National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892-3370, USA.
Anal Bioanal Chem. 2012 Aug;404(2):407-14. doi: 10.1007/s00216-012-6174-5. Epub 2012 Jun 19.
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 (IC(50)s = 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.
鞘磷脂酶是一组水解酶，能够切割鞘磷脂，这种物质是质膜的常见成分，形成神经酰胺和磷酸胆碱。神经酰胺是一种第二信使，存在于几乎所有细胞类型中，调节多种细胞功能，如增殖、分化、凋亡和炎症反应。抑制鞘磷脂酶活性以降低神经酰胺浓度，已成为治疗动脉粥样硬化、病原体感染、炎症、糖尿病和肥胖等多种疾病的潜在方法。为了有效地筛选化合物库以鉴定新的鞘磷脂酶抑制剂，我们开发了一种利用天然底物鞘磷脂的高通量测定法，在 1536 孔板格式中进行。该测定法在 pH 5.0 缓冲液中的信号与基础比值为 6.1 倍，在 pH 6.5 缓冲液中的信号与基础比值为 4.3 倍，表明该测定法非常适合化合物文库筛选。使用该测定法对约 30 万种化合物进行筛选，发现 8 种化合物为鞘磷脂酶抑制剂（IC50 值为 1.7 至 38.2 μM），它们在天然底物测定法和前荧光底物测定法中的活性不同。结果表明，天然底物鞘磷脂酶测定法在筛选鞘磷脂酶抑制剂方面具有稳健性和有效性。
