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A high-throughput sphingomyelinase assay using natural substrate

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Abstract

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.

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Acknowledgments

The authors thank Sam Michael for assistance in robotic screen, Paul Shinn for assistance in compound management, and Seameen J. Dehdashti for critical reading of the manuscript. The authors also thank ATT Bioquest for technical assistance on the assay development and optimization. This research was supported by the Molecular Libraries Initiative of the NIH Roadmap for Medical Research (5U54MH084681-02 and RO3MH093173-01) and the Intramural Research Programs of National Heart, Lung and Blood Institute, National Institutes of Health.

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Authors and Affiliations

  1. National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, 20892-3370, USA

    Miao Xu, Ke Liu, Noel Southall, Juan J. Marugan & Wei Zheng

  2. Lipoprotein Metabolism Section, Cardio-Pulmonary Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892-1508, USA

    Alan T. Remaley

  3. Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310016, China

    Miao Xu

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  1. Miao Xu
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  2. Ke Liu
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  3. Noel Southall
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  4. Juan J. Marugan
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  5. Alan T. Remaley
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  6. Wei Zheng
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Correspondence to Wei Zheng.

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Xu, M., Liu, K., Southall, N. et al. A high-throughput sphingomyelinase assay using natural substrate. Anal Bioanal Chem 404, 407–414 (2012). https://doi.org/10.1007/s00216-012-6174-5

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  • DOI: https://doi.org/10.1007/s00216-012-6174-5

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