title: A rapid assay for assessment of sphingosine kinase inhibitors and substrates.
abstract: Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-(32)P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, K(m), and the K(i) values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors.
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22904, USA. yk2n@virginia.edu
authors.forename: Yugesh
authors.initials: Y
authors.lastname: Kharel
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Mathews
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Kennedy
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Houck
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Macdonald
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Lynch
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000302:isolation & purification; D057166:High-Throughput Screening Assays; D007700:Kinetics; D008246:Lysophospholipids / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012588:Scintillation Counting; D054852:Small Molecule Libraries / Q000737:chemistry; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity; D000099157:Sphingosine Kinase
chemical_list: D000255:Adenosine Triphosphate; D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D010761:Phosphorus Radioisotopes; D017853:Phosphotransferases (Alcohol Group Acceptor); D054852:Small Molecule Libraries; D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate