title: Scintillation Proximity Assay to Detect the Changes in Cellular Dihydrosphingosine 1-Phosphate Levels.
abstract: Compounds that modulate the activity of sphingosine 1-phosphate (S1P)-metabolizing enzymes are expected to be potential therapeutic agents for various diseases. Investigation of their potencies requires not only cell-free but also cell-based assays in which intracellular accumulation/depletion of S1P could be monitored. However, conventional methods have limitations to their simplicity, mainly due to the necessity of a separation process that separates S1P from its related substances. Here, we describe a method utilizing a scintillation proximity assay (SPA) for semi-quantifying intracellular [(3)H]-labeled dihydroS1P ([(3)H]dhS1P), which is also a substrate for S1P-metabolizing enzymes. We found that uncoated yttrium silicate SPA beads could selectively bind to and detect [(3)H]dhS1P rather than [(3)H]dihydrosphingosine (the non-phosphorylated form of [(3)H]dhS1P). Based on this, we developed a novel cell-based assay system which does not require any organic solvent extraction or chromatographic separation, and confirmed its practicality by using siRNA targeting S1P lyase (S1PL) and known S1PL inhibitors as models. Our results demonstrated that this assay is useful for rapid and easy evaluation of S1PL inhibitors, and could be potentially applicable for all compounds that modulate the activity of S1P-metabolizing enzymes.
authors: Modality Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan. otoyo.mamoru.v7@daiichisankyo.co.jp.
authors: Mamoru
authors: M
authors: Ohtoyo
authors: Modality Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan.
authors: Masakazu
authors: M
authors: Tamura
authors: Pain & Neuroscience Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan.
authors: Nobuo
authors: N
authors: Machinaga
authors: Manufacturing Department I, Production Division, Kitasato Daiichi Sankyo Vaccine Co., Ltd., 6-111, Arai, Kitamoto, Saitama, 364-0026, Japan.
authors: Fumihito
authors: F
authors: Muro
authors: Modality Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan.
authors: Ryuji
authors: R
authors: Hashimoto
mesh_terms: D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors*; D000818:Animals; D002478:Cells, Cultured; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004791:Enzyme Inhibitors; D051379:Mice; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D015019:Yttrium / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; C060504:dihydrosphingosine 1-phosphate; D015019:Yttrium; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase); D013110:Sphingosine
keywords: Cell-based assay; Dihydrosphingosine 1-phosphate; S1P lyase; Scintillation proximity assay; Sphingosine 1-phosphate