 4.3-fold in pH 6.5 buffer, indicating a robust assay for compound library screening. A screen of ~300,000 compounds using this assay led to the identification of eight compounds as sphingomyelinase inhibitors (IC50s = 1.7 to 38.2 μM) that exhibited different activities between the natural substrate assay and profluorescence substrate assay. The results demonstrate the robustness and effectiveness of the natural substrate sphingomyelinase assay for screening sphingomyelinase inhibitors.