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A Novel High-Throughput Screening Format to Identify Inhibitors of Secreted Acid Sphingomyelinase

Departments of Molecular Pharmacology and Cardiovascular Research, Berlex Biosciences, Richmond, CA

Kenneth C. Appell

Andrew Cole

Department of Drug Discovery, Pharmacopeia Inc., Princeton, NJ

Anthony Johns

Rene Pagila

Mark A. Polokoff

Departments of Molecular Pharmacology and Cardiovascular Research, Berlex Biosciences, Richmond, CA

Ira Tabas

Departments of Medicine, Anatomy and Cell Biology, and Physiology and Cellular Biophysics, Columbia University, New York, NY

R. Michael Snider

Janet A. Meurer-Ogden

Departments of Molecular Pharmacology and Cardiovascular Research, Berlex Biosciences, Richmond, CA

Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z' factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC₅₀ values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.

Key Words: acid sphingomyelinase • lipid retention • atherosclerosis • high-throughput screening • hydrophobic interaction chromatography

Journal of Biomolecular Screening, Vol. 10, No. 3, 225-234 (2005)
DOI: 10.1177/1087057104272546


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