A high-throughput sphingomyelinase assay using natural substrate

作者：Xu Miao; Liu Ke; Southall Noel; Marugan Juan J; Remaley Alan T; Zheng Wei^*

来源：Analytical and Bioanalytical Chemistry, 2012, 404(2): 407-414.

DOI：10.1007/s00216-012-6174-5

• Acid sphingomyelinase
• ASM inhibitors
• High-throughput screening
• Natural enzyme substrate

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 similar to 300,000 compounds using this assay led to the identification of eight compounds as sphingomyelinase inhibitors (IC(50)s = 1.7 to 38.2 mu M) 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.

• 出版日期2012-8
• 单位浙江大学