TY - JOUR
T1 - A rapid assay for assessment of sphingosine kinase inhibitors and substrates
AU - Kharel, Yugesh
AU - Mathews, Thomas P.
AU - Kennedy, Andrew J.
AU - Houck, Joseph D.
AU - MacDonald, Timothy L.
AU - Lynch, Kevin R.
N1 - Funding Information:
The authors gratefully acknowledge Robert Stoffel (Abbott Laboratories, Worcester, MA, USA) for his suggestion to take advantage of the physiochemical properties of S1P in developing our assay. The authors also thank Jose Tomsig, (University of Virginia Department of Pharmacology) for his comments on the manuscript. This research was supported in part by research grants from the National Institutes of Health ( R01 GM067958 ) and the Ivy Foundation for Biomedical Research.
PY - 2011/4/15
Y1 - 2011/4/15
N2 - 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-32P]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, Km, and the Ki 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.
AB - 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-32P]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, Km, and the Ki 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.
KW - High-throughput assay
KW - Sphingosine 1-phosphate
KW - Sphingosine kinase
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U2 - 10.1016/j.ab.2011.01.003
DO - 10.1016/j.ab.2011.01.003
M3 - Article
C2 - 21216217
AN - SCOPUS:79952448148
SN - 0003-2697
VL - 411
SP - 230
EP - 235
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 2
ER -